Encyclopedia of Toxicology, 4th edition, 9 volume set [4 ed.] 0128243155, 9780128243152

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ENCYCLOPEDIA OF TOXICOLOGY

DEDICATION Dedicated to the remediation of planet earth’s largely human-generated centuries of environmental degradation and its resultant climate crisis, and also to the toxicologists who, via scientific research, application, and communication play a critical role in helping ameliorate and forestall further damage to the natural world and to the health of populations and individuals. Equally dedicated to the elimination of strife, injustice, conflict, and divisiveness among the world’s citizens and to the brave people and movements striving to create a peaceful and lawful realm where freedom is presumed, diversity is valued, and equal opportunity is affirmed. And to my mom, Yetty, who celebrated her 95th birthday in 2023.

ENCYCLOPEDIA OF TOXICOLOGY FOURTH EDITION

EDITOR IN CHIEF Philip Wexler Independent Toxicology Information Specialist and U.S. National Library of Medicine (retired)

VOLUME 1

ASSOCIATE EDITORS Mohammad Abdollahi Tehran University of Medical Sciences (TUMS), Tehran, Iran

Shayne Gad Gad Consulting Services, Raleigh, NC, USA

Helmut Greim Technical University of Munich, Freising-Weihenstephan, Germany

Mary Gulumian North West University, Water Research Unit, South Africa

Evangelia I. Iatrou Laboratory of Toxicology and Forensic Chemistry, Medical School, University of Crete, Heraklion, Crete, Greece

Diana Miguez Latitud - LATU Foundation, Technological Laboratory of Uruguay (LATU), Montevideo, Uruguay

Asish Mohapatra Health Risk Assessment and Toxicology Specialist, Environmental Health Program, Health Canada, Calgary, Alberta, Canada

Sidhartha D. Ray Department of Pharmaceutical & Biomedical Sciences, Touro University College of Pharmacy, NY, USA

Jose Tarazona European Food Safety Authority, Parma, Italy and Spanish National Environmental Health Centre (CNSA)Instituto de Salud Carlos III. Ministry of Science and Innovation. Madrid, Spain

Aristidis Tsatsakis Laboratory of Toxicology and Forensic Chemistry, Medical School, University of Crete, Heraklion, Crete, Greece

Timothy Wiegand University of Rochester Medical Center and Strong Memorial Hospital, Rochester, NY, USA

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge MA 02139, United States Copyright © 2024 Elsevier Inc. All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers may always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-824315-2 For information on all publications visit our website at http://store.elsevier.com

Publisher: Oliver Walter Acquisitions Editors: Clodagh Holland-Borosh and Blerina Osmanaj Content Project Managers: Pamela Sadhukhan and Greetal Carolyn Associate Content Project Managers: Nandhini Mahendran and N. Kiruthigadevi Designer: Miles Hitchen

CONTENTS OF VOLUME 1 List of Contributors for Volume 1

xi

Editor Biographies

xix

Foreword

xxv

Preface A critical appraisal of the toxicological aspects of COVID-19 and its vaccines

xxvii 1

Taxiarchis Konstantinos Nikolouzakis, Daniela Calina, Benjamin Brooks, Anca Docea, and Aristides Tsatsakis

Academy of toxicological sciences

13

Aniqa Niha and Sidhartha D Ray

Acceptable daily intake

15

Joshua P Gray

Accutane

17

Dexter W Sullivan Jr.

Acenaphthene

23

Jeb Reece H Grabato, Amelia B Hizon-Fradejas, Sofia Angela P Federico, and Elmer-Rico E Mojica

Acephate

29

MM Ghazimoradi, M Noruzi, and M Sharifzadeh

Acetaldehyde

35

TR Van Vleet and BK Philip

Acetamide

39

Soheil Mohammadi and Alireza Foroumadi

Acetaminophen

43

Kenneth Pawa and Sidhartha D Ray

Acetamiprid

53

David R Wallace

Acetic acid

61

Mariana Tadros and Sidhartha D Ray

Acetone

67

Miklós Péter Kalapos and Veronika Ruzsányi

Acetonitrile

79

Heriberto Robles

2-Acetylaminofluorene

83

Joshua P Gray

Acetylene

89

Sara Mostafalou

v

vi

Contents of Volume 1

Acetylsalicylic acid

95

Rachel Gorodetsky

ACGIH® (American Conference of Governmental Industrial Hygienists)

99

LM Brosseau

Acids

103

Sai Shiva Krishna Prasad Vurukonda and Agnieszka Saeid

Acifluorfen, sodium salt

109

M Noruzi and M Sharifzadeh

Acrolein

115

E Vilanova and D Pamies

Acrylamide

125

Marzieh Rashedinia and Gholamreza Karimi

Acrylic acid

135

Ya O Mezhuev, Aristides Tsatsakis, and A Taghizadehghalehjoughi

Acrylonitrile

139

JD Schumacher

Acute health exposure guidelines

145

Shelley B DuTeaux

Adiponitrile

153

Ayesha Rahman Ahmed

Advances in physiologically based modeling coupled with in vitro-in vivo extrapolation of ADMET: Assessing the impact of genetic variability in hepatic transporters

159

Dimosthenis A Sarigiannis, Antonios K Stratidakis, and Spyridon P Karakitsios

Adverse outcome pathways: Development and use in toxicology

171

Donna S Macmillan and Catherine Willett

Aerosols

183

John T Szilagyi

A-esterase

189

Ida Adeli, Hosna MohammadSadeghi, and Behnaz Bameri

Aflatoxin

193

Elisha Yagudayev and Sidhartha D Ray

Aging

201

Huihui Wang, Yiying Bian, Siqi Yu, Tong Su, Hongbin Wang, Yuanyuan Xu, and Jingbo Pi

Air pollution: Sources, regulation, and health effects

215

PF Duffney, LW Stanek, and JS Brown

Alachlor

229

JD Schumacher

Alar

235

Kristina D Chadwick and Raja S Mangipudy

Albuterol

239

Samantha E Gad

Alchemy

247

PG Maxwell-Stuart

Alcoholic beverages and health effects

253

Parna Haghparast and Tina N Tchalikian

Aldicarb Virginia C Moser

265

Contents of Volume 1

Aldrin

vii 271

Martin P Boland

Algae

277

Timothy J Wiegand

Alkalis

293

Timothy J Wiegand

Alkyl halides

297

Diana Miguez and Jose V Tarazona

Allyl alcohol

301

TM Shashkova, Ya O Mezhuev, and Aristides Tsatsakis

Allyl formate

305

Sushma Ramsinghani

Allylamine

309

Somayeh Salarinejad and Alireza Foroumadi

Alpha-1 adrenergic receptor antagonists

315

Andrew J Chambers and Kirk L Cumpston

Aluminosilicate fibers

319

Mark J Utell, Joseph J Kuchera, and Paul Boymel

Aluminum

329

Shayne C Gad

Aluminum phosphide

335

Mahshid Ataei, Omid Mehrpour, and Mohammad Abdollahi

Amdro

345

M Noruzi, MM Ghazimoradi, and M Sharifzadeh

American Academy of Clinical Toxicology

349

Timothy J Wiegand and Rachel Gorodetsky

American Association of Poison Control Centers

353

Timothy J Wiegand

American Board of Toxicology

357

Ava Jalshgrari, Vivek Lawana, and Sidhartha D Ray

American College of Medical Toxicology (ACMT)

359

Paul M Wax and Timothy J Wiegand

American College of Toxicology

363

Zumar Smith and Sidhartha D Ray

American Industrial Hygiene Association

367

Haji Bahadar

Americium

369

Irena Malátová and V era Be cková

Ames test

377

Robin C Guy

4-Aminobiphenyl

381

Heriberto Robles

Aminoglycosides

385

Adrian A Palmer and Brian W Skinner

4-Aminopyridine Soheil Mohammadi and Alireza Foroumadi

393

viii

Contents of Volume 1

Amiodarone

399

Jeanna M Marraffa

Amitraz

403

Virginia C Moser

Amitrole

409

Atoosa Karimi Babaahmadi and Maryam Armandeh

Ammonia

415

Carolin Bischoff

Ammonium nitrate

421

Sofia Angela P Federico, Amelia B Hizon-Fradejas, Jeb Reece H Grabato, and Elmer-Rico E Mojica

Amphetamines

427

Timothy J Wiegand

Amyl nitrite

435

A Kubic and M Wahl

Anabolic steroids

439

Mahwish Qureshi and Sidhartha D Ray

Analytical toxicology

445

Shayne C Gad

Ancient warfare and toxicology

451

A Mayor

Androgens

457

Eva Israilova, Davidmierhi Pinkhasov, and Sidhartha D Ray

Anesthetics

463

Samaneh Nakhaee and Omid Mehrpour

Aneuploidy

473

David A Eastmond

Angiotensin converting enzyme (ACE) inhibitors

477

Timothy J Wiegand and Henry A Spiller

Aniline

483

Shayne C Gad

Animal and computational models in toxicology and pharmacology

489

Marzieh Daniali and Mohammad Abdollahi

Animal models

495

Shayne C Gad

Animal venoms in medicine

499

Asieh Karimani, Vahideh Ghorani, and Ramin Rezaee

Animals, poisonous and venomous

505

T Dodd-Butera, M Broderick, and Cecilia Meza

Anthracene

515

Jeb Reece H Grabato, Amelia B Hizon-Fradejas, Sofia Angela P Federico, and Elmer-Rico E Mojica

Anthrax

521

Ryan E Fabian Campusano and Sidhartha D Ray

Antibacterial agents

525

Roberto Maldonado, Vera Bulakhova, Manish Varma, Numair Mukhtar, Dorina Birce, and Sidhartha D Ray

Anticancer therapeutic agents Inna Khodos and Sidhartha D Ray

549

Contents of Volume 1

Anticholinergics

ix 567

Michael Liu

Anti diabetic agents

573

Sidhartha D Ray, Azhar Hussain, Aniqa Niha, Michael Krmic, Ava Jalshgrari, Diana Genis, and Jisha Reji

Antidotes

591

Christy Turco and Sidhartha D Ray

Antifungal agents

603

Gina Bertelli, Monica Sciturro, Sidhartha D Ray, and Mayur S Parmar

Antimicrobial agents

615

Ryan E Fabian Campusano, Rodina Abdelhady, David Guirguis, Silvia Abdelmalak, Mariam Shaker, and Sidhartha D Ray

Antimicrobial resistance – Impact on humans

629

Arjun Bagai, Arathi Kulkarni, and Mayur S Parmar

Antimicrobial resistance and the environment

643

Matí as Giménez, Fernanda Azpiroz, Josefina Vera, and Silvia B Batista

Antimony

653

Shayne C Gad

Antimony trioxide

659

Shayne C Gad

Antiprotozoal medicines

665

Preeti Patel, Amritaparna Sengupta, Ashish Patel, and Sidhartha D Ray

Antiviral agents

691

Kenny Lee, Roberto Maldonado, Saqib Khan, and Sidhartha D Ray

Anxiolytics

715

Parna Haghparast, Thao Nguyen, and Sidhartha D Ray

Apoptosis

731

Sidhartha D Ray, Ningning Yang, Aniqa Niha, and Diana Genis

Apoptosis, necrosis, and other forms of cell death

749

Aarthi Nivasini Mahesh, Nicole Lim Si En, Mei Hsuan Wong, Sidhartha D Ray, and Shruti Bhatt

Applied toxicology at the Agency for Toxic Substances and Disease Registry (ATSDR)

761

Scott Sudweeks, Kai Elgethun, Henry Abadin, Greg Zarus, and Elizabeth Irvin

Aramite

769

Ayesha Rahman Ahmed

Aristolochic acids and aristolactams

775

Michael Heinrich

Arsenic

781

Robert W Kapp Jr

Arsenical vomiting agents

791

Leila Etemad, Mahdi Balali-Mood, and Mohammad Moshiri

Arsine

801

Fred Farris and Mohammed Islam

Arts, crafts, theater, and entertainment

813

Farzaneh Kefayati and Maryam Armandeh

Asbestos

821

Marí a-Belén Nieto, Antonio J Garcí a-Fernández, and Isabel Navas

Asia Pacific Association of Medical Toxicology (APAMT) Mahdi Balali-Mood and Hossein Hassanian-Moghaddam

831

x

Contents of Volume 1

Aspartame

835

Robin C Guy

Astemizole

839

Timothy J Wiegand

Atrazine

845

J Liu and Lucí a Pareja

Atropine

851

Amanda Lofton Scott and Timothy J Wiegand

Avermectin

857

Mohsen Amin and Navid Mirmohammadsadegh

Azamethiphos

867

Raúl A Alzogaray and Eduardo N Zerba

Azathioprine

871

Sara Salcedo, Emma Martí nez-López, and Antonio Juan Garcí a-Fernández

Azinphos-Methyl

881

Prabhakar Mishra, Yuvashree Muralidaran, and Shraddha Bijalwan

Bacillus cereus

889

Kyle Buckley and Janet Grotticelli

Bacillus thuringiensis

893

MC Astuto and I Cattaneo

Barbiturates

903

Caitlin Frohnapple, Flavia Nobay, and Nicole M Acquisto

Barium

911

Shayne C Gad

Batrachotoxin

917

Mersal Danai and Sidhartha D Ray

BCNU (bischloroethyl nitrosourea) induced toxicity

923

Dipan B Ray

Behavioral toxicology

927

Edward D Levin

Belladonna alkaloids

933

Momina Qureshi and Sidhartha D Ray

Benchmark dose

939

M Noruzi, Mohammad Amin Rezvanfar, and Seyed Mojtaba Daghighi

Benfluralin

945

M Noruzi and M Sharifzadeh

Benomyl

951

Hosna MohammadSadeghi, Ida Adeli, and Behnaz Bameri

Benz[a]anthracene

957

Joshua P Gray

Benzene Charles C Barton

961

LIST OF CONTRIBUTORS FOR VOLUME 1 Henry Abadin Office of Innovation and Analytics, Agency for Toxic Substances and Disease Registry, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States Rodina Abdelhady Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Silvia Abdelmalak Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Mohammad Abdollahi Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Nicole M Acquisto University of Rochester Medical Center, Rochester, NY, United States Ida Adeli Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Ayesha Rahman Ahmed Department of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, WA, United States Raúl A Alzogaray Centro de Investigaciones de Plagas e Insecticidas (UNIDEF-CITEDEF-CONICET-CIPEIN), Villa Martelli, provincia de Buenos Aires, Argentina;

Escuela de Hábitat y Sostenibilidad, Universidad Nacional de San Martí n, San Martí n, provincia de Buenos Aires, Argentina Mohsen Amin Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Maryam Armandeh Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran MC Astuto European Food Safety Authority, Parma, Italy Mahshid Ataei Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran Fernanda Azpiroz Sección de Fisiologí a & Genética Bacterianas, Facultad de Ciencias, UdelaR, Montevideo, Uruguay Atoosa Karimi Babaahmadi Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Arjun Bagai Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Clearwater, FL, United States Haji Bahadar Institute of Pharmaceutical Sciences, Khyber Medical University Peshawar, Peshawar, Pakistan

xi

xii

List of Contributors for Volume 1

Mahdi Balali-Mood Mashhad University of Medical Sciences, Mashhad, Iran; Medical Toxicology and Drug Abuse Research Center, Birjand University of Medical Sciences, Birjand, Iran Behnaz Bameri Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Charles C Barton JUUL Inc., Washington, DC, United States Silvia B Batista Laboratorio Microbiologí a Molecular, Departamento de Bioquí mica y Genómica Microbiana, Instituto de Investigaciones Biológicas Clemente Estable, Ministerio de Educación y Cultura, Montevideo, Uruguay Vera Becková National Radiation Protection Institute (SÚRO, v.v.i.), Praha, Czech Republic

Benjamin Brooks Laboratory of Toxicology and Forensic Chemistry, Medical School, University of Crete, Heraklion, Crete, Greece LM Brosseau Colfax South LLC, Minneapolis, MN, United States JS Brown U.S. Environmental Protection Agency, Office of Research and Development, Center for Public Health and Environmental Assessment, Research Triangle Park, NC, United States Kyle Buckley Touro College of Osteopathic Medicine, New York, NY, United States Vera Bulakhova Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Daniela Calina Department of Clinical Pharmacy, University of Medicine and Pharmacy of Craiova, Craiova, Romania

Gina Bertelli Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Clearwater, FL, United States

I Cattaneo European Food Safety Authority, Parma, Italy

Shruti Bhatt Department of Pharmacy, School of Science, National University of Singapore, Singapore, Singapore

Kristina D Chadwick Bristol-Myers Squibb, Early Development Leadership, Princeton, NJ, United States

Yiying Bian Program of Environmental Toxicology, China Medical University, Shenyang, China

Andrew J Chambers Virginia Commonwealth Univeristy Health System, Department of Emergency Medicine, Section of Clinical Toxicology, Richmond, VA, United States

Shraddha Bijalwan Department of Biotechnology, School of Applied Sciences, REVA University, Bengaluru, Karnataka, India Dorina Birce Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Carolin Bischoff Regulatory Toxicology, BASF SE, Ludwigshafen am Rhein, Germany Martin P Boland Leidos Australia, Scoresby, VIC, Australia Paul Boymel Independent Consultant—Technology Management and Material Science, Naples, FL, United States; Worldwide Vice President, Technology, Unifrax I LLC Information, Tonawanda, NY, United States M Broderick California Poison Control System, San Diego, CA, United States

Kirk L Cumpston Virginia Commonwealth Univeristy Health System, Department of Emergency Medicine, Section of Clinical Toxicology, Richmond, VA, United States Seyed Mojtaba Daghighi Faculty of Pharmacy, Pharmaceutical Sciences Research Center (PSRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran Mersal Danai Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Marzieh Daniali Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran

List of Contributors for Volume 1

Anca Docea Department of Toxicology, University of Medicine and Pharmacy of Craiova, Craiova, Romania T Dodd-Butera Azusa Pacific University, Azusa, CA, United States PF Duffney U.S. Environmental Protection Agency, Office of Research and Development, Center for Public Health and Environmental Assessment, Research Triangle Park, NC, United States Shelley B DuTeaux Human Health Assessment Branch, Department of Pesticide Regulation, California Environmental Protection Agency, Sacramento, CA, United States David A Eastmond Department of Molecular, Cell and Systems Biology, University of California, Riverside, CA, United States Kai Elgethun Office of Community Health Hazard Assessment, Agency for Toxic Substances and Disease Registry, National Centers for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States Leila Etemad Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran Ryan E Fabian Campusano Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Fred Farris School of Pharmacy, West Coast University, Los Angeles, CA, United States Sofia Angela P Federico Institute of Chemistry, University of the Philippines Los Baños, College, Laguna, Philippines Alireza Foroumadi Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; Drug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran Caitlin Frohnapple University of Rochester Medical Center, Rochester, NY, United States Samantha E Gad Gad Consulting Services, Raleigh, NC, United States

xiii

Shayne C Gad Gad Consulting Services, Raleigh, NC, United States Antonio J García-Fernández Toxicology and Risk Assessment Group, IMIB-Pascual Parrilla, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain; Area of Toxicology, Department of Health Sciences, University of Murcia, Campus de Espinardo, Murcia, Spain Diana Genis Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States MM Ghazimoradi Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Vahideh Ghorani International UNESCO Center for Health-Related Basic Sciences and Human Nutrition, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran Matías Giménez Laboratorio Microbiologí a Molecular, Departamento de Bioquí mica y Genómica Microbiana, Instituto de Investigaciones Biológicas Clemente Estable, Ministerio de Educación y Cultura, Montevideo, Uruguay; Laboratorio de Genómica Microbiana, Institut Pasteur Montevideo, Uruguay Rachel Gorodetsky D’Youville University School of Pharmacy, Buffalo, NY, United States; University of Rochester Medical Center, Rochester, NY, United States Jeb Reece H Grabato Institute of Chemistry, University of the Philippines Los Baños, College, Laguna, Philippines Joshua P Gray U.S. Coast Guard Academy, New London, CT, United States Janet Grotticelli Touro College of Osteopathic Medicine, New York, NY, United States David Guirguis Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Robin C Guy Robin Guy Consulting, LLC, Lake Forest, IL, United States

xiv

List of Contributors for Volume 1

Parna Haghparast Department of Pharmacy Practice, West Coast University, School of Pharmacy, Los Angeles, CA, United States Hossein Hassanian-Moghaddam Shahid Beheshti University of Medical Sciences, Tehran, Iran Michael Heinrich UCL School of Pharmacy, University College London, London, United Kingdom; China Medical University, Taichung city, Taiwan Amelia B Hizon-Fradejas Institute of Chemistry, University of the Philippines Los Baños, College, Laguna, Philippines Azhar Hussain Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Elizabeth Irvin Office of Community Health Hazard Assessment, Agency for Toxic Substances and Disease Registry, National Centers for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States Mohammed Islam School of Pharmacy, American University of the Health Sciences, Signal Hill, CA, United States Eva Israilova Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Ava Jalshgrari Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Miklós Péter Kalapos Theoretical Biology Research Group, Budapest, Hungary Robert W Kapp Jr BioTox, Lacey Township, NJ, United States Spyridon P Karakitsios Department of Chemical Engineering, Environmental Engineering Laboratory, Aristotle University of Thessaloniki, Thessaloniki, Greece; HERACLES Research Center on the Exposome and Health, Center for Interdisciplinary Research and Innovation, Thessaloniki, Greece Asieh Karimani Department of Pharmacology and Toxicology, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

Gholamreza Karimi Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran; Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran Farzaneh Kefayati Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Saqib Khan Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Inna Khodos Memorial Sloan Kettering Cancer Center, Mortimer B. Zuckerman Research Center, New York, NY, United States Michael Krmic Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States A Kubic Illinois Poison Center, Chicago, IL, United States Joseph J Kuchera Product Stewardship Consultant, Alkegen, Tonawanda, NY, United States; Former Vice President of Product Stewardship, Alkegen, Tonawanda, NY, United States Arathi Kulkarni Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Fort Lauderdale, FL, United States Vivek Lawana North American Science Associates, LLC, Minneapolis, MN, United States Kenny Lee Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Edward D Levin Duke University, Durham, NC, United States Nicole Lim Si En Department of Pharmacy, School of Science, National University of Singapore, Singapore, Singapore J Liu Oklahoma State University, Stillwater, OK, United States

List of Contributors for Volume 1

xv

Michael Liu Department of Pharmacy Practice, Touro College of Pharmacy, New York, NY, United States

Prabhakar Mishra Department of Biotechnology, School of Applied Sciences, REVA University, Bengaluru, Karnataka, India

Donna S Macmillan Humane Society International, Washington, DC, United States

Soheil Mohammadi Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Aarthi Nivasini Mahesh Department of Pharmacy, School of Science, National University of Singapore, Singapore, Singapore Irena Malátová National Radiation Protection Institute (SÚRO, v.v.i.), Praha, Czech Republic Roberto Maldonado Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Raja S Mangipudy Pfizer, Drug Safety R&D and Comparative Medicine, Groton, CT, United States Jeanna M Marraffa Department of Emergency Medicine, Upstate NY Poison Center, Upstate Medical University, Syracuse, NY, United States Emma Martínez-López Area of Toxicology, Department of Health Sciences, University of Murcia, Campus de Espinardo, Murcia, Spain PG Maxwell-Stuart University of St Andrews, St Andrews, UK A Mayor Stanford University, Palo Alto, CA, United States Omid Mehrpour Data Science Institute, Southern Methodist University, Dallas, TX, USA; Michigan Poison & Drug Information Center, Wayne State University School of Medicine, Detroit, MI, United States Cecilia Meza Azusa Pacific University, Azusa, CA, United States Ya O Mezhuev Mendeleev University of Chemical Technology of Russia, Moscow, Russia

Hosna MohammadSadeghi Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Elmer-Rico E Mojica Department of Chemistry and Physical Sciences, Pace University, New York, NY, United States Virginia C Moser Independent Consultant, Apex, NC, United States Mohammad Moshiri Medical Toxicology and Drug Abuse Research Center, Birjand University of Medical Sciences, Birjand, Iran; Department of Clinical Toxicology and poisoning, Imam Reza (p) Hospital, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Sara Mostafalou Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran Numair Mukhtar Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Yuvashree Muralidaran Department of Biotechnology, School of Applied Sciences, REVA University, Bengaluru, Karnataka, India Samaneh Nakhaee Medical Toxicology and Drug Abuse Research Center (MTDRC), Birjand University of Medical Sciences, Birjand, Iran

Diana Miguez Water and Environment Division, Latitud – Fundación LATU, Montevideo, Uruguay

Isabel Navas Toxicology and Risk Assessment Group, IMIB-Pascual Parrilla, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain

Navid Mirmohammadsadegh Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Thao Nguyen Department of Pharmacy Practice, West Coast University, School of Pharmacy, Los Angeles, CA, United States

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List of Contributors for Volume 1

María-Belén Nieto Area of Toxicology, Faculty of Veterinary Medicine, Campus de Espinardo, University of Murcia, Murcia, Spain

Mahwish Qureshi Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States

Aniqa Niha Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States

Momina Qureshi Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States

Taxiarchis Konstantinos Nikolouzakis Laboratory of Toxicology and Forensic Chemistry, Medical School, University of Crete, Heraklion, Crete, Greece

Sushma Ramsinghani University of the Incarnate Word, San Antonio, TX, United States

Flavia Nobay University of Rochester Medical Center, Rochester, NY, United States

Marzieh Rashedinia Department of Pharmacodynamics and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

M Noruzi Faculty of Pharmacy, Pharmaceutical Sciences Research Center (PSRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran Adrian A Palmer Marian University College of Osteopathic Medicine, Indianapolis, IN, United States D Pamies Universidad Miguel Hernández de Elche, Elche, Spain Lucía Pareja Departamento de Quí mica del Litoral, Cenur Litoral Norte, Universidad de la República, Paysandú, Uruguay Mayur S Parmar Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Clearwater, FL, United States Ashish Patel B.J.Medical College, Gandhinagar, Gujarat, India Preeti Patel StatPearls, Monroe, NJ, United States Kenneth Pawa Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States BK Philip AbbVie, North Chicago, IL, United States Jingbo Pi Program of Environmental Toxicology, China Medical University, Shenyang, China Davidmierhi Pinkhasov Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States

Dipan B Ray Experiential Education, Touro College of Pharmacy, New York, NY, United States Sidhartha D Ray Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Jisha Reji Touro College of Osteopathic Medicine, Harlem, NY, United States Ramin Rezaee International UNESCO Center for Health-Related Basic Sciences and Human Nutrition, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran Mohammad Amin Rezvanfar Faculty of Pharmacy, Pharmaceutical Sciences Research Center (PSRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran Heriberto Robles Enviro-Tox Services, Inc., Irvine, CA, United States Veronika Ruzsányi Breath Research Institute, University of Innsbruck, Innsbruck, Austria; Tyrolean Cancer Research Institute, Innsbruck, Austria Agnieszka Saeid Department of Engineering and Technology of Chemical Processes, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland Somayeh Salarinejad Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

List of Contributors for Volume 1

Sara Salcedo Area of Toxicology, Department of Health Sciences, University of Murcia, Campus de Espinardo, Murcia, Spain Dimosthenis A Sarigiannis Department of Chemical Engineering, Environmental Engineering Laboratory, Aristotle University of Thessaloniki, Thessaloniki, Greece; University School for Advanced Study (IUSS), Department of Science, Technology and Society, Environmental Health Engineering, Pavia, Italy; HERACLES Research Center on the Exposome and Health, Center for Interdisciplinary Research and Innovation, Thessaloniki, Greece; National Hellenic Foundation, Athens, Greece JD Schumacher Bristol-Myers Squibb Co., Nonclinical Safety Portfolio Leadership, New Brunswick, NJ, United States Monica Sciturro Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Clearwater, FL, United States Amanda Lofton Scott Allston, MA, United States Amritaparna Sengupta Xcovery Holding Inc, Edison, NJ, United States Mariam Shaker Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States M Sharifzadeh Faculty of Pharmacy, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran TM Shashkova Mendeleev University of Chemical Technology of Russia, Moscow, Russia Brian W Skinner Marian University College of Osteopathic Medicine, Indianapolis, IN, United States Zumar Smith Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Henry A Spiller Kentucky Regional Poison Control Center, Louisville, KY, United States

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LW Stanek U.S. Environmental Protection Agency, Office of Research and Development, Center for Public Health and Environmental Assessment, Research Triangle Park, NC, United States Antonios K Stratidakis University School for Advanced Study (IUSS), Department of Science, Technology and Society, Environmental Health Engineering, Pavia, Italy Tong Su Group of Chronic Disease and Environmental Genomics, China Medical University, Shenyang, China Scott Sudweeks Office of Community Health Hazard Assessment, Agency for Toxic Substances and Disease Registry, National Centers for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States Dexter W Sullivan Jr. Gad Consulting Services, Raleigh, NC, United States John T Szilagyi Bristol-Myers Squibb Co., Summit, NJ, United States Mariana Tadros Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States A Taghizadehghalehjoughi Seyh Edebali University, Faculty of Medicine, Department of Medical Pharmacology, Bilecik, Turkey Jose V Tarazona Risk Assessment Unit, National Centre for Environmental Health, Instituto de Salud Carlos III, Madrid, Spain Tina N Tchalikian Department of Pharmacy Practice, West Coast University, School of Pharmacy, Los Angeles, CA, United States Aristides Tsatsakis Laboratory of Toxicology and Forensic Chemistry, Medical School, University of Crete, Heraklion, Crete, Greece Christy Turco Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Mark J Utell Professor Emeritus of Medicine and Environmental Medicine, Pulmonary and Critical Care Division, University of Rochester Medical Center, Rochester, NY, United States

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List of Contributors for Volume 1

TR Van Vleet AbbVie, North Chicago, IL, United States Manish Varma Touro School of Medicine, New York, NY, United States Josefina Vera Laboratorio Microbiologí a Molecular, Departamento de Bioquí mica y Genómica Microbiana, Instituto de Investigaciones Biológicas Clemente Estable, Ministerio de Educación y Cultura, Montevideo, Uruguay E Vilanova Universidad Miguel Hernández de Elche, Elche, Spain Sai Shiva Krishna Prasad Vurukonda Department of Engineering and Technology of Chemical Processes, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland M Wahl Illinois Poison Center, Chicago, IL, United States David R Wallace Department of Pharmacology, Oklahoma State University Center for Health Sciences, Tulsa, OK, United States Hongbin Wang Group of Chronic Disease and Environmental Genomics, China Medical University, Shenyang, China

Timothy J Wiegand University of Rochester Medical Center and Strong Memorial Hospital, Rochester, NY, USA Catherine Willett Humane Society International, Washington, DC, United States Mei Hsuan Wong Department of Pharmacy, School of Science, National University of Singapore, Singapore, Singapore Yuanyuan Xu Group of Chronic Disease and Environmental Genomics, China Medical University, Shenyang, China Elisha Yagudayev Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Ningning Yang Department of Pharmaceutical Sciences, LECOM School of Pharmacy, Bradenton, FL, United States Siqi Yu Program of Environmental Toxicology, China Medical University, Shenyang, China

Huihui Wang Group of Chronic Disease and Environmental Genomics, China Medical University, Shenyang, China

Greg Zarus Office of Innovation and Analytics, Agency for Toxic Substances and Disease Registry, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, United States

Paul M Wax Emergency Medicine, UT Southwestern School of Medicine, Dallas, TX, United States; American College of Medical Toxicology, Phoenix, AZ, United States

Eduardo N Zerba Escuela de Hábitat y Sostenibilidad, Universidad Nacional de San Martí n, San Martí n, provincia de Buenos Aires, Argentina

EDITOR BIOGRAPHIES Editor-in-Chief

Philip Wexler has published, taught, and otherwise lectured extensively in the United States and abroad in the disciplines of toxicology and toxico-informatics. He is the Editor-in-Chief of four editions of the Encyclopedia of Toxicology (Elsevier. 4th edition 2023) and five editions of Information Resources in Toxicology (Elsevier. 5th edition 2020), as well as Chemicals, Environment, Health: A Global Management Perspective (CRC Press/Taylor and Francis, 2011). He has served as an Associate Editor for Toxicology Information and Resources for Elsevier’s journal, Toxicology and in that capacity, edited special issues on Digital Information and Tools. He is also overseeing and editing an ongoing monographic series on toxicology history. He is one of the senior editors of the Taylor and Francis journal, Global Security: Health, Science, and Policy. A longtime member of the U.S. Society of Toxicology (SOT), he has served as Chair of its World Wide Web Advisory Team and president of its Ethical, Legal, and Social Issues Specialty Section as well as a member of the Education and Communications Work Group of the U.S. CDC/ATSDR’s National Conversation on Public Health and Chemical Exposure. He is a trustee of the Toxicology Education Foundation (TEF). Mr. Wexler is retired from a distinguished U.S. Government career with the National Library of Medicine’s (NLM) former Toxicology and Environmental Health Information Program where he participated in and served as team leader for a spectrum of toxicology databases and initiatives. He is a recipient of SOT’s Public Communication Award, the NLM Regents Award for Scholarly or Technical Achievement, and the Distinguished Technical Communication Award of the Washington chapter of the Society for Technical Communication. During his toxicological downtime, Mr. Wexler writes poetry, with five collections to his credit, and recreationally works in mosaics.

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Associate Editors Mohammad Abdollahi is a highly respected Toxicopharmacological scientist who has made significant contributions to Mechanistic Toxicology. He also focused on Drug and Poison Information Centers and co-established the Tehran Center. He helped TUMS in establishing Digital Journals management system along with the digital library. He was elected and worked for more than 10 years as the Director of the Toxicology Board and the National Society. Through his research, he has identified biomarkers of diseases caused by environmental toxicants. MA’s groundbreaking work has earned him numerous national and international awards, including the prestigious IAS-COMSTECH 2005 and Clarivate Highly Cited 2021 awards and prestigious medals from IFIA for his inventions. His collaborations with various departments on innovative projects, such as the OPCW, WHO, COPE, CINVU/COMSTECH/OIC, UNOG-UNEP-Chemicals (SAICM), and the IAS, are remarkable. Additionally, MA has been recognized as the Best Leader Awardee of Medicine in 2022 and 2023 reported by Research.com. In addition to his research, MA also served as the Associate Editor of the 3rd edition of the “Encyclopedia of Toxicology,” a highly regarded reference book in the field. The Editor-in-Chief of the 3rd and 4th editions is Philip Wexler, a renowned toxicologist, who worked at the National Library of Medicine (NLM) and has written several books on toxicology. The 4th edition of Encyclopedia of Toxicology addresses the challenges of a rapidly changing world. With its comprehensive coverage, it will undoubtedly become the go-to resource for all queries in this field. More information is noted at https://orcid.org/0000-0003-0123-1209. Shayne C. Gad, Ph.D., D.A.B.T., BS Chemistry/Biology Whittier College, Ph.D. Pharmacology/Toxicology University of Texas, 1977, was past president of American College of Toxicology and Roundtable of Toxicology Consultants. He is an expert in neurotoxicology, inhalation toxicology, biocompatibility assessment, statistics and risk assessment, and biopharmaceutical and medical devices safety assessment and development. He successfully prepared and filed 129 IND’s, and was principal of GAD Consulting Services since 1993. He had published 53 books, 75 chapters, 387 abstracts and presentations, and more than 400 sections in large works and encyclopedias. He was a member of SOT, ACT, STP, Safety Pharmacological Society American Statistical; association teacher and adjunct professor at USC and University of Addis Ababa; and has taught courses globally.

Helmut Greim, born in 1935, has studied medicine at the universities of Freiburg and Berlin, Germany. Thereafter, he had research positions in Biochemistry and Pharmacology at the Free University of Berlin and of the Institute of Toxicology, University of Tübingen, 1970–73 as Visiting Research Associate Professor of Pathology, Mount Sinai School of Medicine and Visiting Fellow of Pharmacology, Yale University. After 2 years back in Tübingen, he was appointed as Director of the Institute of Toxicology of the GSF, a federal Research Institute in Munich, in 1975. In 1982, he became Professor and Director of the Institute of Toxicology and Environmental Hygiene, Technical University Munich. He retired from these positions in 2003. His research experience was in the fields of drug metabolism, toxicokinetics, mechanisms of carcinogenic agents, and in vitro test systems. Besides many publications and contributions to textbooks, he has published three German (1996, 2017, 2022), two English (2008, 2019), and one Italian (2000) textbooks in Toxicology (Wiley) and “The cellular response to the genotoxic insult: the question of threshold for genotoxic carcinogens” (the Royal Society of Chemistry, London, 2013). He has organized several workshops on fiber and particle toxicology and on benzene toxicity and as chair of MAK and SCHER of DG SANCO (EU Commission), member of SCOEL and RAC of ECHA, and has been involved in the risk assessment of chemicals.

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Mary Gulumian was the Head of the Toxicology and Biochemistry Section at the National Institute for Occupational Health (NIOH) and thereafter Head of Toxicology Research Projects, at the NIOH. Presently, she holds an honorary Professorial post in the Hematology and Molecular Medicine Department, the University of the Witwatersrand where she has presented courses on Health Risk Assessment and supervised postgraduate students. She has also been appointed as an Extraordinary Professor at the North-West University where she organized workshops on Health Risk assessment. She is the founder member and the past President of the Society for Free Radical Society of South Africa (SFRR-SA), the founder member and President of the Toxicology Society of South Africa (TOXSA), and the founder member and President of the Society of Risk Analysis (SRA)-Africa. Her research interests include hazard identification and communication as well as elucidation of mechanisms of toxicity of micro and nano particles. She received funding from local (DSI and MHSC) and international agencies (EU projects) to conduct research on the topic. She has authored and co-authored numerous scientific publications on this topic and made a great number of keynote and invited presentations at local and overseas conferences. She has also provided expert consultation to industry and government departments on the toxicity of chemicals in the working and ambient environments. Professor Gulumian is a member of the Editorial Board of the Journal, Human and Experimental Toxicology (HET), Toxicology, and Particle and Fibre Toxicology (PFT), and also the Associate Editor of the Journal, Inhalation Toxicology. Evangelia Iatrou was born in Heraklion, Greece, in 1986. In 2008, she graduated from the Department of the Environment, School of the Environment, University of the Aegean. In 2009, she got her master’s degree in environmental and Ecological Engineering at the University of the Aegean. Her master thesis concerned the “Pesticides photodegradation study and assessment of their combined toxicity in Lemna minor.” She has worked in the Laboratory of Toxicology and Forensic Sciences, Medical School, University of Crete for the period 2009–10. Afterward, she turned back at University of Aegean, and she made her doctoral thesis in Environmental Sciences, titled “Study of the fate of antimicrobial substances in artificial wetland systems planted with the Lemna minor organism and investigation of the possibilities of utilization of the produced biomass.” During her Ph.D., she provided lectures as ancillary work for the following courses: Ecotoxicology, Analytical Chemistry, and Wastewater Management. Since then (2017), she is a postdoctoral researcher in Laboratory of Toxicology and Forensic Sciences, Medical School, University of Crete. Evangelia Iatrou has published original articles in peer-reviewed international journals, abstracts, and presentations in national and international congresses. She has participated in national and international congresses and in educational EUROTOX Advanced Course at year 2013. She is also a reviewer in the scientific journals Toxicology Reports and Ecotoxicology and Environmental Safety. Furthermore, Evangelia Iatrou is an adult educator in Second Chance Schools, teaching Environmental Education. She has organized various speeches and awareness raising campaigns on environmental issues in the context of school activities. Diana Míguez is a Principal Research Scientist and Water Program Director at Latitud—LATU Foundation, Technological Laboratory of Uruguay (LATU) since 2017. Pharmaceutical chemist (UDELAR, 1989) and Ph.D. in Water Sciences, Cranfield University, UK, 2014. Thesis: Integrated Risk Assessment of Endocrine Disruptors in the Uruguay River. Dr. Míguez possesses more than 30 years of experience in Analytical Chemistry, Water Science and Technology, Environmental Toxicology, Ecotoxicology, Medical Geology, Sustainability, and Risk Assessment fields. She held prior posts in pharmaceutical and food analyses, and since 1991, she is the head of the Water and Chemicals Department, and senior specialist at LATU.

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Editor Biographies

Asish Mohapatra is a toxicologist and a human health risk assessment specialist with Health Canada. He has a masters’ degree in Life Sciences, a predoctoral degree in Environmental Sciences (chemical toxicology), and a graduate certificate in environmental management from the University of Calgary, and certificates in case works in toxicology and public health risk analysis from Harvard University. His 25 years of work experience includes toxicology and human health risk assessment (HHRA) of industrial chemicals, mines and radiological contaminated sites, chemical fate, transport and transformation and HHRA implications, chemical class specific expertise include metals/metalloids, petroleum hydrocarbons, fluorinated and chlorinated chemicals toxicology, computational systems toxicology, elimination kinetics and persistence of effects and exposure analysis, mechanism of action, mode of action and adverse outcome pathways framework and knowledgebase development and applications; physiological-based pharmacokinetic models review and applications; Emerging informatics tools and new approaches and methodologies (NAMs) developments in collaborative computational toxicology and opensource platforms; toxico-genomics and epigenomics applications; Data fusion tools and methodological applications in statistics, toxicology and risk assessments; climate change health risk assessments, cumulative exposure (chemical and nonchemical stressors) and risk analysis; permafrost affected soils and active layer dynamics. Major reference publication includes Information Resources in Toxicology (2019, fifth edition). Asish is one of the Co-Editor-in-Chief for the journal—Global Security: Health, Science and Policy and a founding member of the OpenTox Association (a global community of practice promoting collaborations and opensource tools and frameworks in support of knowledgebase development, applications and publications). Sidhartha D. Ray, Ph.D., FACN, Past Acting chair, currently serves as a senior professor of Pharmaceutical and Biomedical Sciences at The Touro University College of Pharmacy, New York. Prior to this, he was at the AMS College of Pharmacy of Long Island University, NY, for 18 years and as the founding chair of Pharmaceutical Science department at Manchester University College of Pharmacy at Indiana. Dr. Ray’s academic career spans over 40 years in pharmacy, teaching, research, and service. His research focuses on pharmacology, molecular toxicology, adverse drug reactions, side effects of a number of therapeutically used drugs and environmental chemicals. His service contributions are reflected by his election into multiple international professional organizations, such as the SOT, AACP, ACN, ASPET, and SFRBM. Dr. Ray serves as the Editor-in-Chief of “Side Effects of Drugs Annual” (Elsevier since 2014), and as Associate Editor of “Archives of Toxicology” and OMCL. He is a “Fellow of the American College of Nutrition” since 1999. He has won Teaching Excellence Award (2005 & 2023), Lifetime Scholarly Achievement Award (2008), “Wall-of-Fame” honor (2011), the Society of Toxicology’s “Educator of the Year” national award in 2013, “Outstanding Scholar award” (2014), and Senior Toxicologist award from ASIOA/SOT (2015). In his lifetime, he has mentored numerous PharmD, MS, MD, and PhD students and colleagues in health and life sciences. His 200+ publications have garnered him 8000 Google scholar citations. His key mantra to success has been instilling a “Lifelong Learning” mindset in his mentees. Visit URL: www.sidhartharay.com/ for more details. José V. Tarazona, Doctor in Veterinary Medicine, Ph.D. in Toxicology, Full Member of the Spanish Royal Academy of Veterinary Sciences. Current affiliation: Research Professor and Head of the Risk Assessment Unit at the National Environmental Health Centre, Instituto de Salud Carlos III, the Spanish public research institution on health under the Ministry of Science and Innovation. Main involvements are in EU projects, particularly in the research partnership PARC, collaborating in several activities and leading the project “Quantify effects of PPP and other stressors through landscape risk assessment informing on environmental impacts.” The activities cover human and environmental risk assessments, focusing on environmental pollutants and pesticides,

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and the evolution of the risk assessment paradigm for incorporating New Approach Methodologies (NAMs), contributing to international actions through APCRA, ILMERAC as co-chairing the NAMs Working Group, the European Parliament and the European Partnership for Alternative Approaches to Animal Testing. Previous affiliation: Head of the Pesticides Unit and Senior Scientific Officer at the Scientific Committee and Emerging Risk Unit, European Food Safety Authority, Parma, Italy. Involved in regulatory risk assessments, chairing the EFSA Working Group on Nanomaterials and the EFSA Nanonetwork, coordinating several Scientific Committee Working Groups and EFSA internal projects focused on NAMs. Aristidis Tsatsakis is the Director of the Laboratory of Toxicology and Forensic Sciences of the University of Crete and the University Hospital of Crete, Greece. Furthermore, he is the initiator, founder, scientific director, and head of the spin-off company of the University of Crete, ToxPlus S.A. He has more than 1500 international publications (books, articles, and conference presentations) and holds several patents. He is the coordinator of several European HORIZON projects and has organized several international conferences as chairman. He served as president of the European Federation of European Societies of Toxicology (EUROTOX) from 2014 to 2016. He is the editor-in-chief of the Public Health Toxicology journal and has served as editor of several leading international journals. He has been awarded Honorary Doctorate and Professor in many universities and institutes around the world as well as member of the Academy of Toxicological Sciences of the United States, the World Academy of Sciences and honorary member of many Toxicological Societies such as Bulgaria and Slovakia. In 2018, he was nominated Honorary President of the European Institute of Nutritional Medicine and Honorary Member of EUROTOX. In 2020 and 2021, he was recognized as Highly Cited Researcher in the field of Pharmacology—Toxicology of Biomedical Sciences taking the top position in the list of the most influential researchers, for the field of Toxicology, worldwide. In 2022, he was awarded the EUROTOX Merit Award 2022 by EUROTOX for his significant contribution to the advancement of the field of Toxicology. Recently, Prof. Tsatsakis was elected as a member of the Academia Europaea recognizing his outstanding achievements as a researcher. Timothy J. Wiegand, MD, FACMT, FAACT, DFASAM, holds Board Certification in Medical Toxicology and Addiction Medicine. He completed Toxicology and Clinical Pharmacology fellowship training at the University of California, San Francisco, in 2006 and is currently the Director of Addiction Toxicology at the University of Rochester Medical Center and an Associate Professor of Emergency Medicine in the Department of Emergency Medicine at Strong Memorial Hospital in Rochester, New York, United States. He is also Medical Director of Huther-Doyle Chemical Dependency program in Rochester, New York, and a consultant toxicologist for the SUNY Upstate Poison Center in Syracuse, New York. Dr. Wiegand founded the Toxicology Consult Service for the University of Rochester Medical Center hospitals and affiliate system in 2010 and subsequently the Addiction Medicine consultation-liaison service for the hospital system. Dr. Wiegand has served as Fellowship Director for the URMC Combined Addiction Medicine Fellowship program, housed in Emergency Medicine. He is also the Fellowship Director for the Medical Toxicology Fellowship program which is anticipated to start in July 2023. Dr. Wiegand has served on the Board of Directors for the American College of Medical Toxicology (ACMT), and he is currently serving on the Executive Council of the American Society of Addiction Medicine (ASAM) Board of Directors as Vice President. Dr. Wiegand has authored numerous chapters in textbooks and papers in peer-reviewed journals and is an associate editor of the Encyclopedia of Toxicology, 4th edition.

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FOREWORD As is true of the terrain of every scientific discipline, new toxicological knowledge is uncovered continuously. Like ascending a mountain, features faintly visible come into clarity the higher we rise. Toxicologists are driven to ponder and reveal answers to the mysteries underlying the actions of potentially toxic agents and weighing risks versus benefits in their application in medicine and other fields. Over four editions now, the Encyclopedia of Toxicology has been helping to map that climb by expanding and distilling into readily digestible entries a more and more extensive view of the current landscape of toxicology. Toxicology continues to be rooted in the health and welfare of the human species. A recent emphasis on environmental toxicology is driven by population growth and industry. Toxicology advances as new technologies and techniques are discovered and implemented. These changes can be rapid and, in many cases, alter the traditional path and pace up the mountain. The concept of “alternative” or “new approach methodologies” is complex and the views of those who will use the information generated by such novel tests will only be properly informed by careful evaluation of outcomes. Big data, artificial intelligence, and complex exposures are a few of the specific present-day issues we are grappling with. There is a need for rigorous, open, and reproducible science as we journey up the mountain. The Encyclopedia is one of an armamentarium of tools helping us chart the course to the mountain’s ever-receding crest because, in truth, mistaken sightings are not uncommon and ultimate goals are elusive. This new 4th edition of the Encyclopedia of Toxicology is an important link between condensed dictionary definitions and detailed research papers, reviews and monographs, and is devoted to wide-ranging aspects of the field, both narrow and broad. In addition to its value to the toxicology community, it will play an important role for nontoxicologists’ gathering background knowledge on one topic or another, or simply curious about the role of poisons in science and society. It is my privilege to play a supporting role in scaling the toxicology mountain by contributing this Foreword to the most recently expanded, updated, and welcome 4th edition of the Encyclopedia of Toxicology. A Wallace Hayes, Ph.D., DABT, FATS, FRSB, FACFE, ERT, University of South Florida College of Public Health, Tampa, FL, United States Michigan State University Center for Integrative Toxicology, East Lansing, MI, United States

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PREFACE Ten years have elapsed since the Encyclopedia of Toxicology’s previous edition and the world of toxicology, as the world itself, has not stood still. On a macro level, human-generated global pollution, despite activist and government pleas to curtail its release, and a handful of good faith national efforts, continues virtually unabated. Greenhouse gas emissions, much of which result from the burning of fossil fuels such as coal, oil, vehicular gasoline, and natural gas, blanket the Earth and trap the sun’s heat. Climate change is at a crisis stage with atmospheric and ocean heat, severe storms, drought, a rising ocean, and food insecurity all on the increase. Heat and drought have been among the drivers of an increase in the risk and extent of wildfires, such as in the western United States recently. Indeed, the United Nations’ Intergovernmental Panel on Climate Change (IPCC)’s most recent synthesis report on climate change, released in March 2023, warns of a dire future unless people individually and governments broadly take swift action. Over the years, our exposure to microplastics and other micromaterials has become ubiquitous. More specifically, we find ourselves increasingly exposed to PFAS (per- and polyfluoroalkyl substances), antimicrobials, flame retardants, bisphenols and phthalates, solvents, and metals. Our usage, storage, and disposal habits must change. Drug overdose deaths, driven by synthetic opioids, fentanyl in particular, some prescribed but frequently manufactured illicitly, have attained record levels. The rise has been a result of both intentional and accidental exposures. The U.S. Centers for Disease Control and Prevention’s (CDC) National Center for Health Statistics data show a provisional predicted value count of more than 107,689 drug overdose deaths for the 12-month period ending October 2022. A concerted effort is required to stem the tide of this epidemic, including securing the borders. The passage of the Omnibus Spending Bill in late 2022 included increased federal funding for state opioid response grants to make medications such as buprenorphine and naltrexone more readily available to all who need them. Apart from the opioid crisis, more research, prevention, and treatment resources need to be devoted to the abuse of prescription and over-the-counter drugs, including their synergistic effects with other substances. The sudden appearance of COVID-19 on the world scene at the start of 2020 upturned lives of people everywhere and continues to do so in one way or another. Interestingly, this infectious disease has had several toxicological implications. A broad spectrum of treatments and prevention measures were employed globally. They ranged from repurposed treatments with proven safety profiles to inadequately tested new technologies or clearly bogus and dangerous recommendations such as drinking or injecting bleach. Given the potential for other novel disease outbreaks, a greater emphasis on the toxicological profiling of a wide range of antivirals needs to be initiated well in advance. In addition to new and wider exposures to drugs and other chemicals, people are being subjected to increasing levels of nonionizing radiation, particularly as generated by cell phones, radar, power lines, Wi-Fi (as in routers), as well as any number of existing and yet to be developed smart devices. More toxicological research is imperative to determine and minimize potentially adverse effects. With tensions and hostilities rising within and among nations more than in decades, threats and pursuit of military warfare are increasing. Such conflicts increase the potential for the use of chemical, biological, and nuclear weapons. Even engaging in traditional battles raises the toxicological stakes, as munitions raining down upon “enemy” territory bring, in addition to casualties, swaths of environmental devastation, impacting the water and food supply and other human necessities. Individual targeted attacks in which poisoning is a component of the espionage game is another tactic, ancient in origin, which some governments seem to have never outgrown, to silence dissidents and enemies.

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Preface

High-dose mammalian toxicity testing continues to be a mainstay of identifying toxic agents and will not disappear overnight. Nonetheless, in the name of animal welfare, expense, and limits in interpretation when extrapolating to humans, they have already begun to diminish. The 3 Rs mantra (replacement, reduction, and refinement) has much to commend it and is adhered to by many laboratories. Zebrafish have become the latest animal model darling of toxicity testing, especially in high-throughput early screening. In vitro testing is a mature alternative methodology to testing in animals and is in widespread use. In silico testing uses computer technology as a platform for assessment and read-across is an approach to evaluate a substance’s toxicity based on data gathered from structurally similar compounds. New Approach Methodologies (NAM) for toxicological evaluation are now pervasive in the United States and elsewhere. The International Cooperation on Alternative Test Methods (ICATM) is a multinational organization whose primary goals are to establish cooperation in validation studies, peer reviews, and new alternative test methods and strategies. Two specific toxicology research frontiers, which are certain to experience growth in the coming years, are multiple exposures and epigenetics. Current risk assessments focus largely on individual substances. However, in order to be relevant to real-world scenarios, more attention will be paid to determining risk from combined exposures to chemical, biological, and radiological agents. Epigenetics is the study of the way cells control gene activity without changing the DNA sequence. Its significance for toxicology is that the broad environment, including pollutants and potentially even diet, can alter the epigenome, affecting the way DNA sequences are read. Edition by edition, the Encyclopedia, to encompass the growth in the field, has grown from 749 entries in its first 1998 edition to over 1200 today. Some 75 new entries have been added since the third edition. These include a potpourri of subjects such as COVID-19, in silico toxicology, imaging, microbiome, micro- and nano-plastics, telomeres, data fusion applications, organ-on-a chip, wildfires, gender differences, nurdles, burn pits, toxicology in the ICU, medical devices, nuclear warfare agents, monoclonal antibodies, 3D printers, and Novichok. Virtually every other entry, whether representing a specific agent or class of chemicals, method/ technology/tool, or other broad topic, except for a very limited number of entries, such as those of historical import, have been updated to reflect current research and thinking. Given all the research and applications that have been advanced to strengthen the scientific underpinnings of toxicology, some principles remain ironclad. First articulated in 1538, Paracelsus’ dictum, Alle Dinge sind Gift, und nichts ist ohne Gift; allein die Dosis macht, dass ein Ding kein Gift ist (All things are poison, and nothing is without poison; the dosage alone makes it so a thing is not a poison more colloquially expressed as The dose makes the poison) has remained a succinct and unwavering summation of at least one aspect of toxicology. Similarly, the four widely acknowledged steps of risk assessment (1. hazard identification, 2. hazard characterization, 3. exposure assessment, and 4. risk characterization) have withstood the test of time. The idea of toxicity has infiltrated the psyche of individuals and society as never before, and the term and its cognates have become ever more prevalent buzzwords and are routinely applied to circumstances that have little if anything to do with science. It has become a ready verbal tool to connote negativity of almost any sort. Thus, we hear talk of toxic behaviors, toxic situations, toxic relationships, toxic people, etc. In fact, toxic was the Oxford English Dictionary’s 2018 Word of the Year and has not lost its sparkle and overuse since then. We are no longer in an era when a major Encyclopedia can be a one-person undertaking. Pliny the Elder may have successfully compiled his Naturalis Historia single-handedly in the first century AD, but those days are long gone. That said, I would like first and foremost to thank my 11 Associate Editors, paragons of toxicological wisdom, without whose expertise and commitment, I could not have completed even the first few entries under the letter, A. On the publisher side, Elsevier’s Blerina Osmanaj, the Encyclopedia’ s Acquisitions Editor, as well as Paula Davies and Pamela Sadhukhan, its Content Project Managers played prominent roles. Other Elsevier staff with less visible roles have also done their part to bring the book to fruition and are due an equally well-deserved shout-out. Finally, I am grateful to Prof. A. Wallace Hayes, an exemplar of the scholarly research toxicologist, whom I have known and worked with over decades, for his kind Foreword. Wally’s own extensive toxicology text, while not an Encyclopedia per se, is no less encyclopedic and informative than this book, and makes for a good companion piece. My Associate Editors and I would like to pay tribute to the enduring relevance of toxicology as a scientific discipline impacting our everyday lives. As with any science, it will continue to evolve. On a lighter note, maybe someday toxicologists will eventually reach a consensus on whether moderate amounts of coffee and wine are good for us or not. And with Consumer Reports recently finding that many brands of dark chocolate bars vary widely in their lead and cadmium compositions, when will we ever be safe? As for mercury in fish?—don’t get me started! Encyclopedias are not revised all that often but no doubt, a fifth edition of this one will be warranted before long. Will I, personally, dive in? Time will tell. Meanwhile, Happy Reading in this Fourth! Philip Wexler Editor-in-Chief

A critical appraisal of the toxicological aspects of COVID-19 and its vaccines Taxiarchis Konstantinos Nikolouzakisa, Daniela Calinab, Benjamin Brooksa, Anca Doceac, and Aristides Tsatsakisa, aLaboratory of Toxicology and Forensic Chemistry, Medical School, University of Crete, Heraklion, Crete, Greece; bDepartment of Clinical Pharmacy, University of Medicine and Pharmacy of Craiova, Craiova, Romania; cDepartment of Toxicology, University of Medicine and Pharmacy of Craiova, Craiova, Romania © 2024 Elsevier Inc. All rights reserved.

Introduction Virology Coronavirus virology Variants of concern Alpha (B.1.1.7 lineage) Beta (B.1.351 lineage) Gamma (P.1 lineage) Delta (B.1.617.2 lineage) Omicron (B.1.1.529 lineage) Omicron sublineage B.A.2 Epidemiology Geographic distribution and case counts Transmission Viral shedding and period of infectiousness Prolonged viral RNA detection does not indicate prolonged infectiousness Asymptomatic or presymptomatic transmission Immune response Humoral immunity Cell-mediated immunity Vaccines Antigenic target Vaccine platforms Vaccine-enhanced disease Children vaccination Conclusion References Further reading

2 3 3 3 3 3 4 4 4 4 4 4 4 5 6 6 6 6 6 6 7 7 7 7 9 9 11

Abstract In December 2019, a new viral strain occurred in Wuhan, China, that rabidly took over the globe resulting in the most impactful pandemic of the last 100 years. Soon, the viral strain was identified as a member of the coronaviridae family; the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease was named coronavirus disease 19 (COVID-19). Understanding the virology of this new pathogen and creating effective vaccines and treatments became the foundation of the research efforts to control the pandemic. COVID-19 has demonstrated high levels of transmissibility and significant morbidity and mortality. Vaccines for COVID-19 were developed at an unprecedented speed. While concerns about vaccine safety were widely shared, the COVID-19 vaccines generally proved safe and effective. However, as the virus spread across the globe, it soon became evident that various strains could rise and dominate. Indeed, although a large portion of the global population has been vaccinated, infectiousness remained high, with some mortality decrease. Thus, re-appreciation of SARS-CoV-2 toxicodynamics, infection profiles, and summarization of the available vaccines is still needed to understand possible individual and/or environmental predispositions explaining these trends. Here, we provide a framework to evaluate the toxicology of the many COVID-19 vaccines in light of COVID-19 and its variants. In summary, the COVID-19 vaccines remain safe, effective, and thus recommended even in light of the reduced effectiveness of the vaccine due to the variants.

Keywords virus; RNA; SARS-CoV-2; COVID-19; vaccine

Key points

• •

COVID-19 represents an example of a global healthcare and socioeconomic stressor Effective management of the COVID-19 pandemic requires in-depth understanding of the SARS-CoV-2 virus

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A critical appraisal of the toxicological aspects of COVID-19 and its vaccines

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Over the course of the pandemic new strains of the SARS-CoV-2 virus have emerged, making strategy planning even more difficult Even though vaccination was proposed to serve as the main tool against SARS-CoV-2 spread, it is now clear that a multidisciplinary approach is needed In order to achieve maximum efficacy, various vaccine platforms against the SARS-CoV-2 virus have been employed

Introduction In December 2019, a new viral strain occurred in Wuhan, China. At its first description, COVID-19 was described as pneumonia of unknown cause. The symptomatology of the affected patients included fever, malaise, dry cough, and dyspnea. A previously unknown betacoronavirus was discovered through unbiased sequencing in samples from these patients (Zhu et al., 2020). The isolated pathogen was a novel coronavirus, primarily named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Up to that time, six coronavirus species were known to affect humans. Four (229E, OC43, NL63, and HKU1) are more prevalent and typically cause common cold symptoms (Su et al., 2016). On the contrary, the two other strains (severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV)) are zoonotic in origin and have been linked to sometimes fatal illness (Cui et al., 2019). By January 5 of 2020, the whole genome sequence of 2019-nCoV was completed by Wuhan Institute of Virology, China CDC and Shanghai Public Health Clinical Center of Fudan University (Chan et al., 2020; Zhou et al., 2020) and deposited immediately to the GenBank (Chan et al., 2020). SARS-CoV-2 was found to be structurally and functionally similar to the other members of the family as it is enveloped with a single-stranded RNA genome. The virus has four structural proteins: Spike (S), nucleocapsid (N), envelope (E), and membrane (M) proteins (Fig. 1) (Calina et al., 2021). After almost 3 months, on March 11, 2020, the WHO finally assessed that COVID-19 can be characterized as a pandemic, following the 1918 Spanish flu (H1N1), 1957 Asian flu (H2N2), 1968 Hong Kong flu (H3N2), and 2009 Pandemic flu (H1N1) (Liu et al., 2020). The clinical presentation of COVID-19 ranges from asymptomatic to moderate morbidity to mortality (Chen et al., 2020a). Evidence suggests that COVID-19 manifests mainly as a lower respiratory tract infection, although other G.I. and other locations are also reported (Alimohamadi et al., 2020). While symptoms are similar between variants, the frequency and severity are different (Whitaker et al., 2022). The most common COVID-19 symptoms are consistent with a lower respiratory infection. The most commonly reported symptoms with the alpha strain of COVID-19 included fever (81.2%), cough (58.5%), and fatigue (38.5%) (Ekroth et al., 2022). Less reported symptoms include vomiting, diarrhea, myalgia, hemoptysis, and sore throats (Grant et al., 2020). Other symptoms such as loss of taste and smell are also common (Variants of the Virus | CDC, n.d.; Koshima et al., 1989). The case fatality rate (CFR) for COVID-19 was calculated at 3.4% by the WHO in March of 2020 (seasonal flu is 10,000 mg/L. A 24-h old animals from a clone of Daphnia magna were exposed to the test item in tap water. The LC50 was determined as >10,000 mg/L (Registration dossier. ECHA, n.d.).

Exposure standards and guidelines OSHA has currently set exposure limits for acetamide of 10 ppm (25 mg/m3) for an 8-h TWA. However, these are not currently enforced by the agency. In order to protect workers from adverse health effects due to acetamide exposure, the American Conference of Governmental Industrial Hygienists (ACGIH) set its threshold limit value, at 1 ppm (2.5 mg/m3) for an 8-h TWA. Short Term Exposure Limit (STEL) of 15 ppm (37.5 mg/m3) is also recommended (Department of Labor Logo United Statesdepartment of Labor. ACETAMIDE | Occupational Safety and Health Administration, n.d.).

References Acetamide 60-35-5 wiki. GuideChem. (n.d.). Retrieved November 5, 2022, from https://www.guidechem.com/encyclopedia/acetamide-dic277.html Department of Labor Logo United States department of Labor. ACETAMIDE | Occupational Safety and Health Administration (n.d.). Retrieved December 15, 2020 from https://www. osha.gov/chemicaldata/831. International Agency for Research on Cancer, Re-Evaluation of Some Organic Chemicals, Hydrazine and Hydrogen Peroxide. (n.d.). Retrieved March 30, 2023, from https://www.ncbi. nlm.nih.gov/books/NBK498841/. Moore MM, Gollapudi B, Nagane R, Khan N, Patel M, Khanvilkar T, Roy AM, Ramesh E, Bals B, Teymouri F, Nault R, and Bringi V (2019) The food contaminant acetamide is not an in vivo clastogen, aneugen, or mutagen in rodent hematopoietic tissue. Regulatory Toxicology and Pharmacology 108: 104451. https://doi.org/10.1016/j.yrtph.2019.104451. Nakamura K, Ishii Y, Takasu S, Nohmi T, Shibutani M, and Ogawa K (2020) Lack of in vivo mutagenicity of acetamide in a 13-week comprehensive toxicity study using F344 GPT Delta Rats. Toxicological Sciences 177(2): 431–440. https://doi.org/10.1093/toxsci/kfaa126. Registration dossier. ECHA (n.d.). Retrieved March 31, 2023, from https://echa.europa.eu/cs/registratyion-dossier/-/registered-dossier/17464/1/1. U.S. National Library of Medicine. (2023). Acetamide. National Center for Biotechnology Information. PubChem Compound Database. Retrieved March 31, 2023, from https:// pubchem.ncbi.nlm.nih.gov/compound/Acetamide

Further reading Kennedy GL Jr. (1986) Biological effects of acetamide, formamide, and their monomethyl and dimethyl derivatives. Critical Reviews in Toxicology 17: 129–182. Nakamura K, Ishii Y, Takasu S, Nohmi T, Shibutani M, and Ogawa K (2020) Lack of in vivo mutagenicity of acetamide in a 13-week comprehensive toxicity study using F344 Gpt delta rats. Toxicological Sciences 177(2): 431–440. https://doi.org/10.1093/toxsci/kfaa126. Nault R, et al. (2020) A toxicogenomic approach for the risk assessment of the food contaminant acetamide. Toxicology and Applied Pharmacology 388: 114872.

Relevant websites https://wwwosha.gov/chemicaldata/831. https://wwwepa.gov/sites/default/files/2016-09/documents/acetamide.pdfhttps://echa.europa.eu/registration-dossier/-/registered-dossier/17464/7/7/1. https://inchemorg/documents/icsc/icsc/eics0233.htmhttps://webwiser.nlm.nih.gov/substance?substanceId¼517&identifier¼Acetamide&identifierType¼name&menuItemId¼1& catId¼162.

Acetaminophen Kenneth Pawa and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of K. Shankar, H.M. Mehendale, Acetaminophen, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 26–29, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00215-3.

Chemical profile Uses Background information Routes of exposure Toxicokinetics Mechanism of toxicity In vitro toxicity Cytotoxicity in hepatic cells Cytotoxicity in other cells Acute animal toxicity Acute human toxicity Chronic animal toxicity Cytotoxicity of pulmonary cells in mice Endocrine toxicity in mice Chronic human toxicity Clinical management of acetaminophen induced hepatotoxicity Toxicogenomics Environmental fate Ecotoxicology Other hazards Exposure limits Miscellaneous References

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Abstract Acetaminophen is perhaps the most widely used over-the-counter analgesic and antipyretic medication. It can be found in over 235 products either alone or in combination with other medications for numerous indications. The most common toxic effect of Acetaminophen is acute liver toxicity. Literature indicates that many patients require liver transplant after overexposure to this compound, either accidentally or intentionally. Acetaminophen undergoes extensive phase 1 biotransformation producing a toxic intermediate (NAPQI) which is held responsible for causing extensive liver toxicity. Overdose accounts for more than 56,000 emergency room visits annually, and is implicated in nearly 50% of all acute liver failure in the United States for the past 40 years. A recent American Association of Poison Control Centers (Gummin et al., 2021) annual poisoning data shows acetaminophen alone caused 242 fatalities, and acetaminophen combinations caused 87 fatalities (Gummin et al., 2021). Therefore, this over-the-counter drug remains extremely clinically relevant from human health perspective (USFDA, 2022).

Keywords Acetadote; Acetaminophen; Analgesic; Antipyretic; Biological reactive intermediate; Cyclooxygenase-1 (COX-1); Cyclooxygenase-2 (COX-2); Cyclooxygenase-3 (COX-3); Cell death; Cell injury; Glutathione; Hepatotoxicity; Liver injury; N-acetyl cysteine (NAC); NAPQI: N-Acetyl-para-benzoquinoneimine; Paracetamol (acetaminophen); Reactive oxygen species (ROS); Serum alanine aminotransferase (ALT); Toxicogenomics

Key points

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Acetaminophen is used as an over-the-counter analgesic and antipyretic medication Acetaminophen is sold under various brand names throughout the World, either alone or combination with other drugs, mainly as a pain killer Acetaminophen can be potentially hepatotoxic for individuals when taken alone in large quantities Acetaminophen is not recommended for individuals with a pre-existing liver or kidney condition N-Acetylcysteine (NAC) remains the only antidote for acetaminophen poisoning when administered within hours after exposure Acetaminophen antidote (NAC) is available in many forms including extended release forms

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Acetaminophen

Chemical profile

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Name: Acetaminophen Chemical Abstracts Service Registry Number: 103-90-2 Synonyms: APAP, 40 -Hydroxyacetanilide, P-hydroxyacetanilide, Acetamide N-(4-hydroxyphenyl), N-acetyl-p-aminophenol, N-acetyl-p-aminophenol, P-acetamidophenol; 4-Acetamidophenol, 4-Acetaminophenol, Paracetamol, Tylenol Pharmaceutical Class: Acetaminophen is a synthetic nonopioid congener of acetanilide, a p-aminophenol derivative with analgesic and antipyretic properties. Molecular Formula: C8H9NO2 Chemical Structure:

Acetaminophen

N-acetyl-p-benzoquinone imine (NAPQI)

Uses Acetaminophen is a nonnarcotic analgesic and antipyretic drug. Generally, acetaminophen is used to relieve pain of moderate intensity, defined as pain that occurs in headache and in many muscle, joint, and peripheral nerve disorders (Critical Care Medicine, 2011). Headaches are one of the most common indications for the use of acetaminophen. Acetaminophen is used to treat acute tension headaches and mild to moderate migraine, especially in combination with caffeine and aspirin. Acetaminophen is indicated in chronic pain associated with rheumatoid arthritis, back or hip pain, osteoarthritis, dental pain, or acute pain due to soft-tissue injury. Acetaminophen is currently the drug of choice for pain associated with osteoarthritis and is commonly used in patients who cannot be treated with non-steroidal anti-inflammatory drugs such as patients with bronchial asthma, peptic ulcer disease, hemophilia, salicylate-sensitized people, children under 12, and pregnant or breastfeeding women (James et al., 2011). Acetaminophen is a suitable substitute for aspirin for its analgesic or antipyretic uses in cases where aspirin is contraindicated (gastric bleeding) or when the prolongation of bleeding time caused by aspirin would be a disadvantage. Acetaminophen has been used in studies of pain relief following obstetric and gynecological procedures, including Caesarean section, hysterectomy, tubal ligation, primary dysmenorrhea, and termination of pregnancy. Acetaminophen is also used to manage chronic pain of cancer, postpartum pain, and postoperative pain after minor surgery. In a double-blind crossover study, the analgesic oral butorphanol and acetaminophen in combination, showed additive analgesic effects against moderate to severe pain due to metastatic carcinoma over that of the individual drug. A study done in 2015 with long-acting acetaminophen in postoperative dental pain showed that acetaminophen can be used long term in patients with post-operative dental pain (Good Clinical Practice, 2015). Acetaminophen is also widely used as an antipyretic drug to reduce fever at therapeutic doses. Often, acetaminophen is prescribed to children due to the risk of Reyes syndrome associated with the use of NSAIDS (such as aspirin). Like adults, acetaminophen toxicity exists when used outside of therapeutic ranges. Acetaminophen dosing depends on the child’s age, weight, dosage form, and the child’s other medications. Parents and caretakers of children must consult a healthcare professional for appropriate dosing (Blough and Wu, 2011; Cleveland Clinic, 2020).

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Background information Acetaminophen can be found as the active ingredient, either alone or in combination, in more than 100 over-the-counter products and prescription drugs. The pharmacology and toxicology of this drug have been extensively studied and reviewed. The first clinical use of acetaminophen dates back to 1893 by von Mering as an effective antipyretic with comparable pharmacological effects to antipyrine and phenacetin. However, after a hiatus of almost half a century, acetaminophen was rediscovered as the major metabolite of phenacetin and acetanilide in humans and was marketed in the United States as a combination with aspirin and caffeine in 1950. In the 1960s and 1970s, concerns about gastrointestinal adverse effects of aspirin and methemoglobinemia of acetanilide only led to increased popularity of acetaminophen as a generally safe antipyretic analgesic. Hepatotoxicity of acetaminophen began to be reported in the late 1960s and has been a topic of intense scientific evaluation to this day. There has been speculation of further toxicity such as pulmonary, renal, and endocrine toxicity, but no further toxicity has been proven in humans. Besides potent antipyretic and analgesic actions, acetaminophen shows very weak anti-inflammatory activity. Studies show that acetaminophen reduces levels of prostaglandin metabolites in urine but does not reduce synthesis of prostaglandins by blood platelets or by the stomach mucosa. Acetaminophen is a weak inhibitor of both cyclooxygenase-1 (COX-1) and COX-2 and has been debated to possibly inhibit COX-3 in vitro (cf. Shankar and Mehendale, 2014; USFDA, 2022).

Routes of exposure Acetaminophen is available in several dosage forms, including tablets, capsules, syrups, elixirs, suppositories, and the intravenous route. Oral ingestion is the most common route for both accidental and intentional exposure to acetaminophen. Many studies are being done post 2010 to show the efficacy of intravenous acetaminophen in various clinical uses.

Toxicokinetics Absorption of acetaminophen occurs in the gastrointestinal tract primarily by passive nonionic diffusion and is highly dependent on several factors, including dose, presence of food and other chemicals, mucosal blood flow, age, body weight, time of day, and coexisting disease condition. At pharmacological doses, acetaminophen is absorbed rapidly, with an 88% bioavailability when taken orally. Acetaminophen absorption occurs about 2–4 h post oral administration (Forrest et al., 1982). A large number of studies have evaluated the pharmacokinetic parameters of acetaminophen in humans after oral or intravenous dosing. Most studies consistently report volume of distribution to be between 0.8 and 1.l kg−1, with about 10%–20% of drug bound to red blood cells. Total clearance and plasma half-life with therapeutic doses in healthy subjects were usually 3–5 ml min kg−1 and 1.5–2.5 h, respectively. After supra-pharmacological or toxic doses, absorption may be delayed after producing peak blood concentrations at approximately 4 h post-ingestion. In humans, the majority of acetaminophen is metabolized in the liver to glucuronide and sulfate conjugates that are eliminated in the urine. Estimates in humans from urinary metabolites report 50%–60% as glucuronide conjugate, 25%–44% as sulfate conjugate, and 2–5% of cysteine and mercapturic acid conjugates respectively. In young children, the sulfate conjugate predominates. The water-soluble glucuronide and sulfate conjugates are eliminated via the kidneys. Approximately 1%–5% is eliminated in the urine as unchanged acetaminophen. The half-life of therapeutic dose is 1.5–2.5 h. In overdosed patients, this may be increased to more than 4 h, and may even exceed 12 h in patients with severe acetaminophen-induced liver toxicity. Individuals with pre-existing liver and kidney disease should consult with a physician prior to beginning acute and chronic therapy (Mazaleuskaya et al., 2015). Fig. 1 shows the metabolism, distribution, and excretion of acetaminophen once it is ingested, and the potential for any toxicities when used outside of the recommended doses. This figure indicates the major biotransformation pathways, phase-II conjugation reactions and elimination routes of acetaminophen and its metabolites. Upon metabolism by the cytochrome P450, it produces the biological reactive intermediate (NAPQI) and various reactive oxygen species which covalently bind to macromolecules (such as glutathione) and cause lipid peroxidation of liver cell plasma membranes. This results in oxidative stress and possibly kills hepatocytes via apoptosis, necrosis, and autophagy (Yan et al., 2018). Ultimately, all these events contribute to liver injury and massive leakage of serum ALT. Massive histopathological changes are common after injury.

Mechanism of toxicity Although a major part of the ingested dose of acetaminophen is detoxified, a very small portion is metabolized via the cytochrome P450-mixed function oxidase pathway to a highly reactive biological intermediate known as n-acetyl-p-benzoquinoneimine (NAPQI). NAPQI is normally detoxified by endogenous glutathione to cysteine and mercapturic acid conjugates and excreted in the urine. Recent studies have shown that hepatic P450s, CYP2E1, and to a lesser extent CYP1A2 are responsible for conversion of acetaminophen to NAPQI. In acetaminophen overdose, the amount of NAPQI increases and simultaneously depletes endogenous glutathione stores. A precipitous depletion in glutathione makes the cells vulnerable to toxicity. Several animal studies have shown

46

Acetaminophen

Necrosis

Acetaminophen Ingested

Autophagy Human Liver

CYP 2E1 OxidaƟon ~5-10%

Acetaminophen

Glucuronide and Sulfate Conjugates (non-toxic)

NAPQI (toxic)

GSH DepleƟon

GSHDependent

GlucuronidaƟon SulfaƟon (~ 90%) Urinary ExcreƟon (~ 1-5%)

Hepatocyte Necrosis (OxidaƟve Damage)

Apopcrosis (Mixed Apoptosis and Necrosis)

Cysteine and Mercapturic Acid Conjugates (non-toxic) Apoptosis

Urinary ExcreƟon

Fig. 1 Pathway of acetaminophen induced liver damage.

that, APAP toxicity can be minimized by enhancing hepatic and mitochondrial GSH levels via administering N-acetylcysteine (glutathione is a tripeptide consisting of amino acids: cysteine, glycine and glutamic acid). Time course studies have shown that covalent binding of reactive NAPQI and subsequent toxicity occur only after cellular glutathione stores are reduced by 70% or more of normal. Mitochondrial dysfunction and damage can be documented experimentally in vitro and in vivo models, as early as few minutes after the exposure to the drug, suggesting that this may be a critical factor leading to cellular necrosis. If metabolism remains unchecked, and NAPQI production continues, then NAPQI covalently binds to critical cellular macromolecules in hepatocytes and cause cell death. Recent proteomic studies have identified at least 20 known proteins that are covalently modified by the BRI of acetaminophen- NAPQI. Interestingly, the resulting acetaminophen-cysteine (APAP-Cysteine) protein adducts can be quantified via a HPLC coupled with electrochemical detection (HPLC-EC). Hepatic necrosis and inflammation develop as a consequence of hepatocellular death, which results in development of clinical and laboratory findings consistent with liver failure (Gunawan and Kaplowitz, 2007; James et al., 2009; Hinson et al., 2010; Mazaleuskaya et al., 2015). In the past two decades, several studies have indicated that acetaminophen is a powerful inducer of programmed cell death or apoptosis in addition to necrosis. Acetaminophen-induced apoptosis involves a complex interplay of cell signaling pathways involving multiple intracellular organelles, such as, mitochondria, nucleus, endoplasmic reticulum, and cytoplasm. Key players that propel toxic events leading to various forms of cell deaths are oxidative stress (mediated by BRIs and ROS), intracellular perturbation of Ca2+, and a complex interplay of proteolytic caspases (Ray et al., 1996; Ray et al., 2001; Ray et al., 2006). Recent clinical studies have shown microRNA perturbations during acetaminophen-induced liver injury.

In vitro toxicity Acetaminophen causes cytotoxicity in several cell types; however, the most widely studied cytotoxicity of acetaminophen is in primary hepatocytes or hepatocyte cell lines.

Cytotoxicity in hepatic cells Primary hepatocytes from rats, mice, hamsters, rabbits, dogs, pigs, monkeys, and humans have been shown to be susceptible to acetaminophen in vitro. The cytotoxicity of acetaminophen varies considerably depending on species, presumably due to differences in bioactivation and glutathione status. The most obvious morphological effect of acetaminophen in isolated primary hepatocytes is blebbing of the cell membrane. However, electron microscopy has shown that toxicity is associated with progressive loss of microvilli, mitochondrial abnormalities, and appearance of myeloid bodies. Exposure of primary mouse hepatocytes to concentrations of acetaminophen above 1 mM led to significant lactate dehydrogenase leakage in as soon as 3 h. Cytotoxicity of acetaminophen has also been examined using standard liver cell lines, including, PC12 cells, HepG2 cells, and H4IIEC3 cells, among other cell lines. Immortalized hepatocyte cultures, in many cases, lose their ability to bioactivate acetaminophen and hence

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are resistant to toxicity. Transient or consistent overexpression of drug-metabolizing enzymes (CYP4502E1 and/or CYP4501A2) leads to increased cytotoxicity of acetaminophen. Acetaminophen is also cytotoxic in cultures of rat liver sinusoidal endothelial cells, Kupffer cells, and mouse fibroblasts.

Cytotoxicity in other cells The cytotoxicity of APAP has been demonstrated in cultures of HeLa cells, L929 and 3 T3 murine fibroblasts, chick embryo neurons, rat embryonic and skeletal muscle, peripheral blood lymphocytes, and lung and dermal cells. In addition, cytotoxicity of acetaminophen has been evaluated in the BF-2 fish cell line (see Section “Ecotoxicology”).

Acute animal toxicity A large body of evidence is available examining the acute toxicity of acetaminophen in animal models. Mice and rats have been widely used to study the toxic effects of acetaminophen. Since rats are relatively resistant, the mouse has been the most widely used species to study the mechanisms of acetaminophen toxicity and to examine chemicals that potentiate or protect from the toxicity. Hepatotoxicity and nephrotoxicity are the two main effects associated with acute overdose of acetaminophen. Of these, death in most species is due to acute hepatic failure. LD50 values range from 350 mg kg−1 to 4500 mg kg−1 depending on the species and the route of acetaminophen administration, mice (LD50 350–600 mg kg−1) being more far more sensitive than rats, guinea pigs, and rabbits (LD50 > 3 g kg−1). LD50 values can vary based on the type of species and can differ drastically from human models. In animal models, death occurs by 12 h after acetaminophen exposure. In mice after a toxic dose, general findings in addition to the severe hepatic necrosis include necrotic changes in the kidney, bronchiolar epithelium, testes, lymphoid follicles of the spleen, and small intestine. Cats are particularly susceptible to acetaminophen intoxication because of their impaired glucuronic acid conjugation mechanism and saturation of their sulfate conjugation pathway. The clinical signs associated with experimental acetaminophen administration to cats included cyanosis followed by anemia, hemoglobinuria, icterus, and facial edema. Laboratory findings in acetaminophen-poisoned cats include methemoglobinemia and an elevated serum alanine aminotransferase activity.

Acute human toxicity Hepatotoxicity is the primary toxic insult from acute acetaminophen overdose. Acetaminophen overdose accounts for more than 56,000 emergency room visits and is implicated in nearly 50% of all acute liver failure in the United States. Exposure to toxic doses of acetaminophen may be due to intentional (suicidal) or unintentional (accidental). Data from Parkland Hospital suggest that greater percentages of unintentional overdose victims suffer from fatal consequences compared to persons attempting suicide (with acetaminophen) primarily due to their characteristic late presentation. Data from the U.S. ALF Study Group show that unintentional overdoses (which are more frequent in liver failure cases) were also larger (median dose of 34 g) compared to suicidal doses, being consumed over several preceding days. There is no clear agreement on a maximum tolerated dose of acetaminophen. Most people tolerate 4–8 g day−1 of acetaminophen without any hepatotoxic incidence. However, the risk of severe hepatotoxicity may rise if the 4 g day−1 dose, especially in individuals with unknown idiosyncratic reasons. Thus, the maximum daily dose for humans should not exceed 4 g per day (Lee, 2004; Lee, 2020; Chun et al., 2009; cf. Shankar and Mehendale, 2014). The typical clinical manifestations are secondary to hepatic damage. Plasma concentrations should be obtained to determine the probability of acetaminophen-induced hepatotoxicity. The Rumack-Matthews nomogram is used to assess the risk of hepatotoxicity. Levels more than 150 mg ml−1 of acetaminophen at 4 h post-ingestion are associated with a high probability of development of hepatotoxicity. While not yet clinically available, newer methods of detecting APAP-toxicity include detection of APAP-cysteine adducts via HPLC-EC and via metabolomic analysis of urine samples (early-intervention pharmacometabolomics). The clinical presentation follows four distinct phases. Gastrointestinal irritation, nausea, and vomiting are present in the first 24 h postingestion. The second stage (24–48 h post-ingestion) is characterized by the resolution of the initial symptoms, accompanied by elevations of hepatic transaminases. Cases that progress to stage three develop hypoglycemia, coagulopathies, jaundice, and symptoms consistent with hepatic failure. Surviving patients go through a fourth stage of recovery. As toxicity develops, half-life becomes prolonged and transaminases rise and fall. In instances where reliable history of time of ingestion is not available, calculations of body burden may be useful in deciding treatment (Rumack and Bateman, 2012; cf. Shankar and Mehendale, 2014).

Chronic animal toxicity In a 2-year feed study, there was no evidence of carcinogenic activity of acetaminophen in male F344/N rats that received 600, 3000, or 6000 ppm acetaminophen for 104 weeks (U.S. Department of Health and Human Services, 1993). There was equivocal evidence of carcinogenic activity in female F344/N rats based on increased incidences of mononuclear cell leukemia. Overall, there is

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inadequate evidence in experimental animals for the carcinogenicity of APAP and is not classifiable as a carcinogen of any category. Acetaminophen was non-mutagenic in the Salmonella/mammalian microsome assay at concentrations ranging from 0.1 to 50 mg per plate. In a study to examine the effect of acetaminophen on reproduction and fertility, no changes in the number of pups/litter, viability, or adjusted pup weight were found. Acetaminophen in the diet of Swiss mice reduced weight gain during nursing. Fertility endpoints (ability to bear normal numbers of normal-weight young) were generally not affected (NTP, 1993; cf. Shankar and Mehendale, 2014).

Cytotoxicity of pulmonary cells in mice A survey of patients in the UK found an association between frequent APAP use and signs of asthma (Kennon-McGill and McGill, 2018). In mice, bronchiolar epithelium necrosis has been observed when treated with very large doses of APAP but the data are clearly not relevant for the chronic low-dose exposures that are thought by few investigators to cause asthma and other pulmonary diseases. There is some evidence that low doses of APAP are proinflammatory in the lungs and can potentially lead to bronchiolar damage, however, this remains controversial, as is the link between acetaminophen use and pulmonary toxicity. Chronic exposure to APAP can deplete GSH in the lungs and could be a potential cause of APAP induced respiratory diseases especially if APAP enhances susceptibility to reactive-oxygen species. GSH depletion and increased oxidative stress response genes have been detected in lungs of mice exposed to acutely toxic doses (Kennon-McGill and McGill, 2018).

Endocrine toxicity in mice APAP is deemed safe for pregnant women to use therapeutically to reduce pain and fever. It has been reported that there is an increased risk of cryptorchidism (genital abnormalities) after prenatal exposure to APAP. This finding has been reported in humans in a few studies, which has been linked to anti-androgen activity of APAP. However, the results are inconsistent and difficult to interpret. Other studies that evaluated APAP and other androgen measures like penis width and anogenital distance has shown no effect on the human fetus. However, in rodent models, there has been a connection between APAP use during pregnancy and altered anogenital distance. Overall, there is not a clear relationship between APAP exposure, and halted development and reproductive effect in humans (Kennon-McGill and McGill, 2018).

Chronic human toxicity There is inadequate evidence in humans for the carcinogenicity of acetaminophen and it cannot be categorized to any carcinogen class. The chronic ingestion of excessive amounts of APAP may produce similar toxicity as a large acute dose but in a more insidious fashion. Age, chronic alcohol abuse, and preexisting disease may be contributing factors. The American Academy of Pediatrics considers use of APAP is safe during breast-feeding, and it is classified as a category B chemical by the FDA (studies in laboratory animals have not demonstrated a fetal risk, but there are no controlled studies in pregnant women) (Black and Hill, 2003). Acetaminophen should be given with care to patients with impaired kidney and/or liver function. Acetaminophen should be given with care to patients taking other drugs that affect the liver and patients with history of liver disease.

Clinical management of acetaminophen induced hepatotoxicity Activated charcoal or other gastrointestinal decontamination procedures can be utilized as deemed necessary. Induction of emesis is not recommended as prolonged emesis may interfere with N-acetylcysteine (NAC) therapy. The Rumack-Matthew nomogram is utilized to identify proper course of treatment. Blood acetaminophen concentrations of 150 mg l−1 (or higher) at 4 h post-ingestion indicate severe risk of hepatic failure and are treated with a standard NAC treatment regimen. NAC is a glutathione substitute and prevents hepatic damage by boosting glutathione biosynthesis as well as by quenching NAPQI. If administered within 8–10 h after an acute overdose, NAC reduces the risk of hepatotoxicity to less than 5%. An oral loading dose of 140 mg kg−1 (as a 5% solution in soft drink or juice) is followed by 70 mg kg−1 given orally as a 5% solution in soft drink or juice every 4 h for an additional 17 doses. An alternative intravenous dosing protocol (20 h regimen) for NAC (Acetadote®; Cumberland Pharmaceuticals) can also be used in patients where oral NAC administration is not possible. A loading dose of 150 mg kg−1 NAC (in 200 ml of 5% dextrose in water) is administered over 15 min, followed by 50 mg kg−1 NAC in 500 ml of 5% dextrose over the next 4 h. A final dose of 100 mg kg−1 NAC is administered in 1000 ml of 5% dextrose over a 16 h period. A longer 72 h treatment regimen with intravenous NAC is recommended in the United States. An injectable form and an extended-release form of NAC are available (Acetadote®) for treating patients who developed acetaminophen-induced liver injury. Basic and advanced life-support measures should be utilized as required by the condition of the patient. Studies have also suggested that an increase in alpha-fetoprotein, a surrogate for hepatic regeneration following injury, is strongly associated with a favorable outcome in patients with acetaminophen-induced liver injury and hence may be used as a supplement to existing prognostic criteria (Heard, 2008; Hodgman and Garrard, 2012; Klein-Schwartz and Doyon, 2011; Heard and Dart, 2022) (Table 1).

Acetaminophen Table 1

Acetaminophen clinical management using N-acetylcysteine.

Route

Treatment

Intravenous regimen (Acetadote)

Step 1: Administer 150 mg/kg NAC IV in 200 mL D5W over 60 min Step 2: Administer 50 mg/kg NAC IV in 500 mL D5W over 4 h Step 3: Administer 100 mg/kg NAC IV in 1000 mL D5W over 16 h Total duration: 21 h Total dose: 300 mg/kg divided in 60 min, 4 h, and 16 h increments  In patients weighting less than 30 kg, 20% NAC should be diluted to a concentration of 40 mg/mL Dilute NAC to a final concentration of no more than 5% in a soft drink or juice to reduce risk of vomiting Step 1: Administer 140 mg/kg NAC orally initially Step 2: Administer 70 mg/kg every 4 h orally for an additional 17 doses Total duration: 72 h Total dose: 1330 mg/kg divided in initial dose and 4 h increments for 17 doses  Shorter durations of therapy can be used if there are no elevations in transaminases 36 h post ingestion with no acetaminophen detected in the blood

Oral regimen (Mucomyst)

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Acetaminophen toxicity management with doses and duration of treatment using N-acetylcysteine in both the oral and IV routes. Poison Control Center, U. (2005). Acetylcysteine for acetaminophen overdose. Utox Update 7(1). https://poisoncontrol.utah.edu/newsletters/pdfs/ toxicology-today-archive/Vol7_No1.pdf.

Toxicogenomics Toxicogenomics combines transcript, protein, and metabolite profiling with conventional toxicology to investigate the interaction between genes and environmental stress in disease causation. The patterns of altered molecular expression that are caused by specific exposures or disease outcomes have revealed how several toxicants act and cause disease. One study compared hepatotoxic response to APAP exposure to data obtained from patients suffering from APAP-induced acute liver failure (ALF). The results indicated that several cell lines (hHEP, HepaRG and hSKP-HPC) discern comparable APAP-induced hepatotoxic functions. Pathway analyses further indicated that HepaRG cells show the highest predicted activation of the functional genes related to liver damage, followed by hSKP-HPC and hHEP cells that generated similar results (Winnike et al., 2010; Kienhuis et al., 2011; Driessen et al., 2015; Rodrigues et al., 2016; Yokoyama et al., 2018; Fernandez-Checa et al., 2021).

Environmental fate Acetaminophen was found to be inherently biodegradable and has no bioaccumulation potential. No other information about the environmental fate of acetaminophen is currently available.

Ecotoxicology The acute toxicity of APAP has been examined in several aquatic species. The LC50 value in brine shrimp (Artemia salina) mortality was reported to be 3820 mmol l−1. The EC50 for immobility over a 24 h experiment using water flea (Daphnia magna) was 367 mmol l−1. Acetaminophen is classified as nontoxic or only slightly to moderately toxic in all fish (fathead minnow, Pimephales promelas) and zooplankton species tested. The crustacean fairy shrimp (Streptocephalus proboscideus) appears to be highly sensitive to acetaminophen (average LC50 of 196 mg l−1). Recent studies show that acetaminophen can be teratogenic to the embryo and larvae of catfish Clarias gariepinus when exposed to 0.5, 1, 10 mg/L concentrations for 96 h. As APAP levels increased, hatching rates decreased, and the exposed larvae had an increase in swimming speed about 8 times as much as non-APAP exposed larvae. There were also neurotoxic, cardiotoxic, and teratogenic effects reported among the species. Acute effects of APAP have also been demonstrated on the developmental, swimming performance and cardiovascular activities of the African catfish embryos/larvae Clarias gariepinus sp. (Erhunmwunse et al., 2021). These findings may suggest APAP’s ability to impact the survival of catfish and other aquatic life.

Other hazards Acetaminophen is stable under ordinary conditions of use and storage. In the presence of heat and water, APAP will hydrolyze into acetic acid and p-aminophenol. Incineration can produce carbon monoxide, carbon dioxide, and nitrogen oxides. Flammability: As with most organic solids, fire is possible at very high temperatures or by contact with an ignition source.

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Explosivity: Fine dust dispersed in air in sufficient concentrations, and in the presence of an ignition source, could serve as a potential dust explosion hazard. The minimum concentration for explosion is 0.25 oz. per cubic feet. The recommended fire-extinguishing media are water spray, dry chemical, alcohol foam, or carbon dioxide. Acetaminophen is capable of generating a static electrical charge. Processes involving dumping of APAP into flammable liquid, inert atmosphere in the vessels or temperatures of flammable liquid should be maintained below its flashpoint.

Exposure limits Therapeutic exposure: The total adult daily dose of APAP should not exceed 4 g. Dosages of acetaminophen over 4–8 g day−1 over long periods of time may be associated with higher risk of liver toxicity. Acetaminophen should not be administered for more than 10 days or to young children except under the supervision of a medical professional. Occupational exposure: Mallinckrodt recommends an airborne exposure limit of 5 mg m−3.

Miscellaneous A large number of reports in the scientific literature and public media suggest a potential high risk of liver toxicity could be due to APAP when consumed following alcohol intake. Recent data suggests that only chronic heavy drinkers may be at greater risk of toxicity following an overdose of acetaminophen and that no potentiation of toxicity occurs at therapeutic doses (Prescott, 2000; Tujios and Fontana, 2011). However, acetaminophen use during acute or chronic alcohol exposure remains a controversial topic.

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(2015) A transcriptomics-based hepatotoxicity comparison between the zebrafish embryo and established human and rodent in vitro and in vivo models using cyclosporine-A, amiodarone and acetaminophen. Toxicology Letters 232(2): 403–412. https://pubmed.ncbi.nlm.nih.gov/25448281/. Erhunmwunse NO, Tongo I, and Ezemonye LI (2021) Acute effects of acetaminophen on the developmental, swimming performance and cardiovascular activities of the African catfish embryos/larvae (Clarias gariepinus). Ecotoxicology and Environmental Safety 208: 111482. https://pubmed.ncbi.nlm.nih.gov/33120276/. Fernandez-Checa JC, et al. (2021) Advanced preclinical models for evaluation of drug-induced liver injury - consensus statement by the European Drug-Induced Liver Injury Network [PRO-EURO-DILI-NET]. Journal of Hepatology 75(4): 935–939. Forrest JA, Clements JA, and Prescott LF (1982) Clinical pharmacokinetics of paracetamol. Clinical Pharmakokinetics 7(2): 93–107. https://pubmed.ncbi.nlm.nih.gov/7039926/. Good Clinical Practice Network (2015) Study of long-acting acetaminophen in Postoperative Dental pain. ICHGCP. https://ichgcp.net/clinical-trials-registry/NCT01960114. Gummin DD, et al. (2021) 2018 Annual Report of the American Association of Poison Control Centers’ National Poison Data System (NPDS): 38th Annual Report. Clinical Toxicology 59(12): 1282–1501. https://pubmed.ncbi.nlm.nih.gov/34890263/. Gunawan BK and Kaplowitz N (2007) Mechanisms of drug-induced liver disease. Clinics in Liver Disease 11(3): 459–475. Heard KJ (2008) Acetylcysteine for Acetaminophen Poisoning. The New England Journal of Medicine 359(2): 285–292. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2637612/. Heard KJ and Dart R (2022) Management of Acetaminophen (paracetamol) Poisoning in Children and Adolescents. UpToDate. (UpToDate.com), April 13. https://www.uptodate.com/ contents/management-of-acetaminophen-paracetamol-poisoning-in-children-and-adolescents?search¼Management%20of%20acetaminophen%20(paracetamol)%20poisoning %20in%20children%20and%20adolescents&source¼search_result&selectedTitle¼1150&usage_type¼default&display_rank¼1. Hinson JA, Roberts DW, and James LP (2010) Mechanisms of acetaminophen-induced liver necrosis. Handbook of Experimental Pharmacology 196: 369–405. Hodgman MJ and Garrard AR (2012) A review of acetaminophen poisoning. Critical Care Clinics 28(4): 499–516. https://pubmed.ncbi.nlm.nih.gov/22998987/. James LP, Letzig L, Simpson PM, et al. (2009) Pharmacokinetics of acetaminophen-protein adducts in adults with acetaminophen overdose and acute liver failure. Drug Metabolism and Disposition: The Biological Fate of Chemicals 37(8): 1779–1784. James L, Sullivan JE, and Roberts D (2011) The proper use of acetaminophen. Paediatrics and Child Health 16(9): 544–547. https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC3223888/. Kennon-McGill S and McGill MR (2018) Extrahepatic toxicity of acetaminophen: critical evaluation of the evidence and proposed mechanisms. Journal of Clinical and Translational Research 3(3): 297–310. Kienhuis AS, et al. (2011) Application of toxicogenomics in hepatic systems toxicology for risk assessment: Acetaminophen as a case study. Toxicology and Applied Pharmacology 250(2): 96–107. Klein-Schwartz W and Doyon S (2011) Intravenous acetylcysteine for the treatment of acetaminophen overdose. Expert Opinion on Pharmacotherapy 12(1): 119–130. Lee WM (2004) Acetaminophen and the U.S. acute liver failure study group: Lowering the risks of hepatic failure. Hepatology 40(1): 6–9. https://doi.org/10.1002/hep.20293. Lee WM (2020) Acetaminophen toxicity: A history of serendipity and unintended consequences. Clinical Liver Disease 16(Suppl 1): 34–44. Mazaleuskaya LL, et al. (2015) PharmGKB summary: Pathways of acetaminophen metabolism at the therapeutic versus toxic doses. Pharmacogenetics and Genomics 25(8): 416–426. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498995/. NTP - National Toxicology Program (1993) Toxicology and Carcinogenesis Studies of Acetaminophen in F344/N Rats and B6C3F1 Mice (Feed Studies). Technical Report Series No. 394, NIH Publication No. 93–2849. Prescott LF (2000) Paracetamol, alcohol and the liver. British Journal of Clinical Pharmacology 49(4): 291–301. https://doi.org/10.1046/j.1365-2125.2000.00167.x. Ray SD, Mumaw VR, Raje RR, and Fariss MW (1996) Protection of acetaminophen-induced hepatocellular apoptosis and necrosis by cholesteryl hemisuccinate pretreatment. The Journal of Pharmacology and Experimental Therapeutics 279(3): 1470–1483.

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Ray SD, Balasubramanian G, Bagchi D, and Reddy CS (2001) Ca2+−calmodulin antagonist chlorpromazine and poly (ADP-ribose) polymerase modulators 4-aminobenzamide and nicotinamide influence hepatic expression of BCL-XL and P53 and protect against acetaminophen-induced programmed and unprogrammed cell death in mice. https://pubmed. ncbi.nlm.nih.gov/11461765/. Ray SD, Patel N, Shah N, Nagori A, Naqvi A, and Stohs SJ (2006) Pre-exposure to a novel nutritional mixture containing a series of phytochemicals prevents acetaminophen-induced programmed and unprogrammed cell deaths by enhancing BCL-XL expression and minimizing oxidative stress in the liver. Molecular and Cellular Biochemistry 293(1–2): 119–136. https://pubmed.ncbi.nlm.nih.gov/16902808/. Rodrigues RM, et al. (2016) Toxicogenomics-based prediction of acetaminophen-induced liver injury using human hepatic cell systems. Toxicology Letters 240: 50–59. https://doi. org/10.1016/j.toxlet.2015.10.014. Rumack BH and Bateman DN (2012) Acetaminophen and acetylcysteine dose and duration: past, present and future. Clinical Toxicology 50(2): 91–98. Shankar K and Mehendale HM (2014) Acetaminophen. In: Encyclopedia of Toxicology, 3rd edn, 26–29. https://doi.org/10.1016/B978-0-12-386454-3.00215-3. Tujios S and Fontana RJ (2011) Mechanisms of drug-induced liver injury: from bedside to bench. Nature Reviews. Gastroenterology & Hepatology 8(4): 202–211. U.S. Department of Health and Human Services (1993) Abstract for TR-394. NIEHS. https://ntp.niehs.nih.gov/publications/reports/tr/300s/tr394/index.html?utm_source¼direct& utm_medium¼prod&utm_campaign¼ntpgolinks&utm_term¼tr394abs. USFDA (2022) Acetaminophen. https://www.fda.gov/drugs/information-drug-class/acetaminophen. Winnike JH, et al. (2010) Use of pharmaco-metabonomics for early prediction of acetaminophen-induced hepatotoxicity in humans. Clinical Pharmacology and Therapeutics 88(1): 45–51. https://pubmed.ncbi.nlm.nih.gov/20182423/. Yan M, Huo Y, Yin S, and Hu H (2018) Mechanisms of acetaminophen-induced liver injury and its implications for therapeutic interventions. Redox Biology 17: 274–283. https://doi. org/10.1016/j.redox.2018.04.019. Yokoyama Y, et al. (2018) Comparison of drug metabolism and its related hepatotoxic effects in HepaRG, cryopreserved human hepatocytes, and HepG2 cell cultures. Biological & Pharmaceutical Bulletin 41(5): 722–732. https://pubmed.ncbi.nlm.nih.gov/29445054/.

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Acetamiprid David R Wallace, Department of Pharmacology, Oklahoma State University Center for Health Sciences, Tulsa, OK, United States © 2024 Elsevier Inc. All rights reserved. This is an update of DR Wallace, Acetamiprid, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 30–32, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00091-9.

Chemical profile Background Uses/occurrences Exposure and exposure monitoring Toxicokinetics Mechanism of action/toxicity In vitro toxicity data Acute and short-term toxicity Chronic toxicity Immunotoxicity Reproductive and development toxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Toxicogenomics Clinical management Environmental fate and behavior Ecotoxicity Exposure standards and guidelines References

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Abstract Acetamiprid belongs to the chloronicotinyl neonicotinoid, class of insecticide. Acetamiprid exhibits greater agonistic potency at insect nicotinic acetylcholine receptors compared to mammals. The predominant use for acetamiprid is to control insects that damage leafy plants. It is available as ready-to-use, wettable powder, or water-dispersible granules. Other neonicotinoids (imidacloprid) undergo biotransformation in rodents resulting in a metabolite with higher affinity for the nicotinic receptor compared to (−)-nicotine itself, increasing the chance for toxicity in mammals. Currently, acetamiprid has not demonstrated biotransformation to a toxic metabolite, but recently it was shown that acetamiprid undergoes transepithelial absorption across intestinal cells, potentially resulting in acetamiprid accumulation in the body. Overall, the general belief is that acetamiprid presents low hazard risks to human/vertebrate populations under normal conditions. In situations of high bioaccumulation, toxicity to wildlife may be evident.

Keywords Bioaccumulation; Biotransformation; Chloronicotinyl; Honeybees; Insecticide; Neonicotinoid; Nicotinic acetylcholine receptor

Chemical profile

• • • • • • •

Name: Acetamiprid. Synonyms: Mospilan, Assail, Tristar. Chemical Abstracts Service (CAS) Registry Number: 135410-20-7. Chemical/Pharmaceutical/Other Class: Insecticide—Neonicotinoid (Pyridylmethylamine). Molecular Formula: C10H11ClN4 Chemical Name: (E)-N-[(6-chloro-3-pyridyl) methyl]-N0 -cyano-N-methyl-acetamidine. Chemical Structure: (Fig. 1)

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00267-0

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Acetamiprid

Fig. 1 Structure of Acetamiprid. From ChemSpider (https://www.chemspider.com/Chemical-Structure.184719.html) and reproduced under Creative Commons-Share Alike 3.0 (CC-SA 3.0).

Background Acetamiprid belongs to the neonicotinoid class of insecticides that were developed in the late 1980s. The precise structure of acetamiprid is that of a chloronicotinyl compound and it has been shown to be a potent agonist at the postsynaptic nicotinic acetylcholine receptors in insects (Tomizawa and Casida, 2005). Numerous studies have shown that acetamiprid exhibits a higher affinity for insect nicotinic receptors compared to its affinity at vertebrate receptors. Although acetamiprid has shown to have higher affinity for nicotinic receptors in insects compared to mammals, there have been some reports of imidacloprid (another neonicotinoid) undergoing biotransformation in rodents resulting in a compound that has higher affinity for the nicotinic receptor compared to the levorotatory enantiomeric form, (−)-nicotine. This could potentially lead to toxicity in mammals. The primary use for acetamiprid is to control sucking insects such as aphids, which have been known to attack and damage leafy plants. Acetamiprid is available as a ready-to-use (RTU) formulation in addition to wettable powders (WP) and water-dispersible granules. Formulations for dispersal include 99.5% technical, 70% wettable powder end use, 70% water-soluble packet end use, and 0.006% ready-to-use end use product. Acetamiprid can be applied by ground and/or aerial means with sprayers. The application rate should not exceed 0.55 pounds of active ingredient per acre per season. This concentration will be sufficient to control insect populations. There have been no reports of chronic toxicity or of bioactivation of acetamiprid so far in mammals. There has been at least one report where individuals attempted suicide by ingesting a commercial mixture of acetamiprid. There have been no studies which have examined symptomology in humans following acetamiprid exposure, but based on the recorded symptoms in suicide-attempt patients and in other vertebrate species, it appears that primary symptoms include spasms, respiratory distress, and possibly convulsions (Imamura et al., 2010). A recent report showed that acetamiprid can undergo transepithelial absorption across intestinal cells, possibly resulting in toxicity if acetamiprid accumulates within the body. In general, acetamiprid is a relatively safe insecticide with few reported side effects in vertebrates. The persistence of acetamiprid in the environment is low, with quick degradation by soil microbes, or degradation by exposure to ultraviolet (UV) light in soil or in ground water (Gupta and Gajbhiye, 2007; Gupta et al., 2008). Exposure to sunlight or UV light resulted in a 95% + loss of acetamiprid from the sample and acetamiprid exhibited greater photolability compared to another neonicotinoid, thiacloprid (Gupta et al., 2008). The persistence of acetamiprid in soil is moderate under controlled settings, but in natural settings, the persistence of acetamiprid is reduced in soil under natural sunlight, and moisture, indicating a lower risk for human toxicity (Gupta and Gajbhiye, 2007). The soil microbiome has critical role in the removal of acetamiprid with nearly 95% of the acetamiprid lost after 15 days (Liu et al., 2011).

Uses/occurrences Acetamiprid is used as an insecticide to control sucking-type insects on leafy vegetables, fruits, and termites (US EPA, 2002; Mo et al., 2005; Pesticide Management Regulatory Agency, 2011). In many instances, these insects may be resistant to the effects of organophosphorus and other conventional insecticides. When resistance to organophosphate or organochlorine pesticides occur, the neonicotinoids are a viable option. Resistance to the effects of the neonicotinoids can also develop. Care must be taken in determining a pesticide to overcome the resistance to imidacloprid, with dinotefuran being an effective option. Acetamiprid if used on imidacloprid-resistant aphids, will promote further resistance (Shi et al., 2011).

Exposure and exposure monitoring There are three major sources of acetamiprid exposure: dietary, drinking water, and occupational. Dietary exposure may result from consuming fruits and vegetables commonly found in the local diet. By comparing values for drinking water level of comparison vs. drinking water estimated concentration, a potential toxin can be identified in susceptible populations. The Environmental Protection Agency (EPA) has chosen children 1–6 years of age as a primary risk group. Comparing the values for drinking water level of comparison (DWLOC) and the drinking. water estimated concentration (DWEC) for acute, short/intermediate, and chronic dietary exposure, the DWLOC/DWEC values are 600/17 ppb, 400/4 ppb, and 80/4 ppb, respectively. Since the DWLOC values are all significantly higher than the DWEC values, toxicity is not expected to occur. Occupational exposure can occur with individuals who manufacture or handle acetamiprid. A margin

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of exposure of less than 100 would be considered an occupational risk hazard, and for acetamiprid, the values are greater than 100 except for long-term post-application exposure which is 90. The value will return to 100 one-day after application. Detection of acetamiprid residues on food products, as well as from biological samples, has been accomplished by high-performance liquid chromatography and gas chromatography with mass spectrometry. Recently, immunoassays and the use of liquid chromatography with tandem mass spectrometry have been reported.

Toxicokinetics Acetamiprid is rapidly and extensively metabolized. Metabolites in urine account for 79–86% of the administered dose. Only 3–7% of acetamiprid is collected unchanged in the urine and feces. Demethylation by Phase I biotransformation is the major pathway, with 6-chloronicotinic acid being the major metabolite. Compounds can then undergo Phase II transformation with glycine conjugation representing the major pathway. Acetamiprid, and other neonicotinoids, only weakly interfere with the function of different drug transporters. Transporters that were examined include the organic cation transporter (OCT1 and 2), the organic anion transporter (OAT), and the multidrug and toxin extrusion transporter (MATE1 and 2). In the presence of acetamiprid, only weak changes in transporter functions were described suggesting that interference with drug transport systems is not a means of acetamiprid-related toxicity in humans (Le Vée et al., 2019).

Mechanism of action/toxicity The primary mechanism of acetamiprid toxicity against insects is due to its action at nicotinic cholinergic receptors (Tomizawa and Casida, 2003, 2005). The unique nature of the neonicotinoids as insecticides is that the negatively charged cyano (or nitro) group will specifically interact with a cationic binding region of the nicotinic acetylcholine receptor that is unique to insects. This action will convey selectivity of action against insects and leave mammalian nicotinic receptors relatively unaffected. Acetamiprid in human cell culture demonstrates a high affinity for the a3 nicotinic receptor subtype (Tomizawa and Casida, 1999). The affinity at the a7 nicotinic receptor subunit is nearly 15- to 20-fold higher.

In vitro toxicity data Studies using Caco-2 cells (human colorectal adenocarcinoma cells as a model of the intestinal epithelial barrier) have shown that acetamiprid can be absorbed across the intestinal epithelial layer in a bidirectional function utilizing both active and passive diffusion (Brunet et al., 2008). Caco-2 cells are an excellent model for the intestinal epithelial barrier due to their ability to spontaneously differentiate into a heterogeneous mixture of intestinal epithelial cells.

Acute and short-term toxicity There is little evidence for acetamiprid toxicity in vertebrates. The EPA classifies acetamiprid as both a class II and class III agent (US EPA, 2002). Acetamiprid rating of II was in acute oral studies with rats, II in acute dermal and inhalation studies with rats, and category IV in primary eye and skin irritation studies with rabbits. There is some evidence for contact exposure, dermal irritation, and stomach poisoning following oral ingestion. Animal: Acute studies in laboratory animals, mainly rats, have demonstrated relatively low toxicity potential for acetamiprid. Oral ingestion appears to elicit the most severe toxicological responses. At dosages in excess of 140 mg kg−1, acetamiprid elicited neurotoxic signs, with animals exhibiting disorders of movement and posture. Surviving animals were free of signs on the following day. The LD50 in rats has been reported to be 450 mg kg−1 for acetamiprid. Although requiring a large dose for lethality, acetamiprid is still one of the most toxic of the neonicotinoids. The LD50 for other compounds such as clothianidin, imidacloprid, and thiamethoxam are 10-fold higher in the rat. Acetamiprid was only slightly toxic following inhalation (LC50 >1.15 mg L−1) and weakly toxic following dermal administration (LD50 >2000 mg kg−1 in rabbit). There was minimal or no irritation of eyes or skin. Some metabolites of acetamiprid exhibited greater toxicity than the parent compound. In other species such as birds and fish, acetamiprid is relatively nontoxic with acute LD50 values in birds >2000 mg kg−1 and >100 ppm in fish. Acetamiprid has been reported by multiple investigators to be moderately toxic in bee populations in that if acetamiprid is applied directly over bees. Human: No evidence is available for assessing human outcomes following acute exposure to acetamiprid. Signs associated with acute exposure and limits of exposure have been established using data from animal studies. The symptoms associated with rodent toxicity may be applicable to human symptoms and these include lethargy, respiratory distress, reduced movement, and loss of balance with staggering followed by spasms (National Center for Biotechnology Information, 2021). There have been some reports of acute acetamiprid toxicity in humans following suicide attempts and the symptoms presented by these individuals were similar to what has been reported in other vertebrate toxicity studies with higher concentrations of acetamiprid (Imamura et al., 2010). The

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ingested acetamiprid solutions were 2–18% but contained many other components that may have contributed to the symptoms observed. Accident ingestion, resulting in lower plasma acetamiprid levels, present with nausea, low blood pressure, and hyperglycemia, which represents low dose organophosphate exposure. Treatment of symptoms, maintenance of physiological function coupled with activated charcoal treatment resulted in a full recovery (Todani et al., 2008). Due to the selectivity of acetamiprid for insect nicotinic cholinergic receptors, little human toxicity is expected following normal use.

Chronic toxicity Examination of chronic exposure to acetamiprid has utilized animals and little data on chronic human exposure are available. The selectivity of acetamiprid for insect nicotinic receptors would suggest minimal toxicity following chronic exposure. Animal: Chronic dietary administration of acetamiprid resulted in reduced body and organ weight. Higher doses resulted in neurological dysfunction. Human: No evidence is available for assessing human outcomes following chronic exposure to acetamiprid. Symptoms associated with chronic exposure and limits of exposure have been established using data from animal studies. Due to the selectivity of acetamiprid for insect nicotinic cholinergic receptors, little human toxicity is expected. Concern has risen considering little work has been done with the metabolites of acetamiprid. One of the primary sources of acetamiprid contamination and exposure is through the diet. A study measuring multiple neonicotinoids in China showed that acetamiprid was moderately present and may pose a potential risk to human following long-term consumption and exposure (Yu et al., 2021).

Immunotoxicity Previously, there were no reports of immunotoxicity of acetamiprid in humans/vertebrates or other species. Recent data suggest some degree of immunotoxicity in vitro and in vivo (rats and mice). Acetamiprid (5 mg kg−1/day  2 months) in mice resulted in decreased splenocyte proliferation and immunosuppression (Marzouki et al., 2017). In Wistar rats, a dose of 110 mg kg−1 for 90 days resulted in reduced lymphocyte number and macrophage activity (Shakthi Devan et al., 2015). Another study in neuronal SH-SY5Y cells exposed to 4 mM acetamiprid demonstrated an increase in the expression of mitogen-activated protein kinase-8 (MAPK-8) and stress-related genes, with an increase in apoptosis (Öztas¸ et al., 2021).

Reproductive and development toxicity There were no reports of reproductive toxicity of acetamiprid in humans/vertebrates or other species until the last decade. Recently, the impact of acetamiprid on reproductive and developmental toxicity had increased attention. Babies who were born small for their gestational age compared to size-appropriate for their gestational age, tended to have higher levels of the acetamiprid metabolite, N-des-methylacetamiprid, in their urine compared to appropriate-sized infants (Ichikawa et al., 2019). This finding of the acetamiprid metabolite in the urine highlights the need and the importance for further testing. Exposure of porcine oocytes to acetamiprid at 30 or 100 ppm significantly reduced the nuclear maturation of the oocytes, with multiple chromosomal irregularities (Ishikawa et al., 2015). In male mice and rats, exposure to acetamiprid resulted in a weak reduction in the fertilization ability of sperm and reduced the early embryonic development (Gu et al., 2013). In rat and mouse testes, exposure to acetamiprid (10–30 mg kg−1 b.w.) resulted in an increase in testosterone metabolizing enzymes and altering mitochondrial respiration and adenosine triphosphate production in Leydig cells (Kong et al., 2017; Terayama et al., 2018). A portion of acetamiprid-mediated toxicity is due to elevated oxidative stress, reduction in glutathione redox cycling, and altered sperm function (Mosbah et al., 2018; Guiekep et al., 2019). Although acetamiprid elicited developmental effects that were weaker than other neonicotinoids, it still produced some neurodevelopmental deficits. There was only a weak correlation between the inhibition of neurite growth and Gap-43 expression in PC-12 cells exposed to 10 nM acetamiprid (Christen et al., 2017). In the developing (postnatal days 12–26) brain receiving 5.0 mg kg−1/day acetamiprid, there was a significant reduction in cell number and the number of actively dividing cells (Nakayama et al., 2019). Acetamiprid in a dose of 5 mg kg−1/day from embryonic day 14 to postnatal day 14 showed similar results in cortical tissue from mice (Kagawa and Nagao, 2018). In vivo in utero exposure of acetamiprid, or through lactation resulted in behavioral alterations and a reduction in anxiety responses (Sano et al., 2016).

Genotoxicity There have been no reports of genotoxicity of acetamiprid in humans/vertebrates or other species. There were no positive results in genotoxicity studies using bacterial or mammalian cell assays. There has been a report that indicated that acetamiprid did induce sister chromatid exchange and chromosomal aberrations in human blood lymphocytes (Kocaman and Topaktas, 2007). This study demonstrated that at all of the concentrations and time points examined, acetamiprid significantly reduced the mitotic index in blood lymphocytes, while the nuclear division index was only reduced at 24-h (Kocaman and Topaktas, 2007). At least in blood lymphocytes, acetamiprid has demonstrated evidence for genotoxicity.

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Carcinogenicity Literature searches for the carcinogenicity of acetamiprid yielded no results. In the United States it is categorized as a ‘unlikely to be a human carcinogen’ (US EPA, 2002).

Organ toxicity Human studies are lacking that describe acetamiprid toxicity in individual organ systems. In rats, administered acetamiprid (10–40 mg kg−1/day  60 days) showed significant liver damage as indicated by elevated antioxidant and liver enzyme activities, with a parallel increase in lipid peroxidation, indicative of liver damage (Chakroun et al., 2016).

Interactions Acetamiprid is not known to interact with many compounds. There has been some evidence that the combination of acetamiprid and thiacloprid, two relatively low toxicity neonicotinoids, have a synergistic response (Haas and Nauen, 2021). Within pesticide classes, sulfoximine and neonicotinoids exhibit synergistic responses in at least two bee species, Apis mellifera and Osmia bicornis, which may translate into increase toxicity risk to humans if used together (Azpiazu et al., 2021). Mixtures of the neonicotinoids, acetamiprid, carbendazim, chlorpyrifos, and cyhalothrin, generally increased in toxicity compared to individual neonicotinoid pesticides (Yang et al., 2018). The application of these pesticides was in an earthworm model and do suggest that care must be taken when doing a risk assessment of a pesticide mixture.

Toxicogenomics There has been little human data describing the toxicogenomic effects of acetamiprid. A study using human neuronal cultures describes a synergistic toxic effect when the response to pesticide mixtures is compared with control values (Cheng et al., 2020). A recent report suggests that two neonicotinoids (acetamiprid and thiacloprid) exhibit a synergism with the inhibition of CYP9Q3 and CYP9Q2 in honey bees (Haas and Nauen, 2021).

Clinical management There are no guidelines for acetamiprid toxicity except for symptomatic control (National Center for Biotechnology Information, 2021). Clinical management consists of managing any physical symptoms that are presented after removing the individual from the source of acetamiprid contamination: remove any contaminated clothing and rinse skin or eyes thoroughly with water for 15–20 min. If acetamiprid is inhaled, move the individual to fresh air and monitor breathing, and aid if necessary.

Environmental fate and behavior The primary route of exposure is via diet (food and water) (Yu et al., 2021). Occupational exposure for individuals who work with this insecticide can occur via dermal contact or inhalation. The primary means for measuring acetamiprid in the environment, or in biological samples is liquid chromatography-mass spectrometry (LC-MS) (Hernández et al., 2005). The use LC-MS or one of the variations of the detection systems, is quite expensive which reduces its general use. The development of an enzyme-linked immunosorbent assay (ELISA) with moderate sensitivity has been developed and may provide a rapid and inexpensive means for quantifying acetamiprid in produce and in biological samples (Watanabe et al., 2006; Wang et al., 2012). The persistence of acetamiprid in the environment is low, with quick degradation by soil microbes, or degradation by exposure to UV light in soil or in ground water (Gupta and Gajbhiye, 2007; Gupta et al., 2008). Acetamiprid exhibits a very short half-life in soil. Exposure to sunlight or UV light resulted in a 95% + loss of acetamiprid from the sample and acetamiprid exhibited greater photolability compared to thiacloprid, another neonicotinoid (Gupta et al., 2008). The persistence of acetamiprid in soil is moderate under controlled settings, but in natural settings, the persistence of acetamiprid is reduced in soil under natural sunlight, and moisture, indicating a lower risk for human toxicity (Gupta and Gajbhiye, 2007). It is rapidly degraded by aerobic metabolism. The soil microbiome has critical role in the removal of acetamiprid where nearly 95% of the acetamiprid could be lost after 15 days (Liu et al., 2011). Acetamiprid is stable to hydrolysis at environmental temperatures, and it photodegrades slowly in water. It is transformed moderately rapidly in aerobic aquatic environments, but only slowly in anaerobic aquatic systems. There appears to be minimal effects on drinking water and due to the rapid breakdown, has not demonstrated the ability to bioaccumulate in wildlife. Due to the rapid breakdown of acetamiprid, it is not expected to be persistent in the environment. Metabolites of acetamiprid will pose a greater risk to the environment, but additional work is needed to determine the fate and toxicity of acetamiprid metabolites.

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Ecotoxicity Since acetamiprid is cleared from the environment quickly under ‘normal’ usage due to exposure to, light (photolysis), UV light, or water, the threat to other wildlife is minimal (Gupta and Gajbhiye, 2007; Gupta et al., 2008; Guohong et al., 2009). Only 5% of the original application concentration of acetamiprid is available 15 days after application (Liu et al., 2011). Low environmental concentrations of acetamiprid increases specificity for insects and reduces actions at other unintended organisms. Higher concentrations of acetamiprid have demonstrated toxicity in honey bees by inhibiting P450 enzymes (Decourtye and Devillers, 2010; Haas and Nauen, 2021), but not toxic to different wasp species (Jiang et al., 2019). The inhibition of P450 enzymes, CYP9Q3 in particular, results in the observed synergistic toxicity when bees are also exposed to fungicides (Azpiazu et al., 2021). Recently, Health Canada re-evaluated three neonicotinoids for toxicity to honey bees, and acetamiprid was dropped from that group due to the lower toxic risk (Pesticide Management Regulatory Agency, 2020). Although the risk of acetamiprid is low in the environment, attempts have been made to develop a mechanism for soil remediation. Alternative methods have been investigated for the removal of acetamiprid from the soil and the use of Trametes versicolor (white rot fungus) can remove 20% of the acetamiprid found in soil samples after 7 days (Hu et al., 2021). Due to rapid degradation and the relatively low potency in vertebrate species, acetamiprid is a relatively safe insecticide when applied properly.

Exposure standards and guidelines The EPA has established guidelines for toxicological dose and end points for acetamiprid. Using the No Observable Adverse Effect Limit (NOAEL) and uncertainty factor, the reference dose (RfD) can be calculated. For acute dietary ingestion for infants and children, NOAEL ¼ 10 mg kg−1; RfD ¼ 0.10 mg kg−1 day−1. Chronic dietary exposure for all populations, NOAEL ¼ 7.1 mg kg−1; RfD ¼ 0.07 mg kg−1 day−1. Short- and intermediate-term incidental exposure to infants and children, NOAEL ¼ 15 mg kg−1 day−1 and for adults NOAEL ¼ 17.9 mg kg−1 day−1. Long-term dermal exposure, NOAEL ¼ 7.1 mg kg−1 day−1 with dermal absorption of 30%. Short- and intermediate-term inhalation exposure, NOAEL ¼ 17.9 mg kg−1 day−1 and for long-term inhalation exposure, NOAEL ¼ 7.1 mg kg−1 day−1.

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Kagawa N and Nagao T (2018) Neurodevelopmental toxicity in the mouse neocortex following prenatal exposure to acetamiprid. Journal of Applied Toxicology 38(12): 1521–1528. https://doi.org/10.1002/jat.3692. Kocaman AY and Topaktas M (2007) In vitro evaluation of the genotoxicity of acetamiprid in human peripheral blood lymphocytes. Environmental and Molecular Mutagenesis 48: 483–490. https://doi.org/10.1002/em. Kong D, et al. (2017) Acetamiprid inhibits testosterone synthesis by affecting the mitochondrial function and cytoplasmic adenosine triphosphate production in rat Leydig cells. Biology of Reproduction 96(1): 254–265. https://doi.org/10.1095/biolreprod.116.139550. Le Vée M, et al. (2019) Neonicotinoid pesticides poorly interact with human drug transporters. Journal of Biochemical and Molecular Toxicology 33(10): 1–10. https://doi.org/10.1002/ jbt.22379. Liu Z, et al. (2011) Soil microbial degradation of neonicotinoid insecticides imidacloprid, acetamiprid, thiacloprid and imidaclothiz and its effect on the persistence of bioefficacy against horsebean aphid Aphis craccivora Koch after soil application. Pest Management Science 67(10): 1245–1252. https://doi.org/10.1002/ps.2174. Marzouki S, et al. (2017) Specific immune responses in mice following subchronic exposure to acetamiprid. Life Sciences 188: 10–16. https://doi.org/10.1016/j.lfs.2017.08.022. Mo J, et al. (2005) Toxicity of acetamiprid to workers of Reticulitermes flaviceps (Isoptera: Rhinotermitidae), Coptotermes formosanus (Isoptera: Rhinotermitidae) and Odontotermes formosanus (Isoptera: Termitidae). Journal of Pesticide Science 30(3): 187–191. https://doi.org/10.1584/jpestics.30.187. Mosbah R, Djerrou Z, and Mantovani A (2018) Protective effect of Nigella sativa oil against acetamiprid induced reproductive toxicity in male rats. Drug and Chemical Toxicology 41(2): 206–212. https://doi.org/10.1080/01480545.2017.1337127. Nakayama A, et al. (2019) The neonicotinoids acetamiprid and imidacloprid impair neurogenesis and alter the microglial profile in the hippocampal dentate gyrus of mouse neonates. Journal of Applied Toxicology 39(6): 877–887. https://doi.org/10.1002/jat.3776. National Center for Biotechnology Information (2021) PubChem Annotation Record for Acetamiprid. Hazardous Substances Data Bank (HSDB).pubchem.ncbi.nlm.nih.gov/compound/ Acetamiprid. Öztas¸ E, et al. (2021) Cellular stress pathways are linked to acetamiprid-induced apoptosis in SH-SY5Y neural cells. Biology 10(9): 1–17. https://doi.org/10.3390/biology10090820. Pesticide Management Regulatory Agency (2011) Acetamiprid. Government of Canada2–5. Pesticide Management Regulatory Agency (2020) Update on the Neonicotinoid Pesticides. Government of Canada. Available at: https://www.canada.ca/en/health-canada/services/ consumer-product-safety/reports-publications/pesticides-pest-management/fact-sheets-other-resources/update-neonicotinoid-pesticides.html. Sano K, et al. (2016) In utero and lactational exposure to acetamiprid induces abnormalities in socio-sexual and anxiety-related behaviors of male mice. Frontiers in Neuroscience 10: 1–12. https://doi.org/10.3389/fnins.2016.00228. Shakthi Devan RK, Prabu PC, and Panchapakesan S (2015) Immunotoxicity assessment of sub-chronic oral administration of acetamiprid in Wistar rats. Drug and Chemical Toxicology 38(3): 328–336. https://doi.org/10.3109/01480545.2014.966382. Shi X, et al. (2011) Toxicities and sublethal effects of seven neonicotinoid insecticides on survival, growth and reproduction of imidacloprid-resistant cotton aphid, Aphis gossypii. Pest Management Science 67(12): 1528–1533. https://doi.org/10.1002/ps.2207. Terayama H, et al. (2018) Effect of acetamiprid on the immature murine testes. International Journal of Environmental Health Research 28(6): 683–696. https://doi.org/ 10.1080/09603123.2018.1504897. Todani M, et al. (2008) Acute poisoning with neonicotinoid insecticide acetamiprid. Japanese Journal of Toxicology 21(4): 387–390. Tomizawa M and Casida JE (1999) Minor structural changes in nicotinoid insecticides confer differential subtype selectivity for mammalian nicotinic acetylcholine receptors. British Journal of Pharmacology 127(1): 115–122. https://doi.org/10.1038/sj.bjp.0702526. Tomizawa M and Casida JE (2003) Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors. Annual Review of Entomology 48: 339–364. https://doi.org/10.1146/annurev.ento.48.091801.112731. Tomizawa M and Casida JE (2005) Neonicotinoid insecticide toxicology: mechanisms of selective action. Annual Review of Pharmacology and Toxicology 45: 247–268. https://doi.org/ 10.1146/annurev.pharmtox.45.120403.095930. US EPA (2002) Acetamiprid, Pesticide Fact Sheet. Available at: https://www3.epa.gov/pesticides/chem_search/reg_actions/registration/fs_PC-099050_15-Mar-02.pdf. Wang R, et al. (2012) Highly sensitive and specific detection of neonicotinoid insecticide imidacloprid in environmental and food samples by a polyclonal antibody-based enzyme-linked immunosorbent assay. Journal of the Science of Food and Agriculture 92(6): 1253–1260. https://doi.org/10.1002/jsfa.4691. Watanabe E, et al. (2006) Immunoassay for acetamiprid detection: Application to residue analysis and comparison with liquid chromatography. Analytical and Bioanalytical Chemistry 386(5): 1441–1448. https://doi.org/10.1007/s00216-006-0683-z. Yang G, et al. (2018) Quantitative ecotoxicity analysis for pesticide mixtures using benchmark dose methodology. 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Acetic acid Mariana Tadros and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.D. Pravasi, Acetic Acid, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 33–35, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00216-5.

Chemical profile Introduction Uses Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Animal Human Chronic toxicity animal Human Immunotoxicity Clinical management Ecotoxicology Exposure standards and guidelines Conclusion References Further reading

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Abstract Acetic acid, which is also known as ethanoic acid, is a synthetic carboxylic acid. AcOH and HOAc are the two most common abbreviation of acetic acid (AA). Glacial acetic acid, the pure and water free form of AA, is clear colorless liquid with vinegar odor. The mechanism of action is not fully understood, however, the undissociated form of acetic acid has shown benefits in enhancing lipid solubility thus increasing fatty acid accumulation on the cell membrane and in other cell wall structure. As a weak acid, it can inhibit carbohydrate metabolism which leads to subsequent death of organism. It is used as an antiseptic when in solution, with effectivity against bacteria (streptococci, staphylococci, pseudomonas, enterococci, etc.). Normal routes of exposure are via eyes, skin, and inhalation. It can cause eye, skin, nose, throat irritation; eye & skin burns; skin sensitization; dental erosion; black skin, hyperkeratosis; conjunctivitis, lacrimation; pharyngeal edema, and chronic bronchitis. AA is absorbed from the gastrointestinal tract and through the lungs and almost completely oxidized by tissues. Household vinegar contains up to 8% of AA. It is used in the manufacture of acetic anhydride, cellulose acetate, vinyl acetate monomer, acetic esters, chloroacetic acid, plastics, dyes, insecticides, photographic chemicals, and rubber. Other uses include the manufacture of vitamins, antibiotics, hormones, and organic chemicals, and as a food additive. It may contain up to 700–1200 mg/kg (mg/kg) in wines, up to 860 mg/kg in aged cheeses, and 2.8 mg/kg in fresh orange juice. OSHA standard for airborne acetic acid as an eight-hour, time-weighted average (TWA) is 10 ppm. ACGIH has adopted a threshold limit value (TLV) of 10 ppm. The USFDA has affirmed that acetic acid is generally recognized as safe as a multipurpose food additive. Overexposed individuals should receive supportive care.

Keywords Acetic acid; Cleaning agent; Fermentation; Food additive; Food ingredient; Vinegar

Key points

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Acetic acid (AA) is extensively used in food, chemical and agriculture industry. Although exposure to AA is normally safe, overexposure may have non-fatal toxic consequences. Last two decades have witnessed considerable progress in AA research in biomedical sciences of which many are clinical applications.

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Chemical profile

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Name: Acetic acid Chemical Abstracts Service Registry Number: 64-19-7 Synonyms: Glacial acetic acid, Ethylic acid, Pyroligneous acid, Vinegar acid, Methanecarboxylic acid Molecular Formula: C2H4O2, or CH3COOH Chemical Structure:

Introduction Acetic acid (AA) is known to be used in ancient times. It was known in Greek before the 3rd century BCE. Acetic acid was concentrated from vinegar through distillation by Jabir Ibn Hayyan, or also known as Gaber, in the 8th century. In 1847, Hermann Kolbe (germen chemist) was the first to use inorganic martial to synthesize AA. AA is present throughout nature as a normal metabolite of most biological organisms. Acetic acid may also be released to the environment in a variety of waste effluents, in emissions from combustion processes, and in exhaust from gasoline and diesel engines. AA is produced by the decomposition of solid biological wastes and is readily metabolized by living organisms. Using bacteria (Acetobacter species), large quantities of vinegar are manufactured by fermenting ethanol. Acetic acid’s food industry code is E260 (food additive) and is used as an acidity regulator/condiment. In cell biochemistry, the acetyl group, derived from acetic acid, when bound to coenzyme A, is quintessential to carbohydrate and lipid metabolism. Majority of the acetic acid is manufactured by fermenting methanol, partly by recycling and oxidation of ethanol. Glacial acetic acid is a name for anhydrous (water-free) acetic acid, which is used in the commercial setting. Acetobacter, aka acetic acid bacteria can produce AA, in the form of vinegar in an aerobic environment. Common substrates can be apple-cider, wine, and fermented grain, malt, rice, potato mashes or other forms of starchy masses. Acetobacter utilizes the following chemical reaction to generate acetic acid: C2 H5OH ðethanolÞ + O2 ! CH3 COOH ðacetic acidÞ + H2 O

Uses Large quantities of acetic acid are used to make products such as photographic chemicals, pesticides, microbicide, herbicide, pharmaceuticals, food preservatives, rubber, and plastics. Acetic acid is also present as the main component of vinegar (5–18% of acetic acid), albeit at very low concentrations that are harmless to humans. Acetic acid is used in the manufacture of various acetates, acetyl compounds, cellulose acetate, acetate rayon, plastics, and rubber. It is also used in tanning, as laundry sour, in printing calico, and in dyeing silk. It’s use as solvent in the process of producing TPA (terephthalic acid) and the raw material for Pet (polyethylene terephthalate). Also, it is a solvent for gums, resins, volatile oils, and many other substances. Acetic acid is widely used in commercial organic synthesis. It is also used in formulation of local, ontological, or drops 2% in alcohol to treat infectious diseases of the external ear. Research indicates that acetic acid disinfection (at a 0.34% concentration) may play a role in improving individual symptoms in covid-19 patients when used adjunctively than that of patients not on it. Acetic acid is an FDA approved substance added to food as a sensor for pH control. Also, acetic acid is found to have a direct activation of large conductance calcium and voltage activated K+ channels (BKCa) in detrusor smooth muscle cells. Acetic acid has become a commonly used agent in orthopedic surgery. The proposed roles include biofilm eradication, surgical debridement, postoperative scar reduction and managing soft tissue injuries.

Environmental fate and behavior Acetic acid is present throughout nature as a normal metabolite of both plants and animals. Acetic acid may also be released to the environment in a variety of waste effluents, in emissions from combustion processes, and in exhaust from gasoline and diesel engines. If released to air, a vapor pressure of 15.7 mmHg at 25  C indicates acetic acid should exist solely as a vapor in the ambient atmosphere. Vapor-phase acetic acid will be degraded in the atmosphere by reaction with photochemically produced hydroxyl radicals; the half-life for this reaction in air is estimated to be 22 days. Physical removal of vapor-phase acetic acid from the atmosphere occurs via wet deposition processes based on the miscibility of this compound in water. In acetate form, acetic acid has also been detected in atmospheric particulate material. If released to soil, acetic acid is expected to have very high to moderate mobility based upon measured Koc values, using near-shore marine sediments, ranging from 6.5 to 228. No detectable adsorption was measured for acetic acid using two different soil samples and one lake sediment. Based on a classification scheme and a Koc value of 1.0, acetic acid is not anticipated to adsorb to suspended solids and sediment. Volatilization from water surfaces is not expected based upon Henry’s Law constant of 1.43  10−7 atm-cu m/mol. The pKa of acetic acid is 4.76, indicating that this

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compound will exist predominantly in anion form at an environmental pH range of 5–9. Volatilization from dry soil surfaces may occur based upon the vapor pressure of this compound. Biodegradation in both soil and water is expected to be rapid; a large number of biological screening studies has determined that acetic acid readily under both aerobic and anaerobic conditions (99% degraded in 7 days) and aerobic conditions. Acetic acid also degrades through photodegrade in a slower rate than biodegrades (50% degrades after 21 days. Volatilization from water surfaces is not expected to be an important fate process based on its measured Henry’s law constant. An estimated bacterial colony foraging (BCF) of 20 ml/kg, and 30 min was the interval the rats survived when exposed to concentrated vapor inhalation. While in the case of the inhalation of metered vapor at a concentration of 16,000 ppm for 4 h, the rate of mortality was 1/6 (Smyth et al., 1962). It was, however, lethal to all the rats exposed to 32,000 ppm (Smyth et al., 1962). After inhaling 5% (50,000 ppm, 1 ppm equals to 0.0001%) of acetone by guinea-pigs for 5–8 h, the congestion of spleen, kidneys and lungs was observed, and even pulmonary edema occurred (Sollmann, 1957). When inhaled (12,600 ppm – 50,600 ppm) it was narcotic to rats and mice. The narcotic effect of acetone was delayed of considerably longer duration compared to toluene or ethanol. Both the degree and the rapidity of the induction of narcosis were dose-dependent. Inhaled acetone was fairly distributed in the organs (Bruckner and Peterson, 1981). Male rats exposed to different acetone concentrations (5000–20,000 ppm) for 1 hour, alternating days with air for 6 pairing sessions, a significant decrease was observed in the locomotor activity resembling central nervous system depression in comparison to controls, while place preference was not affected (Lee et al., 2008). No irritation was seen in the case of uncovered application of 0.01 ml undiluted sample of acetone on the clipped skin of rabbits, but 0.005 ml of undiluted acetone results in severe burn on rabbit cornea (Smyth et al., 1962). Application of 1 v/v% acetone in drinking water to male mice for a week did not have any influence on the body weight, liver weight, blood glucose concentration and serum glutamate oxaloacetate transaminase activity, but decreased hepatic reduced

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glutathione levels, while increased CYPIIE1 isozyme activities. Microscopic examinations did not reveal any morphological change in the liver (Kalapos, 1999a). The data of externally added acetone on blood glucose concentrations of animals are contradictory. Some investigations reported on a rise of blood glucose level after acetone addition, while others failed to corroborate this finding (Kalapos, 2003).

Human Acute intoxication cases can be results of either an incidental short-term exposure to acetone at workplace or ingestion happening accidentally or intentionally, mostly as a tool for committing suicide. Under experimental conditions, the exposure to acetone vapor at a concentration of 1000 ppm for 4 or 8 h did not lead to considerable appearance of tension, tiredness or any other complaints, but some mild decrements on behavioral performance tests were seen even at 250 ppm concentration (Dick et al., 1989; Seeber et al., 1992). In contrast, a chamber experiment with 10 male volunteers, 250 ppm acetone for 2 h, reported on only slight throat discomfort (Ernstgråd et al., 1999). Significantly increased white blood cell counts, increased eosinophil counts, and decreased phagocytic activity of neutrophils were reported on in the case of young male volunteers exposed to 500 ppm for a single 6-h exposure, while in other volunteers exposed to 500 ppm for 2 h hematologic parameters were within normal range (Matsushita et al., 1969; DiVincenzo et al., 1973). Injection of acetone to humans resulted in a drop of blood pressure, provoked drowsiness, but did not affect blood glucose concentration (Koehler et al., 1941). Its inhalation caused bronchial irritation and depressive symptoms as producing stupor, dyspnea, fall of body temperature and heart rate, and poor pulse (Sollmann, 1957; Ross, 1973). Occupational exposure to acetone results in its detection in urine samples of exposed workers and there is a linear correlation between environmental concentration in breathing zone and urinary excretion (Ross, 1973; Ghittori et al., 1987; Kawai et al., 1990; Brega et al., 1991; Fujino et al., 1992; Kieswetter et al., 1994). In the case of eight workers exposed to near 1000 ppm (2400 mg/m3) acetone the ratings on scales of well-being and acute symptoms correlated with urine acetone levels, but not with acetone concentrations in the air, stressing the value of individual difference (Kieswetter et al., 1994). Acute occupational exposure to acetone results in nausea and vomiting, drowsiness, eye irritation, poor pulse, weakness or even unconsciousness (Ross, 1973). Symptoms following acute acetone ingestion include nausea, vomiting that may progress to hematemesis and gastric hemorrhage, sedation, respiratory depression, ataxia, and paresthesia. Depression resembles alcoholic stupor. Coughing and bronchial irritation may be the only clues to ingestion of quantities that are too small to produce sedation. Swallowing very high levels of acetone can result in unconsciousness and damage to the skin in the mouth. Unconsciousness and even coma accompany high levels of exposure, usually in the case of suicidal or accidental ingestions. Among laboratory data, high levels of acetone are accompanied with a transient elevation of blood glucose and lactate levels, and increased osmolality and leukocytosis may be seen without any rise of serum levels of hepatic enzymes. Urine test for acetone is strongly positive. Organ damage including liver injury is not evident suggesting that acetone itself does not exert an immediate toxicity to organs in these suicidal cases, if survived (Sollmann, 1957; Gitelson et al., 1966; Zettinig et al., 1997; Kostusiak et al., 2003). Since the function of central nervous system is immediately affected by acetone exposure, it is likely that this is the life-threatening factor in acute intoxication and not the metabolic effects of acetone. Observations with tadpoles suggest that the anesthetic properties of acetone are related to its enhancer effect upon glycine and GABAA receptors, and to the inhibition of NMDA receptors (Yang et al., 2007). The effective concentrations in the case of NMDA and GABAA receptors were extremely high, thus only a glycinergic mechanism seems relevant, but animal data cannot simply be extrapolated to humans.

Chronic toxicity Animals The application of acetone in their drinking water to rodents (rats and mice, male and female) for 13 weeks resulted in depressed body weight gain (at 50,000 and 100,000 ppms), bone marrow hypoplasia (at 100,000 ppm). Nephropathy at 20,000 and 50,000 ppms, as well as hypogonadism at 50,000 ppm developed only in male rats. Acetone treatment during 13-week was associated with relative increases in kidney and liver weights above 20,000 ppm (Dietz et al., 1991). No susceptibility of different age groups of laboratory rats to acetone can be stated. When chickens were treated with 350 mg acetone/kg for 30 days the harmful effect of the compound resulted in an amelioration of hematological parameters, an increase of liver injury verified by enzymatic and morphological analyses, and oxidative stress (Abd El-Rahman et al., 2017). In the case of repeated, daily 4-h inhalation of acetone vapor at concentrations of 12,000 and 16,000 ppms for 10 days to female rats led to a specific alteration of avoidance behavior and a tolerance developed (Goldberg et al., 1964). Although acetone inhalation causes bronchial and corneal irritation, indeed the majority of its actions are depressive symptoms including stupor, dyspnea, fall of body temperature and heart rate, and poor pulse. Its effects are proportional to acetone concentration in the vapor.

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Human The relevance to humans of above described effects observed in animal studies is questionable, therefore it is yet unknown if humans would experience such toxicological effects or not. Noteworthy is, that under occupational environment exposure to acetone frequently happens for longer than couple of weeks, but these findings show exposed workers do not exhibit more serious long lasting complaints and only drowsiness, eye and throat irritation, dizziness, headache or problems with performance are noted. If experienced the symptoms are transient (Tran et al., 2020). However, prolonged or repeated skin contact to acetone may produce severe dermatitis. From methodological point of view, important shortcomings limiting the use of data for assessing health effects of acetone in humans are that chronic studies were usually undertaken in an occupational environment where the coincidental exposure to other chemicals could not be fully excluded and the proper control groups were sometimes lacking. More important is, however, the note that acetone potentiates the toxicity of consumed ethanol, several medicines and chemicals, thus enhance the ignition of the formation of such hazardous materials like methylglyoxal or free radicals.

Immunotoxicity No data available regarding immunological effects in humans after oral exposure to acetone. (The effects of acetone upon hematologic parameters are shown above in the section of Acute and short-term toxicity). Animal studies are also sparse. Exposure of CD-1 male mice to acetone in the drinking water at average doses as high as 1144 mg/kg/day for 28 days did not provided evidence for immunotoxicity (Woolhiser et al., 2006).

Reproductive and developmental toxicity Acetone crosses placenta. Transplacental isopropanol and consequently acetone exposure was documented in a newborn, delivered by an alcoholic female, with weak respiratory effort, and Apgar 2 and 5 at 1 and 5 min, respectively (Wood et al., 2007). In animal studies, developmental toxicity was not reported despite the fact that the occurrence of acetone levels in fetal tissues was shown (López-Sariano and Argiles, 1986; Peinado et al., 1986). The vaginal cytology in rodents was not affected, either (Dietz et al., 1991). However, the exposure to acetone in drinking water for 13 weeks resulted in a depression of sperm motility and increased the incidence of abnormal sperm (Dietz et al., 1991). When small planktonic crustacean (Daphnia magna) was challenged to increasing acetone concentrations (7.9–79 mg/l) in the culture medium for 21 days significant reduction was not observed either in survival or in fecundity. However, abnormal development of antennae was already recorded at the highest concentration in the first subsequent generation and the median effect time (ET50) was 12.5 days. Longer the maternal exposure to acetone, higher the incidence of developmental abnormality (Leoni et al., 2008). In an in vitro study with zebrafish (Danio rerio) embryos, a period dependent susceptibility to acetone was reported on. The most strongly affected (by 0.5%) were the 7 day post-fertilization larvae, while in other stages even 2.5% of acetone was tolerated (Maes et al., 2012). Workers at a plastic production plant and exposed to both styrene and acetone showed an increase in the number of abnormal sperm heads (Jelnes, 1988). Selective surveys for acetone are, however, unavailable (Jensen et al., 2006).

Genotoxicity The genotoxicity of acetone has been thoroughly investigated in laboratory. Acetone of reagent grade was evaluated by the standard plate incorporation method in the Ames Salmonella reverse mutation assay with strains TA98, TA100, TA1535, TA1537, and TA1538. Results were negative in these strains. Negative in vitro results were also gained in sister chromatid exchange assay, SHE cell transformation assay and in DNA repair-deficient bacterial tests (Kalapos, 2014). Acetone was not genotoxic to E.coli (Quintero et al., 2012). Human data specific to acetone are not available. However, studies located regarding genotoxicity associated with occupational exposures to acetone and other volatile substances (e.g. toluene) indicated that the reported effects were predominantly attributed to toluene not acetone (Pitarque et al., 1999; Heuser et al., 2005). These results currently do not raise genotoxicity, but the lack of human data prompts caution.

Carcinogenicity No studies are available that would have been undertaken for longer than 2 weeks period of time on the toxicity and carcinogenicity of acetone to laboratory animals. The U.S. Environmental Protection Agency (USEPA) has stated that there is no sufficient evidence present to classify acetone as carcinogenic to humans. The International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) and National Toxicology Program (NTP) of the National Institute of Health (NIH) have not classified acetone for carcinogenicity.

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Organ toxicity Slight liver hypertrophy and neuropathy were only observable in rodents, when those were exposed to acetone in their drinking water for 13 weeks (Dietz et al., 1991). Shorter exposure did not lead to detectable changes (Kalapos, 1999a).

Interactions A significant toxicological role has to be assigned to acetone. It is not only metabolized by, but also induces the CYPIIE1 isozymes (Gonzalez, 1989), resulting in the production of a more reactive intermediate, methylglyoxal (Kalapos, 1999b). On the other hand, acetone disturbs biotransformation of various of foreign compounds, thus potentiating their toxicity. Being capable of metabolizing a broad range of xenobiotics, such as acetaminophen, aniline, alcohols, benzene and halogenated substrates, the induction of CYPIIE1 gene products, both mRNA and protein, in the liver results in an increased rate of oxidation of xenobiotics, thereby leading to an overproduction of their reactive metabolites, especially catechols, phenols or free radicals (Bánhegyi et al., 1988; Gonzalez, 1989; Zang et al., 1992; Ernstgråd et al., 1999; Joshi and Adhikari, 2019). In addition, acetone depletes glutathione stores, thus hampering the protection against electrophiles (Kalapos et al., 1991a, b).

Toxicogenomics No conclusive research is available.

Clinical management If inhaled intentionally with the purpose of recreational intoxication (usually in combination with other solvents as ingredient of commercial products) or accidentally and breathing is difficult, the person is moved to fresh air and administered oxygen. For skin contact, the contaminated area is washed with water. For eye contact, water is used for flushing. In case of ingestion, mainly with suicidal intent, intubation, breath assisting, gastric lavages and hemofiltration may be performed parallel with forced diuresis. Other feature with clinical relevance is that acetone efforts an antiseizure effect in animal models for epilepsy when administered in millimolar concentrations (Likhodii and Burnham, 2002). No toxicity was noted in these cases. It is suggested that acetone is a factor contributing to the beneficial effects of high-fat low-carbohydrate ketogenic diet used in clinical practice, particularly to control intractable epilepsies in children (Likhodi et al., 2003). In experimental animals no sex differences have been recognized, but it is unknown whether this rule applies to humans, too. Nonetheless, as sensitive populations have to be considered those suffering in diseases in which elevated acetone levels in the plasma may occur (i.e. starvation, diabetes mellitus, alcoholism, inherited disorders of metabolism, etc.).

Environmental fate and behavior Acetone evaporates rapidly, even from water and soil. Once entered the atmosphere, it is degraded by photolysis, a reaction in which free radicals are involved or removed by wet deposits. It is a significant groundwater contaminant because of its miscibility in water (Singh et al., 1994).

Ecotoxicology The LD50 of acetone for fish is 8.3 g/l of water over 96 h (Sollmann, 1957). It may also be biodegraded when released into the soil, since it is consumed by microorganisms, but its bioaccumulation and toxicity to aquatic life are not expected.

Exposure standards and guidelines The National Research Council’s Committee on Toxicology (USA) recommends 1.000 ppm and 200 ppm 24-h emergency exposure limit (24-h EELs) and 90-day continuous exposure limit (90-d CEL) for acetone, respectively. The United States Department of Labor, Occupational Safety and Health Administration recommends the exposure limit of 1.000 ppm for acetone (time-weighted average). Although, the standards and guidelines may differ in various countries, for general industry, shipyards, and construction the occupational exposure between 250 and 1000 ppm is, in general, recommended (ATSDR, 2022). The application guideline for food and water is 0.9 mg/kg/day (ATSDR, 2022).

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Conclusion Acetone is currently not regarded as a genotoxic or mutagenic chemical in humans, and is not classified as a carcinogen agent either. There is no concern for its chronic neurotoxicity, too. Nevertheless, only limited information is available that does not demonstrate conclusive effects either in humans or in experimental animals arising from chronic exposure to acetone. This particularly applies to reproductive toxicity. Therefore, there is a need for further experimental and epidemiological studies that are specific to acetone exposure, making possible to distinguish its effects alone from that of other contaminating chemicals.

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Kumar S, Nayek M, Kumar A, Tandon A, Mondai P, Vijay P, Bhangale UD, and Tyagi D (2011) Aldehyde, ketone and methane emissions from motor vehicle exhaust: A critical review. American Chemical Science Journal 1: 1–27. Landau BR and Brunengraber H (1987) The role of acetone in the conversion of fat to carbohydrate. Trends in Biochemical Sciences 12: 113–114. Landini BE and Bravard ST (2010) Effect of exhalation variables on the current response of an enzymatic breath acetone sensing device. IEEE Sensors Journal 10: 19–24. Lee DE, Pai J, Mullapudui U, Alexoff DL, Ferieri R, and Dewey SL (2008) The effects of inhaled acetone on place conditioning in adolescent rats. Pharmamacology, Biochemistry and Behavior 89: 101–105. Leoni B, Bettinetti R, and Galassi S (2008) Sub-lethal effects of acetone on Daphnia magna. Ecotoxicology 17: 199–205. Likhodi SS, Serbanescu I, Cortez M, Murphy P, Snead OC, and Burnham WM (2003) Anticonvulsant properties of acetone, a brain ketone elevated by the ketogenic diet. Annals of Neurology 54: 219–226. Likhodii SS and Burnham WM (2002) Ketogenic diet: does acetone stop seizures? Medical Science Monitor 8: HY19–24. Lohmann DP, Pennington DG, and McBride LR (1991) Acetone: A hazard to plastic medical products – A case report. Journal of Thoracic and Cardiovascular Surgery 102: 937–938. López-Sariano FJ and Argiles JM (1986) Acetone levels in rat plasma and tissues: Effect of pregnancy and fasting. IRCS Medical Science 13: 161–162. López-Sariano FJ, Alemany M, and Argiles JM (1985) Rat acetoacetic acid decarboxylase inhibition by acetone. International Journal of Biochemistry 17: 1271–1273. Lorenc JD (2003) Inhalant abuse in the pediatric population: A persistent challenge. Current Opinion in Pediatrics 15: 204–209. Maes J, Verlooy L, Buenafe OE, de Witte PAM, Esguerra CV, and Crawford AD (2012) Evaluation of 14 solvents and carriers for screening applications in zebrafish embryos and larvae. PLoS One 7: e43850. Matsushita T, Yoshinume A, Inoue T, Yamada S, and Suzuki H (1969) Experimental studies for determining the mac value of acetone: 1. Biological reactions in the“ one-day exposure” to acetone. Sangyo Igaku 11: 477–484. Mitsui T, Yuza N, Kato N, and Kondo T (1999) Is acetone a normal breath constituent in Japanese? Clinica Chimica Acta 280: 205–208. Mourkides GA, Hobbs DC, and Koeppe RE (1958) The metabolism of acetone-2-C14 by intact rats. Journal of Biological Chemistry 234: 27–30. Müller J (1898) Ueber die Ausscheidungsstätten des Acetons und die Bestimmung desselben in der Athemluft und den Hautausdünstungen des Menschen. Archiv für Experimentelle Pathologie und Pharmakologie 40: 351–362. Musa-Veloso K, Likhodii SS, and Cunnane SC (2002) Breath acetone is a reliable indicator of ketosis in adults consuming ketogenic meals. American Journal of Clinical Nutrition 76: 65–70. Nebelthau A (1897) Beitrag zur Kenttniss der Acetonurie. Zentralblatt für innere Medizin 18: 977–982. Obeidat Y (2021) The most common methods for breath acetone concentration detection: A review. IEEE Sensors Journal 21: 14450–14458. Owen OE, Trapp VE, Skutches CL, Mozzoli MA, Holdtke RD, Boden G, and Reichard GA (1982) Acetone metabolism during diabetic ketoacidosis. Diabetes 31: 242–248. Pappas AA, Ackerman BH, Olsen KM, and Taylor EH (1991) Isopropanol ingestion: A report of six episodes with isopropanol and acetone serum concentration time data. Journal of Toxicology. Clinical Toxicology 29: 11–21. Pedersen KJ (1929) The ketonic decomposition of beta-keto carboxylic acids. Journal of the American Chemical Society 51: 2098–2107. Peinado J, López-Sariano FJ, and Argiles FJ (1986) The metabolism of acetone in pregnant rat. Bioscience Reports 6: 983–989. Petters W (1857) Untersuchungen über die Honigharnruhr. Prager Vierteljahrschrift Praktische Heilkunde 55: 81–94. Pitarque M, Vaglenov A, Nosko M, Hirvonen A, Creus A, and Marcos R (1999) Evaluation of DNA damage by the Comet assay in shoe workers exposed to toluene and other organic solvents. Mutation Research 441: 115–127. Prabhakar A, Quach A, Wang D, Zhang H, Terrera M, Jackemeyer D, Xian X, Tsow F, Tao N, and Forzani ES (2015) Breath acetone as biomarker for lipid oxidation and early ketone detection. Global Journal of Obesity, Diabetes and Metabolic Syndrome 1: 12–19.

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Price D and Rittenberg D (1950) The metabolism of acetone. Journal of Biological Chemistry 185: 449–459. Quintero N, Stashenko EE, and Fuentes JL (2012) The influence of organic solvents on estimates of genotoxicity and antigenotoxicity in the SOS chromotest. Genetics and Molecular Biology 35: 503–514. Reichard GA, Haff AC, Skutches CL, Paul P, Holroyde CP, and Owen OE (1979) Plasma acetone metabolism in the fasting human. Journal of Clinical Investigation 63: 619–626. Righettoni M, Tricoli A, Gass S, Schmid A, Amann A, and Pratsinis SE (2012) Breath acetone monitoring by portable Si:WO3 gas sensors. Analytica Chimica Acta 738: 69–75. Righettoni M, Schmid A, Amann A, and Pratsinis SE (2013) Correlations between blood glucose and breath components from portable gas sensors and PTR-TOF-MS. Journal of Breath Research 7: 037110. Rollo J (ed.) (1798) Cases of the diabetes mellitus with the results of the trials of certain acids, and other substances, in the cure of Lues venereal, 2nd edn. London: T. Gillet. Ross DS (1973) Acute acetone intoxication involving eight male workers. American Occupational Hygiene 16: 73–75. Ruzsanyi V (2013) Ion mobility spectrometry for pharmacokinetic studies – exemplary application. Journal of Breath Research 7: 046008. https://doi.org/10.1088/1752-7155/7/ 4/046008. Ruzsanyi V, Baumbach JI, Sielemann S, Litterst P, Westhoff M, and Freitag L (2005) Detection of human metabolites using multi-capillary columns coupled to ion mobility spectrometers. Journal of Chromatography A 1084: 145–151. Sakami W and Lafaye J M (1950) Formation of formate and labile methyl groups from acetone in the intact rat. Journal of Biological Chemistry 187: 369–378. Schwartz L (1898) Über die Oxydation des Acetons und homologer Ketone der Fettsäurereihe. Archiv für Experimentelle Pathologie und Pharmakologie 44: 168–194. Schwarz K, Pizzini A, Arendacká B, Zerlauth K, Filipiak W, Schmid A, Dzien A, Neuner S, Lechleitner M, Scholl-Bürgi S, Miekisch W, Schubert J, Unterkofler K, Witkovský V, Gastl G, and Amann A (2009) Breath acetone - aspects of normal physiology related to age and gender as determined in a PTR-MS study. Journal of Breath Research 3: 027003. Seeber A, Kieswetter E, and Blaszkewicz M (1992) Correlations between subjective disturbances due to acute exposure to organic solvents and internal dose. Neurotoxicity 13: 265–269. Seymour KJ, Bluml S, Sutherling J, Sutherling W, and Ross BD (1999) Identification of cerebral acetone by 1H-MRS in patients with epilepsy controlled by ketogenic diet. Magnetic Resonance Materials in Physics, Biology and Medicine 8: 33–42. Simpson DM and Beynon RJ (2010) Acetone precipitation of proteins and the modification of peptides. Journal of Proteome Research 9: 444–450. Singh HB, O’Hara D, Herlth D, Sachse W, Blake DR, Bradshaw JD, Kanakidou M, and Crutzen PJ (1994) Acetone in the atmosphere: Distribution, sources, and sinks. Journal of Geophysical Research 99: 1805–1819. Sluis MK, Small FJ, Allen JR, and Ensign SA (1996) Involvement of an ATP-dependent carboxylase in a CO2-dependent pathway of acetone metabolism by Xanthobacter strain Py2. Journal of Bacteriology 178: 4020–4026. Smith D, Ismail K, Diskin AM, Chapman G, Magnay JL, Španel P, and O’Brien S (2006) Increase of acetone emitted by urine in relation to ovulation. Acta Obstetrica et Gynecologica Scandinavica 85: 1008–1011. Smith S, Burden H, Persad R, Whittington K, de Lacy Costello B, Ratcliffe NM, and Probert CS (2008) A comparative study of the analysis of human urine headspace using gas chromatography-mass spectrometry. Journal of Breath Research 2: 037022. Smyth HF, Carpenter CP, Weil CS, Pozzani UC, and Striegel JA (1962) Range-finding toxicity data: List VI. American Industrial Hygiene Association Journal 23: 95–107. Sollmann T (1957) A manual of pharmacology and its application to therapeutics and toxicology, 8th edn. Philadelphia: W.B. Saunders, pp. 878–879. Taylor DG, Trudgill PW, Cripps RE, and Harris PR (1980) The microbial metabolism of acetone. Journal of General Microbiology 118: 159–170. Tran HM, Bui HTM, Toumsang S, Ngo NTB, Nguyen NPT, Nguyen HTM, Nguyen SM, Hara K, and Wangwingwatana S (2020) Occupational symptoms due to exposure to volatile organic compounds among female Vietnamese nail salon workers in Danang city. Journal of Occupational Health 62: e12160. Turner C, Španel P, and Smith D (2006) A longitudinal study of ammonia, acetone and propanol in the exhaled breath of 30 subjects using selected ion flow tube mass spectrometry, SIFT-MS. Physiological Measurement 27: 321–327. Turner C, Walton C, Hoashi S, and Evans M (2009) Breath acetone concentrations decreases with blood glucose concentration in type I diabetes mellitus patients during hypoglycaemic clamps. Journal of Breath Research 3: 046004. Unterkofler K, King J, Mochalski P, Jandacka M, Koc H, Teschl S, Amann A, and Teschl G (2015) Modeling-based determination of physiological parameters of systemic VOCs by breath gas analysis: A pilot study. Journal of Breath Research 9: 036002. van Stekelenburg GJ and Koorevaar G (1972) Evidence for the existence of mammalian acetoacetate decarboxylase: with special reference to human blood serum. Clinica Chimica Acta 39: 191–199. Wang CC, Weng YC, and Chou TC (2007) Acetone sensor using lead foil as working electrode. Sensors and Actuators B: Chemical 122: 591–595. Weiss JM (1958) Acetone: Tonnage by-product. Chemistry and Engineering News 36: 79–82. Wigaeus E, Holm S, and A˚ strand I (1981) Exposure to acetone. Uptake and elimination in man. Scandinavian Journal of Work, Environment and Health 7: 84–94. Wilson MP, Hammond SK, Nicas M, and Hubbard AE (2007) Worker exposure to volatile organic compounds in the vehicle repair industry. Journal of Occupational and Environmental Hygiene 4: 301–310. Wood JN, Carney J, Szczepanski K, Calello DP, and Hurt H (2007) Transplacental isopropanol exposure: case report and review of metabolic principles. Journal of Perinatology 27: 183–185. Woolhiser MR, Houtman CE, and Waechter JM (2006) Acetone in drinking water does not modulate humoral immunity in mice as measured by the antibody, plaque-forming cell assay. International Journal of Toxicology 25: 333–339. Yang L, Zhao J, Milutinovic PS, Brosnan RJ, Eger EI, and Sonner JM (2007) Anesthetic properties of the ketone bodies b-hydroxybutyric acid and acetone. Anesthesia and Analgesia 105: 673–679. Zang CS, Brady JF, and Hong JZ (1992) Dietary effects on cytochrome P450, xenobiotic metabolism, and toxicity. FASEB Journal 6: 737–744. Zettinig G, Watzinger N, Eber B, Henning G, and Klein W (1997) Überlebte Vergiftung nach Einnahme der zehnfachen Letaldosis von Aceton. Deutsche Medizinische Wochenschrift 122: 1489–1492.

Further reading Ruzsányi V and Kalapos MP (2017) Breath acetone as a potential marker in clinical practice. Journal of Breath Research 11: 024002.

Relevant websites http://www.atsdr.cdc.gov :Agency for Toxic Substances and Disease Registry. Toxicological Profile for Acetone. http://www.inchem.org :Acetone: Environmental Health Criteria (from the International Program on Chemical Safety) (EHC 207, 1998). https://www.cdc.gov/niosh/npg/npgd0004.html :National Institute for Occupational Safety and Health. https://echa.europa.eu/substance-information/-/substanceinfo/100.000.602 :European Chemical Agency.

Acetonitrile Heriberto Robles, Enviro-Tox Services, Inc., Irvine, CA, United States © 2024 Elsevier Inc. All rights reserved. This is an update of H Robles, Acetonitrile, Editor(s); Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 40–42, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00624-2.

Chemical profile Background Uses Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Chronic toxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Other hazards Ecotoxicology Exposure standards and guidelines References

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Abstract Acetonitrile is a liquid with an ether-like odor. It is a highly polar, volatile organic compound used as a solvent in many different industrial applications. Acetonitrile is rapidly absorbed from the lungs and intestinal tract. Once absorbed, acetonitrile is metabolized to hydrogen cyanide and it can be acutely lethal when absorbed in high doses. Once acetonitrile is metabolized, the mechanism of action is the same as expected for cyanide poisoning. Signs and symptoms of acute acetonitrile intoxication include chest pain, tightness in the chest, excessive saliva secretion, nausea, emesis, tachycardia, hypotension, short and shallow respiration, headache, restlessness and seizures. Signs and symptoms do not follow immediately after exposure as acetonitrile has to be metabolized to hydrogen cyanide. Sign and symptoms may be delayed by three or more hours after exposure.

Keywords Cyanomethane; Ethane nitrile; Ethanenitrile; Ethyl nitrile; Industrial solvent; Methanecarbonitrile; Methyl cyanide; Volatile organic compounds

Key points

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Acetonitrile is a highly polar, volatile solvent used in many different industrial applications. Exposure to acetonitrile can occur through the oral, dermal, and inhalation routes. Symptoms of poisoning have been observed in persons exposed through these three routes. Acetonitrile can be acutely lethal when absorbed in high doses. Acetonitrile is metabolized to inorganic cyanide. Once metabolized, the mechanism of action is the same as expected for cyanide poisoning. Toxicity is produced by the combined effect of circulating acetonitrile and cyanide. Employees exposed to acetonitrile at potentially hazardous levels should be enrolled in a medical monitoring program. Treatment of acetonitrile poisoning is similar to that of cyanide poisoning (see Cyanide). This includes immediate therapy with 100% oxygen and assisted ventilation, if necessary. Therapy should also include correction of the metabolic acidosis and to combat cyanide poisoning.

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Abbreviations

ACGIH LD50 mg kg−1 mg L−1 mg m−3 NIOSH OSHA ug L−1 USEPA

American Conference of Government Industrial Hygienists Lethal dose 50 Milligrams per kilogram Milligrams per liter Milligrams per cubic meter National Institute of Occupational Safety and Health U.S. Occupational Safety and Health Administration Micrograms per liter United States Environmental Protection Agency

Chemical profile

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Chemicals Abstracts Service Number: 75-05-8 Synonyms: Cyanomethane, Methyl cyanide, Ethane nitrile, Ethanenitrile, Ethyl nitrile, Methanecarbonitrile, Methyl cyanide. Molecular Formula: C2H3N

Background Acetonitrile is a liquid with an ether-like odor. It is a highly polar, volatile solvent used in many different industrial applications. It is widely used in the pharmaceutical, photographic, chemical and analytical industries. It is useful as an industrial solvent for the separation of olefins, polymers, spinning fibers, and plastics. Other uses include the extraction and refining of copper and by-product ammonium sulfate; used for dyeing textiles and in coating compositions; used as a stabilizer for chlorinated solvents; manufacture of perfumes and cosmetics; and as a general reagent in a wide variety of chemical processes (PubChem, 2021).

Uses Acetonitrile is used in the chemical industry as an intermediary in the synthesis of several chemicals and products such as acetophenone, thiamine, acetamidine, and alpha-naphthaleneacetic acid, nitrogen-containing compounds, acrylic fibers, nitrile rubber, pesticides, pharmaceuticals, perfumes and lithium batteries. It is also used as a polar solvent for both organic and inorganic compounds and in nonaqueous titrations (PubChem, 2021).

Environmental fate and behavior If released to ambient air, acetonitrile will remain in the vapor phase where it will be degraded through reaction with photochemically-produced hydroxyl radicals. The half-life of acetonitrile in ambient air has been estimated to be about 620 days (PubChem, 2021). If released to soil, acetonitrile is expected to volatilize rapidly. Biodegradation in soil is not expected to be a major degradation pathway. If released to water, acetonitrile is not likely to adsorb to soil and sediment particles. Acetonitrile is expected to be removed from water bodies through volatilization as the chemical hydrolysis and bioaccumulation potential for this chemical are low (PubChem, 2021).

Exposure and exposure monitoring Exposure to acetonitrile can occur through the oral, dermal, and inhalation routes. Symptoms of poisoning have been observed in persons exposed through these three routes (Integrated Risk Information System (IRIS), 2022). Employees exposed to acetonitrile at potentially hazardous levels should be enrolled in a medical monitoring program (PubChem, 2021). Initially, the employee should undergo a medical examination to establish his/her baseline health conditions. The initial evaluation should include a complete history and physical examination. Persons with history of fainting or convulsive disorders may be at special risk (PubChem, 2021). Examination of the skin (particularly hands and face), eyes, liver, and kidneys should be included. Following initial examination, the employee should be scheduled to undergo annual medical examinations and the above-mentioned tests and examinations should be included (PubChem, 2021). Blood cyanide concentrations exceeding 0.1 mg L−1 of plasma or urine thiocyanate exceeding 20 mg L−1 in workers exposed to acetonitrile are indicative of excessive exposure (European Chemicals Bureau (ECB), 2002).

Toxicokinetics Acetonitrile can be acutely lethal when absorbed in high doses. Acetonitrile is metabolized to a hydroxyl metabolite by cytochrome P450 in the liver (Integrated Risk Information System (IRIS), 2022). Subsequent metabolism through catalase enzymes produces

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inorganic cyanide. Once metabolized, the mechanism of action is the same as expected for cyanide poisoning. Onset of cyanide poisoning may be delayed 3–8 h or more, as metabolism is required to produce the cyanide metabolite. Toxicity may be prolonged for up to 3 days in some cases (European Chemicals Bureau (ECB), 2002).

Mechanism of toxicity Acetonitrile is slowly metabolized by cytochrome P450 in the liver to produce inorganic cyanide (European Chemicals Bureau (ECB), 2002). Toxicity is produced by the combined effect of circulating acetonitrile and cyanide. Cyanide exerts its toxicological effects by disrupting oxygen utilization at the cellular level. The disruption results in decreased oxygen utilization by body tissues and lactic acidosis (European Chemicals Bureau (ECB), 2002).

Acute and short-term toxicity Animal susceptibility to acetonitrile varies by animal species and route of administration. Overall, animal susceptibility is mediated by the animal’s ability to absorb and metabolize acetonitrile into its toxic metabolite, hydrogen cyanide (Integrated Risk Information System (IRIS), 2022). In rats, the oral LD50 has been measured to be around 200 mg kg−1 (European Chemicals Bureau (ECB), 2002). The inhalation LC50 has been determined to be 7500 ppm following an 8-h exposure. The acute dermal lethal dose has been investigated in rabbits. The LD50 through the dermal route has been determined to be 980 mg kg−1 (European Chemicals Bureau (ECB), 2002). Subchronic exposures to low acetonitrile concentrations in the air (665 ppm or less) produced pulmonary inflammation and minor changes in body weights, hematocrit, hemoglobin, and liver and kidney function (Integrated Risk Information System (IRIS), 2022). Toxicological effects of acetonitrile are usually delayed, as the chemical must be metabolized to cyanide. However, exposure to high doses may result in rapidly developing loss of consciousness and respiratory failure (Currance et al., 2007). Signs and symptoms of exposure will be determined by the dose of acetonitrile. Onset of symptoms can be expected to be delayed from 2 h to 12 h as acetonitrile is slowly metabolized to its toxic metabolite, cyanide. Exposure to low doses will produce nausea, salivation, vomiting, headache, and lethargy. Exposure to higher doses may produce cyanide intoxication characterized by irritability, confusion, delirium, paralysis, extreme weakness, lethargy, respiratory depression, metabolic acidosis, tachycardia, shock, coma, seizures, and possibly death (PubChem, 2021).

Chronic toxicity The toxicological effects of acetonitrile have been attributed to the direct effects of the intact molecule combined with the effects of metabolically generated cyanide ions (Integrated Risk Information System (IRIS), 2022). Rats exposed to acetonitrile in air at concentrations ranging from 166 ppm to 665 ppm for 7 h per day for up to 90 days, showed no observable effects at dosed below 330 ppm. At the maximum dose tested (665 ppm), pulmonary inflammation as well as minor kidney and liver changes were noted in some animals (European Chemicals Bureau (ECB), 2002). Dogs and monkeys exposed to acetonitrile in air for 91 days showed minor variations in body weight, hematocrit, and hemoglobin. The animals were dosed acetonitrile at concentrations averaging 350 ppm for 7 h per day, 3 days per week. Autopsy of the animals revealed some cerebral hemorrhaging as well as pigment-bearing macrophages in some animals (Integrated Risk Information System (IRIS), 2022). Epidemiological evidence suggests chronic exposure to acetonitrile results in central nervous system effects including numbness of the extremities and tremors (PubChem, 2021).

Reproductive toxicity As in the case of the acute and chronic toxicological effects, that maternal production of cyanide may contribute to the reproductive and developmental toxicity of acetonitrile. Reproductive studies in laboratory animals show fetotoxicity at high acetonitrile doses. However, no teratogenic effects were observed at any dosage level (Integrated Risk Information System (IRIS), 2022).

Genotoxicity In vitro studies using rat liver microsomes have demonstrated that the conversion of acetonitrile to cyanide is mediated by cytochrome p450 (European Chemicals Bureau (ECB), 2002). Acetonitrile was tested for mutagenicity in the Salmonella/microsome preincubation assay. The test were conducted using up to 5 Salmonella strains and in the presence and absence of rat or hamster liver S-9. All tests were negative for mutagenicity including those run at the maximum dose tested (10 mg/plate; European Chemicals Bureau (ECB), 2002).

Carcinogenicity Male and female rats were exposed to acetonitrile by inhalation at doses ranging from zero up to 400 ppm for 6 h per day, 5 days per week for 2 years. Results of the study were inconclusive regarding the carcinogenic activity of acetonitrile as there was only a

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marginal increased incidence of hepatocellular adenomas and carcinomas in male rats. Furthermore, there was no evidence of carcinogenic activity in the female rats even at exposures as high as 400 ppm. In a similar study using male and female mice exposed to acetonitrile at doses ranging from zero up to 200 ppm by inhalation for 6 h per day, 5 days per week for 2 years, no carcinogenic activity was noted in the animals and doses tested (Integrated Risk Information System (IRIS), 2022). No data are available on its carcinogenic effects in humans. The USEPA has classified acetonitrile as not classifiable as to human carcinogenicity, or Group D.

Clinical management The major goal of treatment is to maintain respiration, blood circulation, and vital signs and to prevent further absorption of acetonitrile into the systemic circulation. If ingested, absorption can be prevented or minimized by instituting gastric lavage or by giving activated charcoal and a cathartic. Gastric lavage is effective only if performed soon after ingestion (Currance et al., 2007). Treatment of acetonitrile poisoning is similar to that of cyanide poisoning (see Cyanide). This includes immediate therapy with 100% oxygen and assisted ventilation, if necessary. Therapy should also include correction of the metabolic acidosis and to combat cyanide poisoning. Cyanide poisoning is treated by the intravenous administration of sodium nitrite and sodium thiosulfate. Care should be taken to maintain treatment for as long as acetonitrile is being metabolized to cyanide (Currance et al., 2007).

Other hazards Acetonitrile is highly flammable and will ignite in the presence of flames, sparks or sufficient heat. Acetonitrile vapors may combine with air to form explosive mixtures. Poisonous gases including hydrogen cyanide and nitrogen oxides are produced in fire (PubChem, 2021).

Ecotoxicology Toxicity thresholds for protozoa, bacteria and green algae have been measured to range from 520 mg L−1 for Microcystis aeruginosa (algae) to 7300 mg L−1 for Scenedesmus quadricauda (PubChem, 2021). The LC50 for acetonitrile in fathead minnow (Pimephales promelas) has been measured to be about 1640 mg L−1 per 96 h in a flow-through bioassay (PubChem, 2021). The European Union Risk Assessment Report on acetonitrile (European Chemicals Bureau (ECB), 2002) summarizes the results of various fish toxicity assays and report that the 24- to 48-h LC50 values were generally higher than 1 g L−1 for Oryzias latipes. The EURAR report also states that the lowest reported 48-h LC50 were 730 and 880 mg L−1 on Cyprinus carpio and Ctenopharyngodon idellus, respectively.

Exposure standards and guidelines OSHA Permissible Exposure Limit ¼ 40 ppm (70 mg m−3). ACGIH 8 h Time Weighted Average ¼ 20 ppm. ACGIH Short Term Exposure Limit ¼ 60 ppm. NIOSH 10-h Time-Weighted Average ¼ 20 ppm (34 mg m−3). The U.S. EPA Integrated Risk Information System has published a reference concentration for acetonitrile of 0.06 mg m−3. Florida State Drinking Water Standard ¼ 42 mg L−1.

See also: Cyanide

References Currance PL, Clements B, and Bronstein AC (2007) Emergency Care For Hazardous Materials Exposure, 3rd revised edn, St. Louis, MO: Elsevier Mosby, 432. European Chemicals Bureau (ECB) (2002) European Union Risk Assessment Report ACETONITRILE. CAS No. 75-05-8, EINECS No. 200-835-2. https://echa.europa.eu/documents/ 10162/764c8da5-79e2-418d-bf1f-ab59592f8cc6 Integrated Risk Information System (IRIS) (2022) Acetonitrile; CASRN 75-05-8. U.S. Environmental Protection Agency (EPA), National Center for Environmental Assessment. https://iris. epa.gov/static/pdfs/0205_summary.pdf PubChem (2021) Phosphorus. https://pubchem.ncbi.nlm.nih.gov/compound/5462309.

Relevant website https://echa.europa.eu/substance-information/-/substanceinfo/100.000.760 :European Chemicals Agency (ECHA).

2-Acetylaminofluorene Joshua P Gray, U.S. Coast Guard Academy, New London, CT, United States © 2024 Elsevier Inc. All rights reserved. This is an update of I. Syed, Acetylaminofluorene, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 46-48, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00217-7.

Chemical profile Background Uses/occurrence Exposure Toxicokinetics (ADME) Mechanism of toxicity Acute and short-term toxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity (pulmonary, neurotox, hepatotox, kidney, endocrine disruption, skin, etc.) Toxicogenomics Environmental fate and behavior Ecotoxicology Exposure standards and guidelines Conclusion Other PubChem URL References Further reading

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Abstract 2-Acetylaminofluorene (2-AAF) is used in the laboratory by toxicologists, cancer biologists, biochemists and other professionals as a positive control in the study of xenobiotic-metabolizing enzymes and the carcinogenesis and mutagenicity of aromatic amines. 2-AAF has been shown to induce a variety of tumors in laboratory animals. 2-AAF itself is a metabolite of 2-aminofluorene, and known human metabolites of 2-AAF are 1-hydroxy-AFF, 5-hydroxy-AFF, and N-Hydroxy-2acetamidofluorene.

Keywords Carcinogenesis; Coal gasification; Cytochrome P450; Glucuronides; Mutagenicity; N-hydroxylation; Nitrous oxide; Pesticide; Photolysis; Skeletal defects

Chemical profile

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Name: 2-Acetylaminofluorene Synonym: N-fluorene-2-yl-acetamide, N-2-fluorenylacetamide, N-fluoren-2-yl-acetamide, 2-Acetylaminofluorene, 2-Acetylaminofluorene, N-(9H-fluoren-2-yl)acetamide. Chemical Abstracts Service Registry Number: 53-96-3 Molecular formula: C15H13NO Chemical Structure:

Image source: https://commons.wikimedia.org/wiki/File:Acetylaminofluorene.png (PUBLIC DOMAIN).

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Background 2-acetylaminofluorene (2-AAF) is a polycyclic aromatic hydrocarbon typically used as a positive control for the induction of carcinogenesis in experimental animals. It is known to induce phase I metabolic enzymes. It is a tan, combustible, crystalline powder at room temperature that is insoluble in water, but soluble in glycols, alcohols, ether, acetic acid, and fat solvents (National Center for Biotechnology Information, 2023).

Uses/occurrence There is no commercial use for 2-AAF. It is primarily used as a model amine-containing aromatic molecule for research. It naturally occurs as a contaminant in coal gasification processes.

Exposure Exposure can occur via inhalation, skin absorption, ingestion, skin, or eye contact, inhalation, ingestion, and dermal contact (The National Institute for Occupational Safety and Health (NIOSH), 2023). Due to its use primarily in academic laboratory settings, the primary risk of occupational exposure is for laboratory workers.

Toxicokinetics (ADME) 2-AAF is bioactivated by cytochrome P450-mediated N-hydroxylation to form hydroxyacetylaminofluorene (Dybing et al., 1979). The acetyl group can also be hydrolyzed to produce 2-aminofluorene (Aune et al., 1985). The acetyl group can also be removed following N-hydroxylation to form N-hydroxy-2-aminofluorene (Razzouk et al., 1982). These products may by conjugated to form sulfate conjugates (60–80%) and glucuronides (10–15%) that are then eliminated in the urine.

Mechanism of toxicity 2-AAF stimulates the expression of CYP1A1 and 1A2 (Ioannides et al., 1993). It is metabolized to form hydroxyl-containing molecules N-hydroxyacetylaminofluorene and 2-aminofluorene which can form adducts with DNA or other molecules (Dybing et al., 1979; Aune et al., 1985; Robertson, 1986). At high doses, 2-AAF is acutely toxic whereas lower doses are carcinogenic.

Acute and short-term toxicity The LD50 of intraperitoneal 2-AAF in 9-week-old male BCC3F1 mice was 2200 mg/kg (Watanabe et al., 2012). The LD50 of intraperitoneal 2-AAF in rats is greater than 200 mg/kg (Marquardt et al., 1985). The oral toxic dose in mice is 810 mg/kg (Haley et al., 1973). In rats, the lowest published toxic dose ranged from 150 to 517.5 mg/kg (Danz et al., 1997; Huggins et al., 1961). Wistar rats implanted with 28-day release pellets containing 2-aminoacetylfluorene (2.5 mg/day for 28 days) experienced less liver injury when treated with bolus doses of carbon tetrachloride or 2-AAF on day 7 than those without implants, suggesting that pre-treatment with low doses offered some protection against subsequent exposure (Sigala et al., 2004).

Reproductive and developmental toxicity 2-AAF induced developmental toxicity in the Frog Embryo Teratogenesis Assay-Xenopus assay partially due to bioactivation by CYP1A1 and 1A2. Detoxification in this model is primarily mediated by epoxide hydrolase and glutathione conjugation (Propst et al., 1997). It is also teratogenic in rats, but in whole rat embryos, induction of neural tube defects required the addition of exogenous NADPH monooxygenase, with 7-hydroxy-acetylaminofluorene being the primary toxic metabolite (Shepard et al., 1983; Faustman-Watts et al., 1985). A single dose of 2-AAF at 0.1 mg/kg in mice on gestation days 8–15 induced skeletal defects, clef lips, cleft palates, and cerebral hernias.

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Genotoxicity 2-Acetylaminofluorene is both a tumor initiator and promoter. Metabolites can form DNA adducts, particularly at the C8 position of guanine bases (Dayanidhi and Vaidyanathan, 2022; Downs et al., 2021; Cai et al., 2018). Sprague-Dawley rats fed 0.02% 2-AAF in their diet for multiple weeks demonstrated increased formation of DNA adducts in the liver and kidney (Bagnyukova et al., 2008). This compound also induces epigenetic changes. Carcinogen-induced epigenetic changes include altered global histone lysine methylation patterns, an increase in histone H3 lysine 9 and histone H3 lysine 27 trimethylation in the promoter regions of several tumor suppressor genes (Pogribny et al., 2011). This led to a dysregulation of cell proliferation and apoptosis. It also induced a reduction of histone H4 lysine 20 trimethylation in male rats and hypermethylation of the p16(INK4A) gene in male rats (Bagnyukova et al., 2008).

Carcinogenicity 2-AAF is reasonably anticipated to be a human carcinogen based on studies performed in experimental animals (National Center for Biotechnology Information, 2023). It is classified as Carc. 1B by the European Chemicals Agency, as an occupational carcinogen by the U.S. Occupational Safety and Health Administration, as a potential occupational carcinogen by the U.S. National Institute for Occupational Safety and Health, but is not classified by the U.S. Environmental Protection Agency for carcinogenicity. 2-AAF is a long-studied inducer of cancer in several tissues including the liver and bladder in several species. Its isomer, 4-acetylaminofluorene, is not carcinogenic, fails to induce P450 1A activity (Ioannides et al., 1993). Liver tumors were induced by dietary administration of 2-acetylaminofluorene to mice, rats, dogs, fish (guppies and zebrafish), and several other species (Allison et al., 1950; Pliss and Khudoley, 1975; Fujii, 1991). Male rats may be more susceptible to 2-AAF-induced hepatocarcinogenesis due to the increased induction of phase I enzymes that bioactivate the compound (Williams et al., 2016). Lung tumors were induced by 2-AAF in mice (Wang et al., 1993). Urinary bladder cancer was induced in newborn hamsters through intraperitoneal injection and feeding (Oyasu et al., 1973, 1972). Indole increased the incidence of bladder tumors in hamsters fed 2-acetylaminofluorene (Matsumoto et al., 1976). Testicular mesotheliomas, liver, and Zymbal-gland tumors were induced in Fischer 344 rats provided 2-aminoacetylfluorene in their diet (Cabral and Neal, 1983). 2-AAF is also a potent tumor promoter; it is frequently used in conjunction with the tumor initiator diethylnitrosamine to promote the development of lesions in the liver of rats (Batinic-Haberle et al., 2021; Castro-Gil et al., 2021).

Organ toxicity (pulmonary, neurotox, hepatotox, kidney, endocrine disruption, skin, etc.) 2-AAF primarily acts as a carcinogen in the liver, bladder, skin, and pancreas (The National Institute for Occupational Safety and Health (NIOSH), 2023). See the Carcinogenicity section for more details.

Toxicogenomics 2-AAF is most highly interactive with the following genes: TRP53, GSTP1, TP53, CDKN1A, ABCB1B, ABCB1, PCNA, BAX, MK167, and CYP1A2. A complete list can be found at the database linked here, together with the 172 papers citing data for this chemical (Comparative Toxicogenomics Database, 2023).

Environmental fate and behavior Environmental releases have remained below 1000 pounds per year since 2003, according to the U.S. Environmental Protection Agency’s Toxics Release Inventory. If released to the air, it will exist in both vapor and particulate phases in the atmosphere. It will be degraded by atmospheric hydroxyl radicals with an estimated half-life of 5 h and by photolysis. If released to the soil, it will have low mobility and low volatility. If released into water, it will adsorb to suspended solids and sediment (reviewed in (National Center for Biotechnology Information, 2023)).

Ecotoxicology 2-AAF causes abnormal development and malformations in Xenopus laevis (EC50 of 6900 mg/L for 96 h; LC50 of 87,000 mg/L for 96 h) (Fort et al., 1989).

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Exposure standards and guidelines Although identified as a potential occupational carcinogen by OSHA, there is no associated PEL. Exposure is to be controlled through engineering controls, work practices, and personal protective equipment.

Conclusion 2-Acetylaminofluorene (2-AAF) is mostly used as an experimental carcinogen which leads to tumor formation in rodents. Recent reports have shown its potential to cause hepatotoxicity. Administration of 2-AAF for several days have been shown to induce hepatic toxicity, inflammation, oxidative stress and hyperproliferation. 2-AAF is popular compound which is used to promote diethylnitrosamine-induced liver cancer in rodent models (Tawfik et al., 2022).

Other https://comptox.epa.gov/dashboard/chemical/details/DTXSID0039227—CompTox Chemicals Dashboard chemical entry for 2-AAF. https://www.cdc.gov/niosh/npg/npgd0007.html—National Institute for Occupational Safety and Health chemical entry for 2-AAF. https://ntp.niehs.nih.gov/ntp/roc/content/profiles/acetylaminofluorene.pdf—National Toxicology Program’s 15th Report on Carcinogens chemical entry for 2-AAF.

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/5897 :National Library of Medicine’s PubChem chemical entry for 2’AAF.

References Allison JB, Wase AW, Leathem JH, and Wainio WW (1950) Some effects of 2-acetylaminofluorene on the dog. Cancer Research 10: 266–271. Aune T, Vanderslice RR, Croft JE, Dybing E, Bend JR, and Philpot RM (1985) Deacetylation to 2-aminofluorene as a major initial reaction in the microsomal metabolism of 2-acetylaminofluorene to mutagenic products in preparations from rabbit lung and liver. Cancer Research 45: 5859–5866. Bagnyukova TV, Tryndyak VP, Montgomery B, Churchwell MI, Karpf AR, James SR, Muskhelishvili L, Beland FA, and Pogribny IP (2008) Genetic and epigenetic changes in rat preneoplastic liver tissue induced by 2-acetylaminofluorene. Carcinogenesis 29: 638–646. Batinic-Haberle I, Tovmasyan A, Huang Z, Duan W, Du L, Siamakpour-Reihani S, Cao Z, Sheng H, Spasojevic I, and Alvarez Secord A (2021) H(2)O(2)-Driven anticancer activity of Mn porphyrins and the underlying molecular pathways. Oxidative Medicine and Cellular Longevity 2021: 6653790. Cabral JR and Neal GE (1983) Testicular mesotheliomas in rats exposed to N-2-fluorenylacetamide (FAA). Tumori 69: 195–199. Cai A, Wilson KA, Patnaik S, Wetmore SD, and Cho BP (2018) DNA base sequence effects on bulky lesion-induced conformational heterogeneity during DNA replication. Nucleic Acids Research 46: 6356–6370. Castro-Gil MP, Sanchez-Rodriguez R, Torres-Mena JE, et al. (2021) Enrichment of progenitor cells by 2-acetylaminofluorene accelerates liver carcinogenesis induced by diethylnitrosamine in vivo. Molecular Carcinogenesis 60: 377–390. Comparative Toxicogenomics Database (2023) 2-Acetylaminofluorene [Online]. Available: https://ctdbase.org/detail.go?type¼chem&acc¼D015073 [Accessed January 9, 2023]. Danz M, Sanger J, Friedrichsen K, and Linss W (1997) 2-Acetylaminofluorene-produced selective cytotoxic damage of a ductal compartment and its repair in the submandibular gland of rats. Cell and Tissue Research 288: 371–379. Dayanidhi PD and Vaidyanathan VG (2022) Understanding the ancillary ligand effect on luminescent cyclometalated Ir(III) complex as a reporter for 2-acetylaminofluorene DNA(AAF-dG) adduct. Journal of Biological Inorganic Chemistry 27: 189–199. Downs TR, Arlt VM, Barnett BC, Posgai R, and Pfuhler S (2021) Effect of 2-acetylaminofluorene and its genotoxic metabolites on DNA adduct formation and DNA damage in 3D reconstructed human skin tissue models. Mutagenesis 36: 63–74. Dybing E, Soderlund E, Haug LT, and Thorgeirsson SS (1979) Metabolism and activation of 2-acetylaminofluorene in isolated rat hepatocytes. Cancer Research 39: 3268–3275. Faustman-Watts EM, Namkung MJ, Greenaway JC, and Juchau MR (1985) Analysis of metabolites of 2-acetylaminofluorene generated in an embryo culture system. Relationship of biotransformation to teratogenicity in vitro. Biochemical Pharmacology 34: 2953–2959. Fort DJ, James BL, and Bantle JA (1989) Evaluation of the developmental toxicity of five compounds with the frog embryo teratogenesis assay: Xenopus (FETAX) and a metabolic activation system. Journal of Applied Toxicology 9: 377–388. Fujii K (1991) Evaluation of the newborn mouse model for chemical tumorigenesis. Carcinogenesis 12: 1409–1415. Haley TJ, Dooley KL, and Harmon JR (1973) Acute oral toxicity of N-2-fluorenylacetamide (2-FAA) in several strains of mice. Proceedings of the Society for Experimental Biology and Medicine 143: 1117–1119. Huggins C, Grand LC, and Brillantes FP (1961) Mammary cancer induced by a single feeding of polymucular hydrocarbons, and its suppression. Nature 189: 204–207. Ioannides C, Cheung YL, Wilson J, Lewis DF, and Gray TJ (1993) The mutagenicity and interactions of 2- and 4-(acetylamino)fluorene with cytochrome P450 and the aromatic hydrocarbon receptor may explain the difference in their carcinogenic potency. Chemical Research in Toxicology 6: 535–541. Marquardt P, Romen W, and Neumann HG (1985) Tissue specific, acute toxic effects of the carcinogen trans-4-dimethylaminostilbene. Archives of Toxicology 56: 151–157. Matsumoto M, Hopp ML, and Oyasu R (1976) Effect of pair-feeding of carcinogen on the incidence of bladder tumors in hamsters. Role of indole, age, and sex. Investigative Urology 14: 206–209. National Center for Biotechnology Information (2023) PubChem Compound Summary for CID 5897, 2-Acetylaminofluorene. https://pubchem.ncbi.nlm.nih.gov/compound/. Oyasu R, Kitajima T, Hopp ML, and Sumie H (1972) Enhancement of urinary bladder tumorigenesis in hamsters by coadministration of 2-acetylaminofluorene and indole. Cancer Research 32: 2027–2033.

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Oyasu R, Kitajima T, Hopp ML, and Sumie H (1973) Induction of bladder cancer in hamsters by repeated intratracheal administrations of 2-acetylaminofluorene. Journal of the National Cancer Institute 50: 503–506. Pliss GB and Khudoley VV (1975) Tumor induction by carcinogenic agents in aquarium fish. Journal of the National Cancer Institute 55: 129–136. Pogribny IP, Muskhelishvili L, Tryndyak VP, and Beland FA (2011) The role of epigenetic events in genotoxic hepatocarcinogenesis induced by 2-acetylaminofluorene. Mutation Research 722: 106–113. Propst TL, Fort DJ, Stover EL, Schrock B, and Bantle JA (1997) Evaluation of the developmental toxicity of benzo[a]pyrene and 2-acetylaminofluorene using Xenopus: modes of biotransformation Stover Group. Drug and Chemical Toxicology 20: 45–61. Razzouk C, Batardy-Gregoire M, and Roberfroid M (1982) Metabolism of N-hydroxy-2-acetylaminofluorene and N-hydroxy-2-aminofluorene by guinea pig liver microsomes. Cancer Research 42: 4712–4718. Robertson IG (1986) The importance of 2-aminofluorene in the mutagenic activation of 2-acetylaminofluorene. Mutation Research 175: 153–157. Shepard TH, Fantel AG, Mirkes PE, Greenaway JC, Faustman-Watts E, Campbell M, and Juchau MR (1983) Teratology testing: I. Development and status of short-term prescreens. II. Biotransformation of teratogens as studied in whole embryo culture. Progress in Clinical and Biological Research 135: 147–164. Sigala F, Kostopanagiotou G, Andreadou I, et al. (2004) Histological and lipid peroxidation changes after administration of 2-acetylaminofluorene in a rat liver injury model following selective periportal and pericentral damage. Toxicology 196: 155–163. Tawfik NG, et al. (2022) Isatin counteracts diethylnitrosamine/2-acetylaminofluorene-induced hepatocarcinogenesis in male wistar rats by upregulating anti-inflammatory, antioxidant, and detoxification pathways. Antioxidants (Basel) 11(4): 699. https://pubmed.ncbi.nlm.nih.gov/35453384/. The National Institute for Occupational Safety and Health (NIOSH) (2023) 2-Acetylaminofluorene [Online]. Available: https://www.cdc.gov/niosh/npg/npgd0007.html. Wang Y, Wang Y, Stoner G, and You M (1993) ras mutations in 2-acetylaminofluorene-induced lung and liver tumors from C3H/HeJ and (C3H x A/J)F1 mice. Cancer Research 53: 1620–1624. Watanabe T, Suzuki T, Natsume M, et al. (2012) Discrimination of genotoxic and non-genotoxic hepatocarcinogens by statistical analysis based on gene expression profiling in the mouse liver as determined by quantitative real-time PCR. Mutation Research 747: 164–175. Williams GM, Duan JD, Iatropoulos MJ, and Perrone CE (2016) Sex differences in DNA damage produced by the carcinogen 2-acetylaminofluorene in cultured human hepatocytes compared to rat liver and cultured rat hepatocytes. Archives of Toxicology 90: 427–432.

Further reading Downs TR, et al. (2021) Effect of 2-acetylaminofluorene and its genotoxic metabolites on DNA adduct formation and DNA damage in 3D reconstructed human skin tissue models. Mutagenesis 36(1): 63–74. https://pubmed.ncbi.nlm.nih.gov/31816077/. Hassanin AH, et al. (2021) Promotive action of 2-acetylaminofluorene on hepatic precancerous lesions initiated by diethylnitrosamine in rats: Molecular study. World Journal of Hepatology 13(3): 328–342. https://pubmed.ncbi.nlm.nih.gov/33815676/. Ali G, et al. (2021) The protective role of etoricoxib against diethylnitrosamine/2-acetylaminofluorene- induced hepatocarcinogenesis in wistar rats: The impact of NF-kB/COX-2/PGE2 signaling. Current Molecular Pharmacology 15(1): 252–262. https://pubmed.ncbi.nlm.nih.gov/34238176/. Barranger A and Hégarat LL (2022) Towards better prediction of xenobiotic genotoxicity: CometChip technology coupled with a 3D model of HepaRG human liver cells. Archives of Toxicology 96(7): 2087–2095. https://pubmed.ncbi.nlm.nih.gov/35419617/. Hassan SK, et al. (2015) 18-b Glycyrrhetinic acid alleviates 2-acetylaminofluorene-induced hepatotoxicity in Wistar rats: Role in hyperproliferation, inflammation and oxidative stress. Human & Experimental Toxicology 34(6): 628–641. https://pubmed.ncbi.nlm.nih.gov/25352648/. Ahmed OM, et al. (2022) Quercetin and naringenin abate diethylnitrosamine/acetylaminofluorene-induced hepatocarcinogenesis in Wistar rats: The roles of oxidative stress, inflammation and cell apoptosis. Drug and Chemical Toxicology 45(1): 262–273. https://pubmed.ncbi.nlm.nih.gov/31665932/.

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Acetylene Sara Mostafalou, Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of S Mostafalou, H Bahadar, Acetylene, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 51–53, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00362-6.

Chemical profile Introduction Background Uses Exposure and exposure monitoring Routes and pathways Human exposure Toxicokinetics Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Neurotoxicity Cardiotoxicity Interactions Toxicogenomics Clinical management Ocular exposure Inhalation Environmental fate and behavior Routes and pathways relevant physico-chemicals properties Partition behavior in water, sediment and soil Environmental persistency Long range transport Bioaccumulation and biomagnifications Ecotoxicology Aquatic ecotoxicity Exposure standards and guidelines Others PubChem URL Conclusion References

90 90 90 90 90 90 90 91 91 91 91 91 91 92 92 92 92 92 92 92 93 93 93 93 93 93 93 93 93 93 93 93 94 94 94 94 94 94 94 94 94

Abstract Acetylene is a colorless, odorless gas used as a closed-system industrial intermediate in the synthesis of other chemicals. Oxyacetylene torches are also used for welding and metal cutting. Based on its physicochemical properties, acetylene exists as a gas in the atmosphere. The potential route of human exposure is through inhalation. Exposure to acetylene causes asphyxiation as it displaces oxygen from the air and results in hypoxia. Decontamination of the patient is needed after being exposed to acetylene. The patient needs to be provided with adequate ventilation. Appropriate management is needed according to the signs and symptoms of the patient.

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Acetylene Keywords Aliphatic hydrocarbon; Asphyxiation; Cytochrome P450; Oxyacetylene; Superoxide dismutase

Highlights

• • •

Acetylene exists as a gas in the atmosphere. Toxic effects include asphyxiation and hypoxia. Adequate ventilation is needed for management of poisoning.

Chemical profile

• • • • • •

Name: Acetylene Chemical Abstracts Service Registry Number: CAS 74-86-2 Synonyms: Acetylene, Dicarbon, Ethine, Ethyne, Vinylene, Welding gas Chemical Class: Aliphatic hydrocarbon CnH2n−2 Molecular Formula: C2H2 Chemical Structure:

Introduction As a hydrocarbon acetylene is considered simplest alkyne composed of two carbon and two hydrogen atoms with the formula C2H2 (Haynes et al., 2016).

Background Acetylene was discovered in 1836 and named by Berthelot in 1860, who studied the properties of this gas. It was used for the first time for lighting in 1892 after the work by Moissan. The oxyacetylene blow pipe was invented in 1901 by Charles, leading to the extensive industrial use of oxyacetylene flame in steel welding, scarfing, surface cementation, oxygen cutting, and hard surfacing (Kim et al., 2023).

Uses About 80% of acetylene production is used as a closed-system manufacturing intermediate for the production of other chemicals. The other chemicals synthesized from acetylene include vinyl chloride monomer, N-vinylcarbazole, 1,4-butanediol, vinyl ethers, N-vinyl-2-pyrrolidone, vinyl fluoride, N-vinylcaprolactam, and vinyl esters. The other use of acetylene as oxyacetylene torches for metal cutting and welding is about 20% (European Chemical Agency (ECHA), 2022). Acetylene had a therapeutic use as an anesthetic, since it gave an immediate recovery without after effects. However, its application as an anesthetic has become limited because of explosive characteristics. At slightly higher concentrations than those for ethylene, acetylene causes CNS depression (Bingham et al., 2001). Anesthesia with acetylene-oxygen has been introduced as a safe alternative to anesthesia with chloroform or ether. This procedure has also no immediate after effects (IUCLID, 2006).

Exposure and exposure monitoring Routes and pathways Minimal consumer exposure is expected when acetylene is used as a closed-system industrial chemical. However, during welding and scarfing, the major route of exposure is through inhalation of oxyacetylene (European Chemical Agency (ECHA), 2022).

Human exposure Human exposure is most likely to occur in workplaces where acetylene is used, and from the atmosphere when released from the other sources. The potential source of exposure for humans is oxyacetylene during welding (European Chemical Agency (ECHA), 2022).

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Toxicokinetics Acetylene can be absorbed into the body by inhalation. After inhalation, acetylene is rapidly absorbed and eliminated from the body in unchanged form. At normal hematocrit and body temperature, the blood-gas partition coefficient of acetylene is 0.833, which indicates that acetylene has a greater tendency to be eliminated from blood. End tidal concentration of acetylene within 10 min of inhalation has been estimated 8.0% of its initial value, indicating that the lungs rapidly excrete acetylene. Although, inhalation has been shown to be the main route of exposure, acetylene can diffuse rapidly from the peritoneal and pleural cavities and also through the skin (European Chemical Agency (ECHA), 2022).

Mechanism of toxicity Acetylene inactivates the cytochrome p450 enzyme by alkylating the prosthetic heme. Exposure to acetylene causes asphyxiation and depletes atmospheric oxygen. Acetylene can also lower the oxygen content of the air in confined areas and cause suffocation. Hypertension due to stimulation of the vasomotor center has been reported by using acetylene-oxygen mixtures during anesthesia (Mostafalou and Bahadar, 2014).

In vitro toxicity data In an in vitro experiment with radiolabeled acetylene, no protein binding was observed, but binding of acetylene to prosthetic heme or the lipids related to the site of reactive metabolites was suggested (IUCLID, 2006).

Acute and short-term toxicity Animal Toxic effects of acetylene in acute and short-term exposures in different animal species have been presented in Table 1.

Human Acetylene at concentrations below a lower explosive limit of 2.5% (25,000 ppm) is not toxic to humans. Varying degrees of temporary and reversible central nervous system depression have been seen after exposure to oxygen containing 10% acetylene. Unconsciousness has been observed in humans after inhaling 33 or 35% acetylene within 7 and 5 min, respectively, and exposure to 80% acetylene has caused complete anesthesia, forced respiration, and hypertension. Generally, dizziness, lethargy, headache, and suffocation are the main symptoms observed in human in acute exposure to acetylene (European Chemical Agency (ECHA), 2022). Harmful effects of impure acetylene include frostbite, increased volume and frequency of respiration followed by unconsciousness Table 1

Acute and short-term toxicities of acetylene in different animal species.

Organism

Acetylene Conc.

Exposure duration

Observed effect

References

Rat Rat Rat

78% 90% 5%

15 min 2h 18 h

EPA (2003) EPA (2003) IUCLID (2006)

Rabbit

70% in oxygen

17 min

Dog

80%

1h

Dog

85% in oxygen

During anesthesia

Cat

80%

During anesthesia

Mammals Mammals Mammals

10% 25% 50%

5–10 min 5–10 min 5–10 min

Anesthesia Respiratory failure # Heme Hepatic accumulation of porphyrin # Carbon dioxide tension " Respiration after anesthesia # Alkali reserve # Carbonic acid concentration " Oxygen-binding capacity of blood " Arterial-oxygen deficit # Blood glucose concentration " Volume and frequency of respiration " Elimination of carbon dioxide Constriction of intestinal blood vessels " Blood flow to peripheral blood vessels Tolerance Intoxication Death

IUCLID (2006) IUCLID (2006)

National Center for Biotechnology Information (NCBI) (2023) IUCLID (2006) Bingham et al. (2001) Bingham et al. (2001) Bingham et al. (2001)

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Acetylene Table 2

Acute toxicity data of acetylene by inhalational exposure (Kim et al., 2023).

Organism

LC50 (pph)

LPTC (pph/min)

LPLC (pph/min)

Dog Rat Mammal Human

85 ND ND ND

ND 78/15 ND 20/4

ND 90/120 50/5 50/5

LC50: median lethal concentration, LPTC: lowest published toxic concentration, LPLC: Lowest published lethal concentration, pph: parts per hundred, ND: not determined.

and motor dysfunction. Nausea, vomiting, cyanosis, irregular pulse, convulsions, and death may be occurred. Acetylene-induced sensitization of myocardium to epinephrine leading to ventricular fibrillation is possible (Andrew, 2008). Acute toxic concentrations of acetylene by inhalational exposure in animals and human have been presented in the Table 2.

Chronic toxicity Animal Sub-chronic and chronic effects of acetylene have been studied in rats, mice, guinea pig, rabbits and dogs. Animals were exposed to acetylene in oxygen at concentrations 25–80% lasting 0.5–2 h/day for periods 1–93 days. The results showed that exposure to lower concentrations of acetylene caused animals slightly sleepy while higher concentration of acetylene caused animals fell asleep 15–20 min after exposure. All the animals recovered from CNS depressant effects of acetylene shortly after cease of exposure, except mice which were died when exposed to acetylene at 50%, 2 h/day for 1–6 days (EPA, 2003). Significant plasma elevation of aspartate and alanine transaminases has been observed in white rabbits after inhaling crude acetylene at concentration of 58,000 ppm for 10 min at 12-h intervals for a period of 3 weeks. In addition, the catalase activity was depressed in the heart, kidney, and liver tissues. A rise in superoxide dismutase was also observed in the heart tissues (Okolie et al., 2005).

Human It is suggested that crude acetylene may have deleterious effects on the blood constituents and vital organs of human in chronic exposures. Death has been recorded in people working in acetylene manufacturing at 40%. Of course, toxicity and death have been attributed to the impurities of crude acetylene like phosphine and arsine (Bingham et al., 2001). Other impurities in acetylene such as hydrogen sulfide, carbon disulfide, or carbon monoxide can also cause toxicity and even death (Andrew, 2008).

Immunotoxicity There is no available evidence on immunotoxicity of acetylene.

Reproductive and developmental toxicity There is no available evidence on reproductive and developmental toxicity of acetylene (European Chemical Agency (ECHA), 2022).

Genotoxicity Sufficient evidence provided by Ames test employing three strains of Salmonella typhimurium (TA97, TA98, and TA100) show that acetylene is not genotoxic (European Chemical Agency (ECHA), 2022).

Carcinogenicity There is no available evidence on carcinogenicity of acetylene.

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Organ toxicity Neurotoxicity At concentrations higher than 10% in air, acetylene primarily acts as a central nervous depressant and asphyxiant in mammalian systems (Kim et al., 2023).

Cardiotoxicity Anesthetic administration of acetylene (20–80%) have been studied in cats and some cardiovascular effects have been recorded. These effects include excitation of vasomotor center leading to increased blood pressure, excitation of vagal center leading to increased pulse frequency, increased amplitude of heart pulse, and displacement of blood flow from intestinal vessels to peripheral vessels (IUCLID, 2006).

Interactions The most recorded interactions of acetylene are those due to coadministration with oxygen for anesthetic applications. Alterations in respiration, blood pressure, CNS depression, and carbon dioxide tension can be resulted from this interaction (Kim et al., 2023).

Toxicogenomics There is no available evidence on toxicogenomics of acetylene.

Clinical management Ocular exposure Remove the patient from the exposure area; irrigate the affected eyes thoroughly with water or 0.9% saline (National Center for Biotechnology Information (NCBI), 2023).

Inhalation After inhaling acetylene gas, the patient needs to be removed from exposure area quickly. The patient needs to be provided adequate ventilation and oxygen if required. Monitor and manage the patient according to the signs and symptoms (National Center for Biotechnology Information (NCBI), 2023).

Environmental fate and behavior Routes and pathways relevant physico-chemicals properties Acetylene is released to the environment through various industrial waste streams of industries. Because of the vapor pressure of acetylene (4.04  104 mmHg at 25  C), it exists in the environment exclusively in the form of gas. The gaseous phase of acetylene is degraded in the environment with photochemically induced hydroxyl radicals; the half-life for this photochemical degradation is approximately 20 days (National Center for Biotechnology Information (NCBI), 2023).

Partition behavior in water, sediment and soil Based on the Henry’s law constant of 0.022 atm-m3mol−1, derived from vapor pressure 4.04  104 mmHg and water solubility 1200 mg L−1, volatilization from moist soil is the major fate process for acetylene. In soil, biodegradation is not expected to be an important fate process for acetylene, as suggested by 0% biochemical oxygen demand in 28 days. Acetylene is not anticipated to be adsorbed by suspended solids and sediments if released to water because of its Koc value. Removal of acetylene from water is expected to be through the volatilization process (Mostafalou and Bahadar, 2014).

Environmental persistency Removal of the acetylene from soil and water is expected to be through the volatilization process. The gaseous phase of acetylene is degraded in the environment with photochemically induced hydroxyl radicals, the half-life for this photochemical degradation is approximately 20 days (European Chemical Agency (ECHA), 2022).

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Long range transport The estimated Koc of acetylene is 38, and based upon this Koc value, acetylene is expected to possess high mobility if released to soil (European Chemical Agency (ECHA), 2022).

Bioaccumulation and biomagnifications The estimated bioconcentration factor (BCF) of 3 for acetylene suggests that the potential for bioaccumulation of acetylene in aquatic organism is low (National Center for Biotechnology Information (NCBI), 2023).

Ecotoxicology Aquatic ecotoxicity Due to its gaseous nature, acetylene rapidly evaporates from aquatic system to air, and limited data are available about the toxic effects of acetylene on fish.

Exposure standards and guidelines Exposure limits recommended by National Institute of Occupational Safety and Health for acetylene is 2500 ppm as a ceiling (European Chemical Agency (ECHA), 2022). Physicochemical properties of acetylene are as follow:

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Melting point: −8.07  C. Boiling point: −8.47  C. Log p (octanol–water): 0.37. Vapor pressure: 5240 mmHg. Henry’s law constant: 0.022 atm-m3 mol−1. Water solubility: 1200 mg L−1.

Others Acetylene is a reactive gas and poses a high risk for fire and explosion. Acetylene reacts with active metals such as copper, silver, and mercury to form explosive acetylide compounds. Impurities such as phosphine may be present in acetylene obtained from calcium carbide, which can have deleterious effects on human health (National Center for Biotechnology Information (NCBI), 2023).

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/Acetylene

Conclusion Asphyxiation and hypoxia are the main toxic effects of acetylene in acute exposures. Adequate ventilation should be considered for the patients poisoned with inhalational exposure to acetylene.

References Andrew, W. (2008) Sittig’s Handbook of Toxic and Hazardous Chemical Carcinogens, Vol. 1: A-H, 2: I-Z., 5th edn. New York: Norwich. Bingham E, Cohrssen B, and Powell CH (2001) Patty’s Toxicology, 5th edn. vols. 1–9. New York, NY: John Wiley & Sons. EPA (2003) High Production Volume (HPV) Challenge Program’s Robust Summaries and Test Plans for Acetylene. Office of Pollution Prevention and Toxics. European Chemical Agency (ECHA) (2022) Substance Information; Acetylene. From: https://echa.europa.eu/substance-information/Acetylene. Haynes WM, Lide DR, and Bruno TJ (2016) CRC Handbook of Chemistry and Physics: A Ready-Reference Book of Chemical and Physical Data, 97th edn. Florida: Boca Raton. IUCLID (2006) Acetylene (74-86-2), 2000 CD-ROM edn. European Chemical Bureau. Kim S, Chen J, Cheng T, Gindulyte A, He J, He S, Li Q, Shoemaker BA, Thiessen PA, Yu B, Zaslavsky L, Zhang J, and Bolton EE (2023) PubChem 2023 update. Nucleic Acids Research 51(D1): D1373–D1380. https://doi.org/10.1093/nar/gkac956. Mostafalou S and Bahadar H (2014) Acetylene. In: Wexler P (ed.) Encyclopedia of Toxicology, 3rd edn., pp. 51–53. Oxford: Academic Press. https://doi.org/10.1016/B978-0-12386454-3.00362-6. National Center for Biotechnology Information (NCBI) (2023) PubChem Compound Summary for CID 6326, Acetylene. From: https://pubchem.ncbi.nlm.nih.gov/compound/Acetylene. Okolie NP, Ozolua RI, and Osagie DE (2005) Some biochemical and haematological effects associated with chronic inhalation of crude acetylene in rabbits. Journal of Medical Sciences 5: 21–25.

Acetylsalicylic acid Rachel Gorodetsky, D’Youville University School of Pharmacy, Buffalo, NY, United States; University of Rochester Medical Center, Rochester, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of R. Gorodetsky, Acetylsalicylic Acid, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 54-55, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00687-4.

Chemical profile Background Uses Exposure and exposure monitoring Routes and pathways Human exposure Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Reproductive and developmental toxicity Genotoxicity Carcinogenicity Clinical management Environmental fate and behavior Exposure standards and guidelines PubChem URL References

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Abstract The use of aspirin to treat fever dates back to antiquity with the use of willow bark and leaves. Ever since the active component of willow bark was isolated and marketed in 1899, it has remained one of the most popular medications for the treatment of fever, pain, and inflammation. Aspirin is consistently listed among the agents most often involved in human exposures, and is considered the causative agent in dozens of deaths each year.

Keywords Acetylsalicylic acid; Activated charcoal; Aspirin; Hemodialysis; Oxidative phosphorylation; Respiratory alkalosis; Salicylates; Sodium bicarbonate; Uncoupling

Chemical profile

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Name: Acetylsalicylic acid Chemical Abstracts Service Registry Number: 50-78-2. Synonyms: Aspirin, ASA, 2-(Acetyloxy)benzoic acid, 2-Carboxyphenyl acetate. Molecular Formula: C9H8O4

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Chemical Structure:

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Background The use of aspirin to treat fever dates back to antiquity with the use of willow bark and leaves. Ever since the active component of willow bark was isolated and marketed in 1899, it has remained one of the most popular medications for the treatment of fever, pain, and inflammation. Aspirin is consistently listed among the agents most often involved in human exposures, and is considered the causative agent in dozens of deaths each year (Gummin et al., 2021).

Uses Aspirin is used as an analgesic, antipyretic, and anti-inflammatory agent.

Exposure and exposure monitoring Routes and pathways Ingestion is the most common route of both accidental and intentional exposures, though rectal exposures have been reported. Toxicity from dermal exposure has not been reported. Intravenous aspirin is available in some countries, though toxicity resulting from this route has not been reported.

Human exposure Human exposures to salicylates in the US are monitored through the National Poison Data System (NPDS), which draws its information from the Poison Center system. In 2020 there were nearly 12,000 single-substance exposures to products containing acetylsalicylic acid (Gummin et al., 2021).

Toxicokinetics Aspirin is rapidly hydrolyzed into salicylate, an active metabolite, in both the gastrointestinal tract and the bloodstream. Both aspirin and salicylate are readily absorbed by passive diffusion across the gastric membrane, with absorption influenced by gastric pH. After a therapeutic dose peak serum concentrations are typically achieved within 1 h, or within 4–6 h for enteric-coated preparations. Aspirin has a volume of distribution of approximately 0.2 L kg−1 and is 90% protein bound. The serum half-life of acetylsalicylic acid is only 15 min, while the half-life of salicylate is approximately 6 h. In overdose, peak serum concentration may be delayed due to pylorospasm, concretions or bezoars, gastric outlet obstruction, or coingestants that slow gastric motility. Peak serum concentrations occurring beyond 24 h post ingestion have been reported. The volume of distribution increases to greater than 0.3 L kg−1, and protein binding decreases to less than 75% in overdose, while the kinetics change from first-order elimination to zero-order, with half lives of 20 h or more (Chyka et al., 2007). Aspirin metabolites are renally eliminated with 10% as salicylic acid, 75% salicyluric acid, 10% phenolic glucuronide, and 5% acyl glucuronide.

Mechanism of toxicity The toxicity of aspirin is multifactorial. Gastrointestinal symptoms such as nausea, vomiting and abdominal pain occur as a result of both local gastric irritation and stimulation of the medullary chemoreceptor trigger zone. Salicylates directly stimulate the respiratory drive in the brain stem, leading to hyperventilation and respiratory alkalosis. Anion gap metabolic acidosis occurs from a buildup of organic acids as well as the uncoupling of oxidative phosphorylation, which results in an imbalance in ATP consumption and production resulting in a net buildup of hydrogen ions. Therefore, aspirin often causes a mixed acid-base status. Furthermore, the uncoupling of oxidative phosphorylation results in failure to produce ATP despite increased oxygen utilization, which leads to heat production and hyperthermia. Aspirin interferes with glucose metabolism and gluconeogenesis, and can cause profound decreases in CSF glucose concentrations despite normal blood glucose concentrations.

Acute and short-term toxicity Animal Animals manifest toxicity to aspirin with similar signs and symptoms to those seen in humans. These may include vomiting and gastric hemorrhage, hyperpnea, respiratory alkalosis, metabolic acidosis, hyperthermia, and seizures (Edwards, 2021).

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Human Acute ingestions of greater than 150 mg kg−1 may result in toxic effects. Early manifestations of toxicity include nausea, vomiting, and tinnitus, followed by hyperventilation with respiratory alkalosis and concomitant metabolic acidosis. Overall serum pH typically demonstrates alkalosis early in the course of toxicity in adult patients, which may convert to predominant acidosis as the patient’s condition deteriorates. Early hyperglycemia may be followed by hypoglycemia or neuroglycopenia. More ominous signs and symptoms associated with severe toxicity include hyperthermia, altered mental status, coma, seizures, cerebral edema, and death. Other symptoms that may be observed include acute lung injury and pulmonary edema, acute renal failure, acute liver injury, and coagulopathies. Signs and symptoms of aspirin toxicity may begin to occur at serum concentrations greater than 30 mg dL−1.

Chronic toxicity Animal Daily doses of acetylsalicylic acid in cats produced toxic hepatitis, vomiting, weight loss, poor appetite in the low-dose group (33–63 mg kg−1 day−1) and anemia, gastric lesions, and death in the high-dose group (81–130 mg kg−1 day−1). High doses of aspirin given to mice on day 6 of gestation produced large incidence of lethal deformities.

Human Chronic salicylism presents similarly to acute toxicity, although more severe symptoms may be present at lower serum concentrations. Chronic salicylism patients will have more profound clinical effects at lower serum salicylate levels compared to patients with acute overdoses. Chronic salicylism is often associated with a delay in diagnosis and higher morbidity and mortality.

Reproductive and developmental toxicity Aspirin readily crosses the placenta, and if given near term, higher concentrations may be found in the neonate than the mother. Low dose aspirin given chronically to pregnant patients may be beneficial in certain circumstances (pregnancies complicated by gestational hypertension or systemic lupus erythematosus) and has not been demonstrated to be harmful to the fetus or neonate (ACOG, 2018). However, full dose aspirin given in the third trimester has been associated with prolonged gestation and labor, premature closure of the ductus arteriosis, and bleeding complications in the neonate. Aspirin used around the time of conception has been associated with an increased risk of spontaneous abortion, and animal models indicate that aspirin may inhibit conception by blocking blastocyst implantation. There is not currently enough information to determine if aspirin is a human teratogen. Maternal aspirin overdose in pregnancy poses a serious threat to the fetus.

Genotoxicity No mutagenic effect has been found (Snyder and Green, 2001).

Carcinogenicity The Carcinogenic Potency Database documents no positive (carcinogenic) experiments with acetylsalicylic acid (National Library of Medicine, 2022). Aspirin is not a known human carcinogen.

Clinical management Basic and advanced life-support measures should be utilized as necessary. Gastrointestinal decontamination with activated charcoal should be considered for appropriate patients. Patients with altered mental status or recurrent vomiting should not be given activated charcoal unless the airway is protected. Delayed absorption of aspirin is common in overdose, and therefore activated charcoal may be administered late, up to 8 h post ingestion, and multiple doses may be beneficial. Correction of fluid and electrolyte disturbances is important. Intravenous sodium bicarbonate should be administered to patients manifesting signs and symptoms of salicylate toxicity with a goal of alkalinizing the urine to a pH of 7.5–8. This increases the urinary excretion of salicylate through ion trapping. Hemodialysis is recommended for patients manifesting severe toxicity such as mental status changes, serum pH less than 7.20, hypoxemia requiring supplemental oxygen, and for patients with serum salicylate levels greater than 100 mg dL−1 in acute overdose or greater than 90 mg dL−1 in the presence of impaired kidney function (Juurlink et al., 2015).

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Environmental fate and behavior Acetylsalicylic acid and its biological metabolites are expected to rapidly hydrolyze and biodegrade in the environment. Salicylic acid, aspirin’s major metabolite, is a naturally occurring compound. Aspirin has been measured in water environments at 6.7 ng/L, an extremely low level that is of no significance to humans (Vulliet and Cren-Olivé, 2011).

Exposure standards and guidelines Recommended dosing of acetylsalicylic acid (aspirin) for adults is generally 325–650 mg as often as every 4 h. Specific dosing varies by the indication. Doses greater than 150 mg kg−1 may result in toxicity. Therapeutic serum concentration is 15–30 mg/dL.

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/2244

References ACOG (2018) Low-dose aspirin use during pregnancy. American College of Obstetricians and Gynecologists. Obstetrics and Gynecology 132: e44–e52. Committee Opinion No. 743. Chyka PA, Erdman AR, Christianson G, et al. (2007) Salicylate poisoning: An evidence-based consensus guideline for out-of-hospital management. Clinical Toxicology 45: 95–131. Edwards SH (2021) Nonsteroidal anti-inflammatory drugs in animals. In: Merck Manual Veterinary Manual. https://www.merckvetmanual.com/pharmacology/inflammation/ nonsteroidal-anti-inflammatory-drugs-in-animals?query¼aspirin. (Accessed March 15, 2022). Gummin DA, Mowry JB, Beuhler MC, et al. (2021) 2020 Annual Report of the American Association of Poison Control Centers’ National Poison Data System (NPDS): 38th Annual Report. Clinical Toxicology 59(12): 1282–1501. Juurlink DN, Gosselin S, Kielstein JT, et al. (2015) Extracorporeal treatment for salicylate poisoning: systematic review and recommendations from the EXTRIP Workgroup. Annals of Emergency Medicine 66(2): 165–181. National Library of Medicine (2022) Carcinogenic Potency Database. National Institutes of Health. https://www.nlm.nih.gov/databases/download/cpdb.html (Accessed March 15, 2022). Snyder RD and Green JW (2001) A review of the genotoxicity of marketed pharmaceuticals. Mutation Research 488(2): 151–169. Vulliet E and Cren-Olivé C (2011) Screening of pharmaceuticals and hormones at the regional scale, in surface and groundwaters intended to human consumption. Environmental Pollution 159(10): 2929–2934.

Relevant website https://www.uptodate.com/contents/salicylate-aspirin-poisoning-in-adults :Salicylate (aspirin) poisoning in adults.

ACGIHW (American Conference of Governmental Industrial Hygienists) LM Brosseau, Colfax South LLC, Minneapolis, MN, United States © 2024 Elsevier Inc. All rights reserved. This is an update of D.L. Dahlstrom, A.B. Bloomhuff, ACGIHW (American Conference of Governmental Industrial Hygienists), Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 178-179, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3. 00584-4.

History ACGIHW today Mission Membership Key activities, publications, databases, and services Related organizations

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Abstract ACGIH® is a 501(c)(3) charitable scientific organization that advances occupational and environmental health and safety (OEHS) with the goal of “Defining Science for OEHS Experts.” ACGIH publications include annual editions of the TLVs® and BEIs® book and work practice guides on Air Sampling Instruments, Bioaerosols Assessment and Control, Industrial Ventilation, and Modern Industrial Hygiene. For more than 80 years, ACGIH® has been dedicated to encouraging interchange of experience and expertise among industrial hygienists and other health and safety professionals by collecting and making accessible information and data that aid them in the fulfillment of their duties. The Board of Directors and Committees consist of members who strive to provide essential, cutting-edge information to government, academia, and corporate facilities throughout the United States, Canada, and countries abroad. In collaboration with the American Industrial Hygiene Association, ACGIH publishes a monthly peer-reviewed journal, the Journal of Occupational and Environmental Hygiene (JOEH). The organization hosts on-line webinars and in-person courses and manages technical committees on bioaerosols, biological exposure indices, industrial ventilation, and threshold limit values for chemical substances and physical agents. ACGIH has more than 3000 members located across the world.

Keywords Environmental health; Industrial hygiene; Occupational Health; Safety; Workplace

History The independent National Conference of Governmental Industrial Hygienists (NCGIH) convened on 27 June 1938, in Washington, DC. Representatives to the conference included 76 members, representing 24 states, three cities, one university, the US Public Health Service, the US Bureau of Mines, and the Tennessee Valley Authority. This meeting was the culmination of concerted efforts by John J. Bloomfield and Royd S. Sayers. NCGIH originally limited its full membership to two representatives from each governmental industrial hygiene agency. In 1946, the organization changed its name to the American Conference of Governmental Industrial Hygienists (ACGIH®) and offered full membership to all industrial hygiene personnel within the agencies as well as to governmental industrial hygiene professionals in other countries. At its first meeting, NCGIH created nine standing committees. The committees were charged with addressing the important industrial hygiene issues of the pre-war era: appraisal methods; relationships with industry, labor, the medical profession, and other agencies; technical standards; education; uniform reporting of occupational diseases and other illnesses among workers; administrative development of state activities; industrial health code; legislation; and personnel. Over the next five decades, some of these committees evolved and expanded, assuming different titles; some became the purview of other organizations or agencies; and some achieved their goals and ended their active roles. Undoubtedly the best known of ACGIH®s activities, the Threshold Limit Values for Chemical Substances (TLV®-CS) Committee was established in 1941 and became a standing committee in 1944. This group was charged with investigating, recommending, and annually reviewing occupational exposure limits for chemical substances. In 1946, the organization adopted its first list of 148 exposure limits, then referred to as Maximum Allowable Concentrations. The term Threshold Limit Values (TLVs®) was introduced in 1956. The first Documentation of the Threshold Limit Values was published in 1962 and is now in its ninth edition. Today’s list of

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TLVs® includes 770 chemical substances and physical agents, as well as 54 biological exposure indices (BEIs®) for selected chemicals. In 1961, ACGIH® began co-sponsoring an annual conference, the American Industrial Hygiene Conference and Exposition (AIHce), with the American Industrial Hygiene Association (AIHA).

ACGIHW today Membership is open to all practitioners in industrial hygiene, occupational health, environmental health, and safety in the US and abroad. In 2016, ACGIH became a 501(c)(3) charitable scientific organization, with the goals of encouraging interchange of experience among industrial hygienists and collecting and making accessible information and data that aids them in the fulfillment of their duties. In September 2000, the organization expanded membership and Board participation to include corporate employees and consultants, in addition to those working in government or academic organizations. Today, ACGIH® has five scientific committees that develop work practice guidelines for bioaerosols, biological exposure indices, industrial ventilation, and threshold limit values for chemical substances and physical agents. The tradition of reliable working committees has served ACGIH® exceptionally well. Through the efforts of its committees, ACGIH® has been able to provide critical information and has recommended practices to industrial hygienists worldwide. This history of sharing knowledge, based on careful study and independent judgment, has garnered international respect and accolades for ACGIH®.

Mission Since its founding in 1938, ACGIH® has gone through many changes. Its membership has grown and diversified; its interests and projects have multiplied; names and faces in the organization have changed. Despite these changes, ACGIH® has not lost sight of its original objectives, which are reflected in today’s organizational mission: the advancement of occupational and environmental health and safety.

Membership ACGIH currently has approximately 3000 members. Individual membership categories include voting, student, emerging professional, and retired.

Key activities, publications, databases, and services ACGIH® supports its mission by developing scientific guidelines for the use of occupational and environmental health and safety professionals and developing and sponsoring numerous educational activities that facilitate the exchange of ideas, information, and techniques. These courses, symposia, webinars, and workshops are all vehicles for achieving the ultimate goal of worker health and safety. The best known of the ACGIH’s committee activities is the Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices book (better known as the TLVs® and BEIs®). These occupational exposure criteria are widely used around the world as the basis for occupational health protection. Today’s list of TLVs® includes 770 chemical substances and physical agents, as well as 54 biological exposure indices (BEIs®) for selected chemicals. Two other ACGIH® committees have created publications that are recognized as the preeminent professional references in their respective fields: Industrial Ventilation: A Manual of Recommended Practice, first published in 1951, and Air Sampling Instruments for Evaluation of Atmospheric Contaminants, which debuted in 1960 and was last published in 2001 (9th edition). The Ventilation Manual, now known as Industrial Ventilation: A Manual of Recommended Practice for Design (the Design Manual), is now in its 30th edition and has a 2nd edition companion, Industrial Ventilation: A Manual of Recommended Practice for Operation and Maintenance. In 2022 ACGIH published Air Sampling Technologies: Principles and Applications, a comprehensive text covering the practical application of modern sampling devices, utilization of sampling results ranging from compliance to application of Bayesian statistics for anticipating problems not traditionally found in active or passive air sampling. This publication describes air sampling instruments applicable to any industrial hygiene program in addition to new advances in equipment and technologies. Other important texts from ACGIH Committees include: A Guide for the Control of Audible Sound Hazards (2020); Air Sampling Instrument Selection Guide: Indoor Air Quality (1998); Assessing EMF in the Workplace: A Guide for Industrial Hygienists (1998); Bioaerosols: Assessment and Control (1999); A Guide for Control of Laser Hazards, 4th Edition (1990); and Particle Size-Selective Sampling for Particulate Air Contaminants (1999). ACGIH® offers approximately 800 publication titles for sale. Topics include industrial hygiene, environmental health, safety and health science, medical/toxicology, hazardous materials/waste, workplace controls, indoor air quality, physical agents, ergonomics,

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distance learning, computer resources, downloadable TLV® and BEI® Documentation and other downloadable products, and professional development. All of ACGIH®s publications can be ordered online at www.acgih.org. In addition to its publications, ACGIH® supports numerous educational activities that facilitate the exchange of ideas, information, and techniques. These courses, symposia, webinars, and workshops are all vehicles for achieving the ultimate goal of worker health and safety. ACGIH®s dedication to information dissemination is also evident through its commitment to the Journal of Occupational and Environmental Hygiene, which it publishes jointly with the American Industrial Hygiene Association (AIHA). In 1998, ACGIH® formed the Foundation for Occupational Health & Safety (FOHS). FOHS was established to complement the work of the American Industrial Hygiene Foundation (AIHF). The FOHS mission includes:

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Sponsoring research, education, and the publication of scientific information. Providing a vehicle for financial support of the improvement and enhancement of occupational and environmental health and safety and the general public health. Disseminating the results of valuable research findings and assuring a heightened quality of continuing education in occupational safety and health.

More information about FOHS can be found atwww.acgih.org/foundation.

Related organizations

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American Industrial Hygiene Association (AIHA) International Occupational Hygiene Association (IOHA) (US) National Institute for Occupational Safety and Health (NIOSH) (US) Occupational Safety and Health Administration (OSHA). The MAK Commission (Germany) Dutch Expert Committee on Occupational Safety (DECOS)

See also: National Institute for Occupational Safety and Health; Occupational Safety and Health Administration; American Industrial Hygiene Association; Industrial hygiene

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Acids Sai Shiva Krishna Prasad Vurukonda and Agnieszka Saeid, Department of Engineering and Technology of Chemical Processes, Faculty of Chemistry, Wroclaw University of Science and Technology, Wroclaw, Poland © 2024 Elsevier Inc. All rights reserved. This is an update of A. Saeid, SSKP Vurukonda, Acids, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 56–58, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00219-0.

Introduction Soil contamination by acids Toxicology of acids on soil microbiota Acid mine effects on microorganisms Bioremediation of acid pollution Microbial consortia for acid mine treatment Conclusion Funding Conflict of interest References

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Abstract Acids and related mining industries generate large amounts of waste pollutants that seriously threaten the environment, land, water, and soil. The toxicity of these acids and/or from mines has serious effects on the microbial population above and below ground. Different traditional methods are frequently ineffective and unsustainable. Cost-effective treatment options include chemical and other approaches; however, they produce different complication issues. The approach for the alternative environmentally friendly method to treat acid contaminations is the main emphasis of the current review. In this context, treating mine waste and recovering dangerous heavy metals is possible through the biological process of utilizing microorganisms offers a competitive alternative. Microorganisms are used to remove, cleanse, or sequester contaminants from mine waste. Microbial consortia function with the interspecies, and their design and applications were discussed.

Keywords Acids; Acid mines; Biological control; Bioremediation; Consortium; Microorganisms; pH; Soils

Key points

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Evaluation of acids and its fundamentals Among various contaminations by acids, soil contamination is most affective Soil microbiota has drastically affected by the acid pollution Alternative and eco-friendly approaches for the bioremediation of soils affected by acids.

Introduction The most crucial chemical notions are acid and acidity, however there is some disagreement about how to classify them. Lewis provided classifications for bases and acids based on their valence electrical structure in 1923. A Lewis base is an electron-pair donor, whereas a Lewis acid is an electron-pair acceptor (Lewis, 1923; Utku et al., 2020). A LA has a low-lying lowest unoccupied molecular orbital (LUMO) in molecular orbital theory, whereas an LB has a higher-energy highest occupied molecular orbital (HOMO) (HOMO). Lewis proposed an equilibrium state between acid and base in which the forward step is the formation of a coordinative covalent bond between HOMO and LUMO and the reverse step is bond heterolysis (Satchell and Satchell, 1969; Welch et al., 2006; Kawahara et al., 2005). As Lewis acidity increases, the equilibrium shifts more toward adduct formation. Lewis’ definitions of acids applied to a wide variety of species, including those classified by the Arrhenius and Brnsted-Lowry theories (Neumüller, 1990). These all-encompassing terms significantly broaden the scope of acid chemistry and provide insight into a wide range of reactions.

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Soil contamination by acids Soil contamination has become one of the most pressing concerns for geotechnical engineers worldwide, owing to the complex chemical reactions that occur between soils and contaminants. Soil contamination can occur as a result of natural or anthropogenic processes (Gratchev and Towhata, 2013). Weathering processes, acid rain, and acid rock drainage are natural processes (3 pH 7), whereas accidental leakage or spillage of a variety of chemicals during transportation or storage is an anthropogenic process (pH 1). Although natural processes influence soil behavior to some extent, anthropogenic processes significantly alter soil engineering behavior, resulting in progressive structural failures. The increasing rate of acid contamination (pH 1) of soils, as well as the consequences for the volume change behavior of these soils, is reflected in the number of reported failures of industrial structures (Yamanaka et al., 2002; Al-Omari et al., 2007; Parfitt et al., 2011). According to a brief review of the literature, the following mechanisms may influence the engineering behavior of soils in an acidic environment. The first mechanism is mineral structure dissolution and cation leaching from the soil. According to Sivapullaiah et al. (2008), cation exchange reactions cause increased swelling in calcitic soil due to sulfuric acid contamination. Spagnoli et al. (2012) reported a significant increase in shear strength in an acidic environment due to Al3+ dissolution, which acts as a coagulant by increasing internal resistance. In Liu et al. (2013), the displacement of cations by hydrogen ions causes a decrease in the liquid limits and swelling index of the three montmorillonitic soils. According to Gratchev and Towhata (2016), calcium carbonate dissolution from kaolinitic soils results in higher liquid limits, plasticity indices, and compression indices. Reported by Bakhshipour et al. (2016), leaching of aluminum and iron from residual soil increases the Atterberg limits, optimum moisture content, and permeability while decreasing strength and maximum dry density. The second mechanism is the change in charge on the edges of the clay particles, which results in more open and flocculated structures (Olphen, 1991). Wang and Siu (2006) confirmed the high compressibility of soils caused by the formation of such structures in an acidic environment. The third mechanism is the adsorption of anions by the soils in an acidic environment. Krebs et al. (1962) reported that, adsorption of sulfates and chlorides reduces the liquid limits of montmorillonite, whereas adsorption of phosphates increases the liquid limits. Similarly, Sivapullaiah (2009) reported that phosphate adsorption reduces the compression indices of homoionic kaolinite and montmorillonite clays. Changes in the diffuse double layer caused by the replacement of exchangeable cations by hydrogen ions in an acidic environment are the fourth mechanism. Gajo and Maines (2007) reported the collapse of the double layer significantly reduces the compressibility of bentonite. According to Shekhtman et al. (1995), the formation of sulfate-based minerals such as gypsum, halotrichite, potash alums, soda alums, tamaruchite, and melanterite during sulfuric acid contamination causes soil heave. Mal’tsev (1998) came to the conclusion that high swelling in acid-contaminated soils can be attributed to mineralogical changes. Similarly, Sivapullaiah et al. (2009) reported that the high induced swelling in black cotton soil contaminated with sulfuric acid is due to mineralogical changes.

Toxicology of acids on soil microbiota Every teaspoon of soil contains hundreds of millions of microorganisms, including bacteria, fungi, protozoa, and nematodes, the vast majority of which are critical to healthy, productive soils (Chaparro et al., 2012). Soil microbes are essential to healthy soil processes and soil quality. Soil microbes regulate many aspects of most nutrient cycles. Without microbes, organic matter decomposition would be impossible, legumes would be unable to fix nitrogen, and ammonia would not be converted to plant-available nitrate. Soil acidity affects many chemical and biological properties of soil, including nutrient availability and metal toxicity (McBride, 1994), which can also affect microbial communities in many ways (Sylvia et al., 2005). The majority of soils on the planet are made up primarily of aluminum-silicate minerals. At neutral pH, these minerals are solid or crystalline, but they have pH-dependent aluminum (Al) solubility. This means that as soil pH falls below 5.5, the amount of Al available to plants and microbes in the soil increases dramatically. Because Al can be toxic to plants, the effects of soil acidity on crop yields are largely due to Al toxicity in acidic soils (Foy, 1984). Soil acidity can have an impact on the structure and function of the soil microbial community both directly and indirectly. The direct interaction of hydrogen ions (H+), which are present in high concentrations when pH is low, with microbial cells can have a variety of effects on microbial communities, including disruption of cell membranes, altered enzyme production, and limited reproduction. This translates to decreased overall microbial function in soil health and productivity (Birgander et al., 2014). Furthermore, soil fungi are favored by low soil pH, which means that as soil pH decreases, the microbial community shifts from a balance of bacteria and fungi to a much more fungi-dominated soil (Rousk et al., 2010). This may allow for an increase in the number of fungal species, particularly pathogenic ones, as well as the invasion of fungal root pathogens, but it also changes the way organic residues decompose. Because soil fungi and bacteria play different roles in the decomposer community and interact to release nutrients to plants, lowering soil pH will alter those relationships, causing soil carbon and plant nutrients to become immobilized, slowing turnover and nutrient release (Rousk et al., 2009).

Acid mine effects on microorganisms Acid mine tailings (AMT) toxicity is caused primarily by acidic pH and heavy metal content. The synergistic effect of acidic pH and heavy metals increases heavy metal bioavailability, thereby increasing biotoxicity. Heavy metal exposure from mine waste has been

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linked to a variety of disorders and diseases (Ayangbenro and Babalola, 2017). AMT alters and destroys ecosystems, deteriorates landscapes, reduces biodiversity, and increases pollution in the environment. As a result, physical habitats for microorganisms are being reduced and destroyed, as well as nutritional, ecological, and evolutionary issues (Ayangbenro and Babalola, 2017), fragmentation of habitat and intrusion by feral organisms and weed species (Jain et al., 2016). This has serious consequences for the ecosystem’s balance and the resulting disruption of the food web, which has a negative impact on aboriginal organisms’ lifestyles. Stress responses are induced by unfavorable conditions in AMT waste, such as toxic metals and metalloids and low pH, resulting in characteristic changes in microbial cell morphology and assembly (Chakravarty and Banerjee, 2008). These modifications prevent cell growth. Acid also denatures microbial enzymes, halting metabolic functions. This results in the loss of macromolecule building blocks that are required for growth. Rain’s hydronium ions also mobilize toxins like aluminum, resulting in the leaching of essential nutrients and minerals like magnesium that are required for growth (Davies and Mundalamo, 2010). Furthermore, those organisms that are unable to tolerate low pH changes are destroyed, resulting in loss of diversity.

Bioremediation of acid pollution Acid mining is typically avoided by adding alkaline materials to sulfide-rich mine wastes. The procedure is a chemical-neutralizing treatment that promotes acid neutralization, reduced metal solubility, and consequent metal retention in solution via precipitation. The addition of alkaline materials accelerates the oxidation of ferrous iron in AMT waste. This raises the pH of the solution and causes metal precipitation as hydroxides and carbonates. These traditional treatment processes, however, necessitate significant financial resources in terms of capital and operating costs. Most of these processes frequently involve the use of chemicals, which can cause pollution and necessitate additional cleanup (Hilson and Murck, 2001), and some of these techniques are not sustainable. As a result, looking for low-cost treatment with better contaminant removal becomes an option. Currently, the most cost-effective and environmentally friendly method for treating AMT waste is to use plants and microorganisms, though this has not always been successful due to geographical and/or technological constraints. In some cases, biological reactors may require a constant biomass supply feed to function, and if such a feed is not available, such reactors will not function (Hilson and Murck, 2001; Ayangbenro et al., 2018). Bioremediation is regarded as a viable alternative to conventional remediation techniques, and sulfate-reducing microorganisms have been used in the treatment of acid mine waste. Remediation refers to the removal of an issue, and when it is associated with the removal of an ecological issue, such as soil and groundwater contamination, it is referred to as bio-remediation. Bioremediation is a mechanism that employs living microorganisms to reduce or prevent natural contamination (Cristaldi et al., 2020). It is an advancement toward the elimination of toxins from the climate, restoring the first characteristic environmental factors and preventing further contamination. Instead of simply relocating the problem, bioremediation can provide a permanent in-situ solution to contamination. This technique can be used to remove heavy metals, metalloids, or other inorganic pollutants from soil or water (Ali et al., 2013). When compared to other engineering techniques, it is a cost-effective, efficient, novel, eco-friendly, and solar-powered technology with high public acceptance. Phytoremediation is the most well-known bioremediation technique. Phytoremediation is the direct use of green plants and their associated microorganisms to stabilize or reduce contamination in soils, sludges, sediments, surface water, or ground water. To mitigate the toxic effects of pollutants, this technique applies plant interactions (physical, biochemical, biological, chemical, and microbiological) to contaminated sites. It is a supplementary technology that can be used alongside or in place of mechanical conventional clean-up technologies, which frequently require large capital investments and are energy intensive. Low contaminant concentrations over large cleanup areas and at shallow depths provide especially favorable conditions for phytoremediation. Depending on the pollutant type (elemental or organic), there are several mechanisms involved in phytoremediation (accumulation or extraction, degradation, filtration, stabilization, and volatilization) (Kuiper et al., 2004). Elemental pollutants (toxic heavy metals and radionuclides) are mostly removed by extraction, transformation and sequestration.

Microbial consortia for acid mine treatment Microbial consortia are known to accelerate complicated tasks that allow microorganisms to survive in hostile environments such as AMT waste and can be more resilient to environmental fluctuations (Brune and Bayer, 2012). They have successfully been applied to real-world problems, facilitating complex functions involving inter-species interactions that allow them to survive in acid mine environments (Keller and Surette, 2006). They interact in the syntrophic degradation of complex substances, which allows for complete metabolic reactions between two or more organisms, without which energy cannot be obtained without the cooperation of the other (Zhou et al., 2011). The combination of specific properties of these organisms and interacting metals allows for the simultaneous removal of toxic metals and sulfate from contaminated environments. These organisms contain a wealth of genetic information that can be used to develop modified microbiomes that concentrate metals and sulfate (Dunbar, 2017). These organisms also provide a variety of resistance mechanisms, which aid in remediation. Highly resistant microorganisms with natural or engineered degradation pathways are required for effective application of these organisms. Once an initial consortium has been established, evolution theory may be used to elucidate novel species interactions, resulting in increased microbial consortium productivity and stability (Brune and Bayer, 2012). However, the field of synthetic biology is evolving, and next-generation

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technologies will require more systematic and integrated efforts. The field of single cell genomics and metagenomics will be a useful tool for understanding the genetic diversity of organisms that have yet to be cultured in various environments, as well as for understanding the functional roles of these organisms in such environments (Zhou et al., 2011). The use of these organisms for the treatment of AMT waste is an appropriate method for removing persistent pollutants from the environment. While synthetic biology continues to produce a number of engineered consortiums for bioremediation, there are concerns about their unknowable effects on the ecosystem (Lorenzo, 2017).

Conclusion It is noted that we are only now beginning to understand and, as a result, fully exploit natural diversity for biodegradation and bioremediation of acids or acid contaminations. New microorganisms or microbial consortia for degradation have been discovered, as have new methods for discovering the broad flexibility of microorganisms. Aside from using natural diversity, artificial evolution of enzymes and pathways will undoubtedly lead to improved biocatalysts and high-throughput methods of screening for available remediation methods. Through this brief review, studies to understand the interaction between acid toxicology, its effects on the survival and activities of microorganisms in the environment, and biochemical and genetic engineering studies must intersect. Such crossfeeding will lay the groundwork for successful interventions into environmental processes, leading to optimized bioremediation strategies.

Funding This research was funded by the NCN/Poland Grant No. 2021/42/E/ST10/00379.

Conflict of interest Authors declare no conflict of interest.

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Kuiper I, Lagendijk EL, Bloemberg GV, and Lugtenberg BJ (2004) Rhizoremediation: A beneficial plant-microbe interaction. Molecular Plant-Microbe Interactions 17(1): 6–15. https:// doi.org/10.1094/MPMI.2004.17.1.6. PMID: 1471486. Lewis GN (1923) Valence and the structure of atoms and molecules. American Chemical Monograph Series 172. Liu Y, Gates WP, and Bouazza A (2013) Acid induced degradation of the bentonite component used in geosynthetic clay liners. Geotextiles and Geomembranes 36: 71–80. Lorenzo V (2017) Synthetic microbiology: From analogy to methodology. Microbial Biotechnology 10: 1264–1266. https://doi.org/10.1111/1751-7915.12786. Mal’tsev AV (1998) Theoretical and experimental investigations of the effect or aggressive wetting on various types of bed soils. Soil Mechanics and Foundation Engineering 35(3): 83–86. McBride MB (1994) Environmental Chemistry of Soils. New York: Oxford University Press. Neumüller B (1990) Book Review: Inorganic Chemistry. By DF Shriver, PW Atkins and CH Langford. Wiley 1069–1070. Olphen VH (1991) An Introduction to Clay Colloid Chemistry: For Clay Technologists, Geologists and Soil Scientists, 2nd edn. New York: Wiley. Parfitt MK, Jones DJ, and Garvin RG (2011) Structural, construction, and procedural failures associated with long-term pyritic soil expansion at a private elementary school in Pennsylvania. Journal of Performance of Constructed Facilities 25(1): 56–66. Rousk J, Brookes P, and Bååth E (2009) Contrasting soil pH effects on fungal and bacterial growth suggest functional redundancy in carbon mineralization. Applied and Environmental Microbiology 75(6): 1589–1596. Rousk J, Bååth E, Brookes PC, Lauber CL, Lozupone C, Caporaso JG, and Fierer N (2010) Soil bacterial and fungal communities across a pH gradient in an arable soil. The ISME Journal 4(10): 1340–1351. Satchell DP and Satchell RS (1969) Quantitative aspects of the Lewis acidity of covalent metal halides and their organo derivatives. Chemical Reviews 69(3): 251–278. Shekhtman LM, Baranov VT, and Nesterenko GF (1995) Building deformations caused by the leakage of chemical reagents. Soil Mechanics and Foundation Engineering 32(1): 32–36. Sivapullaiah PV (2009) Effects of soil pollution on geotechnical behaviour of soils. In: Proceedings of the Indian Geotechnical Conference, Guntur, India, 17–19. Sivapullaiah PV, Prasad BG, and Allam MM (2008) Volume change behavior of calcitic soil influenced with sulfuric acid. In: GeoCongress 2008: Geotechnics of Waste Management and Remediation, 819–826. Sivapullaiah PV, Prasad BG, and Allam MM (2009) Effect of sulfuric acid on swelling behavior of an expansive soil. Soil and Sediment Contamination 18(2): 121–135. Spagnoli G, Rubinos D, Stanjek H, Fernández-Steeger T, Feinendegen M, and Azzam R (2012) Undrained shear strength of clays as modified by pH variations. Bulletin of Engineering Geology and the Environment 71: 135–148. Sylvia DM, Fuhrmann JJ, Hartel PG, and Zuberer DA (2005) Principles and Applications of Soil Microbiology (No. QR111 S674 2005). Upper Saddle River, NJ: Pearson Prentice Hall. Utku Y, Thomas ARH, Meng W, and Michael PS (2020) Polymer-supported Lewis acids and bases: Synthesis and applications. Progress in Polymer Science 0079-6700111: 101313. https://doi.org/10.1016/j.progpolymsci.2020.101313. Wang YH and Siu WK (2006) Structure characteristics and mechanical properties of kaolinite soils. I. Surface charges and structural characterizations. Canadian Geotechnical Journal 43(6): 587–600. Welch GC, Juan RR, Masuda JD, and Stephan DW (2006) Reversible, metal-free hydrogen activation. Science 314(5802): 1124–1126. Yamanaka T, Miyasaka H, Aso I, Tanigawa M, Shoji K, and Yohta H (2002) Involvement of sulfur-and iron-transforming bacteria in heaving of house foundations. Geomicrobiology Journal 19(5): 519–528. Zhou J, He Q, Hemme CL, Mukhopadhyay A, Hillesland K, Zhou A, He Z, Van Nostrand JD, Hazen TC, Stahl DA, Wall JD, and Arkin AP (2011) How sulphate-reducing microorganisms cope with stress: Lessons from systems biology. Nature Reviews Microbiology 9: 452–466. https://doi.org/10.1038/nrmicro2575.

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Acifluorfen, sodium salt M Noruzia and M Sharifzadehb, aFaculty of Pharmacy, Pharmaceutical Sciences Research Center (PSRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran; bFaculty of Pharmacy, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of Y.R. Rodriguez, Acifluorfen, Sodium Salt, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 59–62, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01158-1.

Chemical profile Background Uses Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Chronic toxicity Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Exposure standards and guidelines References Further reading

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Abstract Acifluorfen is an herbicide. In nature, sodium Acifluorfen decomposes into Acifluorfen, which is toxic to plant cell membranes. It can be used before or after the annual broadleaf weeds and grasses have emerged to suppress them. Acifluorfen peroxidizes lipids in membranes due to its capacity to inhibit the enzyme protoporphyrinogen oxidase. Orally, topically, or inhaled administration of sodium Acifluorfen has been observed to have negligible acute toxicity. Damage to the liver is the most common type of chronic toxicity effect. This herbicide is probably carcinogenic in humans.

Keywords Acifluorfen; Blazer; Peroxidizing herbicides

Key points

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Introduction of Acifluorfen Acifluorfen uses fate in the environment Acifluorfen toxicokinetic Mechanism of action of Acifluorfen Acifluorfen toxicity (acute and chronic, reproduction and developmental, genotoxicity, carcinogenicity, and immunotoxicity) Acifluorfen Ecotoxicity and exposure guideline

Chemical profile

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Name: Acifluorfen, Sodium salt Molecular formula: C4H7C1F3NO3 Chemical Abstracts Service Registry Number: 62476-59-9 (Acifluorfen-Sodium) 50594-66-6 (Acifluorfen)

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00395-X

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Synonyms: Blazer, Acifluorfen, Carbofluorfen, 5-(2-Chloro-alpha, alpha, alpha-trif1uoro-p-tolyloxy)- 2-nitrobenzoic acid (IUPAC), RH-6201, Sodium 5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoate Chemical structure:

Background In 1980, the Rohm and Haas Company registered sodium Acifluorfen in the United States under the trade name Blazer. The US Environmental Protection Agency (EPA) evaluated pesticide products containing sodium Acifluorfen in 2003 to see if they qualified for pesticide re-registration. Sodium Acifluorfen pesticides that meet conditions for re-registration, such as stricter labeling standards to reduce the likelihood of spread, would be re-registered. Twelve compounds containing sodium Acifluorfen have been approved for use (Rodriguez, 2014).

Uses Sodium Acifluorfen belongs to the nitro phenyl ether family and diphenyl ethers. This herbicide can potentially damage plant cell membranes rapidly and prevents the contents of the plant from moving freely. Once within the cell, Acifluorfen causes the formation of free electrons (which require light) and peroxides, which in turn damage the cell membrane. After damaging the membrane, sodium Acifluorfen breaks down into Acifluorfen (or Acifluorfen acid) and is released into the environment. Sodium Acifluorfen can be employed alone or in combination with other comparable chemicals. Fluid, pre-mixed, and soluble concentrates are a few examples of the many formulations. Aircraft and ground-based dispersion and band treatment systems apply chemicals to crops. Both commercial and household applicators will employ for targeted applications. Crops often benefit from the nonselective nature of Acifluorfen since it is utilized for pre- and after-emergent weed and grass management on annual broadleaf weeds. Annual agricultural usage of sodium Acifluorfen in the United States at 300,000 pounds from 2001 to 2007. Soybean crops received an estimated two hundred thousand pounds of agricultural consumption. Sodium acifluorfen is used to cultivate rice, strawberries, peas, and peanuts. Although sodium Acifluorfen is approved in the home, it can only be used for spot therapy in prepared formulations. Sodium Acifluorfen affects either weeds or grasses; however, its use in private homes is minimal compared to agricultural settings (Rodriguez, 2014).

Environmental fate and behavior Acifluorfen has an organic carbon/water partition coefficient (KOC) ranging from 44 to 684, an approximate vapor pressure of 1.5
10−8 mmHg, and a pKa of 2.07. Henry’s law constant for sodium Acifluorfen equals 6.03
10−11 atm-m3/mol based on the vapor pressure of 1.33
10−5 mmHg. The octanol/water partition coefficient (Kow) is 1.55, while the water solubility of sodium Acifluorfen at 20  C is 62.07 g per 100 ml. Also, sodium Acifluorfen exists as the anion at pH levels between 5 and 9 due to its pKa, 3.86. Sodium Acifluorfen is not likely to evaporate into the air from wet surfaces and is expected to emerge in the environment as an anion. In organic carbon and clay-rich soils, anion forms do not adsorb as effectively as their neutral counterparts. Based on its calculated vapor pressure, Acifluorfen is expected to be present in the air as both a gas and a solid. There is little chance of Acifluorfen volatilizing from either dry or wet soil. Acifluorfen has incredibly high to low mobility in soil. Acifluorfen is released into the environment directly. There is a close relationship between adsorption and desorption and site-specific soil characteristics, including pH and mineral content. Acifluorfen acid is a Lactofen breakdown byproduct, a different herbicide used in farming and forestry. The predicted half-life of Acifluorfen in aerobic aquatic environments is 117 days, whereas, in aerobic soil environments, it ranges from 108 to 200 days. Acifluorfen has a half-life of around 2.75 days under anaerobic conditions. Half-lives for Acifluorfen in water range from 21.7 to 352 h when exposed to light (i.e., water depth, sediment-free, pH). It is anticipated that sodium Acifluorfen will be carried through application, dispersion, and leaching. Surface water may be exposed to sodium acifluorfen through discharge and erosion from treated soil. Bioaccumulation of Acifluorfen (Kow ¼ 1.55) or sodium Acifluorfen (Kow ¼ 3.70) in marine animal is not predicted (Rodriguez, 2014).

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Exposure and exposure monitoring Sodium Acifluorfen may enter the environment via several waste streams during manufacturing. Due to its resistance to abiotic hydrolysis, Acifluorfen will remain in groundwater for an extended period. Another possible route for Acifluorfen to enter the water is the runoff of contaminated groundwater onto surface water. Acifluorfen might remain in surface water if it is not photodegraded. Runoff is a possible transmission pathway to aquatic and ground species due to its propensity to remain in surface waters. Inhalation and skin contact with sodium Acifluorfen can occur in the workplace during production or application. According to the product information for sodium Acifluorfen, when working with this substance, one should wear long sleeves, long pants, and chemically resistant gloves. Using a mask is not a must. A study of the exposure of workers who handle substances as part of their jobs found no cause for alarm. The active substance can be absorbed via the skin if the public uses items containing it. Individuals using sodium Acifluorfen in residential areas such as walkways and patios are at risk of exposure. It is generally agreed that there is minimal to no danger to human health following exposure. Sodium Acifluorfen and lactofen, which degrades into Acifluorfen, are two registered herbicides that are discharged directly into the environment. Soil, surface water, and groundwater have all been found to contain Acifluorfen. Fifty-six out of 283 soil samples (collected to depths of 0.6 m) contained traces of sodium Acifluorfen. Up to 10 months after treatment, tests still showed residues. These findings suggest that residues can enter groundwater through normal environmental pathways. Acifluorfen residues were first detected in 1992 and recorded in the Groundwater Monitoring Studies Summary Database (PGWDB); from 0.003 to 0.025 microgram per liter, Acifluorfen was found in just 4 of 1185 analyzed samples. The National Water Quality Assessment program of the United States Geological Survey (USGS) evaluated water quality throughout the country from 1993 to 2007. Only 173 of the 13,524 samples had detectable residues (both estimated and measured). Ten groundwater samples showed residual levels, with a high of 0.33 microgram per liter. The highest estimated residual was 2.2 micrograms per liter, and the maximum observed residue was 1.1 micrograms per liter from the other 163 samples taken from surface waters. Maximum residue data (measured and approximated) was gathered from surface water and groundwater sources near agricultural fields (Rodriguez, 2014).

Toxicokinetics The toxicokinetic features of Acifluorfen are poorly understood, and just a tiny amount of data is available in the literature. Animal investigations have shown that Acifluorfen mainly distributes to the liver and kidneys. Some absorption through the body can occur through lipid membranes after exposure to the skin, lungs, or GI tract since it is moderately more lipid soluble. The Kow indicated a short biological half-life and fast excretion in urine, but more data on the rate of metabolism or excretion, or the identification of metabolites, are needed. Acifluorfen does not accumulate in the body and is also not very toxic when ingested, applied topically, or inhaled (Rodriguez, 2014).

Mechanism of toxicity Protoporphyrinogen oxidase is an enzyme that catalyzes the dehydrogenation of protoporphyrinogen IX to protoporphyrin IX. Acifluorfen suppresses this process. When exposed to light, protoporphyrin can generate highly reactive oxygen radicals and cause membrane lipid peroxidation. Lipid peroxidation can start a chain reaction that leads to lipid membrane breakdown. Cell membrane dysfunction caused due to lipid peroxidation. Sodium Acifluorfen mainly affects the liver and the kidneys. While some evidence suggests that cells can make cytochrome P450 for sodium Acifluorfen detoxification, this is far from certain. Furthermore, more research is needed to be confirmed (Rodriguez, 2014).

Acute and short-term toxicity The acute toxicity of sodium Acifluorfen is minimal when administered orally, topically, or inhaled. Damage to the eyes caused by sodium Acifluorfen is permanent. Although it is not a skin sensitizer, it can trigger an allergic reaction if it comes into contact with skin often or for an extended period. When administered to rabbits in large doses, sodium Acifluorfen produces minor skin and significant eye irritation. According to the US EPA Toxicity Category classification, sodium Acifluorfen is classified as Category II for acute dermal and oral toxicity, Category I for acute eye irritation, and Category IV for acute inhalation toxicity (Rodriguez, 2014).

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Chronic toxicity The liver is a leading organ of toxicity for Acifluorfen. Studies in rats and mice using subchronic feeding revealed liver toxicity symptoms, such as a rise in liver weight and an increase in the rate of cellular hypertrophy, along with a loss in animal body weight. Chronic feeding causes reversible liver injury in rodents and dogs, as seen by acidophilic cell growth and liver weight gain in these studies of animals. Nephritis/pyelonephritis and kidney weight growth were expected outcomes of long-term orally experiments in rats, mice, and dogs (Rodriguez, 2014). Dose and time-dependent Acifluorfen toxicity, especially in a mixture of other pesticides, in exposure to chronic shallow doses, and also in a vitamin deficiency state, cause central nervous disorders like increased anxiety and locomotor activity problems in rodents (Tsatsakis et al., 2019, 2021; Sergievich et al., 2020).

Immunotoxicity There is a lack of information about immunotoxicity. Some individuals have adverse reactions after exposure to sodium Acifluorfen on the skin for an extended period or after being exposed to it repeatedly. This outcome indicates the possibility of an immunological response; nevertheless, more research is required to determine a definite dose response (Rodriguez, 2014).

Reproductive toxicity A reduction in fetal body weight and a rise in morphological abnormalities have been seen in rats exposed to sodium Acifluorfen. A study of rat reproduction spanning two generations found no changes in reproductive parameters but increased in pup mortality and kidney abnormalities. In rats exposed to sodium Acifluorfen, the signs of maternal toxicity were increased salivation, chromorhinorrhea, piloerection, and urine-stained abdominal fur (Rodriguez, 2014). More study is required to complete its reproductive toxicity and endocrine disruption profiles.

Genotoxicity Researchers found no evidence of genotoxicity when testing Acifluorfen. Sodium Acifluorfen showed mild mutagenic in the Ames test and induced metabolic activation and tumors; however, this is not definite evidence of genotoxicity. No indication of induced mutations has been found in studies of Acifluorfen in its different purities. Researchers have tested the substance at purities of approximately 42.8% (Rodriguez, 2014).

Carcinogenicity According to EPA, Sodium Acifluorfen is “likely to be carcinogenic to humans at doses high enough to cause the biochemical and histopathological changes in livers of rodents, but unlikely to be carcinogenic at doses below those causing these changes.” Non-cancer dietary risk values were below 100% level of concern (EPA, 2022). After 18 months of B6C3Ft mice exposure to 0, 626, 1250, and 2500 ppm, Male mice showed a rise in liver tumors, and weight loss was observed in males and females. At the highest dose, male mice significantly lost body weight and developed more liver tumors than control mice. Only female mice experienced a significant weight loss at the maximum dose (Rodriguez, 2014). Sodium Acifluorfen, affecting the constitutive androstane receptor (CAR; NR1I3), can cause hepatic tumor development in rodents (Kuwata et al., 2016). No carcinogenic consequences are to be expected through residential uses. Constant exposure in the workplace is not predicted. Due to the low frequency of sodium Acifluorfen applications, cancer risks to workers are not often considered (Rodriguez, 2014). Except for all findings, more research is required to determine the carcinogenic potential of this compound and its relevance to humans.

Clinical management Human exposure information is inadequate. However, deaths have been reported at 80 mg/kg, and health consequences can be seen from 50 mg/kg. Skin contact and inhalation are the most common ways that humans are exposed. When skin contact occurs, the contaminated clothing must be removed, the area washed, and the process repeated as necessary. In inhalation exposure, the person should be taken to an area with fresh air, given oxygen, and helped to breath whenever necessary. The possibility of harm from residues on treated crops is relatively low. Even when a person has taken more than 40 mg/kg of a contaminated product, stomach decontamination with the activated charcoal solution should be performed (Rodriguez, 2014).

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Ecotoxicology Sodium Acifluorfen is only moderately harmful to fish and invertebrates. In the research, the acute LC50 of sodium Acifluorfen in rainbow trout (Oncorhynchus mykiss) was 17 mg/l. Fathead minnow (Pimephales promelas) were used in a long-term investigation that concluded the NOAEC to be less than 3.4 m/1. The water bug (Daphnia Magna) had the lowest LC50 (28. l mg/l) in the acute experiment employing technical grade sodium Acifluorfen for invertebrates. There is a lack of information about long-term invertebrates. Sodium Acifluorfen is somewhat hazardous to saltwater fish due to its acute toxicity. Acute studies showed that the LC50 for sheepshead minnow (Cyprinodon variegatus) was 39 mg/l. Long-term studies of estuarine and marine fish are impossible due to a lack of data. There is some indication that acute exposure to sodium Acifluorfen seems toxic to estuarine and marine invertebrates. The mysid shrimp (Americamysis Bahia) has an LC50 of 3.8 mg/1 in an acute investigation. Estuarine and marine invertebrates that live for long periods do not have any data accessible. Sodium Acifluorfen poses little to no threat to plants. When tested at 355 micrograms per liter, equal to the maximum label rate, sodium Acifluorfen did not affect algal growth after 5 days of exposure in a Tier I trial. In Tier II research, the nonvascular aquatic plant Selenastrum capricornutum was found to have the highest NOAEC (more than 265 ppm). Duckweed has an EC50 of 378 ppb, making it the most sensitive water vascular plant studied at that concentration. The extent to which nontarget plants away from the location might be harmed is unknown. According to the available data, sodium Acifluorfen is only moderately toxic to birds (rat LC50 ¼ 325 mg/kg) and only slightly toxic to mammals (rat LC50 ¼ 1540 mg/kg). Sodium acifluorfen is almost safe for birds (quail and mallard LC50 > 10,000 ppm) in both acute and chronic studies (Rodriguez, 2014).

Exposure standards and guidelines The US threshold for sodium Acifluorfen residues has been 0.05 ppm. Acifluorfen sodium tolerances were noticed in 40 CFR 180.383. No Codex maximum residue level MRLs for Acifluorfen has been set. However, in Canada, a maximum allowable level (MRL) of 0.02 ppm has been established for post-emergence use of Acifluorfen to suppress soybean weeds. Further, the MRL for pesticides in dry soybeans in Brazil is 0.02 ppm (Rodriguez, 2014). Time-limited tolerances for residues of Acifluorfen sodium on sugar, beet, and roots are established at 0.1 ppm (EPA, 2022). Furthermore, Acifluorfen does not have a safety threshold for contamination and Total Maximum Daily Loads. Health Advisories for a kid weighing 10 kg is 2 mg per liter daily for 10 days, with a reference dose of 0.01 mg/kg daily and a drinking water equivalent of 0.4 mg mg/l (Rodriguez, 2014). EPA assumed acute dietary intake through water and food to sodium acifluorfen likely consumes 4.0% of the acute population adjusted dose for females 13–49 years old, the most exposed population. Acute cumulative risk is identical to acute dietary risk. Long-term exposure to sodium acifluorfen from water and food will be using 63% of the chronic population adjusted dose for babies 1-year-old, the most exposed population. Therefore, the chronic cumulative risk is comparable to the chronic dietary risk. Based on these risk evaluations, the EPA has determined a low probability that the general public, infants, and children will experience any adverse effects from prolonged exposure to sodium acifluorfen residues. Tolerable amounts of sodium acifluorfen residues were found in all crops. All crops treated with sodium acifluorfen were present at tolerance levels in all commodities for which tolerances have been set or proposed. Estimated drinking water concentrations of sodium acifluorfen for short-term exposures were expected to be 66.7 ppb in surface water and 146 ppb in groundwater, according to data from the Pesticide Root Zone Model Ground Water (PRZM GW) (EPA, 2022).

References EPA (2022) Sodium Salt of Acifluorfen; Pesticide Tolerances for Emergency Exemptions [Online]. Environmental Protection Agency Docket Center (EPA/DC), West William Jefferson Clinton Bldg: Office of Pesticide Programs Regulatory Public Docket (OPP Docket). EPA-HQ-OPP-2021-0604, FRL-9657-01-OCSPP. Kuwata K, Inoue K, Ichimura R, Takahashi M, Kodama Y, Shibutani M, and Yoshida M (2016) Involvement of mouse constitutive androstane receptor in acifluorfen-induced liver injury and subsequent tumor development. Toxicological Sciences 151: 271–285. Rodriguez Y (2014) Acifluorfen, seodium salt, Encyclopedia of Toxicology, 3rd edn. Elsevier. Sergievich AA, Khoroshikh PP, Artemenko AF, Zakharenko AM, Chaika VV, Kodintsev VV, Stroeva OA, Lenda EG, Tsatsakis A, and Burykina TI (2020) Behavioral impacts of a mixture of six pesticides on rats. Science of the Total Environment 727: 138491. Tsatsakis A, Tyshko NV, Docea AO, Shestakova SI, Sidorova YS, Petrov NA, Zlatian O, Mach M, Hartung T, and Tutelyan VA (2019) The effect of chronic vitamin deficiency and long term very low dose exposure to 6 pesticides mixture on neurological outcomes–A real-life risk simulation approach. Toxicology Letters 315: 96–106. Tsatsakis A, Tyshko NV, Goumenou M, Shestakova SI, El’vira OS, Zhminchenko VM, Zlatian O, Calina D, Pashorina VA, and Nikitin NS (2021) Detrimental effects of 6 months exposure to very low doses of a mixture of six pesticides associated with chronic vitamin deficiency on rats. Food and Chemical Toxicology 152: 112188.

Further reading https://www.federalregister.gov/documents/2022/03/31/2022-06817/sodium-salt-of-acifluorfen-pesticide-tolerances-for-emergency-exemptions.

Relevant websites http://www.epa.gov :U.S. Environmental Protection Agency. Search for Acifluorfen-sodium https://pubchem.ncbi.nlm.nih.gov :PubChem. Search for Acifluorfen-sodium

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Acrolein E Vilanova and D Pamies, Universidad Miguel Hernández de Elche, Elche, Spain © 2024 Elsevier Inc. All rights reserved. This is an update of D. Pamies, E. Vilanova, Acrolein, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 63–68, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00466-8.

Chemical profile Background Uses Environmental fate and behavior Exposure routes and pathways Physicochemical properties Partition behavior in water, sediment, and soil Environmental persistence (degradation/speciation) Bioaccumulation and biomagnification Exposure and exposure monitoring Routes and pathways Human exposure Environmental exposure Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Animals Dermal effects Respiratory effects Oral effects Human Dermal and ocular effects Respiratory effects Oral effects Chronic toxicity Animals Human Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Aquatic organism toxicity Terrestrial organisms toxicity Other hazards Exposure standards and guidelines Miscellaneous Further reading

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Abstract Acrolein is a clear, colorless, or yellow liquid with a strong odor. It dissolves in water very easily and quickly changes to a vapor when heated. It is highly flammable and very volatile and quickly combusted in air. Acrolein is used as an intermediate in the manufacture and synthesis of many organic chemicals, as a biocide, in the cross-linking of protein collagen in leather tanning, as a tissue fixative in histological samples, in the manufacture of colloidal forms of metals, and in the production of perfumes. Acrolein is produced as a by-product of combustion of organic compounds and be heating in food processing. Acrolein is endogenous formed by normal lipid oxidation and metabolism of a-hydroxyamino acids, and by biotransformation of some drugs. This endogenous contribution appears to contribute substantially to the exposome, and should be considered in risk assessment and to understand its toxicity and implication to some important human neurodegenerative diseases, cholesterol homeostasis and insulin resistant in diabetes. Affected Organ Systems are cardiovascular (heart and blood vessels), hematological (blood-forming), ocular (eyes), respiratory (from the nose to the lungs). Acrolein affects

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principally the respiratory tract, producing irritation in the mucous membranes of the sensory nerve endings. It can be a pulmonary irritant and lachrymatory agent. No carcinogenic, mutagenic, or reproductive effects have been found in humans, although evidence of carcinogenicity in experimental animals and mechanistic evidences have been reported.

Keywords Aldehydes; Biocide; Leather tanning; Organic combustion; Pulmonary irritant; Tobacco smoke; Water toxicant

Chemical profile

• • • •

Name: Acrolein Chemical Abstracts Service Registry Number: 107-02-8 Common Synonyms: Acrylaldehyde 2-Propenal, prop-2-enal, acroleina Molecular Formula: C3H4O

•

Chemical Structure:

Background Acrolein was first produced as a commercial product in the 1930s through the vapor-phase condensation of acetaldehyde and formaldehyde. Another method was developed in the 1940s, which involved the vapor-phase oxidation of propylene. In the 1960s, some advances were found in propylene oxidation process by the introduction of bismuth molybdate-based catalysis, and that became the primary method used for the commercial production of acrolein. Some bioproducts formed for this reaction are acrylic acid, carbon oxides, acetaldehyde, acetic acid, formaldehyde, and polyacrolein. In World War I, it was used as a chemical weapon (pulmonary irritant and lachrymatory agent). Commercial acrolein contains 95.5% or more of the compound, the main impurities being water (C variant (∗5 or ∗15 haplotypes) may be associated with impaired hepatic uptake and potentially increase the plasma concentration of OATP1B1 substrates, whereas the SLCO1B1∗1B haplotype may be associated

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with enhanced hepatic uptake and reduced plasma concentrations of some OATP1B1 substrates in Caucasians. Note that genetic differences are found among different racial and ethnic backgrounds. The low-activity haplotypes ∗5 and ∗15 have a combined allele frequency of approximately 15–20% in Europeans, 10–15% in Asians, and 2% in sub-Saharan Africans, whereas, the enhanced uptake haplotype ∗1B has an allele frequency of approximately 26% in Europeans, 39% in South/Central Asians, 63% in East Asians, and as high as 77% in sub-Saharan Africans. As transporters can influence the deposition of drugs and genetic variation in transporters do exist among populations, these give rise to intervariation in response of the same drug.

In vitro assessment of transporter activity Tissue volumes and tissue blood flows are essential components of a PBPK model. Correlations between tissue volumes and tissue blood flows should be considered when modeling interindividual variability in drug distribution using PBPK models. There are drug-related characteristics (such as ability to cross membranes, bind to plasma proteins, partition into red blood cells, tissues, or fat, and its specific affinity to influx or efflux transporter proteins) which can influence the dynamics of distribution to various tissues and concentration-time profile of drugs. Many of these can be measured in vitro and used for IVIVE purposes. Current IVIVE approaches for assessing the distribution of the drugs to various tissues involve estimation of tissue-to-plasma partition coefficient (Kp:t) based on affinity to lipids and binding to common proteins present in the tissue interstitial space combined with in vitro measurements of blood and plasma protein binding. Separation of the passive permeability and transporter-mediated flux is an essential element of in vitro cell-based studies as these can be used to predict the behavior of the drug in vivo (using IVIVE). The active transport kinetics is often described by Michaelis-Menten kinetics (Km, Vmax, or CLint, T). The effects of unbound fraction at the binding site cannot be directly measured in most cases; however, delineation via application of models is becoming more popular. These systems together with appropriate modeling are now routinely used to elucidate passive and active transporter processes acting to influence drug permeability. Caco-2 cells grown to confluent monolayers in bicameral filter systems endogenously express the majority of the relevant transporters also expressed in the human intestine in vivo. Alternatively, cell line includes Madine Darby Canine Kidney (MDCK II) and Lilly Laboratories Cells—Porcine Kidney Nr. 1 (LLC-PK1), selected due to their low endogenous transporter expression of mdr1, bcrp, and mrp2. Inhibition of active processes can be an issue knowing the lack of specificity of transporter inhibitors for certain isoforms. Recently, research has focused on using mathematical models to describe drug concentration-time courses at transporter binding sites in order to accurately determine the intrinsic kinetics of active processes. Using a global kinetic approach to simulate the time course of drug concentrations in multiple compartments of the experimental system has been reported in the literature. The models in the last few years have become increasingly more mechanistic (and hence more complex) involving up to five compartments within in vitro system to describe the characteristics assumed to be important to the mechanics of drug transport within a two-chamber filter system like the Snap- or Transwell systems. A modeling approach for assessing in vitro data can help with recognizing processes that have not been identified to facilitate further work on these as yet unidentified mechanisms acting on the drug. They can help to identify the outliers and supply further guidance toward issues that require extra attention. Examples are provided below to show practical applications.

‘PBPK’ modeling of hepatic transport PKs deal with quantitative assessment of the fate of drugs in the body. Mathematical models are necessary to describe and predict concentration-time profiles from data obtained by measuring the drug level in biological fluids such as blood, plasma, and urine. The models can range from simple compartmental analysis to sophisticated PBPK incorporating population-based interindividual variability in physiological parameters. In PBPK modeling describing liver uptake, the diffusion of the compound into the liver will be limited by the blood flow into the organ as the compound is assumed to not exhibit any permeability limitations into the tissue, and this is referred to as a perfusion-limited organ model. If a compound displays a permeability limitation in the organ, blood flow will no longer be a rate-limiting step and the concentration in the organ will rather be dependent on its ability to permeate the tissue, and as described earlier this can be a net effect of diffusion into the tissue, active transporter uptake or efflux. In order to describe and model the latter scenario, a permeability-limited liver model is required. The permeability-limited liver model divides the liver into three compartments: extracellular water (EW), intracellular water (IW) and capillary blood, where the distribution between the compartments is dynamic as described in Fig. 3. Sinusoidal transporters will alter the rate at which the drug is transferred between EW and IW, ktEW-in and ktIW-out. Transporter kinetics are generally described as an intrinsic clearance equal to the maximal rate (Vmax) divided by the Michaelis constant (Km) under linear conditions. Km is the substrate concentration at which the rate reaches half of its maximum value (Vmax). Transport-mediated uptake or efflux is generally described as an intrinsic clearance (CLint) equal to the maximal rate (Vmax) divided by the Michaelis constant (Km) under linear conditions using Michaelis-Menten type equations (Vmax ¼ maximum flux; Km ¼ substrate concentration giving half Vmax). A reduction in Vmax corresponds to a reduced capacity of active transport.

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Fig. 3 The permeability limited liver model. See text for detailed description.

The CLint is a concentration- and time-dependent variable (Eq. 4), where CLint is in mL min−1, Vmax is in per million hepatocytes (pmol) min−1, and Km in mM, respectively (Eq. 4): V CL max K m + Cuint

(4)

where Cu is the unbound concentration at the transporter site. Hence for uptake transporters, it is the unbound concentration in the EW and for efflux transporters, it is the unbound concentration in the IW. Usually measuring the unbound concentrations is a challenging task, hence in silico methods are developed to estimate these values. In order to predict tissue distribution, recently mechanistic equations have been developed that incorporate compound lipophilicity, binding of the compound to plasma and tissue macromolecules, and levels of phospholipids and neutral lipids in plasma and tissues. These models have been further developed accounting for protein binding in the EW and incorporating acidic phospholipids binding for strong bases (pKa >7.0). These equations are developed assuming steady-state conditions and instantaneous equilibrium. However, attempts are made to adapt these equations to nonequilibrium conditions and develop models describing transporters’ functionality in different organs such as the liver, the brain, and the heart. Using a similar approach and without assuming the equilibrium condition, permeability-limited models and equations are developed and implemented within the Simcyp Simulator (since V10). These models are used to estimate the drug unbound fractions in intercellular and EW and predict transporters functionality in different organs including liver, brain, and kidney assuming:

• • • • • •

The tissue is divided into three compartments, namely vascular space and EW and IW spaces. The vascular and extracellular compartments are in instantaneous equilibrium though the total concentration in these can be different. Only unionized and unbound species can permeate (either passively or actively) through the plasma membrane. The movement of the unbound unionized species from the capillary bed (vascular space) to the EW is not a rate-limiting process and the perfusion-limited transition only happens between the EW and IW spaces. Passive permeability at the canalicular side of the liver plays a negligible role in biliary secretion. Since there are no reports suggesting the presence of drug uptake transporters at the canalicular membrane these are not considered.

The developed models have been used successfully to predict the PK profiles and transporter-mediated interaction of repaglinide, cyclosporine A, clarithromycin, and trimethoprim. These models are also used to simulate the cases described in the following section. Modeling the transporter activity as a consequence of genetic polymorphism can therefore be done through altering the maximum flux of the transporter and simulating the impact on organ and tissue concentrations as a result of these.

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Simulating the EFFECT of OATP1B1 polymorphism on the exposure of statins In the following case, how the impact of genetic polymorphism of SLCO1B1 and OATP1B1 activity and subsequently the TKs of statins can be investigated through a PBPK simulation approach and further give important theoretical insights into the PK parameters governing the systemic and organ exposure of hepatic transporter substrates through sensitivity analysis of OATP1B1 affinity and transport rate is described. Utilizing a full PBPK distribution model, based on Rodgers and Rowland, coupled with a permeability-limited liver model allowing the implementation of transporter liver uptake, incorporated into the population-based PBPK simulator Simcyp v11 (Simcyp Ltd., Sheffield), concentration time profiles in liver plasma and muscle tissue of atorvastatin, pravastatin, and simvastatin immediate release were simulated at a low, medium, and high therapeutic doses. A range of affinities and rate of transport values of OATP1B1 substrates were collated from the literature. Figs. 4 and 5 display the distribution of Km and Vmax, respectively, for reported substrates. The ranges of Vmax and Km values served as a basis for designing the sensitivity analysis simulation study, providing naturally occurring ranges. The genotypic polymorphism was implemented through altering Vmax assigning it a value of one-third of the original value or three times larger to emulate the impact of genetic polymorphism. Keeping Km constant and reducing the transporter activity (Vmax) led to a reduction in the amount of substrate drugs being transported into the hepatocytes. Increased transporter activity led to an increase in drug concentration in the liver, whereas a less active transporter resulted in a higher proportion of drug circulating in plasma. Thus, when a higher amount of drug was taken up into the liver, less would remain in circulation; subsequently, less would be distributed to the muscle tissue. At a high Km, the substrate affinity to the transporter is high. The available OATP1B1 transporters will be highly occupied by the substrate drug potentially causing saturation. In this scenario, the rate of transport will only have a minor impact on the overall transport.

Atorvastatin acid Atorvastatin acid is mainly metabolized in the liver by CYP3A4 displays a relatively low passive diffusion clearance (PSdif) into the hepatocytes of 0.017 mL min−1 per million cells (CLpd). Due to the low PSdif, the plasma concentration was highly governed by the OATP1B1 transporter activity.

Fig. 4 Scatter plot of apparent Km (mM) values of endogenous and exogenous OATP1B1 substrates where symbol and color coding differentiate substrates.

Fig. 5 Scatter plot of Vmax (pmol min−1 mg protein−1) values of endogenous and exogenous OATP1B1 substrates where symbol and color coding differentiate substrates.

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Fig. 6 Simulated atorvastatin acid immediate release 40 mg concentration (ng mL−1) time profiles in liver (unbound intracellular concentration), plasma, and muscle tissue at a Km of 0.4 mM and a Vmax of 400 pmol min−1 million cells−1.

Drug concentration in muscle was highly affected by altering the transporter activity. At a Km of 0.4 mM and a Vmax of 400 pmol min−1 per million hepatocytes cells−1, there was an eightfold difference in the muscle concentration when altering the transporter activity related to the OATP1B1 polymorphism (Fig. 6). The difference in liver concentration of drug caused by altered transporter activity was less significant when compared with muscle tissue concentration. As atorvastatin acid is metabolized by CYP3A4, an increase in active hepatic drug uptake will be counteracted in the liver by an increase in metabolic clearance, maintaining similar exposure in the liver (Fig. 6).

Pravastatin Pravastatin displays a PSdif of 0.397 mL min−1 per million cells. The simulated exposure of pravastatin in muscle and liver was higher as compared to plasma. Simulations utilizing parameters reported in literature showed that genotype variation had a reduced impact on the exposure in plasma, liver, and muscle as compared to atorvastatin acid. These findings were similar to the results presented by Kusuhara and Sugiyama, simulating an increase in plasma concentration with a reduced transporter activity whereas drug concentration in plasma was increased and the changes in liver were less significant (Fig. 7).

Simvastatin Simvastatin displays a high passive diffusion rate of approximately 74 mL min−1 per million hepatocytes cells and the distribution of simvastatin to the liver can therefore be considered perfusion limited. For this reason, the exposure of simvastatin was not affected by altered OATP1B1 transporter activity. Although being highly distributed to the muscle, muscular toxicity is more likely to occur (Fig. 8).

Fig. 7 Simulated pravastatin immediate release 20 mg concentration (ng mL−1) time profiles in liver (unbound intracellular concentration), plasma, and muscle tissue at a of Km 0.4 mM and a Vmax of 400 pmol min−1 million cells−1.

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Fig. 8 Simulated simvastatin immediate release 40 mg concentration (ng mL−1) time profiles in liver (unbound intracellular concentration), plasma, and muscle tissue at a Km of 0.4 mM and a Vmax of 400 pmol min−1 million cells−1.

Conclusion Using the transporter OATP1B1 and statins as examples, it has been shown how genotypic transporter polymorphism can alter the drug concentration in plasma, liver and muscle to varying extents depending on the PK properties of the substrate. Therefore, there is no simple algorithm for estimating the effect of transporter polymorphism on toxicokinetics, thus forming/providing a compelling argument for the use of PBPK modeling to assess toxicity as a result of transporter polymorphism and simulation in the toxicokinetics of polymorphic transporter activity. In conclusion, it was demonstrated that membrane transporter polymorphism can contribute to variability in toxicological effects. In vitro assays and IVIVE coupled with PBPK modeling and simulation can provide high-throughput, cost-efficient methods for predicting the toxicokinetics of xenobiotics in man and have the potential of replacing many animal toxicity studies. In silico methods may further be in a better position in predicting active transport effects on the disposition of xenobiotics and their toxicity as compared to current animal models due to observed interspecies variability in abundance and activity of transporters, where accurately predicting the local tissue concentrations may be key to understanding transporter driven toxicity.

References Audouze K, Sarigiannis D, Alonso-Magdalena P, Brochot C, Casas M, Vrijheid M, Babin PJ, Karakitsios S, Coumoul X, and Barouki R (2020) Integrative strategy of testing systems for identification of endocrine disruptors inducing metabolic disorders—An introduction to the OBERON project. International Journal of Molecular Sciences 21(8): 2988. Chu X, Bleasby K, and Evers R (2013) Species differences in drug transporters and implications for translating preclinical findings to humans. Expert Opinion on Drug Metabolism & Toxicology 9(3): 237–252. Elsby R, Hilgendorf C, and Fenner K (2012) Understanding the critical disposition pathways of statins to assess drug–drug interaction risk during drug development: It’s not just about OATP1B1. Clinical Pharmacology & Therapeutics 92(5): 584–598. Harper CR and Jacobson TA (2007) The broad spectrum of statin myopathy: From myalgia to rhabdomyolysis. Current Opinion in Lipidology 18(4): 401–408. Neuvonen PJ, Niemi M, and Backman JT (2006) Drug interactions with lipid-lowering drugs: Mechanisms and clinical relevance. Clinical Pharmacology & Therapeutics 80(6): 565–581. Papadaki KC, Karakitsios SP, and Sarigiannis DA (2017) Modeling of adipose/blood partition coefficient for environmental chemicals. Food and Chemical Toxicology 110: 274–285. Sarigiannis D and Karakitsios S (2019) Advancing chemical risk assessment through human physiology-based biochemical process modeling. Fluids 4(1): 4. Sarigiannis DA, Tratnik JS, Mazej D, Kosjek T, Heath E, Horvat M, Anesti O, and Karakitsios SP (2019) Risk characterization of bisphenol-A in the Slovenian population starting from human biomonitoring data. Environmental Research 170: 293–300. Yu AM, Ingelman-Sundberg M, Cherrington NJ, Aleksunes LM, Zanger UM, Xie W, Jeong H et al. (2017) Regulation of Drug Metabolism and Toxicity by Multiple Factors of Genetics, Epigenetics, lncRNAs, Gut Microbiota, and Diseases: A Meeting Report of the 21st International Symposium on Microsomes and Drug Oxidations (MDO), Acta Pharmaceutica Sinica B 7(2): 241–248.

Further reading Andersen ME, Clewell HJ, Carmichael PL, and Boekelheide K (2011) Can case study approaches speed implementation of the NRC report: ‘Toxicity testing in the 21st century: A vision and a strategy?’. ALTEX 28: 175–182. Dickinson GL and Rostami-Hodjegan A (2008) Building virtual human populations: Assessing the propagation of genetic variability in drug metabolism to pharmacokinetics and pharmacodynamics. In: Bertau M, Mosekilde E, and Westerhoff HV (eds.) Biosimulation in Drug Development, First edn., pp. 425–446. Weinheim: Wiley. Giacomini KM and Sugiyama Y (2005) Membrane transporters and drug response. In: Brunton LL, Lazo JS, and Parker KL (eds.) Goodman & Gillman’s: The Pharmacological Basis of Therapeutics, 11th edn., pp. 41–70. Columbus, OH: McGraw-Hill. Gui C and Hagenbuch B (2009) Role of transmembrane domain 10 for the function of organic anion transporting polypeptide 1B1. Protein Science 18: 2298–2306. Jamei M, Dickinson GL, and Rostami-Hodjegan A (2009) A framework for assessing inter-individual variability in pharmacokinetics using virtual human populations and integrating general knowledge of physical chemistry, biology, anatomy, physiology and genetics: A tale of ‘bottom-up’ vs ‘top-down’ recognition of covariates. Drug Metabolism and Pharmacokinetics 24: 53–75.

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Kusuhara H and Sugiyama Y (2009) In vitro-in vivo extrapolation of transporter-mediated clearance in the liver and kidney. Drug Metabolism and Pharmacokinetics 24: 37–52. Niemi M (2007) Role of OATP transporters in the disposition of drugs. Pharmacogenomics 8: 787–802. Niemi M (2009) Transporter pharmacogenetics and statin toxicity. Clinical Pharmacology and Therapeutics 87: 130–133. Pasanen MK, Neuvonen PJ, and Niemi M (2008) Global analysis of genetic variation in SLCO1B1. Pharmacogenomics 9: 19–33. Poulin P and Theil FP (2002) Prediction of pharmacokinetics prior to in vivo studies. 1. Mechanism-based prediction of volume of distribution. Journal of Pharmaceutical Sciences 91: 129–156. Poulin P and Theil FP (2002) Prediction of pharmacokinetics prior to in vivo studies. II. Generic physiologically based pharmacokinetic models of drug disposition. Journal of Pharmaceutical Sciences 91: 1358–1370. Rodgers T and Rowland M (2007) Mechanistic approaches to volume of distribution predictions: Understanding the processes. Pharmaceutical Research 24: 918–933. Rostami-Hodjegan A (2012) Physiologically based pharmacokinetics joined with in vitro-in vivo extrapolation of ADME: A marriage under the arch of systems pharmacology. Clinical Pharmacology and Therapeutics 92(1): 50–61. Shiran MR, Proctor NJ, Howgate EM, et al. (2006) Prediction of metabolic drug clearance in humans: In vitro-in vivo extrapolation vs. allometric scaling. Xenobiotica 36: 567–580. van de Waterbeemd H and Gifford E (2003) ADMET in silico modelling: Towards prediction paradise? Nature Reviews. Drug Discovery 2: 192–204. Watanabe T, Kusuhara H, Maeda K, Shitara Y, and Sugiyama Y (2009) Physiologically based pharmacokinetic modeling to predict transporter-mediated clearance and distribution of pravastatin in humans. The Journal of Pharmacology and Experimental Therapeutics 328: 652–662.

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Adverse outcome pathways: Development and use in toxicology Donna S Macmillan and Catherine Willett, Humane Society International, Washington, DC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of C. Willett, Adverse Outcome Pathways: Development and Use in Toxicology, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 95–99, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01244-6.

Introduction Adverse outcome pathways (AOPs) The adverse outcome pathway framework Constituents of an AOP Development of AOPs The OECD AOP development program The AOP-KB The AOP-Wiki The AOP Portal Third party tools Principles of AOP development The AOP development process Utility of AOPs How are AOPs used? Skin sensitization Steroid (estrogen receptor)-mediated reproductive toxicity COVID-19 (CIAO-COVID) OECD IATA case studies project Other applications of AOPs Outlook References

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Abstract Adverse Outcome Pathways (AOPs) are simplified biological pathways that describe the essential steps initiated by a molecular perturbation (e.g. exposure to a chemical, radiation, or a virus), through a series of consequential key events, to an eventual toxic response (adverse outcome). Many AOPs have been developed and are stored in the AOP-Wiki, a central repository based on crowd-sourcing; several case examples are presented. AOPs have been used to develop new approach methodologies (NAMs), defined approaches (DAs), integrated approaches to testing and assessment (IATA), and to further elucidate the biological processes of disease.

Keywords Adverse outcome pathway (AOP); Integrated approach to testing and assessment (IATA); Pathway-based toxicology; Systems biology; Systems toxicology; Toxicity pathway

Key points

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AOPs are a framework for organizing, relating, and assessing biological information The OECD is spearheading the development of AOPs globally, but the framework is open to all The primary applications have thus far been in toxicity and disease assessment e.g. skin sensitization, COVID-19, reproductive toxicity Widespread adoption of the AOP framework will improve chemical safety assessment and medical research

Abbreviations ‘Omics AO

Combined reference to genomics, transcriptomics, proteomics, metabolomics Adverse outcome

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AOP CIAO DA E2 EAGMST EMM ER ET&C IATA IE IPCS JRC KE KER MIE MoA NAM OECD REACH SETAC VTG

Adverse outcome pathway Modeling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway framework Defined approach Estrogen Extended Advisory Group on Molecular Screening and Toxicogenomics Environmental and Molecular Mutagenesis Estrogen receptor Environmental Toxicology and Chemistry Integrated approaches to testing and assessment Intermediate event International Program on Chemical Safety European Commission Joint Research Centre Key event Key event relationship Molecular initiating event Mode-of-action New approach methodologies Organisation for Economic Cooperation and Development Registration, Evaluation, Authorisation and Restriction of Chemicals Society of Environmental Toxicology and Chemistry Vitellogenin

Introduction Adverse Outcome Pathways (AOPs) were devised as a way to solve a longstanding problem in toxicology—the need for efficient generation of species-relevant biological information for the potential toxicological effects of a large number of chemicals. After several decades of experience in characterizing chemical toxicity through animal testing, the large number of relatively uncharacterized chemicals already in the environment, the need to generate extensive information for regulatory programs such as the European Union’s Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) program (European Union, 2006), the high cost and slow pace of animal experimentation and the need to improve the certainty of safety decisions made it necessary to rethink the traditional approach to hazard and risk assessment. At the same time, advances in biological understanding as well as experimental technologies (e.g. ‘omics tools, stem cell culturing, reconstructed tissues), allowed the contemplation of dramatically different approaches to understanding disease and toxicology than traditionally animal tests. One such approach couples existing knowledge of normal biology with new chemical and biological information about the consequences of disturbing that biology, leading to a structured, transparent, and hypothesis-based approach to predicting adverse outcomes (AOs) resulting from those perturbations. This general approach has been variously termed Mode-of-Action (MoA), Toxicity Pathway, and AOP approaches. The idea of incorporating mechanistic biochemical information into toxicological assessment is not new. It began with dose-response modeling efforts and the development of MoA frameworks, such as those developed to determine the human relevance of the MoA of pesticides and industrial chemicals leading to carcinogenicity (Sonich-Mullin et al., 2001) and later, non-carcinogenic toxicity (Boobis et al., 2008). Common MoA pathways used in drug development were also created and applied to human disease (Landesmann et al., 2012). The notion of toxicity pathways as articulated by the National Research Council in 2007 in its report, Toxicity Testing for the 21st Century: a Vision and a Strategy, took this concept a bit further by envisioning a system-wide network of pathways leading to a predictive, hypothesis-driven assessment paradigm for toxicity in general (National Research Council, 2007). The goals of this new approach were to improve efficiency and decrease uncertainty in risk and hazard evaluations and in 2010, this concept was further formalized for toxicological assessment for both human health and ecological endpoints as the AOP framework (Ankley et al., 2010).

Adverse outcome pathways (AOPs) The adverse outcome pathway framework The AOP framework was proposed as a way to organize information on the biological and toxicological effects of chemicals, with a view to using this knowledge for further research and risk assessment (Ankley et al., 2010). The framework allows for the integration of all types of information at different levels of biological organization, from molecular to population level, to provide a rational, biologically based argument (or series of hypotheses) to predict the outcome of an initiating event.

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Fig. 1 An illustrative representation of an AOP with several examples at different levels of biological organization. Schematic adapted from Revised Guidance Document on Developing and Assessing Adverse Outcome Pathways (OECD, 2017).

Constituents of an AOP An AOP consists of two elements, key events (KE) and the relationships between them, key event relationships (KERs). KE are essential and measurable elements in a biological pathway: blockage of a KE results in blockage of the pathway. There are two special types of KE, the molecular initiating event, (MIE), which is the initial interaction with a chemical or other perturbation agent, and the AO, the ultimate result of the initial perturbation that is propagated through the intermediate KEs. KERs are the description of how a change in the upstream KE is related to a change in the downstream KE. An individual AOP consists of one MIE, one AO, and any number of intermediate KE. The basis of the AOP concept is that toxicity begins with an initial interaction of a chemical with a biomolecule (e.g. a protein, receptor), the MIE. The MIE then triggers a sequential progression of intermediate KE that eventually leads to an AO. The AO is at the organism, target tissue or organ level for most human health endpoints and at the individual or population level for environmental endpoints. For example, Fig. 1 illustrates an AOP that begins with exposure to a chemical. The MIE in this AOP could be interaction of this chemical with a receptor or DNA, which would then trigger a cellular response (e.g. activation of a gene) and an organ response (e.g. altered function). This sequence of KEs would then give rise to an AO such as impaired reproduction or lethality at organism level, or altered sex ratio and/or even extinction at population level. In reality, biological processes are actually an interrelated network of multiple processes, and an MIE can be associated with a number of AOs (similarly an AO can result from a number of different MIEs). As a pragmatic choice to streamline the development and use of AOPs, they were defined as a more-or-less linear pathway from one MIE to one AO (OECD, 2017). KEs and KERs are designed to be useful in multiple contexts, and it is through the use of the same KE and/or KER in more than one pathway that creates networks within the AOP framework that more closely resemble biology. It is these networks that will be most helpful in predicting possible adverse outcomes associated with MIE that are perturbed by chemical exposure.

Development of AOPs The OECD AOP development program AOP development began with a group of like-minded individuals, mostly at the US Environmental Protection Agency (US EPA), who created the initial AOP-Wiki, a structured database for storing and organizing biological information in the form of AOPs, and created the first few AOPs (Edwards and Preston, 2008; Ankley et al., 2010; Villeneuve et al., 2013). Based on the promise of the AOP framework for improving chemical safety assessment, in 2012, the Organisation for Economic Cooperation and Development (OECD) introduced the AOP Development Program. This program is designed to manage and facilitate AOP development and is overseen by the Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST). There are several subgroups of the EAGMST responsible for developing and maintaining the software, providing and updating guidance, reviewing AOPs in development, and developing materials to educate stakeholders about AOPs and AOP development (OECD, 2021a).

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The AOP-KB The EAGMST oversees development and maintenance of the AOP knowledge base (AOP-KB), a central repository of knowledge and collaboratively developed AOPs, and from which it is possible to access related third-party tools. The AOP-KB contains three elements, the AOP-Wiki, the AOP Portal and Effectopedia (although Effectopedia is not currently supported, AOP-KB, 2022).

The AOP-Wiki The AOP-Wiki is a software platform designed to collect (via crowd-sourcing) and organize available biological knowledge as individual AOPs using a Wiki interface. Principles and instructions for developing AOPs in the AOP-Wiki are provided in the Developer’s Handbook, which is updated regularly and is available from the AOP-Wiki. The AOP-Wiki is a joint effort between the European Commission Joint Research Centre (JRC) and the US EPA and is currently supported by the EAGMST and the Society for the Advancement of Adverse Outcome Pathways (SAAOP). The AOP-Wiki is publicly accessible; anyone can read any of the content. To author an AOP, it is necessary to register so that the changes made to the AOP-Wiki can be tracked and the author can be contacted if the administrators have questions. Registration can be made via a link on the Wiki.

The AOP Portal The AOP Portal is the main entry point for the AOP-KB and is a search engine for all AOPs in the knowledge base. It provides links to AOPs in the AOP-Wiki.

Third party tools o AOPXplorer ▪ A computational tool developed by the US Army Engineer Research and Development Center that enables automated graphical representation of AOPs and their associated networks (US Army Engineer Research and Development Center, 2017). o AOP-DB ▪ An online application developed by the US EPA that supports the discovery and development of putative and potential AOPs. It aggregates relationships between AOP-gene targets, chemical, disease, tissue, ontologies and gene interactions to characterize the impacts of chemicals to human health and the environment (US EPA, 2021).

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Intermediate Effects Database o A database under development by the JRC and will contain chemical related data. The database is derived from non-apical endpoint methods with the aim of understanding how individual compounds (stressors) trigger MIEs and/or KEs.

Principles of AOP development For the scientific community to fully benefit from a knowledge base containing AOPs developed by many authors, it is important to be consistent in development of AOPs. To this end, a set of five core principles have been proposed by experienced AOP developers: (1) (2) (3) (4)

AOPs are not chemical specific AOPs are modular and composed of reusable components—notably KEs and KERs an individual AOP, composed of a single sequence of KEs and KERs, is a pragmatic unit of AOP development and evaluation networks composed of multiple AOPs that share common KEs and KERs are likely to be the functional unit of prediction for most real-world scenarios (5) AOPs are living documents that will evolve over time as new knowledge is generated These principles, and best practices, should be considered when developing AOPs to address related challenges and tackle uncertainties (Villeneuve et al., 2014a,b).

The AOP development process Development of an AOP can begin from any location along the pathway—where you start will depend on the developer’s perspective or expertise. For example, a chemist will likely begin with the MIE and focus on the early part of the pathway, with the probable goal of improving chemical categorization; a bioinformatician would likely begin in the middle, with a series of gene or protein expression associations or ’fingerprints’, and link outward to either the associated MIE or AO; a pathologist would begin with an AO and work ’upstream’ to link to the causal KEs. A developer would then generally begin with a comprehensive literature search to begin collecting as much information as is available about the biology of interest. A full description of a KE should include relevant species, cellular/tissue location and developmental stage or sex specificity. The OECD Guidance Document on Developing and Assessing AOPs (OECD, 2017), the Users’ Handbook supplement to the Guidance Document on Developing and Assessing AOPs (OECD, 2018b), and the evergreen Developer’s Handbook available from the AOP-Wiki (AOP-Wiki, 2022) provide detailed information on how to review and organize the collected information as an AOP in the AOP-Wiki. This guidance defines terms and describes each of the entry fields for the MIE, KE and AO pages. It also describes a

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weight-of-evidence approach to evaluate the evidence for each KER and the overall confidence in the AOP. The weight of evidence approach applies modified Bradford-Hill criteria; e.g. evaluating biological plausibility, empirical support (dose-response, temporality, and incidence) for KERs, and essentiality of KEs (Becker et al., 2015). If at any point, the author(s) of an AOP requires assistance, the SAAOP is available to provide advice or answer questions and can be contacted via the AOP-Wiki. The AOP-Wiki Forum is another source of information and advice, also accessible from the AOP-Wiki (AOP-Wiki, 2022). If there is interest in regulatory application of the AOP, or the author(s) would like a more thorough review, there are now two options: (1) the AOP can be submitted to OECD for inclusion in the OECD AOP development workplan. Coaching in AOP development may be offered to the authors by EAGMST and a compliance check is undertaken, to ensure consistency with the AOP principles and various guidance documents. Next, a scientific review of the AOP is carried out by a group of subject matter experts (OECD, 2021a). Once the AOP is considered the current state of the science by expert review, it is submitted for endorsement by OECD and, if endorsed, published on their website as part of the OECD Series on Adverse Outcome Pathways (OECD, 2022b). (2) the AOP can be submitted to a scientific journal. Recently, the OECD have collaborated with two scientific journals, Environmental Toxicology and Chemistry (ET&C), a journal published by the Society of Environmental Toxicology and Chemistry (SETAC), and Environmental and Molecular Mutagenesis (EMM), the journal of the Environmental Mutagenesis and Genomics Society, to contribute to the scientific review of relevant AOPs. This collaboration is intended to increase the rate of AOP scientific reviews; increase submissions for scientific articles; and enable double recognition through AOP publications in the scientific literature and at the OECD. Several open access AOP reports are now available; two in ET&C (Schmid et al., 2021; Song and Villeneuve, 2021) and one in EMM (Cho et al., 2022).

Utility of AOPs How are AOPs used? The AOPs currently under development differ in detail and complexity, nevertheless, they all have the potential to improve the hazard and risk assessment process. The level of certainty and completion necessary depends on the intended use of the AOP (Fig. 2). A. Chemical grouping or categories/structure-activity relationships AOPs are often used to inform chemical grouping or categories/structure-activity relationships (SAR). For this application, characterization of the MIE is particularly important as well as general support for linkage to biological activity. Well-characterized MIEs are particularly useful for read-across and grouping approaches (Escher et al., 2022; OECD, 2020). B. Hazard identification/prioritization For hazard identification and/or prioritization, assays may be developed that measure the MIE or the KEs in the AOP. Here, understanding which pathway the MIE is likely to take is important, and there should be some evidence of linkage to the AO, along with substantiation of one or more intermediate KEs. This approach is exemplified by the EPA, where their researchers have developed several in vitro AOP-based assays to prioritize potential endocrine disrupting chemicals (Judson et al., 2017, 2018, 2020). C. Integrated strategy design For integrated strategy design, better information on some intermediate KE and stronger linkage to an AO is helpful. Some characterization of related AOPs is also valuable. A few well characterized KE in a pathway will support identification of KEs for which tests can be developed and used in an integrated strategy design. The tests would address a number of critical KEs with strong evidence of the MIE-AO link, thereby maximizing the useful information gained from minimal in vitro testing. One example of this is the OECD’s recently published Guideline No. 497 Defined Approaches for Skin Sensitisation (OECD, 2021b). D. Initial risk assessment Once there is a shift from hazard to risk, the function of the KER becomes important. As quantitative information is added to KERs, early KEs in an AOP may be used directly for risk assessment, without the need to assess the later steps in the pathway (Villeneuve et al., 2013). The quantitative KER may be able to predict a quantitative point-of-departure for the adverse outcome. E. Predictive system for toxicology Finally, a predictive system is supported by quantitative descriptions of the major KER in the relevant network(s) have been elucidated and that is still somewhere in the future. At this stage, chemical assessment will be streamlined and toxicology transformed from a purely empirical to a predictive science.

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Fig. 2 Several ways AOPs can be used to inform toxicological hazard and risk assessment are illustrated here and described in the text.

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Skin sensitization One application of AOPs is exemplified by the application of AOP information to substantiate the regulatory use of in vitro methods for skin sensitization. Skin sensitization is a relatively simple biological process yet involves multiple cell types and KE. The AOP for skin sensitization initiated by covalent binding to proteins was published by the OECD in 2014 (OECD, 2014) (Fig. 3). The AOP consists of four key events which occur after any potential skin metabolism (which may activate/deactivate/have no effect on the chemical) and penetration of the electrophilic chemical (hapten) into the skin. The MIE is the nucleophilic attack of a chemical electrophile by skin proteins (e.g. cysteine and lysine residues) to form a covalent hapten-protein complex. This covalent hapten-protein complex stimulates a cellular response—the induction and activation of cytokines by dendritic cells and keratinocytes in the skin, the second and third key events in the AOP, respectively. KE4 consists of activation and proliferation of T-cells in the lymph nodes which then leads to the adverse outcome (AO) of skin sensitization in the organism (following a second exposure to the chemical). Several new approach methodologies (NAMs) replicate KE1-KE3 and have been published as OECD Test Guidelines (OECD, 2018a, 2021c, 2022c). Results from these assays are typically not used as stand-alone predictions for skin sensitization but can be combined with other information sources (e.g. physicochemical properties, in chemico/in vitro assays, in silico models) in an integrated approach to testing and assessment (IATA). For regulatory application, several different defined approaches (DAs) for skin sensitization have been developed (OECD, 2021b). A DA consists of a fixed data interpretation procedure that stipulates which, and how, information sources are used in the DA to ensure reproducibility, reduce subjectivity, and are designed to overcome individual limitations of the information sources used. These DAs can be used alone or within a larger IATA to replace the murine local lymph node assay (LLNA) (Fig. 4).

Steroid (estrogen receptor)-mediated reproductive toxicity An example of the application of AOPs to predictive modeling is an AOP developed for aromatase inhibition leading to reproductive dysfunction – AOP 25 (Villeneuve et al., 2013; AOP-Wiki., 2021a). In this AOP, chemicals that inhibit aromatase set off a cascade of reactions that results in decreased estrogen synthesis (E2), leading to a decrease in a key protein involved in reproduction, with impaired gametogenesis and subsequent impaired reproduction and population declines. The KERs for several steps in this pathway have been described quantitatively, allowing a series of models to be built that can be used to predict the adverse outcomes at the individual and population levels by measuring the MIE (Conolly et al., 2017). Another AOP, estrogen receptor antagonism leading to reproductive dysfunction – AOP 30 (AOP-Wiki., 2021b), shares several KEs as well as the AO of AOP 25. Chemicals that bind the estrogen receptor (ER) can either mimic or block estrogenic activity (act as agonists, or antagonists, respectively) by changing expression levels of ER responsive genes (Fig. 5). In AOP 30 the MIE is inhibition of the ER, which directly inhibits expression of vitellogenin (VTG), causing the same impairment of reproductive function as AOP 25. Chemicals with potential endocrine activity have been a subject of growing concern, and several regulatory programs around the world have proposed including endocrine activity in regulatory requirements. These related estrogen AOPs, especially the quantitative KERs and predictive models built for AOP 25, demonstrate the potential strength of AOPs for designing efficient assessment schemes to address new safety concerns.

COVID-19 (CIAO-COVID)

CIAO, Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway (AOP) framework (CIAO, 2022), is an example of the application of AOPs to furthering biomedical understanding and interventions. The CIAO project, coordinated by the JRC, was created to develop a network of AOPs describing the biology underlying the effects related to severe acute respiratory syndrome (SARS) viruses, particularly SARS-CoV-2 (COVID-19) (Nymark et al., 2021). The project brings together over 75 scientists

Fig. 3 Adapted from the adverse outcome pathway for skin sensitization initiated by covalent binding to proteins (OECD, 2014).

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Fig. 4 The 2o3 and ITS defined approaches for skin sensitization published in OECD Guideline No. 497. Modified from OECD (2021) Guideline No. 497: Defined approaches on skin sensitisation. OECD Guidelines for the Testing of Chemicals, Section 4, Paris: OECD Publishing, doi: 10.1787/b92879a4-en.

Fig. 5 A simplified representation of two AOPs associated with steroid (estrogen receptor)-mediated reproductive toxicity. The KER for AOP 25 have been described quantitatively and predictive models exist for this AOP.

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globally, who each contribute their expertise into relevant working groups. Most of the working groups focus on developing AOPs based on adverse outcomes, such as anosmia, stroke, and/or respiratory issues with the overarching aim to assemble the AOPs into a network with shared KEs. Other working groups have been investigating modulating factors that influence the severity of the disease, systematic review approaches (both manual and automated) to collect and extract information from the scientific literature, and investigations beyond purely biological phenomena (Wittwehr et al., 2021; Clerbaux et al., 2022b). Establishing the disease progression of COVID-19 using these key biological events and knowledge gained from the other working groups will increase scientific understanding of COVID-19 and provide insight into potential targets for therapeutic approaches. Several AOPs developed from this interdisciplinary project have been published and more are expected in the near future (Vinken, 2021; Pistollato et al., 2022; Clerbaux et al., 2022a).

OECD IATA case studies project In 2015 the OECD launched the Integrated Approaches to Testing and Assessment (IATA) Case Studies Project (OECD, 2022a). IATA are designed to answer a defined question in a specific regulatory context using an iterative approach, while also accounting for uncertainty. IATA can be flexible, judgment-based approaches (e.g. grouping and read-across) or can be more structured, rule-based approaches (e.g. defined approaches). The AOP concept can be used as a framework to develop IATA that combine results from multiple associated information sources [e.g. (Q)SAR, read-across, in chemico, in vitro, ex vivo, in vivo and/or ‘omic technologies (e.g. toxicogenomics)]. The AOP or AOP network of interest can also be used to systematically characterize the biological and toxicological relevance of novel methods in predicting an AO (OECD, 2016). Since its inception, more than 20 case studies have been submitted and reviewed by the IATA Case Studies Project and after each annual review cycle, a considerations document describing the lessons learnt from the review experience is published alongside the approved case studies (OECD, 2022a).

Other applications of AOPs Investigations into the biological mechanisms underpinning toxicity and disease using the AOP framework continues to evolve at a fast pace and is a rich area of research. A series of AOPs have been developed for developmental and neurotoxicity (Bal-Price et al., 2017; Sachana et al., 2021; Ramšak et al., 2022) leading to the creation of an in vitro test battery which has been used as the basis for an AOP-informed IATA for regulatory decision making (EFSA Panel on Plant Protection Products and their Residues, 2021). AOPs have also been developed to: support genotoxic modes of action (Sasaki et al., 2020); fill regulatory gaps in the identification of non-genotoxic carcinogens (Jacobs et al., 2020), identify MIEs leading to death from breast cancer (Benoit et al., 2022), understand the mechanistic link between liver disease and preterm birth (Waspe and Beronius, 2022), design AOP-targeted assays for the fish early life stage test (Scholz et al., 2018) and understand the current knowledge on congenital microcephaly after exposure to ionizing radiation (Jaylet et al., 2022) – and many more are under development.

Outlook AOP development is gaining momentum and the framework holds great promise for improving not only chemical safety but medical research. The AOP community has grown significantly in recent years, reflecting strong leadership by the OECD, the strength of early successes, and global interest in the promise of such a comprehensive knowledge base. The AOP framework can greatly improve not only the assessment of exposure to potential hazardous elements (e.g. chemicals, radiation, etc.), but allow better understanding of disease and provide more effective interventions. To achieve this promise, larger concerted and coordinated effort is required to develop AOPs that cover additional pathways and outcomes of interest—or ’biological space’—analogous to the CIAO project, for example. Additional resources are needed to carry out the scientific review of AOPs; for example, more journals taking part in the scientific review and publication of AOPs in alignment with OECD processes. As we have shown, AOPs are currently being used to inform chemical grouping or categories and structure-activity relationships, design integrated testing strategies, identify KEs for which non-animal tests can be developed, design predictive models for adversities of regulatory concern, and in general aid in increasing the certainty of interpretation of both existing and new information. As AOP networks continue to be developed for new areas of biology, these will become increasingly useful for designing predictive IATA to address regulatory concerns for chemical safety as well as new approaches for medical intervention. One potential application of the AOP framework that has yet to be explored is aiding in research design. As AOPs are built on a comprehensive survey of the current state of knowledge about an area of biology, they also uncover what is not known and can assist in prioritizing areas of research. The AOP framework provides a structure for a simplified yet comprehensive publicly harmonized knowledge base for biological information. The structure it provides is amenable to crowd-sourcing, to scientific review, and public access to biological knowledge. The AOP-Wiki provides a software platform for carrying out the organization, visualization, interconnectivity, and storage of this information, as well as access to a community of users and the AOB-KB provides an interface with third-party tools. As the AOP community expands the biological coverage of the AOP-Wiki and associated tools, the AOP framework will become increasing useful to a wide variety of applications, from improved safety assessment of chemicals and other potential hazards, improved medical interventions and better focused research.

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Aerosols John T Szilagyi, Bristol-Myers Squibb Co., Summit, NJ, United States © 2024 Elsevier Inc. All rights reserved. This is an update of W.E. Achanzar, R.S. Mangipudy, Aerosols, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 100–101, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00223-2.

Background Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity Acute and short term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Other hazards Exposure standards and guidelines References

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Abstract The category of aerosols encompasses an array of chemical and biological toxicants. The fate of aerosols in the environment are dependent upon the physical and chemical properties of the particular agent and its formulation therein. Exposure to aerosols occurs through dermal contact and inhalation, although ingestion can occur as well. The toxicological effects of aerosols can range from local dermal irritation to respiratory distress. Chronic exposure to certain aerosols can lead to chronic respiratory disease and tumorigenesis. The specific toxicological response will be contingent upon the toxicant in question and the extent of exposure.

Keywords Aerosol; Dermal exposure; Electronic cigarette smoke; Fossil fuels; Respiratory exposure; Wood smoke

Key points

• • • •

Aerosols are airborne suspensions of solids or liquids formed intentionally or unintentionally that can increase distribution and exposure of a toxicant. Aerosolized toxicants are absorbed primarily through inhalation or dermal contact, although ingestion can occur in food, drinks, or swallowed air. The mechanisms and effects of an aerosol are largely dependent upon the specific substance in question as well as the size, shape, and density of the aerosolized particles. There is a well-established clinical correlation between long term exposure to certain aerosols and chronic lung diseases such as pulmonary fibrosis, emphysema, chronic obstructive respiratory disorder, asthma, and lung carcinoma.

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Background Aerosols are airborne, often colloidal, suspensions of solid particulate matter or liquid droplets and include mists, dusts, fogs, fumes, sprays, among others. The category of aerosols incorporates both natural (such as wood smoke) and synthetic (such as electronic cigarette vapor) sources. Aerosols can be formed intentionally, as in the use of common household spray cans, or unintentionally, such as in the burning of fossil fuels. In order to classify an aerosol, the agent in question must be stable in airborne suspension for a sufficient length of time to enable its detection. The specific fate and toxicity of an aerosol depend largely on the specific chemical(s) contained but are also contingent upon the atmospheric conditions (i.e., temperature, air circulation), the shape and size of the particles, and, as with all toxicants, the length and extent of exposure (Islam et al., 2020).

Environmental fate and behavior The environmental fate of an aerosol will be dependent on the chemical in question and its physical properties. Depending on the size, shape, and density of the particulate matters in suspension, aerosols can remain in the atmosphere or succumb to gravity and fall, where the airborne agents can be incorporated into ground soil or water sources, for instance.

Exposure and exposure monitoring Human exposure to aerosols primarily occurs via contact of the skin, eyes, or respiratory tract although other routes, such as ingestion, do occur. Environmental aerosol monitoring is performed through air sampling by passive or active capture systems (Oh et al., 2020). These systems are often designed to capture particles in a specified range of sizes and are placed in a particular indoor or outdoor area for a predetermined amount of time. Careful consideration should be given to the physical properties of the aerosol formulation to ensure accuracy, such as volatility and dispersal rate. Depending on the aerosolized agents under investigation, monitoring in biological fluids, such as sputum or blood, to assess human or animal exposure may also be performed.

Toxicokinetics Aerosolized toxicants are absorbed primarily through inhalation or dermal contact, although ingestion can occur in food, drinks, or swallowed air. Absorption through inhalation exposure is influenced by respiratory tract geometry and an aerosol’s physical and chemical properties (i.e. size distribution, shape, density, partition coefficient) (Islam et al., 2020). Solid particulate matter in the lungs may be phagocytized by immune cells or expectorated through the muco-ciliary escalator to reduce their systemic absorption. Dermal absorption is dependent upon the skin permeability of the agent in question and its specific formulation, as well as the surface area of exposure and thickness of the skin in contact with the toxicant. Once systemic exposure occurs, the distribution, excretion, and metabolism of an aerosol will be contingent largely upon the specific toxicants being studied.

Mechanism of toxicity The mechanism of toxicity for a particular aerosol is contingent upon its specific physical properties and chemical constituents as well as the exposure route. For instance, inhalation of sulfur mustard aerosol will induce a genotoxic response in the lung through DNA alkylation and cross-linking (Cheng et al., 2021; Khan et al., 2017). This results in cell death and initiates a local immune response to resolve the area of injury (Laskin et al., 2019; Weinberger et al., 2011). In some cases, pulmonary toxicity may not result from chemical interactions but rather physical interactions. Aerosolized solid particulates trapped in alveolar space will be phagocytized, which damages the surrounding tissue. If the particles cannot be broken down, this causes frustrated phagocytosis that leads to oxidative stress and chronic injury (Padmore et al., 2017). As the cycle continues and tissue is repeatedly damaged and repaired, this increases the risk for localized neoplasia (Wang et al., 2017). Dermal toxicity to aerosols can include immune responses, which manifest in a number of ways including rash, swelling, itch, or physical damage that can be due, for instance, to the cross linking of proteins or saponification of lipid membranes by acidic or caustic aerosols, respectively (Anderson and Meade, 2014).

Acute and short term toxicity Animal A diverse array of aerosolized compounds has demonstrated acute toxicity in animals. The specific effects observed are dependent on the toxicant in question and include injury to respiratory organs and the dermis, neurological symptoms, and death (Anderson and Meade, 2014; Phalen et al., 2008).

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Human The specific constituents of an aerosol and their formulation will determine the particular acute toxic effects in humans (Ho et al., 2020).

Chronic toxicity Animal The particular chronic effects of aerosols in exposed animal models encompasses a range of toxicities and is dependent on their physical properties and chemical constituents. Toxicities observed with various aerosolized compounds include respiratory fibrosis, developmental effects, and tumorigenesis (Feron et al., 2001; Yang et al., 2022).

Human The specific aerosol under investigation will dictate its chronic effects in humans who are exposed. For instance, there is a well-established clinical correlation between long term exposure to certain aerosols and chronic lung diseases such as pulmonary fibrosis, emphysema, chronic obstructive respiratory disorder, asthma, and lung carcinoma (Anderson and Meade, 2014; Czekala et al., 2021).

Immunotoxicity Certain aerosolized toxicants elicit an allergic hypersensitivity response upon dermal exposure or allergic asthma with inhalation (Bert et al., 2021). The potential for immunotoxicity will depend on the specific aerosol components. This could, for instance, occur with an aerosolized toxicant that forms protein adducts which create neoantigens that elicit antibody production and promote an immune response upon repeated exposure.

Reproductive toxicity The incidence of reproductive toxicity for a given aerosol will depend on its specific constituents and their formulation.

Genotoxicity Some aerosolized toxicants that humans are exposed to are genotoxic, such as certain aromatic hydrocarbons produced in the burning of fossil fuels (Pardo et al., 2020). The potential for genotoxicity will depend on the specific aerosol components and their chemical mechanisms in the cell, although aerosol genotoxicity is often the result of the formation of DNA adducts (Lindberg et al., 2011).

Carcinogenicity Long term human exposure to certain aerosolized toxicants can elicit carcinogenesis. Such ascertain components of electronic cigarette vapor or traditional cigarette smoke (Ward et al., 2020; Hess et al., 2017). The potential for carcinogenicity will depend on the specific aerosol components and a wide array of genetic and environmental factors for an individual.

Clinical management Supportive care must be instituted for patients accidentally or intentionally exposed to aerosol contents via topical, inhalation, or oral routes (Fuchs et al., 2021; Klompas et al., 2021). Aerosols can induce local or systemic toxicity of the hematopoietic system, liver, and kidneys. Monitoring complete blood count, urinalysis, and liver and kidney function tests is suggested for patients with significant exposure. Exposed individuals should have a careful, thorough medical history and physical examination performed, looking for any abnormalities. Exposure to chemicals with a strong odor often results in such nonspecific symptoms as headache, dizziness, weakness, and nausea. Many chemicals cause irritation of the eyes, skin, and respiratory tract. Ocular exposure should always be proceeded by flushing the eyes with an appropriate eye wish if available for several minutes. In severe cases respiratory tract irritation can progress to acute respiratory distress syndrome (ARDS)/acute lung injury, which may be delayed in onset for up to 24–72 h in some cases

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(Belej and Aviado, 1975). Irritation or burns of the esophagus or gastrointestinal tract are also possible if caustic or irritant chemicals are ingested. If respiratory tract irritation or respiratory depression is evident in a patient, monitor arterial blood gases and perform a chest X-ray and pulmonary function tests. Dermal exposure should be treated with topical administration of exposure-specific countermeasures. For instance, allergic hypersensitivity dermatitis that is brought about through aerosol exposure should be treated with topical anti-inflammatory agents such as hydrocortisone. There may be additional measures that need to be taken to address the toxic effects of specific aerosol components.

Ecotoxicology Certain aerosols are known to be harmful to ecological systems. For example, the intentional use of aerosolized insecticides is known to reduce insect populations which can impact the entire food chain of an ecosystem. The environmental risk of aerosols will depend on the specific toxicant in question.

Other hazards Apart from the direct toxicity of certain aerosols, many aerosols, such as hydrocarbon droplet suspensions, are also flammable and/or explosive. Aerosolization of a flammable liquid will likely increase the risk of combustion. Particular consumer use aerosols such as spray paint also have abuse potential due to the temporary neurological effects they induce.

Exposure standards and guidelines Standards and guidelines for environmental and occupational exposure are contingent upon the specific toxicant under consideration.

See also: Coal tar; Occupational toxicology; Anthrax; Occupational Safety and Health Administration; Respiratory toxicology; Air pollution: Sources, regulation, and health effects; Recommended exposure limits; Carcinogenesis; Sulfur mustard; Non-lethal weapons; Skin; VX; Cosmetics and personal care products; Chemical warfare; Asbestos; Pesticides and its toxicity

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Oh HJ, Ma Y, and Kim J (2020) Human inhalation exposure to aerosol and health effect: Aerosol monitoring and modelling regional deposited doses. International Journal of Environmental Research and Public Health 17(6): 1923. https://doi.org/10.3390/ijerph17061923. Padmore T, Stark C, Turkevich LA, and Champion JA (2017) Quantitative analysis of the role of fiber length on phagocytosis and inflammatory response by alveolar macrophages. Biochimica et Biophysica Acta - General Subjects 1861(2): 58–67. https://doi.org/10.1016/j.bbagen.2016.09.031. 27784615. PMC5228597. Pardo M, Li C, He Q, et al. (2020) Mechanisms of lung toxicity induced by biomass burning aerosols. Particle and Fibre Toxicology 17: 4. Phalen RF, Oldham MJ, and Wolff RK (2008) The relevance of animal models for aerosol studies. Journal of Aerosol Medicine and Pulmonary Drug Delivery 21(1): 113–124. https:// doi.org/10.1089/jamp.2007.0673. 18518837. Wang CY, Liu LF, Liu XL, Chen WJ, and He GP (2017) Mechanisms of lung cancer caused by cooking fumes exposure: A minor review. Chinese Medical Sciences Journal 32(3): 193–197. https://doi.org/10.24920/J1001-9294.2017.026. 28956747. Ward AM, Yaman R, and Ebbert JO (2020) Electronic nicotine delivery system design and aerosol toxicants: A systematic review. PLoS One 15(6), e0234189. Weinberger B, Laskin JD, Sunil VR, Sinko PJ, Heck DE, and Laskin DL (2011) Sulfur mustard-induced pulmonary injury: Therapeutic approaches to mitigating toxicity. Pulmonary Pharmacology & Therapeutics 24(1): 92–99. https://doi.org/10.1016/j.pupt.2010.09.004. 20851203. PMC3034290. Yang M, Tian F, Tao S, Xia M, Wang Y, Hu J, Pan B, Li Z, Peng R, Kan H, Xu Y, and Li W (2022) Concentrated ambient fine particles exposure affects ovarian follicle development in mice. Ecotoxicology and Environmental Safety 231: 113178. https://doi.org/10.1016/j.ecoenv.2022.11317835026587.

Relevant websites https://www.nasa.gov/centers/langley/news/factsheets/Aerosols.html :National Aeronautics and Space Administration. https://www.cdc.gov/niosh/topics/aerosols/aerosols_overview.html :National Institute for Occupational Safety and Health. http://www.osha.gov/ :Occupational Safety and Health Administration. https://www.epa.gov/aegl :U.S. Environmental Protection Agency.

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A-esterase Ida Adeli, Hosna MohammadSadeghi, and Behnaz Bameri, Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of L.G. Sultatos, A-esterase, Editor (s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 102–103, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00092-0.

Abstract A-esterase enzymes, also called paraoxonase (PON), are a group of enzymes responsible for the metabolism and detoxification of different chemicals such as organophosphate insecticides and nerve gases. Three paraoxonase enzymes, PON1, PON2, and PON3, are produced in the human body. Although all three enzymes encoding genes are adjacently located on chromosomes, PON1 and PON3 are synthesized in the liver and released into the bloodstream, and PON2 is an intracellular enzyme found in several tissues. Apart from being engaged in the metabolism pathways of different compounds, PONs partake in the modulation of various diseases, including cardiovascular diseases, diabetes, and several neurological disorders.

Keywords A-esterase; Arylester hydrolase; Arylesterase; Organophosphate hydrolase; Organophosphate insecticides; Oxon; Paraoxonase; Polymorphism; Toxicity

The enzyme A-esterase or in other terms paraoxonase, aryl-ester hydrolase, arylesterase, or organophosphate hydrolase, can detoxify various substances (previous version), including paraoxon (CAS#311-45-5), chlorpyrifos oxon (CAS#5598-15-2), diazoxon (CAS#962-58-3), and nerve gases such as sarin (CAS#107-44-8), soman (CAS#96-64-0) (Costa et al., 2003), Dichlorvos (CAS#62-73-7), and etc. (Wang et al., 2011). There is also the term “B”-esterase, which unlike “A” esterase is inhibited by organophosphates and is incapable of hydrolyzing them (Mackness and Mackness, 2015). The most crucial paraoxonase and the most studied one, PON1, can hydrolyze a wide variety of lactones, arylesterase, organophosphate insecticides, nerve gases, glucuronide drugs, and estrogen esters. Its prominent enzymatic role in the body is to reduce oxidative stress and protect low-density lipoproteins (LDLs). Besides, it can hydrolyze toxic metabolites of OP compounds, and because of this, it has attracted growing interest and attention in human biomonitoring studies of OP-exposed populations (del Carmen et al., 2019). There are three paraoxonase enzymes in humans called PON1, PON2, and PON3. All three enzymes encoding genes are adjacently located on the long arm of human chromosome 7 (Kowalska et al., 2015; Meneses et al., 2019; White and Anantharamaiah, 2017). PON1 is synthesized and secreted by the liver (Shokri et al., 2020; Tajbakhsh et al., 2017; Estrada-Luna et al., 2018; Ponce-Ruiz et al., 2017; Merwin et al., 2017; Jamwal et al., 2021; Mahrooz et al., 2019) and bound to high-density lipoprotein (HDL) in the blood (Tajbakhsh et al., 2017; Estrada-Luna et al., 2018; Ponce-Ruiz et al., 2017; Jamwal et al., 2021; Mahrooz et al., 2019; Morris et al., 2021). This enzyme is also expressed in the kidney and the intestine (Estrada-Luna et al., 2018). Although the mechanism by which PON1 can cross the blood-brain barrier (BBB) is unknown, it is identified within the cerebrospinal fluid (CSF) and several tissues, namely the brain and the spinal cord (Mahrooz et al., 2019). X-ray crystallography of human serum paraoxonase-1 has indicated a six-bladed propeller structure that each blade is made of four sheets (Kowalska et al., 2015; Shokri et al., 2020). PON1 also has two calcium ions (Ca2+); one is located at the base of the active site, at the top of the structure, which is believed to play a key role in PON1 catalytic activity (Mackness and Mackness, 2015). The other one, placed in the center, is necessary for the enzyme stability (Kowalska et al., 2015; Shokri et al., 2020; Mackness, 2008). So, the presence of these two calcium ions is essential to the enzyme since their removal with chelating agents could irreversibly destroy its activity and stability at the same time (Shokri et al., 2020). Additionally, there is a part in PON1 enzyme-containing hydrophobic amino acids, which is important in HDL binding. It seems that the activity of the enzyme toward organophosphates could differ by modulating the hydrophobicity characteristic of the PON1 active site (Estrada-Luna et al., 2018; Mackness, 2008). Environmental contributors such as diet, lifestyle, physical activity, age, pharmaceuticals, and epigenetic and genetic factors regulate the serum level of PON1 and its activity (Shokri et al., 2020; Merwin et al., 2017). As an instance, the proatherogenic condition hinders the expression of PON1. Otherwise, taking vitamin C, E, and moderate alcohol consumption raises serum levels (Merwin et al., 2017). Lipopolysaccharide (LPS) and cytokines decrease the synthesis of PON1 in the liver within the inflammatory conditions (Tajbakhsh et al., 2017). Moreover, systemic inflammation causes downregulation in PON1 activity through increased levels of TNF-a and pro-inflammatory cytokines (Furlong et al., 2016). PON1 encoding gene has two remarkable polymorphisms at positions 55 and 192. Polymorphism at position 192 contains two forms, including 192R (arginine) and 192Q (glutamine) (Shokri et al., 2020; Jamwal et al., 2021; Camps et al., 2021; Marsillach et al., 2016). The two mentioned forms represent different rates of catalytic characteristics. The 192R form owns higher paraoxonase

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and arylesterase activity, while the 192Q form has been identified to have more lactonase activity (Shokri et al., 2020). Apart from two introduced polymorphisms, plenty of others have been identified in the non-coding region of the PON1 gene. Cytosine to thymidine substitution at position 107 is of great importance and gives rise to PON1 serum levels (Shokri et al., 2020). Aforementioned, the serum level and concentration of PON1 are related to age. Studies have indicated that PON1 levels of newborns are nearly 25–33% of adults, and 6 months to 2 years is needed for PON1 enzyme activity of a newborn to reach to the adults level (Camps et al., 2021; Marsillach et al., 2016). Due to several studies conducted on animals and humans, PON1 can determine the toxicity of certain organophosphates. As mentioned before, PON1 can hydrolyze the oxygen analogs of multiple common OP insecticides (e.g., paraoxon, diazoxon, chlorpyrifos oxon). It has been indicated that low PON1 levels may lead to higher susceptibility and sensitivity to OP toxicity, although several other factors, including absorption and distribution rates of OPs rates of activation and detoxication by different metabolic pathways, influence the overall toxicity outcome (Salari et al., 2021). As a result, it is proposed that those with high activity levels of PONs would be resistant to exposures of parathions and paraoxons (Priyanka et al., 2019). The PON proteins are capable of presenting various types of hydrolytic activity such as lactonase activity (e.g., against homocysteine (CAS# 454-29-5), thiolactone), arylesterase activity (e.g., against phenylacetate (CAS#122-79-2)), and organophosphates activity (e.g., against paraoxon). The PON1 protein exhibits all of the activities mentioned above, while PON2 and PON3 have only shown lactonase activity. According to some authors, PON3 can also display limited arylesterase activity (del Carmen et al., 2019). PON2 and PON3 enzymes are other types of paraoxonases, which are not of great importance as PON1s. Both PON2 and PON3 proteins exhibit antioxidant properties like PON1 and play an essential role in retarding the oxidative modification of low-density lipoprotein (LDL) and cell membranes (del Carmen et al., 2019). PON2 weighs approximately 43-kDa and is ubiquitously expressed inside the cells, but unlike PON1 and PON3, it is not present in plasma (Mahrooz et al., 2019), and its gene expression is exclusively intracellular (Morris et al., 2021). PON2 is mainly localized in the astrocytes of dopaminergic regions, but it is also expressed in several organs, such as the liver, lungs, placenta, testis, and heart. Within the cell, PON2 is located in the nuclear envelope, endoplasmic reticulum, and in the inner mitochondrial membrane, in proximity with complex III of the electron transport chain (Wang et al., 2011; Mackness and Mackness, 2015; Mackness, 2008). Studies have indicated that the PON2 enzyme is a critical protein in mitochondria for functioning correctly and plays an important role in reducing oxidative stress (Tajbakhsh et al., 2017). On the other hand, PON2 is involved in defending the body against inflammatory reactions (Costa et al., 2003) and can regulate the process of macrophage differentiation; In fact, PON2 can switch an M1 to an M2 phenotype in macrophages (Mackness and Mackness, 2015). Discussing PON2’s effects on human’s immune system, it is thought that PON2 exerts prominent lactonase activity, resulting in a reduction of quorum sensing, biofilm formation, and bacterial virulence (Mackness and Mackness, 2015). PON2 enzyme is considered to significantly reduce lipid peroxides (free radical lipid) in macrophages and inhibits LDL oxidation; thereby, the expression of PON2 increases in these cells during oxidative stress (del Carmen et al., 2019). PON3 is a 40-kDa glycoprotein expressed in the endoplasmic reticulum of intestinal cells and mitochondria of selected tissues (Furlong et al., 2016). Similar to PON1 in some aspects; it is also synthesized in the liver and to a lower extent in the kidney, associated with HDL particles and indicate anti-oxidant solid properties (Kowalska et al., 2015; Meneses et al., 2019; White and Anantharamaiah, 2017; Tajbakhsh et al., 2017; Salari et al., 2021); Unlike PON1, it cannot hydrolyze organophosphate compounds, whereas it can hydrolyze statin lactones at a much higher catalytic efficiency than PON1 (Priyanka et al., 2019). PON3 possesses paraoxonase and esterase activities; however, these are significantly reduced compared to PON1 (Tajbakhsh et al., 2017). It is reported that PON3’s concentration decreases with growing older (Priyanka et al., 2019) and in certain autoimmune diseases (Zimetti et al., 2021); conversely, increased serum levels of PON3 protein have been reported in chronic liver disease, HIV infection, and atherothrombotic disease and patients with sepsis (Zimetti et al., 2021). All three forms of the PON enzymes affect inflammatory conditions in the body; and play roles in the process of modulating and pathogenesis of certain diseases, including atherosclerosis and other cardiovascular diseases, diabetes, several neurological disorders such as Parkinson’s disease (PA), Alzheimer’s disease (AD), and Multiple Sclerosis (MS) (previous version). The role of PONs in cancer is being investigated, and overexpression of PON2 and PON3 has been observed in cancer cells; as a result, these proteins may be involved in tumor survival and stress resistance in different types of cancer (Bacchetti et al., 2019).

References Bacchetti T, Ferretti G, and Sahebkar A (2019) The Role of Paraoxonase in Cancer. Seminars in Câncer Biology. Elsevier. Camps J, Castañé H, Rodríguez-Tomàs E, Baiges-Gaya G, Hernández-Aguilera A, Arenas M, et al. (2021) On the role of paraoxonase-1 and chemokine ligand 2 (CC motif ) in metabolic alterations linked to inflammation and disease. A 2021 update. Biomolecules 11(7): 971. Costa LG, Richter RJ, Li W-F, Cole T, Guizzetti M, and Furlong CE (2003) Paraoxonase (PON 1) as a biomarker of susceptibility for organophosphate toxicity. Biomarkers 8(1): 1–12. del Carmen X-GM, Herrera-Moreno JF, Medina-Díaz IM, Bernal-Hernández YY, Rothenberg SJ, Barrón-Vivanco BS, et al. (2019) Relationship between internal and external factors and the activity of PON1. Environmental Science and Pollution Research. 26(24): 24946–24957. Estrada-Luna D, Ortiz-Rodriguez MA, Medina-Briseño L, Carreón-Torres E, Izquierdo-Vega JA, Sharma A, et al. (2018) Current therapies focused on high-density lipoproteins associated with cardiovascular disease. Molecules. 23(11): 2730. Furlong CE, Marsillach J, Jarvik GP, and Costa LG (2016) Paraoxonases-1, -2 and -3: What are their functions? Chemico-biological interactions. 259(Pt B): 51–62. Jamwal S, Blackburn JK, and Elsworth JD (2021) PPARg/PGC1a signaling as a potential therapeutic target for mitochondrial biogenesis in neurodegenerative disorders. Pharmacology & Therapeutics. 219: 107705. Kowalska K, Socha E, and Milnerowicz H (2015) The role of paraoxonase in cardiovascular diseases. Annals of Clinical & Laboratory Science. 45(2): 226–233. Mackness B (2008) The paraoxonases their role in disease development and xenobiotic metabolism. Springer.

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Mackness M and Mackness B (2015) Human paraoxonase-1 (PON1): Gene structure and expression, promiscuous activities and multiple physiological roles. Gene. 567(1): 12–21. Mahrooz A, Mackness M, Bagheri A, Ghaffari-Cherati M, and Masoumi P (2019) The epigenetic regulation of paraoxonase 1 (PON1) as an important enzyme in HDL function: The missing link between environmental and genetic regulation. Clinical biochemistry. 73: 1–10. Marsillach J, Costa LG, and Furlong CE (2016) Paraoxonase-1 and early-life environmental exposures. Annals of global health. 82(1): 100–110. Meneses MJ, Silvestre R, Sousa-Lima I, and Macedo MP (2019) Paraoxonase-1 as a regulator of glucose and lipid homeostasis: Impact on the onset and progression of metabolic disorders. International journal of molecular sciences. 20(16): 4049. Merwin SJ, Obis T, Nunez Y, and Re DB (2017) Organophosphate neurotoxicity to the voluntary motor system on the trail of environment-caused amyotrophic lateral sclerosis: The known, the misknown, and the unknown. Archives of toxicology. 91(8): 2939–2952. Morris G, Puri BK, Bortolasci CC, Carvalho A, Berk M, Walder K, et al. (2021) The role of high-density lipoprotein cholesterol, apolipoprotein A and paraoxonase-1 in the pathophysiology of neuroprogressive disorders. Neuroscience & Biobehavioral Reviews. 125: 244–263. Ponce-Ruiz N, Murillo-González F, Rojas-García A, Mackness M, Bernal-Hernández Y, Barrón-Vivanco B, et al. (2017) Transcriptional regulation of human paraoxonase 1 by nuclear receptors. Chemico-biological interactions. 268: 77–84. Priyanka K, Singh S, and Gill K (2019) Paraoxonase 3: Structure and its role in pathophysiology of coronary artery disease. Biomolecules. 9(12): 817. Salari N, Rasoulpoor S, Hosseinian-Far A, Razazian N, Mansouri K, Mohammadi M, et al. (2021) Association between serum paraoxonase 1 activity and its polymorphisms with multiple sclerosis: A systematic review. Neurological Sciences. 42(2): 491–500. Shokri Y, Variji A, Nosrati M, Khonakdar-Tarsi A, Kianmehr A, Kashi Z, et al. (2020) Importance of paraoxonase 1 (PON1) as an antioxidant and antiatherogenic enzyme in the cardiovascular complications of type 2 diabetes: Genotypic and phenotypic evaluation. Diabetes research and clinical practice. 161: 108067. Tajbakhsh A, Rezaee M, Rivandi M, Forouzanfar F, Afzaljavan F, and Pasdar A (2017) Paraoxonase 1 (PON1) and stroke; the dilemma of genetic variation. Clinical biochemistry. 50(18): 1298–1305. Wang N-N, Yuan L, Dai H, Han Z-K, and Zhao M (2011) Effect of PON1 on dichlorvos toxicokinetics. Emergency Medicine Journal. 28(4): 313–315. White CR and Anantharamaiah G (2017) Cholesterol reduction and macrophage function: Role of paraoxonases. Current opinion in lipidology. 28(5): 397. Zimetti F, Adorni MP, Marsillach J, Marchi C, Trentini A, Valacchi G, et al. (2021) Connection between the altered HDL antioxidant and anti-inflammatory properties and the risk to develop Alzheimer’s disease: A narrative review. Oxidative Medicine and Cellular Longevity. 2021.

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Aflatoxin Elisha Yagudayev and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of M.A. Tirmenstein, R. Mangipudy, Aflatoxin, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 104–106, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00224-4.

Chemical profile Background Uses/occurrence Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity In vitro toxicity Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Toxicogenomics Diagnosis, detection and clinical management Environmental fate and behavior Ecotoxicology Other hazards Exposure standards and guidelines Conclusion References

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Abstract Aspergillus flavus and Aspergillus parasiticus are the two well-known fungal species that produce aflatoxins. Aflatoxins are mycotoxins that are potent human liver carcinogens. Aflatoxin B1 is metabolized to a reactive epoxide (aflatoxin 8,9-epoxide) primarily by the CYP450 (1A2, 3A4) monooxygenase system. Once formed, the epoxide can react further to form DNA adducts (aflatoxin-N7-guanine) and induce mutations and cancer. The most common mutation seen is a G ! T transversion in codon 249 of the p53 tumor suppressor gene. Additional mechanisms of toxicity include inhibition of protein, RNA, and DNA synthesis as well as the formation of Reactive Oxygen Species in glutathione deficiency. The Joint FAO/WHO Expert Committee on Food Additives estimates that regular consumption of staple foods that contain at least 1 mg/kg Aflatoxin B1 or exposures between 20 and 120 mg/kg body weight per day over 1–3 weeks can result in acute aflatoxicosis. Aflatoxin toxicity is associated with hepatocellular damage and necrosis, cholestasis, hepatomas, acute hepatitis, periportal fibrosis, hemorrhage, jaundice, fatty liver changes, immunomodulation, malnutrition, cirrhosis in malnourished children, and Kwashiorkor. There is no antidote for aflatoxin poisoning and supportive care remains the mainstay of poisoning management.

Keywords Aflatoxin 8,9-epoxide; Aflatoxin; Aflatoxin B1; Aspergillus flavus; Bisfuranocoumarin; Carcinogen; CYP4501A2, CYP4503A4; Food contaminants; Fungal toxin; Mycotoxin

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Aflatoxin, a potent fungal toxin, is a carcinogen that contaminates a large portion of the World’s food supply. Major aflatoxin producers are Aspergillus flavus and Aspergillus parasiticus. Aflatoxin is metabolized by CYP4501A2/3A4 in the liver, and it can form DNA adducts which can initiate carcinogenic process in the body. Animal studies suggest aflatoxin exposure may increase risk for prematurity and pregnancy loss, and the fetus could be affected by maternal exposure through direct toxicity as well as indirect toxicity. Chronic exposure to aflatoxin can cause impaired growth and development especially in children, and in adults, it can cause malnutrition, immunomodulation, hepatocellular carcinoma presenting as weight loss, abdominal mass, anorexia, nausea, vomiting, bleeding, psychosis, etc. Aflatoxin exposure can be deemed as an occupational hazard for those who work in the food and agriculture industry. Chronic toxicity occurs by consuming small amounts of aflatoxins but over a prolonged period of time. Aflatoxin toxicity is one of the major causes of liver cirrhosis and hepatocellular carcinoma in developing countries. Despite stringent regulations and advanced strategies to reduce toxin level, the presence of high levels of aflatoxin is very common in food supplies throughout the World, which indicates that exposure to the population at large still remains largely unchecked. There is no antidote for aflatoxin poisoning and supportive care remains the mainstay of poisoning management.

Chemical profile

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Chemical Abstracts Service Registry Number: 1162-65-8 Molecular Formula (Aflatoxin B1): C17H12O6 Synonyms: Aflatoxins B1; B2; B3; B4; G1; G2; M1; M2 Chemical/Pharmaceutical/Other Class: Mycotoxins Chemical Structure (Aflatoxin B1):

Background Aflatoxins are naturally occurring bisfuranocoumarin compounds produced from the molds Aspergillus flavus and Aspergillus parasiticus. Aflatoxins were first discovered after 100,000 healthy turkeys in the southeastern region of England suddenly died in the spring of 1960. This became known as Turkey X Disease. Researchers found that the source of death was the consumption of aflatoxin-contaminated feed from Brazil. Reports indicate that 20 different types of aflatoxins exist; however, the most dangerous forms to humans and animals are the B1, B2, G1, and G2 subtypes (Pickova et al., 2021). The aflatoxins are highly fluorescent. The B refers to blue and the G signifies green fluorescence. M aflatoxins are fungal metabolites present in milk, most commonly in areas which are highly exposed to aflatoxin, being that aflatoxin M1 is a product of aflatoxin B1 metabolism. Aflatoxin B1 is the most common and potent of the aflatoxins. Crops that are affected by aflatoxin contamination include cereals (maize, sorghum, rice, wheat), oilseeds (peanut, sunflower, soybean, cotton), spices (chili peppers, black pepper, coriander, turmeric, ginger), and tree nuts (almond, coconuts, brazil nuts, walnuts, pistachio). Aflatoxin can also be found in the milk, eggs, and meat from animals fed contaminated feed (Khan et al., 2021).

Uses/occurrence Aflatoxins have no industrial or therapeutic use and are chiefly a source of contamination of food crops, especially in nuts.

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Exposure and exposure monitoring A. flavus and A. parasiticus are most commonly found in high temperature and high humidity areas which are conducive to the mold’s growth. They often grow on dead and decaying vegetation, leading to contamination of surrounding food crops. Aflatoxins are estimated to destroy over 25% of the world’s crops each year. Crops can become contaminated both before and following harvesting, with the former most commonly seen in maize, cottonseed, peanuts, and tree nuts, and the latter occurring most frequently to coffee, rice, and spices. Warm and humid storage environments are a common cause of post-harvest contamination. The United States Food and Drug Administration (USFDA) considers the contamination of agricultural products with aflatoxins to be unavoidable, with the goal being minimizing exposure to humans and animals. Ingestion of contaminated produce is the most common route of exposure. However, levels of exposure vary between countries, with mean aflatoxin dietary exposures usually being less than 1 ng/kg body weight per day in developed countries, compared to some sub-Saharan African countries such as Uganda, Kenya, and Gambia, where some data shows exposures being over 100 ng/kg body weight per day (Joint FAO/WHO report, 2017). A variety of methods are available to detect aflatoxin contaminated products. Widely used methods include HPLC-MS for regulatory control oversight in large laboratories, as well as rapid test kits such as ELISA for factories to test their product. Because of the lack of even distribution of molds and aflatoxins throughout stored crop, several organizations have developed protocols for sampling procedures. For example, the Codex Alimentarius Commission has set protocols to be used for certain nuts and fruits in determining the maximum levels of aflatoxins in those products. The Food and Agriculture Organization of the United Nations (FAO) has likewise developed a mycotoxin sampling tool which can be accessed online. Dermal contact is another method of exposure, which can occur in occupational settings through handling and processing of aflatoxin-contaminated crops with low-level respiratory exposures to aflatoxin-contaminated dust particles. Dust levels of aflatoxin collected at different animal feed production sites ranged from not detectable to 8 mg kg−1 (PubChem).

Toxicokinetics Aflatoxins are well absorbed orally. Exposure to human skin results in slow absorption. Aflatoxins are rapidly cleared from blood. Sixty-five percent of an initial dose of aflatoxin B1 is removed from the blood within 90 min and excreted primarily in the bile. The plasma half-life of aflatoxin is short, and it is excreted slowly as multiple moieties as a result of extensive metabolism. When estimated in human liver homogenates, the parent compound had an estimated half-life of 13 min. In vitro liver metabolism studies have shown five different types of metabolic pathways for aflatoxin B1: reduction, hydroxylation, hydration, O-demethylation, and epoxidation. All of these products contain hydroxide groups that allow them to be conjugated with glucuronic acid and sulfate. Aflatoxin B1 (AFB1) is metabolized into many metabolites including AFL, AFM1, AFQ1, AFP1, AF 8,9-epoxide, and AFB2a. In contrast to other metabolites, aflatoxin M1 (AFM1) retains acute oral toxicity (Micromedex).

Mechanism of toxicity Aflatoxin B1 is metabolized to a reactive epoxide (aflatoxin 8,9-epoxide) primarily by the P450 monooxygenase system. In humans, the epoxidation reaction is catalyzed by CYP1A2 and CYP3A4. Once formed, the epoxide can react further to form DNA adducts (aflatoxin-N7-guanine) and induce mutations and cancer (Benkerroum, 2020b). The most common mutation seen is a G ! T transversion in codon 249 of the p53 tumor suppressor gene. The epoxide can also cause damage by binding to RNA and proteins, leading to metabolic dysregulation. It was also found to lead to inhibition of protein, RNA, and DNA synthesis. Alternatively, the epoxide can be detoxified by glutathione conjugation via glutathione S-transferase. Depletion of glutathione stores could be a likely result in generating toxicity due to Reactive Oxygen Species (ROS) (Marchese et al., 2018). AFB1 has been shown to induce apoptosis in microglia cells through oxidative stress by activating NF-kB signaling pathway (Zhou et al., 2022). This observation is based on the earlier finding that AFB1 can be neurotoxic via Reactive Oxygen Species Generation, DNA Damage, Apoptosis, and cell cycle arrest at S-phase (Huang et al., 2020) (Fig. 1).

In vitro toxicity In vitro data has demonstrated that the carcinogenicity of aflatoxin B1 primarily results from its activation by the CYP450 system in the liver. AFM1 was found to bind to microsomes in the absence of metabolism, whereas AFB1 needed a competent metabolic activation system to bind to microsomes. Another study reported activation of human HRAS proto-oncogene with AFB1 exposure, suggesting that these genes may also play a role in aflatoxin’s carcinogenic properties (Marchese et al., 2018). Extremely low doses of AFB1 (0.5–1.0 pg/mL) in human monocyte cultures were shown to decrease phagocytosis and microbicidal activity against Candida albicans, as well as diminish the release of proinflammatory cytokines such as IL-1, IL-6, and TNF-alpha (IARC, 2002).

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Fig. 1 Aflatoxin Metabolism and Mechanism of Toxicity. Aflatoxin B1 is metabolized into the following primary metabolites: by reduction into aflatoxicol, hydroxylation into aflatoxin Q1 and aflatoxin M1, hydration into Aflatoxin B2a, and O-demethylation into Aflatoxin P1. It also undergoes metabolism by CYP1A2 and CYP3A4 to form Aflatoxin exo-8,9-epoxide, its most reactive and cancer-inducing metabolite. The epoxide can either be inactivated by glutathione (GST) or undergo adduct formation into Aflatoxin-N7-guanine, the reactive substance that is responsible for development of hepatocellular cancer. The epoxide can also bind to RNA and proteins leading to metabolic dysregulation or can form reactive oxygen species (ROS) in the presence of glutathione depletion, resulting in oxidative stress. Bioactivation pathway designed by Yagudayev and Ray.

Acute and short-term toxicity Animal The aflatoxin oral LD50 in mice is 9 mg/kg and the LD50 following intraperitoneal injection is 9500 mg/kg. In monkeys, the aflatoxin oral LD50 was found to be 2200 mg/kg. Acute effects included behavioral changes such as somnolence and altered food intake, as well as a negative impact on the liver, leading to jaundice and other effects (PubChem). Animals which rapidly metabolize Aflatoxin B1 forming aflatoxin-albumin adducts have been found to be more susceptible to aflatoxin toxicity. These include animals such as turkeys, sheep, dogs, rats, and pigs. Other animals such as mice and monkeys are more resistant (Benkerroum, 2020a).

Human Acute aflatoxicosis can be caused by ingestion of high doses of aflatoxin over a short period of time. Symptoms of acute toxicity include nausea, vomiting, abdominal pain, lethargy, and jaundice. The Joint FAO/WHO Expert Committee on Food Additives estimates that regular consumption of staple foods that contain at least 1 mg/kg Aflatoxin B1 or exposures between 20 and 120 mg/kg body weight per day over 1–3 weeks can result in acute aflatoxicosis (Joint FAO/WHO report, 2017). Acute exposures to aflatoxins resulting in toxicity have been reported in several counties across the globe. The first such report in a human was in 1967, were a 15-year-old Ugandan boy died due to consumption of cassava meal contaminated with high concentrations of aflatoxin (1700 mg/kg). The boy had been eating the meal for 22 days prior to his death due to liver failure (Benkerroum, 2020a). In 1974, in Western India, more than 200 villages experienced an outbreak of a disease affecting humans that was characterized by jaundice, rapidly developing ascites, portal hypertension, and a high mortality rate, with death usually resulting from massive gastrointestinal tract bleeding. The disease was associated with the consumption of badly molded corn containing between 6.25 and 15.6 ppm aflatoxin (average daily intake per victim of 2–6 mg of aflatoxin) (Palmgren and Ciegler, 1983).

Chronic toxicity Animal Edema and necrosis of hepatic and renal tissues are characteristic of chronic aflatoxin toxicity in experimental animals. Neurological symptoms and neurotoxicity have also been observed in animals administered aflatoxin (Benkerroum, 2020b). In carcinogenicity

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studies, aflatoxin produces liver tumors in mice, rats, fish, ducks, marmosets, tree shrews, and monkeys after administration by several routes. In rats, cancers of the colon and kidney were also noted (IARC, 1987). Chickens who consumed feed contaminated with aflatoxin were found to have impaired reproduction, increased susceptibility to disease, as well as inferior eggshell quality and productivity. Cattle presented primarily with kidney and liver damage, as well as reduced milk production and lack of normal wight gain (Joint FAO/WHO report, 2017).

Human Aflatoxin poisoning is difficult to diagnose early in humans. The two most common methods of detecting aflatoxin levels include testing the urine for aflatoxin metabolites, which are only present within 24 h of exposure, and measuring the level of an Aflatoxin B-albumin complex in the blood serum, which can show signs of exposure over weeks or months. The first clinical symptoms observed are anorexia and weight loss. Aflatoxins are associated with hepatocellular damage and necrosis, cholestasis, hepatoma, acute hepatitis, periportal fibrosis, hemorrhage, jaundice, fatty liver changes, cirrhosis in malnourished children, and Kwashiorkor. The risk of hepatocellular carcinoma increases 30-fold in HBV-infected individuals (Marchese et al., 2018). Chronic consumption has also been associated with impaired growth and development in children (Benkerroum, 2020b). There is evidence of transplacental transport of aflatoxin by the fetoplacental unit (IARC, 2002). Aflatoxins are proven human carcinogens.

Immunotoxicity Animal studies have shown that aflatoxins play an immunomodulatory role, suppressing immune function by affecting T-cell-dependent immunity in several animal species including cattle, poultry, pigs, mouse, rat, and rabbits. Aflatoxins have been shown to affect macrophages, dendritic cells, and natural killer cells by reducing their expression of cytokines and chemokines, as well as down-regulating transcription of toll-like receptors. Aflatoxins were also shown to inhibit the complement system in many animals (Benkerroum, 2020b).

Reproductive and developmental toxicity Animal studies have reported that aflatoxin exposure during pregnancy can lead to fetal abnormalities, fetal growth retardation, fetal loss as well as a slight decrease in the number of live births. Pregnant rabbits fed 0–100 mg aflatoxin/kg body weight per day were reported to have a decreased percentage of live fetuses, impaired organ development, and skeletal abnormalities (Smith et al., 2017). In vitro exposure of rat embryos to aflatoxin B1 induced neural tube defects (IARC, 1993). Analysis of aflatoxins in maternal and cord blood samples has also demonstrated the transplacental transport of aflatoxins in humans (IARC, 2002). Although suspected of playing a role in the early onset of liver cancer in some populations, prenatal exposure has not been demonstrated to be a significant route of exposure to the aflatoxins. It has also been shown that there is an association between aflatoxin consumption during pregnancy and development of maternal anemia and low birthweight of the baby. These effects have been attributed to aflatoxin’s cytotoxic effects, which leads to lysis of red blood cells and interfering with nutrient absorption, which is vital for proper fetal growth (Benkerroum, 2020b). In vitro and in vivo studies indicate that high aflatoxin exposures of 0.5–1.0 mg/kg body weight causes RBC lysis in dogs, rabbits, catfish, and poultry (Smith et al., 2017). Chronic aflatoxin consumption has been associated with impaired growth and development in children, likely due to the aforementioned interference with essential micronutrients, and its effect on protein synthesis (Benkerroum, 2020b). Aflatoxins and active carcinogenic metabolites are excreted in breast milk (Aflatoxin M1). The metabolite was found to be present in the mother’s milk 12–24 h after consumption of aflatoxin-contaminated food; however, levels rapidly decreased and were undetectable three days following consumption of the contaminated food (Maleki et al., 2015). It has also been reported that male rats fed Aflatoxin-B1 developed testicular degeneration and impaired spermatogenesis. This is most likely due to AFB1 binding to the StAR protein, which negatively impacts cholesterol transport into the mitochondria resulting in a decrease in testosterone production. However, it is yet to be determined whether this decrease is sperm count and quality results in decreased fertility in these rats (Supriya et al., 2014).

Genotoxicity Aflatoxin B1 is metabolized into aflatoxin exo-8,9-epoxide as well as aflatoxin endo-8,9-epoxide, with the former being significantly more reactive and toxic than the latter (Marchese et al., 2018). The aflatoxin epoxide undergoes alkylation forming DNA adducts. This results in the formation of three DNA lesions which are primarily responsible for aflatoxin’s genotoxicity. These three are aflatoxin-N7-guanine, apurinic DNA molecule, and AFB1-FAPy, with the latter reported to be the most mutagenic. These lesions result in interference with gene expression and DNA integrity. The lack of effective and rapid DNA repair results in a transverse mutation, namely a G ! T transversion in codon 249 of the p53 tumor suppressor gene. This change in protein structure renders it incapable of binding to DNA molecules, thus losing its ability to perform cell-cycle arrest and apoptosis when necessary (Benkerroum, 2020b).

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Carcinogenicity There is sufficient evidence in humans for the carcinogenicity of aflatoxin B1 and aflatoxin mixtures. Naturally occurring aflatoxins such as aflatoxin B1 are classified by the IARC as “carcinogenic to humans” (Group 1). Aflatoxin M1 is classified as “possibly carcinogenic to humans” (Group 2B). There is also sufficient evidence for the carcinogenicity of aflatoxin mixtures and for aflatoxins B1, G1, and M1 in animals. There is “limited evidence” for the carcinogenicity of aflatoxin B2 and “inadequate evidence” for aflatoxin G2 in animals (IARC, 2002).

Organ toxicity See ‘Acute and short-term toxicity’ section above.

Interactions The pretreatment of rats with an alkaloid found in black pepper, piperine, was found to enhance (3)H-aflatoxin B1 radioactivity in plasma and tissues compared to control. However, piperine had no effect on hepatic (3)H-aflatoxin B1 DNA binding in vivo (Allameh et al., 1992). Indole-3-carbimol, a compound derived from cruciferous vegetables, was found to inhibit AFB1 hepatocarcinogenesis in trout and rats when given prior to and in conjunction with AFB1 but had the opposite effect of promoting carcinogenesis in both species when given continuously following AFB1 administration (Dashwood et al., 1991).

Toxicogenomics Six genes have been identified as being a part of the specific epigenetic footprint of Aflatoxin B1. These are the TXNRD1, PCNA, CCNK, DIAPH3, RAB27A and HIST1H2BF genes. These epigenome and transcriptome changes were noted due to their persistent expression and change in DNA methylation after exposure to AFB1. These genes play a role in AFB1-DNA adduct formation, changes in cell growth, and impairment of effective DNA damage response mechanisms, thus contributing to the development of aflatoxin-induced hepatocellular carcinoma (Rieswijk et al., 2016; Khan et al., 2021).

Diagnosis, detection and clinical management Acute aflatoxin toxicity should be treated with decontamination procedures and good supportive care. For adults, this includes administration of 25 to 100 g of activated charcoal as an aqueous slurry mixture preferably within 1 h of ingestion. Phenobarbital can also be considered as it has been shown to have a protective effect against AFB-induced toxicity, carcinogenicity, and DNA binding in vivo. With chronic ingestion, measures to decrease GI absorption are unlikely to be of much benefit. Therefore, the primary treatment in these cases remains supportive in nature. Elevation of serum alkaline phosphatase is a good indicator of aflatoxin toxicity. Hepatic function should also be monitored throughout (Micromedex; Dhakal and Sbar, 2021).

Environmental fate and behavior Aflatoxin will exist solely in the particulate phase if released into the ambient atmosphere based on estimated vapor pressure values (1.6  10–10 to 7.7  10–11 mmHg) at 25  C. Particulate-phase aflatoxins would be expected to be removed from the atmosphere by wet and dry deposition. Direct photolysis is also possible since aflatoxins absorb light in the environmental UV spectrum. If released to soil, the aflatoxins are expected to have low to no mobility based upon a Koc range of 682 to 23,170. Aflatoxins are not expected to volatilize from dry soil surfaces based upon their vapor pressures. If released into water, the aflatoxins may adsorb to suspended solids and sediment based upon the available Koc values. Estimated BCF values of 2–3 suggest the potential for bioconcentration in aquatic organisms is low. Aflatoxin may also be degraded by photolysis at soil and water surfaces. Aflatoxins may also undergo hydrolysis in the environment due to their cyclic ester functionality. Under environmental conditions (pH 5–9), this hydrolysis may take months to years to occur (PubChem).

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Ecotoxicology Besides being toxic to humans and damaging to a significant portion of the world’s crops, aflatoxins are highly toxic to many animal species, including fish and birds. This is of especial concern in animals that might have been exposed to contaminated feed and in those living in areas with high rates of aflatoxin exposure (Khan et al., 2021; Mughal et al., 2021).

Other hazards Epidemiological and experimental data suggest a synergistic effect of aflatoxin and hepatitis B virus on hepatocellular carcinoma formation (Marchese et al., 2018).

Exposure standards and guidelines FDA recommended levels for aflatoxins in human and animal foods. Product or animal

Total aflatoxin action level (mg kg − 1)

Human food Milk Beef cattle Swine greater than 100 lb Breeding beef cattle, swine, or mature poultry Immature animals Dairy animals

20 0.5 (aflatoxin M1) 300 200 100 20 20

The FDA takes action and removes any product from the market that is found to contain aflatoxin levels at or above these limits (FDA, 2000).

Conclusion Aflatoxin exposure to humans remain a potential health hazard. Exposure can occur directly or indirectly through varied mechanisms. The aflatoxins are undoubtedly the most documented of all mycotoxins and have a wide product presence. While the aflatoxins have at times been detected in most agricultural commodities, their presence is of particular significance in corn, cottonseed, groundnuts, and tree nuts. The economics of many developing countries rely heavily on the export of specific agricultural raw materials and due to insufficient drying equipment, coupled with generally humid atmospheric conditions can lead to unacceptable levels of aflatoxins in harvested groundnuts, palm kernels, and corn. Overall, a significant proportion of the world’s variety of grains, oil seed supply and diverse agricultural products will be contaminated with the AFB1. Globally, countries are inter-dependent on each other for food grains one way or the other which opens up possibilities of migration of AFB1 contaminated food products from one part of the World to another. There have been many strategies to prevent and reduce or detoxify of aflatoxins in grains, oilseeds and miscellaneous other food products with varying levels of effectiveness (Mutegi et al., 2013; Rushing and Selim, 2019; Valencia-Quintana et al., 2020; Fan et al., 2021; Jiang et al., 2021; Jallow et al., 2021; Khan et al., 2021; Yadav et al., 2021).

References Allameh A, et al. (1992) Piperine, a plant alkaloid of the piper species, enhances the bioavailability of aflatoxin B1 in rat tissues. Cancer Letters 61(3): 195–199. Benkerroum N (2020a) Aflatoxins: Producing-molds, structure, health issues and incidence in Southeast Asian and Sub-Saharan African countries. International Journal of Environmental Research and Public Health 17(4): 1215. Benkerroum N (2020b) Chronic and acute toxicities of aflatoxins: Mechanisms of action. International Journal of Environmental Research and Public Health 17(2): 423. Dashwood RH, et al. (1991) Promotion of Aflatoxin B1 carcinogenesis by the natural tumor modulator indole-3-carbinol: influence of dose, duration, and intermittent exposure on indole-3-carbinol promotional potency. Cancer Research 51(9): 2362–2365. https://pubmed.ncbi.nlm.nih.gov/1901761/. Dhakal A and Sbar E (2021) Aflatoxin Toxicity. Stat Pearls. Internet https://www.ncbi.nlm.nih.gov/books/NBK557781/. Fan Y, et al. (2021) Research progress on the protection and detoxification of phytochemicals against aflatoxin B 1-Induced liver toxicity. Toxicon 195: 58–68. https://pubmed.ncbi. nlm.nih.gov/33716068/.

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FDA (2000) FDA guidance for industry: Action levels for poisonous or deleterious substances in human. Food and Animal Feed. https://www.fda.gov/regulatory-information/search-fdaguidance-documents/guidance-industry-action-levels-poisonous-or-deleterious-substances-human-food-and-animal-feed. Huang B, et al. (2020) Aflatoxin B1 induces neurotoxicity through reactive oxygen species generation, DNA Damage, Apoptosis, and S-Phase Cell Cycle Arrest. International Journal of Molecular Sciences 21(18): 6517. https://pubmed.ncbi.nlm.nih.gov/32899983/. IARC Monograph (1987) IARC monographs on the evaluation of carcinogenic risks to humans. suppl. 7 In: Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs Volumes 1 to 42. Lyon: International Agency for Research on Cancer. IARC (2002) IARC Report. https://monographs.iarc.who.int/wp-content/uploads/2018/06/mono82.pdf. - IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Jallow A, et al. (2021) Worldwide aflatoxin contamination of agricultural products and foods: From occurrence to control. Comprehensive Reviews in Food Science and Food Safety 20(3): 2332–2381. https://pubmed.ncbi.nlm.nih.gov/33977678/. Jiang Y, et al. (2021) Aflatoxin in dairy cows: Toxicity, occurrence in feedstuffs and milk and dietary mitigation strategies. Toxins (Basel) 13(4): 283. https://pubmed.ncbi.nlm.nih.gov/ 33920591/. Khan R, et al. (2021) Aflatoxin biosynthesis, genetic regulation, toxicity, and control strategies: A review. Journal of Fungi (Basel) 7(8): 606. https://pubmed.ncbi.nlm.nih.gov/34436145/. Maleki F, et al. (2015) Exposure of infants to Aflatoxin M1 from mother’s breast milk in Ilam, Western Iran. Osong Public Health and Research Perspectives 6(5): 283–287. https:// pubmed.ncbi.nlm.nih.gov/26929911/. Marchese S, et al. (2018) Aflatoxin B1 and M1: Biological properties and their involvement in cancer development. Toxins 10(6): 214. IARC Monograph (1993) Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins. Lyon: IARC. Mughal MJ, et al. (2021) Aflatoxin B 1 induced systemic toxicity in poultry and rescue effects of selenium and zinc. Biological Trace Element Research 178(2): 292–300. https:// pubmed.ncbi.nlm.nih.gov/28064414/. Mutegi C, et al. (2013) Incidence of aflatoxin in peanuts (Arachis hypogaea Linnaeus) from markets in Western, Nyanza and Nairobi Provinces of Kenya and related market traits. Journal of Stored Products Research 52: 118–127. https://www.sciencedirect.com/science/article/pii/S0022474X12000793. Palmgren MS and Ciegler A (1983) Aflatoxins. Plant and Fungal Toxins. In: Keeler RF and Tu AT (eds.) Handbook of Natural Toxins. vol. 1, pp. 299–323. New York: Marcel Dekker. ch 8. Pickova D, Ostry V, Toman J, and Malir F (2021) Aflatoxins: history, significant milestones, recent data on their toxicity and ways to mitigation. Toxins 13(6): 399. Publication, I.O. & The International Agency for Research on Cancer (2002) Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene. France: World Health Organization, 171–284. Rieswijk L, et al. (2016) Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma. Toxicology 350-352: 31–39. Rushing BR and Selim MI (2019) Aflatoxin B1: A review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification method. Food and Chemical Toxicology 124: 81–100. https://pubmed.ncbi.nlm.nih.gov/30468841/. Smith LE, Prendergast AJ, Turner PC, Humphrey JH, and Stoltzfus RJ (2017) Aflatoxin exposure during pregnancy, maternal anemia, and adverse birth outcomes. The American Journal of Tropical Medicine and Hygiene 96(4): 770–776. Supriya C, Girish BP, and Reddy PS (2014) Aflatoxin B1-induced reproductive toxicity in male rats. International Journal of Toxicology 33(3): 155–161. Valencia-Quintana R, et al. (2020) Environment changes, aflatoxins, and health issues, a review. International Journal of Environmental Research and Public Health 17(21): 7850. https://pubmed.ncbi.nlm.nih.gov/33120863/. WHO (2017) Evaluation of certain contaminants in food: eighty-third report of the Joint FAO/WHO Expert Committee on Food Additives. Geneva, Switzerland: World Health Organization. Yadav N, et al. (2021) An overview of nanomaterial based biosensors for detection of Aflatoxin B1 toxicity in foods. Food and Chemical Toxicology 152: 112201. https://pubmed.ncbi. nlm.nih.gov/33862122/. Zhou Y, et al. (2022) Aflatoxin B1 induces microglia cells apoptosis mediated by oxidative stress through NF-kB signaling pathway in mice spinal cords. Environmental Toxicology and Pharmacology 90: 103794. https://pubmed.ncbi.nlm.nih.gov/34971797/.

Relevant websites www.micromedexsolutions.com :IBM Micromedex. Aflatoxins https://pubchem.ncbi.nlm.nih.gov/compound/Aflatoxin-B1 :WHO, Aflatoxin B1 (compound) https://www.who.int/foodsafety/FSDigest_Aflatoxins_EN.pdf :WHO Food Safety Digest: Aflatoxins

Aging Huihui Wanga,∗, Yiying Bianb,∗, Siqi Yub, Tong Sua, Hongbin Wanga, Yuanyuan Xua, and Jingbo Pib, aGroup of Chronic Disease and Environmental Genomics, China Medical University, Shenyang, China; bProgram of Environmental Toxicology, China Medical University, Shenyang, China © 2024 Elsevier Inc. All rights reserved.

Introduction Hallmarks of aging Genomic instability Telomere attrition Epigenetic alteration Loss of proteostasis Dysregulated nutrient sensing Mitochondrial dysfunction ROS and mitochondrial dysfunction Mitochondrial integrity and biogenesis Mitohormesis Cellular senescence Stem cell exhaustion Altered intercellular communication Aging-related disorders Factors affecting aging Genetic factors Diseases Chronic diseases and treatments Infections Drugs and drug abuse Lifestyle Smoking Alcohol consumption Physical activity Circadian clocks Environmental exposures Radiation and ultraviolet Chemicals exposure Psychosocial stress and depression Prevention/intervention Disease intervention and use drugs properly Healthy lifestyle Eradication of tobacco use Restriction of drinking Regular physical activity Regular circadian clocks Restriction of nutrition Keep in a good mood Reduce exposure to harmful environmental factors Summary References

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Abstract Aging is a phenomenon in which the body’s physiological and psychological adaptability to the environment gradually decreases and tends to total loss. The hallmarks of aging include genomic instability, telomere attrition, epigenetic alteration, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and

∗

These authors contributed equally to this work.

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https://doi.org/10.1016/B978-0-12-824315-2.00070-1

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altered intercellular communication. Aging is an independent risk factor for many chronic disorders, such as cardiovascular diseases, malignant neoplasms and neurological diseases. In contrast, various diseases, genetic defects, poor lifestyle choices, environmental exposures, psychosocial stress and depression are known factors accelerating aging and thus could be intervention targets against aging and/or aging-related disorders.

Keywords Aging; Environmental exposure; Epigenetic alteration; Genomic instability; Loss of proteostasis; Mitochondrial dysfunction; Psychosocial stress; Telomere attrition

Key points

• • • •

The primary theories and key hallmarks of aging are summarized. The major factors affecting aging and aging-related diseases are introduced. The major environmental exposures affecting aging are highlighted. The complex nature and intervention strategy of aging are emphasized.

Introduction Aging is a phenomenon by which the body’s physiological and psychological adaptability to the environment gradually decreases and tends to be lost. Essentially, aging is an inevitable result of time in many pathological, physiological and psychological processes. Aging can be divided into two types: physiological or pathological aging. The former refers to a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death, while the latter refers to aging changes caused by various external factors, including various diseases. In practice, it is difficult to distinguish between the two due to the complex nature of aging.

Hallmarks of aging Hundreds of theories about aging have been proposed over the years. In 1889, Weismann put forward the wear and tear theory (Weismann, 1889), which was subsequently followed by a duration of life concept (Loeb and Northrop, 1917; Alpatov and Pearl, 1929), a theory of rate-of-living (Pearl, 1928) and then a cross-linking theory (Bjorksten, 1942). The free radical theory was initially proposed by Harman in 1956. In this theory, it is believed that aging is probably due to the accumulation of harmful effects of various biochemical free radicals, mainly oxygen free radicals, on biological tissues, and the antioxidant system cannot counterbalance the excessive accumulation of these free radicals (Harman, 1956). Later, an updated theory, the mitochondrial theory of free radicals was proposed by Mique and co-workers in 1980. They put forth the mitochondrial free radical aging theory based on the process of mitochondrial electron transport chain (Miquel et al., 1980). According to this notion, senescence is the result of mitochondrial damage caused by reactive oxygen species (ROS) after mitosis, a process verified in many laboratories. The free radical-related theories of aging have been successfully used to explain most aging related phenomena, including the loss of immune response, lipofuscin accumulation, somatic cell mutation, and macromolecular error overproduction. Beyond this, the aging telomere theory was proposed that when a cell divides to produce a new cell, the telomere at the end of the chromosome will become shorter until the telomere reaches a critical length leading to cell senescence. Therefore, the telomere will become shorter with the aging of the cell and the rate of telomere loss becomes a measure of biological aging (Oeseburg et al., 2010; Olovnikov, 1973; Olovnikov, 1996).

Genomic instability Significance evidence shows that genomic instability accelerates during aging in various ways. When subjected to endogenous or exogenous physical, chemical, biological and other stimuli, the genome can be damaged, which can be reflected in many aspects, including errors in nuclear DNA, mitochondrial DNA, and nuclear structure (Hoeijmakers, 2009). If the damage cannot be repaired by DNA repair mechanisms, it can accumulate and promote aging. Somatic mutations associated with aging were mainly found in nuclear DNA and mitochondrial DNA. Mitochondrial DNA lacks protective histones and has limited repair efficiency, so it is a prime target for somatic mutations (Linnane et al., 1989). Studies have shown that mitochondrial mutations in senescent cells are often caused by replication errors early in life, rather than by oxidative damage (Ameur et al., 2011). In addition, changes in the structure of the nuclear layer, the production of an abnormal progesterone subtype A (Ragnauth et al., 2010; Scaffidi and Misteli, 2006) and decreased levels of lamin B1 (Freund et al., 2012) can be seen during aging. Therefore, it could be feasible to delay aging via intervention which strengthen the stability of genomes in the nucleus and mitochondria.

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Telomere attrition Normal aging in mammals is accompanied by telomere shortening. This phenomenon was first discovered by Hayflick in 1961 through use of in vitro cultured fibroblasts cells with limited proliferation, known as replication senescence (Hayflick and Moorhead, 1961). Every time cell mitosis occurred, a segment of the telomere sequence was lost, and when the telomere length shortens enough, the cell stops dividing. This leads to aging and eventual. In addition, telomerase activity is also associated with aging (Oeseburg et al., 2010). Telomerase is a reverse transcriptase found in most tissues and cells, such as germ cells and inflammatory cells. There is evidence that elevated telomerase slows aging (Jaskelioff et al., 2011). Therefore, telomere length and telomerase activity are related to cell senescence.

Epigenetic alteration Epigenetic changes, including DNA methylation, histone modification, and chromatin remodeling, always occur in aging. DNA methylation levels decline with aging (Maegawa et al., 2010). Epigenetic changes are secondary to telomere shortening and are often reversible. Methylation of histones is found during aging in invertebrates. Deletion of the histone methylation complex and inhibition of the histone methylase have been shown to extend lifespan (Greer et al., 2010; Siebold et al., 2010; Jin et al., 2011). In addition, DNA hypomethylation occurs with aging, but there has been no direct experimental evidence that altering DNA methylation patterns can prolong life. Epigenetic remodeling of chromatin is also functionally associated with aging. Epigenetic changes in normal or pathological aging can cause changes in chromatin structure such as loss or redistribution of heterochromatin. In addition, key chromatin proteins (e.g., heterochromatin 1aHP1a) or chromatin remodeling factors (e.g., polycomb group protein or NuRD complex) are found to decrease in senescent cells (Pegoraro et al., 2009; Pollina and Brunet, 2011). Thus, the link between epigenetics and aging also provides the theoretical basis for a new anti-aging therapy involving reversing epigenetic changes, although the specificity of any such intervention might be a critical step that would be hard to achieve at present.

Loss of proteostasis The development of aging and associated diseases is often related to an imbalance of protein homeostasis, and damaged proteins often help in the recognition of aging. Protein stability is influenced by molecular chaperones and proteolytic systems, including autophagy-lysosome and ubiquitin-proteasome systems. When proteins are misfolded or unfolded, they have been associated with several age-related pathological processes such as Alzheimer’s disease, Parkinson’s disease and cataracts (Powers et al., 2009). The proteolytic system can then eliminate these proteins. Chaperone-mediated protein stability and proper folding and specific chaperone synthesis of cytoplasm and organelles are lost during aging (Calderwood et al., 2009). In addition, two quality control systems for protein homeostasis also decline in function during aging (Rubinsztein et al., 2011; Tomaru et al., 2012). This also supports the idea that disrupted protein homeostasis is a feature of aging.

Dysregulated nutrient sensing There is strong evidence that reducing nutritional signals can extend life. Altered insulin/IGF-1 (IIS) pathway, AMPK pathway, mTOR pathway, and FOXO pathway have been found to extend the lifespan of drosophila and mice. IIS is one of the most conserved aging control pathways in evolution. Studies have shown that IIS downregulates a defensive response that regulates cell growth and metabolism in the context of system damage. FOXO and mTOR, which is the target of IIS, are also conserved in evolution (Barzilai et al., 2012; Fontana et al., 2010; Kenyon, 2010). In fact, down-regulation of IIS signals, nutrient abundance and metabolic synthesis contribute to healthy aging. Alternatively, up-regulation of AMPK and sirtuins signals, nutrient deficiency and catabolic metabolism contribute to healthy aging (Lopez-Otin et al., 2013).

Mitochondrial dysfunction Mitochondrial function has a profound impact on the aging process, and mitochondrial dysfunction accelerates aging in mammals.

ROS and mitochondrial dysfunction The mitochondrial free radical theory of aging proposes that the progressive mitochondrial dysfunction that occurs with aging leads to increased ROS production, leading to further mitochondrial deterioration and cell damage (Harman, 1965). However, other studies have shown that ROS can trigger proliferation and survival in response to physiological signals and stress conditions, and the main role of ROS is to activate compensatory homeostasis responses. As time-related aging increases, both cell stress and damage increase, and ROS levels increase synchronously to maintain survival. However, above a certain threshold ROS levels deviate from their original homeostatic purpose and result in exacerbating rather than mitigating age-related damage (Hekimi et al., 2011).

Mitochondrial integrity and biogenesis Increased damage and reduced turnover in mitochondria may contribute to the aging process due to lower biosynthesis and reduced clearance. Mitochondrial dysfunction accelerates aging in a variety of ways, such as influencing apoptosis, interfering with

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intracellular signaling, and triggering inflammatory responses (Kroemer et al., 2007; Green et al., 2011; Raffaello and Rizzuto, 2011). Interestingly, endurance training and alternate-day fasting can improve a healthy lifespan by avoiding mitochondrial degradation, possibly in part through autophagy induction, but autophagy induction is not the only mechanism by which a healthy lifestyle delays aging (Castello et al., 2011; Safdar et al., 2011).

Mitohormesis The relationship between dysfunction and excitement during aging have been investigated extensively. Findings include that severe mitochondrial dysfunction is disease-causing, but mild respiratory dysfunction may prolong life (Haigis and Yankner, 2010). This process is involved in mitochondrial excitation. For example, metformin, a mild mitochondrial toxin, delays the aging of nematodes and prolongs life span through nuclear factor erythroid-related factor 2 (NRF2)-mediated compensatory stress (Onken and Driscoll, 2010). Secondly, mitochondrial uncoupling can also increase the life span of drosophila and mice. However, it is not clear whether extend life can be accomplished by improving mitochondrial function (Caldeira Da Silva et al., 2008; Fridell et al., 2009). Because mitochondrial dysfunction is the broad basis for various other cellular processes, some researchers believe that mitochondrial dysfunction is not a biomarker of aging.

Cellular senescence It is widely believed that cellular senescence is the cause of body aging because the number of senescent cells increases with age. However, cellular senescence is not a common feature of aging tissues. Cellular senescence, a phenomenon first identified by Hayflick using in vitro cultured fibroblasts, is associated with telomere shortening (Hayflick and Moorhead, 1961). Importantly, there are also some aging processes unrelated to telomere shortening, such as inhibitor of CDK4/alternative reading frame (INK4a/ ARF) site and p53-induced aging. Cell senescence induced by INK4a/ARF sites and p53 is considered to be a beneficial compensatory response. Nevertheless, cell senescence will become harmful and exacerbate overall senescence as cell loss increases and tissue regeneration becomes exhausted (Collado et al., 2007).

Stem cell exhaustion One of the most obvious features of aging is a decline in tissue regeneration. For example, immune senescence causes hematopoietic function decrease with age, leading to the decrease of immune cell production and the increase of myeloid tumors (Shaw et al., 2010). Stem cell depletion has been found in almost all adult stem cells (Molofsky et al., 2006; Gruber et al., 2006; Conboy and Rando, 2012). Stem cell failure is the result of a multitude of age-related damage, including telomere shortening, accumulation of DNA damage and overexpression of cycle suppressor proteins, and clearly one of the culprits of tissue and organ aging (Rossi et al., 2007; Janzen et al., 2006). Although insufficient stem cell proliferation is not conducive to the maintenance of the organism, excessive proliferation will accelerate stem cell depletion, resulting in harmful effects (Rera et al., 2011). Therefore, it makes sense to explore drugs to improve stem cell function and thereby delay aging. This concept is feasible because studies have shown that stem cell regeneration reverses aging phenotypes at the organ level (Rando and Chang, 2012).

Altered intercellular communication Senescence also has to do with cell communication. Because the aging of one organ can lead to the aging of other organs, while conversely anti-aging operations for one tissue can also improve the aging of other tissues. There are many different cell-cell communication pathways involved in aging, of which inflammatory the senescence-associated secretory phenotype (SASP) is the most typical one (Birch and Gil, 2020). Neurodegenerative diseases such as Alzheimer’s and Parkinson’s are associated with chronic inflammation. Given all this, it makes sense to repair cellular communication to delay aging. Fortunately, studies have found that genetic, pharmaceutical and nutritional interventions that can ameliorate these cellular communication deficits (Freije and Lopez-Otin, 2012; Rando and Chang, 2012). In summary, there are nine most recognized hallmarks of aging, which include four major markers (genomic instability, telomere wear, epigenetic changes, and protein homeostasis imbalances) that are responsible for aging damage, three antagonistic markers (downregulation of nutritional perception, mitochondrial dysfunction, and lower senescence) corresponding to injury in a hormetic manner, and two comprehensive markers (stem cell depletion and changes in intercellular communication) directly impacting tissue homeostasis and function (Fig. 1).

Aging-related disorders Many diseases are related to aging. Twenty-three percent of the total global burden of disease is found older populations (people aged 60 years and older). Of particular note, cardiovascular diseases, malignant neoplasms, chronic respiratory disease, neurological and mental disorders account for 30.3%, 15.1%, 9.5%, 7.5% and 6.6% of disease burden, respectively in those aged populations (Prince et al., 2015). It is believed that aging can deteriorate the dysfunction or disorders of multiple organ systems

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Fig. 1 The key hallmarks of aging include four major markers that are responsible for aging damage, three antagonistic markers corresponding to injury in a hormetic manner, and two comprehensive markers directly impacting tissue homeostasis and function.

throughout the body, including the cardiovascular system, skin, liver, lung, kidney and urinary system, eyes, nervous system and endocrine system. Particular disease examples impacted by aging are summarized below: a. Cardiovascular diseases (CVD): It is well known that the morbidity of CVD increases with age including coronary artery disease (CAD), myocardial ischemia reperfusion injury (MIRI), and ischemic heart disease. Among these ischemic heart disease accounts for 77.7 million Disability Adjusted Life Years (DALYs) in older people in 2010. DALY burden in older people was forecast to increase by 34.7% from 2004 to 2030 (Prince et al., 2015). b. Malignant neoplasms: The expected increase in cancer incidence in aged persons will have substantial economic and social impacts globally. Among men aged 85 years and older, prostate and lung cancers are the most common causes of cancer death. While in women, breast cancer is the leading cause of cancer death (Jemal et al., 2011). c. Chronic respiratory disease: There is a progressive deterioration in lung function with age, resulting in an increased prevalence of chronic obstructive pulmonary disease (COPD). COPD accounted for 43.3 million DALYs in older people in 2010. The global burden was forecast to increase by 89% from 2004 to 2030, and now is the 3rd leading cause of death worldwide (World Health Organization, 2020). d. Neurological disease: Various neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Lewy body dementia and amyotrophic lateral sclerosis (ALS), are clearly related to aging. It is worth mentioning that Alzheimer’s disease in about 90% of cases affects individuals from the age of 65 (Herrup, 2015) and its prevalence doubles each 5 years, generating a time-dependent exponential increase (Querfurth and LaFerla, 2010). e. Metabolism disorders: Endocrine system diseases, type 2 diabetes mellitus (T2DM) in particular, and diabetic complications, including diabetes macroangiopathy, diabetic retinopathy (DR), diabetic nephropathy (DN), diabetic foot ulcers (DFU), and diabetic peripheral neuropathy (DPN), develop with the accumulation of senescent cells. Diabetes mellitus accounted for 22.6 million DALYs in older people in 2010 which is a huge challenge to the economy and society. The burden on older people is forecast to increase by 96% from 2004 to 2030 (Prince et al., 2015). f. Articulations: Articulations can be affected by aging, inducing osteoarthritis (OA) and intervertebral disc degeneration (IDD). Fully 30–50% of adults over the age of 65 years suffer from OA (Shane Anderson and Loeser, 2010), and its prevalence increases with age. In addition, OA often occurs in multiple joints including hands, spine, hips and knees and over 80% adults who have OA have it in more than one joint. g. Other chronic diseases: Other common age-related organ and system dysfunction seen include aged skin, liver impairment, renal dysfunction and visual impairment. Aged skin appears thin, finely wrinkled, with uneven pigmentation and brown spots (lentigines), lax, as well as leathery. Aging can impair liver and kidney function, inducing dysfunction. Furthermore, age-related visual impairment accounted for 10.4 million DALYs among older people in 2010. Visual impact of aging includes age-related macular degeneration (AMD), glaucoma and cataracts. Globally, AMD ranks third as a cause of blindness after cataract and glaucoma. Furthermore, advanced AMD is rare before the age of 55, and much more common in persons of 75 years and older. Furthermore, the highest prevalence of advanced AMD occurs after 80-years old (Prince et al., 2015).

Factors affecting aging Aging is the time-dependent functional decline and decreased adaptability of a living organism to external stimuli that can be accelerated by various factors including genetics, disease status and drug intake, unhealthy lifestyle, environmental exposures and psychological disorders (Fig. 2A).

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Fig. 2 The diagram of factors affecting aging and possible intervention strategies. (A) Factors affecting aging includes genetic factors, various diseases, unhealthy lifestyles, environmental exposures as well as psychosocial stress and depression. (B) Point-to-point intervention strategies targeting disease prevention and proper treatment, healthy lifestyle, and reducing exposure to harmful environmental factors.

Genetic factors Recent numerous studies have explored the relationship between genes and aging. For example, Hutchinson-Gilford Syndrome (HSPS) caused by lamin A/C gene mutation is a congenital genetic disorder, which appears to be progressive geriatric degeneration in infancy. In contrast, 35 cloned genes, such as longevity assurance gene 1 (LAG1), longevity-assurance gene cognate (LAC1), oncogene (RAS1 and RAS2), prohibitins (PHB1 and PHB 2), have been identified that determine longevity in the model organisms and even mammals (Jazwinski, 2000).

Diseases Chronic diseases and treatments Many chronic diseases can accelerate aging. For example, it has been suggested that cancer can contribute to the aging process (Calcinotto et al., 2019). Furthermore, chemotherapy can accelerate immunosenescence in post-chemotherapy patients. HIV, depression and other psychosocial stress, as well as chronic infection and chronic inflammatory diseases, such as arthritis, atherosclerosis, celiac disease, vasculitis, COPD, lupus, and psoriasis, have been found accelerating aging (Cho et al., 2019; Jimenez et al., 2018; Swinburn et al., 2005; Berti et al., 2019; Wang and Bennett, 2012; Alexopoulos, 2005).

Infections In accordance with chronic infections, Gavazzi and Krause (2002) pointed to a vicious circle: aging might cause infections; the infection could in turn accelerate the aging process. The underlying mechanisms include pathogen-dependent tissue destruction, accelerated cellular aging through increased turnover and the enhanced inflammation. Within the review of Cohen and Torres (2017) it was noted that HIV-infected patients suffered from accelerated aging. One possible contributor to this process was likely increased cellular senescence and the other was alteration in gut microbiota composition diversity and function, both which occur in HIV patients as in aging (Desai and Landay, 2018). Furthermore, studies have shown that aging is related to COVID-19 and its adverse health outcomes from ICU admission to death (Chen et al., 2021).

Drugs and drug abuse Several anticancer drugs, including aphidicolin, cisplatin, carboplatin and many others, incite cellular senescence in tumor and other normal tissues (Ewald et al., 2010). Indeed, other clinical drugs, such as D-galactose, amphetamines and opioids, exacerbate aging by increasing oxidative stress in multiple organs such as in the kidney and brain (Womack and Justice, 2020; Azman and Zakaria, 2019). In contrast, some drugs such as resveratrol, rapamycin and others show reverse effects and can extend lifespan (Selvarani et al., 2021; Li et al., 2018). Approximately 35 million people have drug addiction and/or need treatment for addiction (World Drug Report, 2019). The usage of addictive substances including amphetamine and its congeners, cannabis, cocaine and opiates has been demonstrated to accelerate aging by increasing oxidative stress, cell senescence, and other aging-related processes (Sarafian et al., 2006; Sekine et al., 2008; Pomierny-Chamiolo et al., 2013).

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Lifestyle Bad living habits such as poorly constructed diet, irregular life style, lack of exercise, excessive fatigue, uncontrolled smoking or drinking, bad eating and poor hygiene habits all can accelerate aging.

Smoking The smoking of cigarettes represents an important accelerator of the aging process, both directly through complex mechanisms mediated prevalently by excessive formation of free radicals, and indirectly by favoring the appearance of various pathologies in which cigarette smoke is a recognized risk factor. The WHO has reported that cigarette smoking is responsible for the shortening of life expectancy by an average of 7 years and of disease-free life time by 14 years (WHO, 2004). In addition, metals such as lead, arsenic and cadmium in tobacco can lead to tissues and organism aging.

Alcohol consumption Excessive alcohol intake is another factor contributing to early aging and aging-related diseases such as cancer and cardiovascular disease (WHO, 2018). According to WHO, about 1.8 million deaths around the world are due to excessive alcohol consumption. Some studies have found that alcohol is related to telomere length shortening, epigenetic aging, and disruption of bone marrow-derived mesenchymal stem cells.

Physical activity The appropriate amount of physical activity has benefits for multiple organs and systems, in both healthy people and people with diseases like diabetes, Alzheimer’s disease, and depression. High physical activity turns out to be correlated with telomere length and telomerase activity and can slow down aging. While endurance runners show a contrary phenomenon-shorter telomere length. In addition, exercise has a positive effect on the immune system and muscle mass (Larzelere et al., 2011).

Circadian clocks The circadian clock, which is important for maintaining tissue homeostasis, affects several processes associated with aging and is closely intertwined with several signaling pathways linked to aging. Furthermore, aging reduces the stability of circadian rhythms, which can change behavior and molecular rhythms. These circadian clock-dependent behavioral and molecular changes in turn further accelerate the process of aging (Welz and Benitah, 2020).

Environmental exposures The impacts of environmental exposure on aging can be complicated, and can include the rate of aging, the health status of the aged, and the response of the aged compared with the young (National Research Council (U.S.). Committee on Chemical Toxicity and Aging, 1987). Age-related diseases (e.g., cancer, atherosclerosis, diabetes, osteoarthritis, osteoporosis, cataracts, hearing loss, amyotrophic lateral sclerosis, Parkinson’s disease, and senile dementia of the Alzheimer’s type) are often coupled with underlying environmental exposures (Liu et al., 2021). Environmental exposures are mainly divided into physical pollution, chemical exposure and psychosocial factors exposure, for which here we have sorted out distinct aspects below. However, different environmental exposure factors may well be mixed. For example, physical and chemical factors may exist simultaneously in air pollution and water pollution.

Radiation and ultraviolet Radiation is a major factor affecting human aging in the environment and can affect aging in various ways. Radiation is mainly divided into two types: ionizing radiation and non-ionizing radiation. The a-rays, b-rays, g-rays, X-rays, protons and neutrons belong to ionizing radiation, while infrared, ultraviolet (UV), microwave and laser belong to non-ionizing radiation. The effect of ionizing radiation on longevity has been vigorously studied starting in the late 1940s. Several studies have shown that radiation can accelerate aging and induce degenerative health effects and mortality (Radman, 2016; Shen and Tower, 2019; Hernández et al., 2015). More recently, radiation has been associated with a much wider spectrum of age-related diseases, including cardiovascular disease (Baselet et al., 2019). Some diseases of old age, such as diabetes, are notably absent as a radiation risk. Ionizing radiation, especially high linear-energy-transfer radiation, produces more double-strand breaks (DSBs), clustered lesions and genomic instability than endogenous sources of ROS (Richardson, 2009). Exposure of cells to ionizing radiation causes oxidative events, which change the atomic structure through direct interaction of the radiation with target macromolecules or through water-radiolysis products (Azzam et al., 2012). In irradiated cells, the levels of these reactive oxygen substances may increase due to disturbances in oxidative metabolism and chronic inflammatory responses, leading to long-term effects of exposure to ionizing radiation on genome stability (Azzam et al., 2012). Oxidative stress caused by the accumulation of ROS can cause damage to lipids, proteins, nucleic acids and organelles, causing cell aging, which is one of the core mechanisms of radiation-induced skin aging (Kammeyer and Luiten, 2015). Non-ionizing radiation (herein mainly using UV light as an example) directly damages DNA by inducing the formation of damage to DNA, typically leading to mutations (Rastogi et al., 2010). The two major types of DNA damage caused by UVB (280–315 nm) and UVC (45 mg/L) can lead to telomere length reduction and low naive T cell production (Song et al., 2019). Other studies have shown arsenic trioxide significantly induces the senescence of human articular chondrocytes by increasing the activity of senescence-associated b-galactosidase (SA-b-Gal) and the protein expression of p16, p53 and p21. Arsenic induces phosphorylation of end-stage kinase (p38 and JNK), and inhibitors of p38 and JNK significantly reverse the senescence of chondrocytes caused by arsenic. Arsenic can also increase IL-1a, IL-1b, TNF-a and CCL2 mRNA expression, trigger NF-kB signaling and the induction of SASP (Chung et al., 2020). The main exposure routes of cadmium are ingestion, inhalation, or skin absorption. The main way of occupational cadmium exposure is through inhalation of cadmium dust and smoke. For the general population, the main way of exposure to cadmium is from eating contaminated food or smoking. The toxic mechanism of cadmium to the human body is very complicated, including oxidative stress, abnormal gene expression, disturbances of signal pathways and abnormal enzyme activities (Balali-Mood et al., 2021). The length of telomeres is considered to be closely related to aging. Studies have shown that the higher the mother’s exposure to cadmium before delivery, the shorter the telomeres of the newborn’s cord blood (Khoshhali et al., 2020). Cadmium also inhibits ATPase, lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities (Mao et al., 2011). Furthermore, cadmium increases the level of ROS and lipid peroxidation in the body, thereby inducing DNA damage and interacting with DNA repair mechanisms, promoting the occurrence of aging (Méndez-Armenta et al., 2003). In addition, cadmium may also damage the function of cell mitochondria, and malfunctioning mitochondria are related to aging and many diseases (Gobe and Crane, 2010). POPs: POPs include pesticides, industrial and technical chemicals, by-products of industrial processes, etc. Among pesticides, organochlorine pesticides (OCPs) are one of the major categories of POPs in the environment. OCPs in food may cause the body to develop cardiovascular problems such as hypertension, which can accelerate aging (Lind and Lind, 2012). Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in industrial products are two kinds of POPs with similar structures, physical and chemical properties. Both of them can cause endocrine disorders, diabetes, obesity and other diseases, promoting the aging process (Blanc et al., 2021; Kaw and Kannan, 2017; Green et al., 2021). By-products of industrial processes such as polychlorinated dibenzodioxins (PCDD) and polychlorinated dibenzofuran (PCDF) cause cancer, diabetes, endocrine disorders, hypertension, glucose intolerance and cardiovascular disease. These POPs are present in the air and food, affecting the physiological functions of the organism, accelerating the disease process, as well as causing the organism to age more rapidly.

Psychosocial stress and depression Psychosocial status is strongly related to aging and longevity. Appropriate psychosocial pressure can stimulate the brain to produce a chemical substance called neurotrophic factor, strengthen the power connection between brain neurons, and appropriately delay the decline of brain function (McEwen et al., 2015). In addition, appropriate psychosocial pressure can regulate the immune system, thereby temporarily increasing the immune level (Peters et al., 2021). But excessive or constitutive psychosocial pressures have the opposite effects. Studies have shown that long-term excessive psychosocial stress can disrupt the body’s endocrine system, thereby

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making people irritable, anxious, changing their normal circadian clock, promoting the aging process. The possible mechanism is interference with an aging regulator called Klotho, a gene involved in the aging process in mammals (Kuro, 2019). In addition, some studies have shown that long-term excessive psychosocial stress can lead to various health outcomes related to aging by promoting inflammation and oxidative stress, such as depression in later life, cognitive decline, Alzheimer’s disease, atherosclerosis and metabolic syndrome (Silberman et al., 2016; Schneiderman et al., 2005). Recent epidemiological evidence indicates that excessive psychosocial stress can be considered as a risk factor for certain types of cancer and cause mitochondrial dysfunction during cellular aging (Kruk et al., 2019). Depression is a common disorder related to metabolic, cardiovascular, and neurological diseases, associating with the acceleration of aging. Depression involves altered hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoid activity, leading to excitotoxicity, diminished insulin signaling, intraneuronal gluco-privation, accumulation of intracellular calcium, telomere shortening and decrease in telomerase activity (Gold et al., 2020; Juruena et al., 2004; Xie et al., 2018). Furthermore, Han et al. have demonstrated that higher methylation aging in Major Depressive Disorder is present in both blood and brain, and that higher epigenetic aging largely overlaps with the same underpinnings associated with chronological aging (Han et al., 2018).

Prevention/intervention Aging is the main risk factor for chronic diseases and disability in human societies has a great impact on social and health care expenditures. At present aging and, eventually, death are unavoidable. Nevertheless, research efforts on aging-associated diseases to extend life span have not stopped. Studies have pointed out that a healthy lifestyle, especially regular aerobic exercise and certain dietary patterns, is considered a “first-line” strategy to prevent and/or treat aging. Some studies also show that reducing exposure to harmful chemicals in the environment can significantly delay the aging process. In addition, exposure to the right amount of sunlight may reduce breast cancer, colorectal cancer, high blood pressure, cardiovascular disease, metabolic syndrome, multiple sclerosis and other diseases closely related to aging. Several effective ways to prevent or intervene in aging are discussed below (Fig. 2B).

Disease intervention and use drugs properly Reducing the occurrence of chronic inflammation, hypertension and hyperglycemia is an effective means to reduce aging. Inflammation is now considered to be a biomarker for accelerated aging and one of the hallmarks in aging biology (Kanapuru and Ershler, 2009). For example, in contrast to acute, transient inflammation, chronic inflammation is linked to numerous age-related diseases such as cancer, type 2 diabetes, cardiovascular disease, neurodegenerative diseases and frailty (Fougère et al., 2017). Reducing inflammation can help slow down aging. Similarly, hypertension and hyperglycemia can lead to vascular endothelial injury and microvascular lesions, leading to the formation of atherosclerosis. In the absence of intervention, both hypertension and hyperglycemia can destroy the homeostasis of the internal environment, and cause multiple organ dysfunction, thus leading to the occurrence of more rapid aging (Spinas et al., 2014). Therefore, the control of blood pressure and blood sugar is an effective way to delay aging. And controlling body weight, blood lipids, reduce oxidative stress can similarly play positive roles in generally prolonging life. In the treatment of chronic diseases, drugs should be used properly: Some drugs in the process of treatment can also produce side effects, accelerating aging (Ham et al., 2020). Drugs of abuse, including cocaine, opiates, and methamphetamine, have been shown to promote pre-mature aging (Dowling et al., 2008). Recently, emerging evidence indicates that abused drugs can dysregulate autophagy which, in turn, plays a key role in neuro-inflammation (microglial/astrocyte activation). Based on the linkage between inflammation and the aging process, upregulation of neuro-inflammation due to autophagy/mitophagy dysregulation may play a critical role in pre-mature aging during chronic HIV infection in the context of abused drugs (Guo and Buch, 2019). Similarly, hormonal drugs can damage the skin barrier and inhibit the activity of immune cells, making the body susceptible to environmental hazards. It is now clear that all non-steroidal anti-inflammatory drugs (NSAIDs) are associated with varying degrees of the cardiovascular and gastrointestinal risk (Marsico et al., 2017; García-Rayado et al., 2018). And some NSAIDs may cause adverse nervous system reactions, such as dizziness, headache, tinnitus, drowsiness, insomnia, and even abnormal sensation, hallucinations, tremors, etc. (Clark and Ghose, 1992). In addition, some anti-cancer drugs will damage other organs, accelerate the other organs’ functional decline and aging. Therefore, we should carefully read the drug instructions before taking the medicine, and strictly follow the doctor’s advice. These can reduce the adverse reactions of drugs to a certain extent, and help reduce aging.

Healthy lifestyle Eradication of tobacco use

Smoking accelerates the aging process, with 10 years of “smoking” being enough to create additional aging markers. This is in part because nicotine causes narrowing of blood vessels in the outermost layer of the skin, while other chemicals in tobacco damage collagen and elastin, which are essential for skin to stay elastic (Dhall et al., 2016). In addition, smoking can also speed the decline of the function of several organs and systems, as the lung, liver and cardiovascular system, and thereby accelerate aging (MaCnee, 2009).

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Restriction of drinking Alcohol consumption leads to epigenetic changes that accelerate aging. Epigenomic association analysis (EWAS) has shown that alcohol consumption is significantly associated with DNA methylation at 24 CpG sites (Stephenson et al., 2021). In addition, some studies have also shown that alcohol consumption increases the burden of the liver, causing different degrees of liver damage resulting in a variety of hepatic diseases (Lackner and Tiniakos, 2019). Furthermore, neural lesions are correlated with consumption of alcohol (Nunes et al., 2019), and there appears to be no “safe” level of alcohol consumption. In conclusion, alcohol consumption may accelerate aging by acting at different levels, such as genetic, tissue and organ level.

Regular physical activity It’s worth noting that persons who exercise regularly have better muscle mass and hormone levels than those who do not exercise. Usually, the thymus deteriorates and the number of T cells produced decreases with aging. But studies have shown the thymus produces similar numbers of “immune weapon” T cells in aging sports enthusiasts as there are in the thymus of young people (Woods et al., 2003). Moreover, moderate overload of cellular oxidation pathways can enhance ATP synthesis ability and prevent the decline of cell reserve ability.

Regular circadian clocks Circadian rhythms are generated by intrinsic cellular mechanisms that control a large array of physiological and metabolic processes. There is erosion in the robustness of circadian rhythms during aging, and disruption of this “clock” by genetic ablation of specific genes is associated with the emergence aging-related characteristics. Importantly, environmental conditions are thought to modulate the aging process. For example, caloric restriction is a very strong effector capable of delaying aging. Intracellular pathways implicating nutrient sensors, such as silent mating-type information regulation 2 homolog (SIRTs) and mTOR complexes, impinge on cellular and epigenetic mechanisms that control the aging process (Orozco-Solis and Sassone-Corsi, 2014). Strikingly, accumulating evidence indicates that these pathways are involved in both the modulation of the aging process and the control of the clock. Hence, innovative therapeutic strategies focused on controlling the circadian clock and the nutrient-sensing pathways might be beneficial in slowing the negative effects of aging.

Restriction of nutrition Studies report that the restriction of caloric intake increase lifespan in mice, rats and primates (Swindell, 2012). Notably, dietary restriction increases not only the maximum lifespan but also suppresses the development of age-associated diseases. Studies have also elucidated the relationship between mTOR (target of rapamycin) and dietary restriction. mTOR is a versatile protein that acts as a major hub integrating signals from growth factors, nutrient availability, energy status and various stressors. These signals regulate several outputs that include mRNA translation, autophagy, transcription and mitochondrial function, and have been shown to mediate an extended lifespan (Bjedov and Rallis, 2020; Laberge et al., 2015).

Keep in a good mood A good mood means an optimistic attitude towards life and positive thinking. The psychological aspects of happiness include positive emotions, optimism and satisfaction with life, which are increasingly considered to be beneficial for the heart, vascular disease and impact longevity. Growing literature associates positive well-being with better cardiovascular health, a lower incidence of CVD in healthy people, and a reduced risk of adverse outcomes in existing CVD patients (Boehm et al., 2020; Thompson et al., 2020; Hernandez et al., 2018). In the Whitehall Phase II British Civil Service Cohort, nearly 8000 middle-aged participants answered questions about their optimism (i.e., their positive expectations for their future) and emotional vitality (that is, active participation in the world, emotional regulation, and overall feeling). Those with a higher level of happiness and satisfaction in life had a lower risk of coronary heart disease (about 20–30% reduction after 5 years) than those who were not optimistic and satisfied. Studies have shown that a good mood helps us secrete related neurotransmitters (such as dopamine, glutamate, and norepinephrine, etc.), face the world’s pressure and maintain the normal function of the nervous system (Serretti and Artioli, 2004). A good mood can also reduce the occurrence of inflammation, to a certain extent, and reduce the occurrence of inflammation-related aging (Kim et al., 2016). Although research on the mental health and health of the elderly is still at an early stage, there is evidence that the higher the happiness of the elderly, the greater the probability that their life will be longer.

Reduce exposure to harmful environmental factors In our daily lives, excessive exposure to chemical pollutants such as arsenic compounds, organophosphorus pesticides, heavy metals, and algal toxins will accelerate the aging process. In this regard, we should drink water that meets the national water quality standards as much as possible, and avoid eating contaminated seafood and animal offal, whose heavy metal content easily exceeds most standards. Agricultural workers should pay attention to their protection when spraying pesticides, including as wearing appropriate approved masks and protective clothing. Before cooking, generally food should be washed to prevent a large number of chemical contaminants from entering the body through the digestive tract. Through the above several measures, the influence of environmental chemical pollutants on the body’s aging can be attenuated.

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Summary Although multiple theories of aging have been proposed over time, none of them can universally explain all the phenomena seen in aging. Furthermore, factors affecting aging are complex, increasing difficulty of aging prevention and intervention. From a toxicological point of view, older people are often more susceptible than young people to chemical toxicity, including from toxicants in the environment, or toxicity after administration of medicines. More attention should be given to aging research and geriatric toxicology.

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Air pollution: Sources, regulation, and health effects PF Duffney, LW Stanek, and JS Brown, U.S. Environmental Protection Agency, Office of Research and Development, Center for Public Health and Environmental Assessment, Research Triangle Park, NC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of Lindsay Wichers Stanek, James S. Brown, Air Pollution: Sources, Regulation, and Health Effects, Reference Module in Biomedical Sciences, Elsevier, 2019, ISBN 9780128012383, https://doi.org/10.1016/B978-0-12-801238-3.11384-4. Disclaimer: The views expressed in this book chapter are those of the author(s) and do not necessarily represent the views or the policies of the U.S. Environmental Protection Agency.

Types and sources of air pollutants Regulation of air pollution Health effects of air pollution Ozone Sulfur oxides Particulate matter Nitrogen oxides Carbon monoxide Lead Traffic related pollution Future directions and control strategies References Further reading

215 216 217 218 219 221 222 223 223 225 225 226 228

Abstract Air pollution results from both natural (e.g., fires, volcanoes, and wind-blown dust) and anthropogenic sources. The particles and gases that comprise air pollution are known to cause adverse health effects in humans. Importantly, subpopulations of people such as those in different life stages (older adults and children) and people with preexisting disease (e.g., asthmatics), or some sociodemographic characteristics (e.g., minority populations) have been shown to be at greater risk from exposure to inhaled ambient air pollutants. To reduce the disease burden related to ambient air pollution, regulations have been promulgated in many countries throughout the world. Evidence from epidemiology and toxicology studies suggests that reductions in air pollution levels yield improvements in health outcomes.

Keywords Air Quality Index; Air toxics; Carbon monoxide; Carboxyhemoglobin; Clean Air Act; Clinical studies; Emissions; Environmental Protection Agency; Fossil fuels; Gasoline; Gas–particle interactions; Lead; National ambient air quality standards; Nitrogen oxides; Nonattainment; Ozone; Particulate matter; Stationary and mobile sources; Sulfur dioxide; Temperature inversion

Types and sources of air pollutants Both anthropogenic and natural sources contribute to the particles and gases that pollute our ambient environment. Episodic natural events such as fires, wind erosion, dust storms, and volcanic eruptions produce considerable amounts of particulate matter (PM) and gases including mineral ash, soil, pyrolysis products of combustion, carbon monoxide, and carbon dioxide. Although these ‘natural’ particles and gases can have significant global effects such as short- and long-term alterations in weather conditions and climate, little can be done to alter the contribution of pollutants from natural sources. The continued interest in understanding the generation and health effects of anthropogenic air pollutants stems largely from the fact that measures can be taken to control these pollutants and thus reduce the associated adverse health effects. Such control measures are often promulgated and regulated at the governmental level based on comparisons of the tangible (e.g., increased morbidity and mortality, financial) and intangible (e.g., quality of life) costs to society and the impact on the environment (e.g., animals, vegetation, and climate) versus the cost of implementing control measures. Both stationary and mobile sources contribute to PM and gases that make up polluted urban and rural environments. Electric utilities and power plants represent the major stationary point sources of pollution. Industrial processing plants such as smelters and waste disposal and recycling facilities also produce a wide range of PM and gases (EPA, 2021a). Because these pollution sources are stationary, significant differences in both quantity and makeup of regional air pollution have been observed. For example, sulfur

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present in fossil fuels (primarily in coal) that have historically been used for power production and heating in the northeastern and midwestern United States (U.S.) resulted in the acid components of ambient air particles in this region being primarily sulfates. A major portion of ambient air pollution results from gasoline- and diesel-powered automobile and truck emissions. The mobile and ubiquitous nature of motor vehicles makes their pollution products widespread in developed countries. While the presence of lead (Pb) in automobile exhaust has been eliminated globally with Algeria stopping sales of leaded automotive fuel July 2021 (Rukikaire, 2021), the contribution of motor vehicles to ambient concentrations of other pollutants like nitrogen oxides, hydrocarbons, and carbon monoxide in urban atmospheres is great. Indeed, transportation sources were responsible for 54% of the nitrogen oxides and 63% of the carbon monoxide emissions in the U.S. in 2020 (EPA, 2020c). In developing countries throughout Asia and Africa, where biomass burning is prevalent, transportation sources make up a smaller fraction of the total emissions. Sunlight can drive a series of chemical reactions involving nitrogen oxides and hydrocarbons (a process known as photochemical oxidation) which result in secondary pollutants such as ozone. Ambient ground level ozone is a major health concern for both urban and rural dwellers. Ozone can also have ecological effects including visible foliar injury, reduced plant growth, reproduction and yield, as well as reductions in agricultural crop yield (e.g., soybeans) and tree growth, in particular in cottonwoods and the black cherry [see Chapter 8 of EPA (2020b)]. Regardless of the source of primary and secondary air pollutants, meteorological conditions play a significant role in the formation and transport of gases and PM. One well-documented historical example occurred when sulfur dioxide released from industrial point sources in the northeastern U.S. formed acidic aerosols. These acidic precursors underwent long-range transport via a high-pressure air system resulting in sulfate and strong acidic aerosol concentrations in rural southeastern Canada where no local sources were present (Smith-Palmer and Wentzell, 1986). More recently, transcontinental transport has been documented for PM, ozone, and ozone precursors. Dust clouds derived from storms in Asia are known to be transported to other continents [e.g., Jaffe et al., 2005; Uno et al., 2011]. Asian anthropogenic emissions can be transported across the Pacific Ocean and have been recognized to contribute as much as 5–7 ppb ambient ozone surface concentrations in the western U.S. (Zhang et al., 2008). Thus, movement of masses of air can reduce ambient levels of pollutants in one region at the expense of air quality in another region. Meteorological conditions also influence the creation of photochemical smog. Prolonged stagnation of air masses can lead to the accumulation of primary and secondary pollutants. Inversions occur when cooler air is trapped beneath a blanket of warm air, resulting in stagnant weather patterns. In southern California and valleys, for example, the combination of inversions, sunlight, and motor vehicle emissions drives the photochemical reaction of trapped precursors and often results in high ozone concentrations.

Regulation of air pollution Reductions in ambient concentrations of some, but certainly not all, air pollutants have taken place over the last several decades. The situation in the U.S. will be used as example throughout the article; similar patterns have been observed in Europe and other developed countries. In the U.S., the Environmental Protection Agency (EPA) is the primary federal agency responsible for promulgating and regulating air pollution standards. National Ambient Air Quality Standards (NAAQS) have been established for six outdoor air pollutants (i.e., the criteria pollutants): lead, carbon monoxide, ozone (indicator for a mix of photochemical oxidants formed from natural and anthropogenic precursor emissions), nitrogen dioxide (indicator for nitrogen oxides), sulfur dioxide (indicator for sulfur oxides), and PM. The NAAQS (Table 1) are periodically reviewed and updated as appropriate, based on available scientific Table 1

National ambient air quality standards.

Pollutant

Primary standard level

Averaging timea

Nonattainment populationb

Carbon monoxide

35 ppm (40 mg m−3) 9 ppm (10 mg m−3) 0.15 mg m−3 0.053 ppm 0.100 ppm 0.070 ppmc 0.12 ppmd 12 mg m−3 35 mg m−3 150 mg m−3 0.075 ppm

1h 8h Rolling 3-month average Annual average 1h 8h 1h Annual average 24 h 24 h 1h

0

Lead Nitrogen dioxide Ozone PM2.5 PM10 Sulfur dioxide

9.8 0 133.3 22.0 8.6 1.8

a

Each standard also has a form which defines the air quality statistic that is to be compared to the level of the standard in determining whether an area attains the standard. Millions of persons living in counties with air quality levels not meeting NAAQS, based on 2021 population estimates and rounded to the nearest 100,000 (EPA, 2021b).

b c

The 0.070-ppm O3 standard became effective in December 28, 2015.The previous standards are not revoked and remain in effect for designated areas.

d

The EPA revoked the 1-h ozone standard in most areas, although some areas have been continuing obligations under that standard (“antibacksliding”).

Air pollution: Sources, regulation, and health effects Table 2

217

Air Quality Index (AQI) for reporting daily air quality.

Health concern

AQI value

AQI colors

Meaning

Good Moderate

0–50 51–100

Green Yellow

Unhealthy for sensitive groups Unhealthy

101–150

Orange

Air quality is satisfactory, and air pollution poses little or no risk. Air quality is acceptable. However, there may be a risk for some people who are unusually sensitive to air pollution. Members of sensitive groups may experience health effects. The general public is less likely to be affected.

151–200

Red

Very unhealthy Hazardous

201–300 301–500

Purple Maroon

Some members of the general public may experience health effects; members of sensitive groups may experience more serious health effects. Health alert: The risk of health effects is increased for everyone Health warning of emergency conditions: Everyone is more likely to be affected.

research evaluating the health and welfare effects of pollutant exposures. Those areas where monitored pollutant concentrations exceed the NAAQS can be designated as nonattainment areas. Based on 2010 population estimates, nearly 130 million people in the U.S. currently live in a county where the NAAQS for at least one of the six criteria air pollutants were exceeded (EPA, 2021b). To protect the public from air pollution-related health effects, the EPA and organizations such as the American Lung Association recommend people consider air quality and their individual sensitivities when planning outdoor activities (specifically ozone and PM). The U.S. Air Quality Index (AQI) is a tool to simplify reporting air quality to the public and is generated from the levels of criteria pollutants in the air. Criteria pollutant levels are associated with a number which corresponds to the associated health concerns, if any, for both the general public and those susceptible to the effects of air pollution (Table 2). Reporting of the daily AQI is required for metropolitan regions with populations greater than 350,000 people and has led to the widespread dissemination of current and forecasted AQI values. Evidence shows some success of this program in helping susceptible populations (especially children, older adults, asthmatics) avoid outdoor activities and associated adverse health effects from episodes of high air pollution (Neidell, 2009). Control and reduction of ambient air pollutants in the U.S. have been met with varying degrees of success. Since 1970, total emission of Pb from anthropogenic sources has decreased by 86%. Unleaded gasoline now accounts for nearly all gasoline sales. This change has virtually eliminated highway vehicle sources as emitters of lead and reduced ambient lead levels by greater than 97% (EPA, 2013a). Likewise, stationary point source emissions of lead, primarily industrial smelters in the U.S., have dropped by greater than 95% over the last three decades, although significant emissions may exist with individual smelters (EPA, 2020c). Currently, the major source of airborne Pb emissions in the U.S. is from piston-engine aircraft operating on leaded fuel. Over the past decades, programs in reducing gaseous pollutants have been very successful for carbon monoxide (70% decrease in total emissions). Regulations requiring the use of low or ultralow sulfur diesel fuel have contributed greatly to the decrease in sulfur dioxide emissions (92%). The reduction program for nitrogen dioxide has also been successful during a time when total motor vehicle miles in the U.S. have increased substantially. Changes in ambient levels of nitrogen oxides and volatile organic compounds have resulted in some success in reducing ambient levels of ozone. The long-term trend for ozone concentrations is downward, although meteorological conditions appear to modify peak ozone levels monitored throughout the U.S. (e.g., high ozone levels during summers with hot, dry conditions; high ozone levels during the winter in snow covered Intermountain West oil fields). In summary, legislative efforts in the U.S. have been successful in reducing ambient air pollution over the last three decades and similar efforts in the European Union (EU) and Hong Kong have also resulted in significant improvements in air quality. Often, these legislative changes must be country or region specific because of important differences in emission sources. For example, diesel cars are much more prevalent in European countries than in the U.S., whereas biomass burning is a major emission source in developing countries. The EU and the World Health Organization (WHO) each develop air pollution guidelines and source emission reduction plans to decrease air pollution and the associated adverse health effects. In countries where biomass burning is used for cooking and heating purposes, limited but effective intervention programs have shown that ventilation changes can directly improve the indoor air pollution levels (Kumar et al., 2021). Reduction of emissions from mobile and stationary point sources has proved effective at reducing ambient air pollution levels but enacting regulatory changes has often proven challenging in the U.S. due to conflicting interests of business, state, and federal regulations, and enforcement agencies. While progress has been made in reducing ambient pollutant levels, air quality management is still needed as, in the U.S., millions of people lived in counties which exceeded the NAAQS standards in 2021 (Table 1).

Health effects of air pollution There is strong evidence that many air pollutants play a causal role in adverse health effects. Major challenges for environmental health scientists are to identify the acute and long-term adverse health effects of ambient air pollution, pinpoint the relevant concentrations at which these effects occur, and determine sensitive subpopulations. The determination of sensitive populations is

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especially important in characterizing risk, as federal regulation in the U.S. requires protection of public health with an adequate margin of safety. In general, a great deal more is known about the acute effects of exposures to ambient air pollutants in humans, which is supported by controlled exposure studies of humans and animals, than is known about the chronic effects, which are inferred from associations found in epidemiology studies and are supported by shorter-term experimental studies that provide biological plausibility. The following discussion outlines the findings of epidemiologic, controlled clinical, and animal studies which have examined the adverse health effects of outdoor air pollutants.

Ozone Ground level ozone is produced from a chemical reaction between oxides of nitrogen and carbon containing compounds including CO, methane (CH4), and volatile organic compounds. Ozone precursors are generated from anthropogenic sources such as automobile vehicle use, industrial processes and farming operations, however, natural sources, such as wildfires, are also significant sources of ozone precursors. The formation of ozone from its precursors is stimulated by ultraviolet radiation. Therefore, ozone formation is highest in urban areas with elevated precursor levels and during times of the year when solar radiation is highest. Exposure to ozone is a major health concern in urban and rural communities throughout the U.S. Nationally, average 8-h ozone concentrations have decreased by 25% from 1990 to 2020. Much of the improvement in ozone levels has occurred since 2002 due largely to reductions in emissions of oxides of nitrogen (an ozone precursor). Based on the evidence integrated across controlled human exposure studies, animal toxicology studies, and epidemiologic studies, there is clear, consistent evidence of a causal relationship between short-term exposure (i.e., from days to weeks) to ozone and respiratory effects. Human clinical studies show that controlled exposures to ozone cause predictable, transient decrements in lung function (McDonnell et al., 2013), symptoms of breathing discomfort (Adams, 2006; Schelegle et al., 2009), lung inflammation (Devlin et al., 1991; Mudway and Kelly, 2004), and epithelial damage and permeability (Kehrl et al., 1987; Que et al., 2011). Increases in airway responsiveness (a condition in which the conducting airways have enhanced bronchoconstriction following exposure to variety of stimuli, e.g., allergens, cold air, sulfur dioxide) have also been reported. For example, Horstman et al. (1990) observed a dose dependent increase in airway responsiveness to methacholine in young healthy adult men exposed to as little as 80 ppb ozone following 6.6-h exposures. Animal studies of controlled ozone exposure also show lung function impairment [see Section 3.1.4.1.2 of EPA (2020b)], increased airway reactivity [see Section 3.1.4.3.2 of EPA (2020b)], and impaired lung defenses [see Section 3.1.4.4.2 of EPA (2020b)] following ozone exposure with many looking into mechanistic underpinnings of the observed results. These findings provide biological plausibility for the population-level associations seen in epidemiologic studies between short-term ambient exposure to ozone and asthma exacerbation (Silverman and Ito, 2010), respiratory-related hospitalizations (Katsouyanni et al., 2009; Cakmak et al., 2006), and emergency department visits (Darrow et al., 2011; Tolbert et al., 2007). The magnitude of respiratory effects (e.g., decrements in pulmonary function and symptomatic responses) is generally a function of ozone concentration, minute ventilation rate (volume of air inhaled per minute), and exposure duration. Any physical activity will increase minute ventilation and therefore the dose of inhaled ozone. For healthy young adults exposed in a controlled clinical study at rest for 2 h, 500 ppb is the lowest ozone concentration reported to produce a statistically significant group mean decrements in lung function compared to no ozone exposure (Horvath et al., 1979), however, a recent study reported statistically significant effects on lung function in resting individuals at a much lower concentration (average of 70 ppb) after a long period of exposure (6.6 h) (Hernandez et al., 2021). The magnitude of observed lung function decrements is larger when subjects perform moderate, quasi-continuous exercise (typically intermittent periods of brisk walking) during the exposure period (60–70 ppb). Following these prolonged exposures to 60–70 ppb ozone with intermittent exercise, statistically significant decrements in lung function, increases in respiratory symptoms, and pulmonary inflammation have been reported (Adams, 2006; Schelegle et al., 2009; Kim et al., 2011). These longer exposure duration studies, in large part, were the basis for EPA’s decision to lower the level of the ozone NAAQS to 70 ppb in 2015. Although there is a relatively rapid recovery in pulmonary function and respiratory symptoms over a few hours following exposure, inflammatory responses occur shortly after exposure and persist for at least 1 day. An influx of neutrophils and an increase in many mediators including eicosanoids, neutrophil elastase, and cytokines have been measured in bronchoalveolar lavage fluid recovered from subjects exposed for 6.6 h to 80 ppb (i.e., near ambient concentration) of ozone (Devlin et al., 1991). In otherwise young healthy adults exposed for 2–8 h to ozone, controlled clinical studies have demonstrated a large degree of intersubject variability in lung function decrements, respiratory symptom responses, inflammation, airway responsiveness, and altered epithelial permeability. The magnitude of increases in inflammation, airway responsiveness, and epithelial permeability, in response to ozone exposure, do not appear to be correlated, nor are these responses correlated with changes in lung function (Balmes et al., 1996). However, these responses to ozone tend to be reproducible within a given person over a period of several months indicating differences in the intrinsic responsiveness of individuals (Hazucha et al., 2003; Holz et al., 1999). It should be noted that even when group mean responses are small and seem physiologically insignificant, some intrinsically more responsive individuals experience distinctly larger effects under the same exposure conditions. For example, small group mean changes (e.g., 2–3%) in forced expiratory volume in 1 s (FEV1) have been observed in healthy young adults exposed to 60 ppb ozone for 7 h. Despite this, 10% of the group experienced FEV1 decrements in excess of 10% under these conditions, (Schelegle et al., 2009). Therefore, within the general population, a proportion of people experience greater than average responses and may be at increased risk of more adverse health effects [see Section 3.1.4.1.1 of EPA (2020b)].

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With repeated ozone exposures over several days, lung function and respiratory symptom responses become attenuated in both healthy individuals and asthmatics, but this attenuation of response is lost after about a week without exposure (Kulle et al., 1982; Linn et al., 1982; Horvath et al., 1981). Airway responsiveness also appears to be somewhat attenuated with repeated ozone exposures in healthy individuals, although this adaptation process tends to take longer to develop and persists longer than the effects on lung function (Gong et al., 1997; Kulle et al., 1982; Folinsbee et al., 1994). Some indicators of pulmonary inflammation are attenuated with repeated ozone exposures, whereas other markers, such as epithelial integrity and damage, do not show attenuation, suggesting continued tissue damage during repeated ozone exposure. Both respiratory symptoms and decrements in pulmonary function have neural components and these responses decrease with increasing age beyond young adulthood. However, other cell and molecular responses to ozone exposure likely exist and may even increase as antioxidant defenses change with increasing age. The respiratory effects observed in short-term controlled exposure studies are similar to those reported during exposure to ambient air. Decrements in lung function have been noted in a series of summer camp studies where children were exposed to ambient ozone during normal outdoor play activity with lung function measured in the afternoons (Kinney et al., 1996). Compared to short-term controlled chamber studies, greater decrements in lung function were observed in the camp studies when the data were normalized for ozone concentration. Many factors may explain the greater response in the camp study, but a possible reason is the simultaneous exposure to ambient co-pollutants such as acid aerosols. Epidemiologic studies have found strong correlations between respiratory symptoms such as cough, throat irritation, and chest discomfort and ambient ozone levels [see Chapter 6 of EPA (2013b)]. Exacerbations of asthma, increases in hospital admissions for respiratory infections, and excess mortality have also been reported to be associated with short-term ozone air pollution episodes. Age, sex, ethnicity, socioeconomic status indicators (i.e., educational attainment, income level, employment status), and preexisting disease (e.g., diabetes, asthma) may increase susceptibility to ozone-related mortality. Thus, many epidemiologic and clinical studies provide evidence that adverse respiratory effects occur following exposure to ozone at ambient concentrations. Population-based epidemiologic studies examining the effects of long-term ozone exposure (i.e., from months to years) show associations with long-term reductions in lung function, development of asthma, and pathological changes [see Chapter 7 of EPA (2013b) and Section IS.4.3.2 of EPA (2020b)]. Limited new epidemiologic evidence also suggests that long-term exposure to ozone increases the risk of premature mortality, however, the ability of epidemiologic studies alone to establish cause and effect is hampered by confounding factors such as co-pollutants. Animal studies using concentrations well above the current NAAQS reveal that the centriacinar region of the airways and the nasal cavity are the most sensitive to pathological changes induced by chronic ozone. In addition, rodent models of allergy have shown enhanced lung injury, inflammation, and allergy responses when exposed to ozone. Studies of infant primates show changes in immune responses similar to asthma and the development of irreversible changes to the structure of the distal (deep) lung that decrease pulmonary function following exposures at rest to 500 ppb ozone (Fanucchi et al., 2006). The animal studies provide coherence and biological plausibility to explain the morphologic and effects on asthmatics seen in epidemiologic studies. Overall, strong evidence shows that both short- and long-term exposure to ozone at ambient concentrations is associated with respiratory morbidity, with some evidence of premature respiratory-related mortality.

Sulfur oxides Several species of sulfur oxides, such as sulfur dioxide (SO2), sulfur monoxide (SO), sulfur trioxide (SO3), and disulfur monoxide (S2O), exist in the gaseous phase of ambient air. For regulatory purposes, SO2 is used as the indicator species for all sulfur oxides due to its relative abundance in the atmosphere, importance in atmospheric chemistry, and known links to human health effects. SO2 can be formed as a primary air pollutant or secondarily through reduction reactions of sulfur containing compounds. Stationary industrial sources are the largest source of SO2, in particular through the burning of fossil fuels (primarily coal) which contain trace amounts of sulfur. Sulfur oxides are also generated from natural events like volcano eruptions and wildfires. Significant and, on occasion, disastrous adverse health effects have accompanied acute air pollution episodes involving reducing-type pollutants. In the middle of the twentieth century, multiple day thermal inversions resulted in a smog containing high levels of PM and sulfur dioxide in Donora, Pennsylvania (Oct. 1948) and London, England (Nov. 1948; Dec. 1952; Jan. 1956; Dec. 1957; Jan. 1959; and Dec. 1962). Excess mortality accompanied each of these pollution episodes and has been attributed to the smoke and sulfur dioxide generated by coal combustion. For London, the maximum 24-h pollutant concentrations during smog episodes were generally in the range of 1700–4500 mg/m3 for PM in the form of black smoke and 1800–4500 mg/m3 (0.7–1.7 ppm) for SO2 [see Table 14–1 of (EPA, U.S., 1982)]. The highest daily and hourly sulfuric acid concentrations are believed to be about 350 mg/m3 and 678 mg/m3, respectively, in December 1962 [see Section 2.5.2 of EPA (1989)]. The 1948 Donora smog episode was less well characterized, but daily SO2 concentrations are thought to have been as high as 1100 mg/m3 (0.4 ppm) [see Section 14.3.1.1 of EPA (1982)]. These deadly smog episodes highlighted the need for increased air pollution regulation. From 1990 to 2020, the national 1-h average concentration of SO2 in the U.S. has decreased 92% (Table 3). Epidemiologic, clinical, and animal studies have confirmed that exposures to both PM and sulfur oxides result in adverse health effects. In these studies, adverse effects have been observed during pollution episodes in which the gas and particle concentrations were lower than the thermal inversion conditions mentioned above. Delineating the relative contribution of PM and sulfur oxides to these adverse effects is difficult because of the chemical-physical association of sulfur oxides and particles. This section is limited

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Changes in emission estimates for the U.S., 1990–2020.

Pollutant

30-Year trend

Carbon monoxide Nitrogen oxides (NOx) Sulfur dioxide Volatile organics PM10 PM2.5 Lead

69% decrease 68% decrease 92% decrease 48% decrease 31% decrease 39% decrease 86% decrease

to the current state of knowledge on sulfur oxides and acid aerosol-related health effects. The next section discusses PM-related effects. Controlled laboratory studies using human subjects and test animals have demonstrated that sulfur dioxide can produce functional and pathological changes. These changes include increases in airway resistance and impaired mucus clearance (Andersen et al., 1974; Park et al., 2001). In general, the concentrations of sulfur dioxide necessary to produce these changes are greater than those encountered in the ambient environment. A notable exception is the bronchoconstrictive effect of sulfur dioxide on atopic and asthmatic subjects as demonstrated by reductions in FEV1 and increases in specific airway resistance (sRaw). Inhalation of 0.2 to 0.6 ppm sulfur dioxide for 5–10 min in combination with moderate exercise causes bronchoconstriction, shortness of breath, and cough in these sensitive individuals [see review in Section 5.2.1.2 of EPA (2017)]. Interestingly, bronchoconstriction will rapidly resolve if exercise is stopped despite continued exposure to concentrations up to 1 ppm (Linn et al., 1984). Similar bronchoconstrictive effects in normal (nonatopic) individuals occur only after exposure at much higher concentrations (>1 to 5 ppm) of sulfur dioxide (Linn et al., 1987). Because of increasing evidence of a subpopulation of individuals sensitive to near ambient peak levels of sulfur dioxide (i.e., asthmatics), the decades-old U.S. EPA annual NAAQS for sulfur dioxide was changed in 2010 to a 1-h standard with a level of 75 ppb. This switch to a 1-h standard and the revoking of the long-term standard was done to protect public health by reducing exposures to high short-term concentrations. Considerable attention has been placed on the contribution to health effects of sulfur oxide in the context of deposition onto airborne particulate matter to form acid aerosols (EPA (1989)). The carbon, mineral, and heavy metal-based particles produced during fossil fuel combustion and industrial processes promote the conversion of sulfur dioxide to sulfuric acid. Recognition of sulfur dioxide–particle interactions came as a result of findings garnered from a number of epidemiology and animal toxicology studies and the characterization of sulfuric acid, ammonium sulfate, and ammonium bisulfate associated with atmospheric particles. This makes separating the health effects of PM and those related to sulfur oxides difficult. Because of this, scientific evidence for sulfur oxides and PM are often reviewed in tandem, as in the case of the 1982 U.S. EPA Air Quality Criteria Document for Particulate Matter and Sulfur Oxides EPA (2017). Epidemiologic evidence from both Europe and North America suggests that acid aerosols formed by gas–particle interactions in the atmosphere have played a major role in the adverse health effects seen during severe and moderate pollution episodes [see Section 5.2.1 of EPA (2017)]. The pollution-associated increases in mortality observed in London from 1958 to 1972 were more closely associated with acid aerosol concentrations than other pollutants such as smoke and sulfur dioxide. In the U.S. and Canada, cross-sectional analyses have demonstrated that ambient sulfate concentrations and PM concentrations are predictors of excess mortality and hospital admissions due to air pollution, but the strongest associations are typically found with fine particle mass (i.e., nominally particles smaller than 2.5 mm in diameter). A seminal prospective cohort study, known as the Six Cities Study, found that increased mortality from cardiopulmonary deaths and lung cancer was strongly associated with sulfate and particulate concentrations (Dockery et al., 1993). This same study demonstrated that the incidence of bronchitis in children was correlated with ambient levels of acid aerosols. Similarly, in northern Europe, an acidic pollution (PM and SO2) episode in 1985 was linked with significant excesses in respiratory mortality and morbidity, particularly in adults with preexisting respiratory disease (Wichmann et al., 1989), and with decrements in pulmonary function in children that persisted for 2 weeks after the event (Dassen et al., 1986). In a 1986 study of a Canadian children’s summer camp, acid aerosol episodes (PM, H2SO4, and O3) corresponded to decreases in lung function (Raizenne et al., 1989). In summary, a large body of evidence suggests that acid aerosols can play a significant role in the adverse health effects attributed to air pollution. Animal studies have demonstrated that exposure to near ambient concentrations of sulfuric acid produces both conducting airway and alveolar changes including increased airway resistance, airway hyperresponsiveness, and alterations in clearance mechanisms and macrophage function [see Section 5.2.1.7 of EPA (2017)]. Controlled human exposures to acid aerosols, however, have demonstrated few pulmonary effects at concentrations below 500–1000 mg m−3. The effects reported after acute exposures to sulfuric acid aerosols have largely been observed in atopic subjects, are small in magnitude, and are readily reversible. Therefore, there is a research need to explain the difference between the results of epidemiologic studies and the paucity of data demonstrating adverse health effects in controlled human studies. One possible cause of this discrepancy is the type of acid aerosols used in the

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laboratory studies. Although pure sulfuric acid droplets are used almost exclusively in controlled exposures, ambient acid aerosols are chemically complex and are proposed to be composed of a core consisting of carbon, minerals, or heavy metals surrounded by acidic (sulfuric or nitric acid) surface material.

Particulate matter PM consists of a complex range of chemically and physically diverse substances that exist in the atmosphere as discrete suspended particles (liquid droplets or solids). PM is frequently classified by size according to its nominal median aerodynamic diameter (measured in micrometers [mm]). The U.S. EPA, EU, and WHO have set national and regional standards to protect against the health and welfare effects associated with exposures to ambient fine and coarse particles. Fine particles are generally considered to have nominal diameters less than or equal to 2.5 mm and are designated as PM2.5. Inhalable coarse particles are those between 2.5 and 10 mm and are designated as PM10–2.5; PM10 (particles with nominal diameters less than or equal to 10 mm) is the indicator used in the U.S. for the coarse particle NAAQS. These size classifications have been accepted worldwide and the WHO also uses PM10 and PM2.5 size classifications for their PM air quality guidelines. PM with nominal diameters less than about 0.1 mm is referred to as ultrafine particles (UFPs), which is consistent with the International Organization for Standardization (ISO) definition of a nanoparticle as an object with all three external dimensions in the nanoscale, i.e., from approximately 0.001 to 0.1 mm (ISO, 2008). PM in all size ranges has contributions from both primary sources (i.e., emitted directly into the atmosphere) and secondary processes (i.e., formed in the atmosphere from precursor emissions) from anthropogenic and natural sources. Both primary and secondary PM2.5 comes predominantly from combustion processes related to transportation, power generation, and industry. Constituents of PM2.5 include sulfates, nitrates, elemental and organic carbon, metals, and crustal material. PM10–2.5 comes mainly from primary abrasion (e.g., road dust) and crushing processes as well as airborne soils and pollens. UFPs in urban environments are derived from primary combustion-related sources and secondary nucleation (i.e., growth of droplets around existing nuclei) of sulfuric acid vapor, ammonia, and certain organic compounds. Local sources, as well as meteorological and topographic conditions, can strongly influence the degree of spatial variability in PM. Depending on size, PM remains suspended in the atmosphere for anywhere from minutes to weeks and is ultimately removed from the atmosphere through processes involving dry and wet deposition. The current U.S. national 24-h standard level for PM10 is 150 mg m−3. The current national 24-h standard level for PM2.5 is 35 mg m−3, with an annual standard level of 12 mg m−3. Importantly, annual PM10 emissions decreased 30% between 1990 and 2020, with the change in annual PM2.5 emissions decreasing 35% for the same period. The study of health effects associated with exposure to PM has been a major focus of research in the U.S., Europe, and Asia in recent decades. The U.S. EPA has recently reviewed the available literature on the emissions and the effects of PM on human health and the environment in the Integrated Science Assessment (ISA) for Particulate Matter EPA, U.S. (2019). This ISA provides the scientific foundation for EPA’s NAAQS for PM2.5 and PM10 and contains a critical review of recent evidence for effects of PM on human health and welfare. Population-based studies have consistently reported associations between short-term exposures to PM2.5 and cardiovascular and respiratory hospital admissions in numerous regions throughout the U.S., Europe, and Asia [Sections 5.1.6.1 and 6.1.6.1 of EPA (2019)]. Similarly, epidemiologic findings demonstrate that short-term exposures to PM2.5 are associated with increases in total mortality and mortality due to cardiovascular and respiratory causes [Sections 5.3.7 and 6.1.9 EPA (2019)]. Long-term exposures to PM2.5 have been associated with total and cardiovascular mortality, accelerated development of atherosclerosis, decrements in lung function growth, increased respiratory symptoms, and asthma development [see Sections 5.2, 6.2.4, and 6.2.10 of EPA (2019)]. Children and racial minorities appear to be most susceptible to the effects of PM although other factors like age, preexisting disease state, and certain genetic factors may also play a role in determining sensitivity to PM effects [see chapter 12 of EPA (2019)]. The health effects resulting from exposure to PM10–2.5 are much less certain due to the more limited body of scientific evidence available. Although epidemiologic studies provide some evidence of positive associations between short- and long-term PM10–2.5 exposures across various health endpoints, the methods used to estimate PM10–2.5 concentrations and subsequently assign exposures to PM10–2.5 have not been systematically evaluated in the peer-reviewed literature. Compared to PM2.5, greater spatial variability in PM10–2.5 concentrations also contributes to uncertainty about using PM10–2.5 concentrations as a surrogate for human exposure. Additionally, in animal studies using rodents, only a small portion of inhaled PM10–2.5 penetrates through the nasal passages to reach the lungs (Asgharian et al., 2014). This necessitates particle delivery by intra-tracheal instillation or nasal aspiration in rodent studies, complicating the extrapolation to humans. Also, only a small fraction of PM10–2.5 actually enters the lower respiratory tract of humans. Brown et al. (2013) estimated 50% particle penetration (i.e., the particle size at which half of the particles are removed prior to reaching a lung region) into the lower respiratory tract at an aerodynamic diameter of around 3 mm for adults and 5 mm for children. Considering that PM10 sampling has 50% penetration of 10 mm particles (i.e., half of 10 mm particles are collected by the sampler, with lower collection efficiency for particles >10 mm and higher collection efficiency for particles 4.0 ppm (Baldauf et al., 2008). With increasing distance from the roadway, these levels drop off exponentially. In-vehicle CO concentrations are typically between two and five times higher than ambient concentrations. Significant exposures to CO can also occur indoors because there are many indoor combustion sources that emit CO. Cigarette smoke is an important indoor contributor to CO exposure. Carbon monoxide is classified as a chemical asphyxiant. Its detrimental effects are mediated by its ability to bind with hemoglobin to form carboxyhemoglobin (COHb) or other oxygen carrying or utilizing proteins. COHb reduces the carrying capacity of hemoglobin for oxygen, which impairs the release of oxygen to the tissues and results in hypoxia. Carbon monoxide is produced endogenously through heme degradation, metabolism of drugs, and degradation of unsaturated fatty acids, inhaled solvents, and other xenobiotics [see Section 4.5 of EPA (2010a)]. In nonsmoking human subjects, COHb levels are generally 10%) (Zevin et al., 2001). Effects on the cardiovascular system have been demonstrated in controlled human exposure studies among CO-exposed individuals with coronary artery disease. Consistent decreases in the time to onset of exercise-induced angina and electrocardiogram changes have been observed following 1-h CO exposures (average CO concentrations of 120 and 250 ppm) that result in COHb levels in the range of 2% and 4% (Anderson et al., 1973; Allred et al., 1991). In epidemiologic studies showing associations between short-term CO exposures (mean CO concentrations 5000 ppm in young mallard ducks and bobwhite quail. Slightly toxic to honey bees (LD50 >36 mg per bee). Slightly to moderately toxic on an acute basis to freshwater aquatic animals (LC50/EC50 1–33 ppm). Highly to moderately toxic to freshwater aquatic animals on a chronic basis (NOEC 0.1 ppm, LOEC 0.2 ppm). Moderately toxic to saltwater fish (LC50 3.9 ppm), moderately toxic to saltwater mysid (LC50 2.4 ppm), and moderately toxic to shellfish (EC50 1.6 ppm). Highly toxic to aquatic plants (based on a single species tested: NOEL ¼ 0.35 ppb, LOEL ¼ 0.69 ppb, EC50 ¼ 1.64 ppb). See Rotterdam Convention (2011) for further ecotoxicology data.

Exposure standards and guidelines

• • • • •

Acceptable daily intake is 0.0025 mg kg−1 day−1. 8-h time weighted average is 1 mg m−3. Maximum contaminant level is 0.002 mg L−1. Reference dose is 0.01 mg kg−1 day−1 See Environmental Protection Agency (1998), Lu (1995), and Gadagbui et al. (2010).

Alachlor

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Miscellaneous The Material Safety Data Sheet should always be referred to for detailed information on handling and disposal.

Conclusion Alachlor is a preemergence herbicide that has low soil persistence but can persist years in underwater aquifers. Routes of human exposure include inhalation, dermal, ocular, and oral ingestion. It is corrosive and causes skin and eye irritation. Alachlor is a confirmed animal carcinogen with unknown relevance to humans. Recent large human epidemiology studies of individuals exposed to alachlor have been conducted and describe potential associations between alachlor and several human diseases, though with conflicting evidence between studies.

See also: Common mechanisms of toxicity in pesticides; Pesticides and its toxicity

References Andreotti G, Hoppin JA, Hou L, Koutros S, et al. (2015) Pesticide use and relative leukocyte telomere length in the agricultural health study. PLoS One 10(7): e0133382. Biagini RE, Henningsen GM, MacKenzie B, Sanderson WT, et al. (1993) Evaluation of acute immunotoxicity of alachlor in male F344/N Rats. Bulletin of Environmental Contamination and Toxicology 50: 266–273. Environmental Protection Agency (1998) Reregistration Eligibility Decision—Alachlor. Gadagbui B, Maier A, Dourson M, Parker A, et al. (2010) Derived reference doses (RfDs) for the environmental degradates of the herbicides alachlor and acetochlor: results of an independent expert panel deliberation. Regulatory Toxicology and Pharmacology 57(2–3): 220–234. Geret F, Burgeot T, Haure J, Gagnaire B, Renault T, Communal PY, and Samain JF (2013) Effects of low-dose exposure to pesticide mixture on physiological responses of the Pacific oyster, Crassostrea gigas. Environmental Toxicology 28(12): 689–699. Heydens WF (1998) Summary of toxicology studies with alachlor. Journal of Pesticide Science 24: 75–82. Hou L, Andreotti G, Baccarelli AA, Savage S, et al. (2013) Lifetime pesticide use and telomere shortening among male pesticide applicators in the Agricultural Health Study. Environmental Health Perspectives 121(8): 919–924. James KA and Hall DA (2015) Groundwater pesticide levels and the association with Parkinson disease. International Journal of Toxicology 34(3): 266–273. Kidd H and James DR (eds.) (1991) The Agrochemicals Handbook, 3rd edn. Cambridge: Royal Society of Chemistry Information Services. Kier LD, Heydens WR, Lau H, Thake DC, and Wilson AGE (1996) Genotoxicity studies of alachlor. Toxicologist 30: 231. Kim H, Min J, Park J, Lee S, and Lee J (2011) Erythema multiforme major due to occupational exposure to the herbicides alachlor and butachlor. Emergency Medicine Australasia 23(1): 103–105. Kronenberg JM, Fuhremann TW, and Johnson DE (1988) Percutaneous absorption and excretion of alachlor in rhesus monkeys. Fundamental and Applied Toxicology 10: 664–671. Lebov JF, Engel LS, Richardson D, Hogan SL, et al. (2016) Pesticide use and risk of end-stage renal disease among licensed pesticide applicators in the Agricultural Health Study. Occupational and Environmental Medicine 73(1): 3–12. Leet X, Acquavella J, Lynch C, Anne M, et al. (1996) Cancer incidence among alachlor manufacturing workers. American Journal of Industrial Medicine 30: 300–306. Lerro CC, Andreotti G, and Koutros S (2018) Alachlor use and cancer incidence in the agricultural health study: An updated analysis. Journal of the National Cancer Institute 110(9): 950–958. Lo YC, Yang CC, and Deng JF (2008) Acute alachlor and butachlor herbicide poisoning. Clinical Toxicology (Philadelphia, Pa.) 46(8): 716–721. Lu FC (1995) A review of the acceptable daily intakes of pesticides assessed by the World Health Organization. Regulatory Toxicology and Pharmacology 21: 351–364. Mahaboonpeeti R, Kongtip P, Nankongnab N, Tipayamongkholgul M, Bunngamchairat A, Yoosook W, and Woskie S (2018) Evaluation of dermal exposure to the herbicide alachlor among vegetable farmers in Thailand. Annals of Work Exposures and Health 62(9): 1147–1158. Naito H, Nagae M, Okahara S, Maeyama H, et al. (2011) Prolonged convulsion after intoxication of alachlor herbicide (Lasso): A case report. Chudoku Kenkyu 24(1): 35–38. Rotterdam Convention (2011) Rotterdam Convention on the prior informed consent procedure for banned or severely restricted chemicals. Decision Guidance Document—alachlor. Shrestha S, Parks CG, Goldner WS, Kamel F, et al. (2018) Pesticide use and incident hypothyroidism in pesticide applicators in the agricultural health study. Environmental Health Perspectives 126(9): 97008. Wan N and Lin G (2016) Parkinson’s disease and pesticides exposure: New findings from a comprehensive study in Nebraska, USA. The Journal of Rural Health 32(3): 303–313. Weichenthal S, Moase C, and Chan P (2010) A review of pesticide exposure and cancer incidence in the agricultural health study cohort. Environmental Health Perspectives 118(8): 1117–1125.

Further reading Heydens WF, Lamb IC, and Wilson AGE (2010) Chloroacetanilides. In: Krieger R (ed.) Hayes’ Handbook of Pesticide Toxicology. 3rd edn., vol. 1, pp. 1753–1769. London: Elsevier Inc. Hudson RH, Tucker RK, and Haegele MA (1984) Handbook of Toxicity of Pesticides to Wildlife. Resource Publication 153 Washington, DC: US Department of the Interior, Fish and Wildlife Service. Johnson WO, Kollman GE, Swithenbank C, and Yih RY (1978) RH 6201 (blazer): A new broad spectrum herbicide for postemergence use in soybeans. Journal of Agricultural and Food Chemistry 26: 285–286.

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Alar Kristina D Chadwicka and Raja S Mangipudyb, aBristol-Myers Squibb, Early Development Leadership, Princeton, NJ, United States; b Pfizer, Drug Safety R&D and Comparative Medicine, Groton, CT, United States © 2024 Elsevier Inc. All rights reserved. This is an update of K.D. Chadwick, R.S. Mangipudy, Alar, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 110–111, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00226-8.

Chemical profile Background and uses Environmental fate and behavior Exposure Toxicokinetics Acute and short-term toxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Exposure standards and guidelines Further reading

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Abstract Alar (CAS 1596-84-5) is a pesticide used on ornamental plants. There are no longer any registered food uses for alar in the United States due to concerns over potential carcinogenicity via its degradant, unsymmetrical dimethylhydrazine (UDMH). Potential exposure would be limited to dermal or inhalation routes during or after application to plants.

Keywords Alar; Daminozide; Herbicide; Pesticide; Unsymmetrical dimethylhydrazine (UDMH)

Key points

• • •

Alar is a pesticide registered for use on ornamental plants. Due to concerns over potential carcinogenicity via its degradant, there are no longer any registered food uses for alar in the United States. Alar is of very low acute and subacute toxicity based on oral or inhalation routes of exposure. There is a slightly greater degree of acute toxicity with dermal exposure.

Abbreviation UDMH

Unsymmetrical dimethylhydrazine.

Chemical profile

• • • • •

Name: Alar Chemical Abstracts Service Registry Number: 1596-84-5 Synonyms: aminozide; daminozide; DMSA; B-995; kylar; aminocide Chemical Formula: C6H12N2O3 Chemical Structure:

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Background and uses Alar is a systemic growth regulator approved in the United States for use on ornamental plants such as chrysanthemums, poinsettias, and bedding plants located in enclosed structures such as greenhouses. Alar reduces internode elongation; induces heat, drought, and frost resistance; and produces darker foliage and stronger stems as well as earlier and multiple flowers and fruits. A spray is often applied at the rate of 1500-10,000 ppm. It is a systemic agent (i.e., taken up by the fruit) and cannot be removed by washing or peeling. In 1984 the US Environmental Protection Agency (EPA) initiated a special review of products containing alar based on concerns that alar and its degradant, UDMH, caused tumors. As a result, the sole registrant, Uniroyal Chemical Company, voluntarily canceled all food-use registrations for Alar in 1989. EPA determined that the remaining non-food uses of Alar did not pose an unreasonable risk to humans.

Environmental fate and behavior Alar does not degrade following contact with water but degrades rapidly in soil resulting in volatile compounds (including formaldehyde) and bound residues; therefore mobility is not considered a concern. In greenhouse studies, alar persistence ranged from 3 to 4 days in different soils. Since alar is registered for greenhouse use only, agricultural run-off into groundwater is not expected to be a concern.

Exposure Exposure should be limited to dermal and inhalation based on approved usage.

Toxicokinetics A breakdown product of alar is an asymmetrical 1,1-dimethylhydrazine that is excreted renally.

Acute and short-term toxicity Alar is of very low acute and subacute toxicity based on oral or inhalation routes of exposure. There is a slightly greater degree of acute toxicity with dermal exposure. EPA considers that alar will not pose unreasonable risks or adverse effects to humans or the environment. The primary toxic effect seen in animals includes ptosis, central nervous system (CNS) depression, gastrointestinal irritation, and possibly liver function abnormalities.

Reproductive toxicity Alar has been shown to produce some maternal toxicity at high doses but did not produce developmental or reproductive toxicity.

Genotoxicity Neither alar nor UDMH have been shown to be mutagenic.

Carcinogenicity Concerns about carcinogenicity with UDMH and therefore alar led to the withdrawal of alar for food-use in 1989 and the revoking of food tolerances (maximum residue limits) by the EPA in 1990. Two-year bioassays suggested that alar was carcinogenic in female rats (adenocarcinomas of the endometrium of the uterus) and increased vascular tumors in the livers of male mice. UDMH caused a slight increase in liver tumors in rats and liver vascular tumors in mice. Since the presence of UDMH is dependent on alar, alar is classified by the EPA as a “possible human carcinogen.”

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Clinical management No human cases have been reported so treatment recommendations are speculative. Dermal contamination probably requires no treatment other than decontamination. Dietary exposure is not anticipated since alar is no longer approved for food use, however if ingested, treatment by emesis, gastric lavage, and/or activated charcoal may be indicated. Patients should be monitored for CNS depression, ptosis (eyelid drooping), and liver function abnormalities if significant amounts (>8 g) have been consumed.

Ecotoxicology Alar is considered to have very low acute toxicity to mammals, birds, and freshwater fish. It is slightly toxic to aquatic invertebrates.

Exposure standards and guidelines Worker protection standards should be followed including appropriate personal protective equipment and restricted entry intervals.

Further reading Edgerton LJ (1967) Colorimetric determination of alar residues in apples. Journal of Agricultural and Food Chemistry 15(5): 812–813. https://doi.org/10.1021/jf60153a021. Fan AM (1989) Pesticides and food safety. Regulatory Toxicology and Pharmacology 9(2): 158–174. Finkel AM (1992) Alar: The aftermath. Science 255(5045): 664–665. IPCS: INCHEM: http://www.inchem.org/documents/jmpr/jmpmono/v89pr05.htm. Kimm VJ (1991) Alar’s risks. Science 254(5036): 1276. Kirkland D (2010) Further analysis of Ames-negative rodent carcinogens that are only genotoxic in mammalian cells in vitro at concentrations exceeding 1 mM, including retesting of compounds of concern. Mutagenesis 25(6): 539–553. Marshall E (1991) A is for apple, alar, and alarmist? Science 254(5028): 20–22.

Relevant websites https://www3.epa.gov/pesticides/chem_search/reg_actions/reregistration/fs_PC-035101_1-Sep-93.pdf :US EPA website (daminozide). https://archive.epa.gov/epa/aboutepa/daminozide-alar-pesticide-canceled-food-uses.html :US EPA website. https://pubchem.ncbi.nlm.nih.gov/compound/Daminozide :Pubchem.

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Albuterol Samantha E Gad, Gad Consulting Services, Raleigh, NC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.E. Gad, Albuterol, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 112–115, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00809-5.

Chemical profile Background Uses/occurrence Exposure Routes and pathways Human exposure Environmental exposure Toxicokinetics (ADME) Mechanism of toxicity Acute and short-term toxicity Animal Human Chronic toxicity Human Reproductive and developmental toxicity Animal Human Genotoxicity Carcinogenicity Organ toxicity Interactions Clinical management Environmental fate and behavior Routes and pathways and relevant physicochemical properties Partition behavior in water, sediment, and soil Solubility Volatility Adsorption Partitioning Atmospheric fate Terrestrial fate Aquatic fate Environmental persistency (degradation/speciation) Hydrolysis Photolysis Biodegradation Long-range transport Ecotoxicology Aquatic Daphnid PubChem URL References

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Abstract Albuterol (CAS # 18559-94-9) is a short-acting b2-adrenergic agonist that is primarily used as a bronchodilator agent to treat asthma or other pulmonary diseases. It causes smooth muscle relaxation. Off-label uses include treatment of hyperkalemia and prevention of premature labor; it is found in metered dose inhalers, unit doses for nebulizers, and as an oral syrup and tablets.

Keywords Acidosis; Salbutamol; b-adrenergic receptor Encyclopedia of Toxicology 4th Edition

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Chemical profile






Name: Albuterol Synonyms: Salbutamol, Ventolin, Proventil, Apo-Salvent, Novo-Salmol, Albuterol sulfate; Volmax Chemical Abstracts Service Registry Number: 18559-94-9 Molecular Formula: C13H21NO3 Chemical Structure:

Background Albuterol is a short-acting b2-adrenergic agonist that is primarily used as a bronchodilator for the treatment of asthma or other pulmonary diseases. It is prepared as a racemic mixture of the R(−) and S(+) stereoisomers. The stereospecific preparation of R(−) isomer of albuterol is referred to as levalbuterol. Albuterol may be used for the treatment of hyperkalemia and is found in metered dose inhalers, unit doses for nebulizers, and as an oral syrup and tablets. Albuterol is a selective b2-adrenergic agonist that primarily causes smooth muscle relaxation. With therapeutic use, adverse effects with albuterol therapy include tachycardia, tremor, hyperactivity, nausea, and vomiting. Toxicity may result from overstimulation of b-adrenergic activity. In addition, b-adrenergic selectivity is lost, so b-1 effects can be seen. With mild-to-moderate toxicity poisoning/exposure, tachycardia, hypertension, tachypnea, tremor, agitation, nausea, vomiting, hypokalemia, and hyperglycemia may occur. Severe effects include hypotension, dysrhythmias, seizures, and acidosis and are likely to occur only after ingestion (Tilley et al., 2023).

Uses/occurrence Albuterol is used as a bronchodilator in the treatment of asthma or other pulmonary diseases. Off-label uses include treatment of hyperkalemia and in the prevention of premature labor. The oral adult dose is 2–4 mg three to four times a day. Oral doses for children are 0.1–0.2 mg kg−1. The inhalational dose is typically 0.1–0.15 mg kg−1 per dose or 0.5 mg kg−1 h−1 for continuous administration. b-Adrenergic receptors mediate the effects of the sympathetic nervous system throughout the body. b2 Receptors are found on vascular, bronchial, gastrointestinal, and uterine smooth muscle as well as skeletal muscle, hepatocytes, and also the myocardium. Albuterol stimulates adenyl cyclase which catalyzes cyclic adenosine monophosphate (AMP) from adenosine triphosphate (ATP). This mediates bronchodilation and smooth muscle relaxation through activation of protein kinases, leading to phosphorylation of proteins, which in turn increases bound intracellular calcium. The reduced availability of intracellular ionized calcium inhibits actin–myosin linkage, leading to the relaxation of smooth muscle. b2-Adrenergic receptors in the lung also inhibit secretions and decrease histamine release. Stimulation of b2-adrenergic receptors found on the uterine smooth muscle inhibits the onset of labor. There are no well-controlled studies showing evidence that oral albuterol will stop preterm labor or prevent labor at term and albuterol has not been approved for the management of preterm labor. Serious adverse reactions, including pulmonary edema, have been reported following administration of albuterol to women in labor (Tilley et al., 2023).

Exposure Routes and pathways Oral, inhaled, and dermal.

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Human exposure Occupational exposure to albuterol may occur through dermal contact with this compound at workplaces where albuterol is produced or used. National Institute for Occupational Safety and Health (National Occupational Exposure Survey, 1981–83) has statistically estimated that 744 workers (603 of these are female) are potentially exposed to albuterol in the United States.

Environmental exposure Monitoring data indicate that the general population may be exposed to albuterol via ingestion of drinking water and through the use of pharmaceutical products containing albuterol.

Toxicokinetics (ADME) Nebulized albuterol has been found more effective than systemic administration. Oral albuterol is readily absorbed from the gut. Sulfate conjugation is the primary metabolic pathway; it is transformed in the liver. There appears to be no direct biotransformation of albuterol in the lungs. Most of an inhaled dose is deposited on the pharynx after inhalation and then swallowed. Albuterol, as both the sulfate and sulfate conjugates (metabolite and unchanged drug), is eliminated via the kidneys. Albuterol follows first-order kinetics. The half-life is 3–5 h with oral dosing, 2–7 h with inhalation, and 5.5–6.9 h with intravenous dosing. Maximum brochodilation occurs within 0.5–2 h, for oral forms in 2–3 h, and sustained-release forms in 6 h. Absorption may be delayed in large overdoses, especially with sustained-release formulations (Libretto, 1994).

Mechanism of toxicity Tachycardia occurs as a reflex to the drop in mean arterial pressure (MAP) or as a result of b-1 stimulus. b-Adrenergic receptors in the locus coeruleus also regulate norepinephrine-induced inhibitory effects, resulting in agitation, restlessness, and hand tremor. Stimulation of nonpulmonary b2 receptors may lead to an increase in heart rate, QTc interval prolongation, nonspecific T-wave changes, skeletal muscle tremor, and slight increases in blood glucose and nonesterified fatty acids. Hypokalemia is more pronounced in patients receiving intravenous albuterol. Hypotension is also known to occur mostly in overdose. The buildup of cyclic AMP in the liver stimulates glycogenolysis and an increase in serum glucose. In skeletal muscle, this process results in increased lactate production. Direct stimulus of sodium/potassium ATPase in skeletal muscle produces a shift of potassium from the extracellular space to the intracellular space. Relaxation of smooth muscle produces a dilation of the vasculature supplying skeletal muscle, which results in a drop in diastolic and MAP. Myocardial ischemia and infarction have been associated with excessive tachycardia in elderly patients. The skin may be warm and pink with evidence of diaphoresis (Libretto, 1994; Tilley et al., 2023).

Acute and short-term toxicity Signs and symptoms of overdose include exaggeration of common adverse reactions, particularly angina, hypertension, hypokalemia, and seizures. Cardiac arrest may occur.

Animal Albuterol appears to be relatively benign in animals, similar to human. Agitation, vomiting, and lethargy may be seen. In rats, the oral LD50 was more than 2000 mg kg−1, and inhalation LC50 could not be determined.

Rodent LD50, intraperitoneal

167–295 mg kg

Rodent LD50, intravenous Rodent LD50, oral Rodent LD50, subcutaneous

48.7–57.1 mg kg 660–2707 mg kg 737–2500 mg kg

Human A review of albuterol overdoses revealed that up to 20 times, the oral daily dose produced no deaths. The effects of albuterol overdose are usually mild and benign, although they can be prolonged. Cardiovascular effects are usually limited to a sinus

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tachycardia and widened pulse pressure. Although there may be a drop in diastolic pressure, the systolic pressure is maintained by increased cardiac output from the trachycardia. Transient hypokalemia can result, caused by a shift of extracellular potassium to the intracellular space with total body stores of potassium generally remaining normal. A transient metabolic acidosis can be seen due to increased lactate production. Restlessness, agitation, tremors apprehension, dizziness, nausea, vomiting, and dilated pupils are common in albuterol overdose. Diabetes mellitus has been reported in less than 3% of patients receiving albuterol sulfate inhalation aerosol in clinical trials (Libretto, 1994).

Child

TDLo

Oral

0.6–1.85 mg kg−1

Human Man Human

TDLo TCLo TDLo

Intravenous Inhalation Oral

0.006 mg kg−1 0.036 mg kg−1 per 6 h 1.6–5.714 mg kg−1

Chronic toxicity Human Continued dependence of salbutamol tablets taken in high doses (30–40 tablets daily and 48–64 mg day−1) has led to symptoms of toxic psychosis in one elderly woman and paranoid psychosis in a 52-year-old man. For up to 90 years, 100-mg inhalations of salbutamol daily has been used by asthmatics, who increased doses because they “needed it” and wanted to “feel good.” Long-term tolerance develops to bronchodilator action, tremor, tachycardia, prolongation of QTc interval, hyperglycemia, hypokalemia, and the vasodilator response.

Reproductive and developmental toxicity Animal A study in Stride Dutch rabbits at oral doses of 50 mg kg−1 (approximately 25 times the maximum recommended daily oral dose for adults on a milligram per square meter basis) found cranioschisis in 7 of 19 (37%) fetuses.

Human During worldwide marketing experience, various congenital anomalies, including cleft palate and limb defects, have been reported in the offspring of patients being treated with albuterol. Some of the mothers were taking multiple medications during their pregnancies. No consistent pattern of defects can be discerned, and a relationship between albuterol use and congenital anomalies has not been established. Standard nonclinical DART studies in rats and rabbits have not revealed any adverse effects. Albuterol and albuterol sulfate/ipratropium bromide are classified as Food and Drug Administration Pregnancy Category C by the manufacturer.

Genotoxicity No evidence of mutagenicity (Libretto, 1994).

Carcinogenicity Chronic exposure or carcinogenicity studies on Sprague–Dawley rats for 2 years at dietary doses of 2, 10, and 50 mg kg−1 of body weight (corresponding to 1/2, 3, and 15 times, respectively, the maximum recommended daily oral dose for adults on a milligram per square meter of body surface area basis of 2/5, 2, and 10 times, respectively, the maximum recommended daily oral dose for children on a milligram per square meter basis) found significant dose-related increases in the incidence of benign leiomyomas for the mesovarium. Albuterol would thus be considered to have tumorigenic (but not carcinogenic) potential at higher doses (Brambilla et al., 2013).

Organ toxicity Renal.

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Interactions Following single-dose intravenous or oral administration of albuterol to healthy individuals who had received digoxin for 10 days, a 16–22% decrease in serum digoxin concentration was observed. Epinephrine and other orally inhaled sympathomimetic amines may increase sympathomimetic effects and risk of toxicity. MAO inhibitors and tricyclic antidepressants may cause serious cardiovascular effects and risk of toxicity. Propranolol and other beta blockers may antagonize effects of albuterol.

Clinical management The threshold dose for the development of three or more signs of toxicity is 1 mg kg−1 or 3–10 times the recommended daily dose. Toxicity is short lived and does not require specific therapy or hospital admission in most cases. Children have survived overdoses as large as 100 mg and adults have survived doses up to 240 mg without serious complications. Activated charcoal effectively adsorbs albuterol. The hypokalemia produced reflects a transient shift in potassium location rather than a true deficit of potassium; external replacement therapy is rarely necessary, but can be added to intravenous fluids to support the heart if electrocardiographic changes are noted. A conservative approach to tachycardia is recommended since arrhythmias beyond an increase in rate have not occurred with overdose. Support of blood pressure and control of tachycardia are major therapeutic interventions. The presence of other dysrhythmias or hypotension indicates a more severe poisoning. If hypotension is present, intravenous fluid should be used initially. If the hypotension does not respond, a b-adrenergic blocking agent can be used. First-line choices include esmolol or propranolol since the hypotension is often primarily due to the tachycardia. Alternatively, a vasopressor with pure alpha activity such as phenylephrine can be used. Tachycardia can also be treated if necessary with a beta blocker, but this is rarely warranted. Premature ventricular contractions rarely require treatment. Methylxanthine and other sympathomimetic overdoses can present in a similar manner. Symptoms may occur after inhalation, but seem to be less common and less serious than when significant amounts have been ingested. Some decontamination may be accomplished by mouth rinsing for materials left on the oral surfaces after use of an inhaler. Material absorbed via inhalation should be treated as with an oral exposure. If ocular exposure occurs, irrigate exposed eyes with copious amounts of room temperature water for at least 15 min. If irritation, pain, swelling, lacrimation, or photophobia persist, the patient should be seen in a health care facility. For dermal exposure, remove contaminated clothing and wash exposed area thoroughly with soap and water. A physician may need to examine the area if irritation or pain persists (Spangler, 1989).

Environmental fate and behavior Albuterol’s production and use as a bronchodilator may result in its release to the environment through various waste streams.

Routes and pathways and relevant physicochemical properties Melting point: 151  C (Lunts) and 157–158  C (Collins), Octanol/water partition coefficient: log Kow ¼ 0.64 (estimated), Water solubility: 1.43  104 mg L−1, Henry’s law constant ¼ 6.4  10−16 atm-m3 mol at 25  C, Soil sediment sorption (log Koc): −1.6 to −1.15, measured.

Partition behavior in water, sediment, and soil Solubility This material contains an active ingredient that for environmental fate predictions has solubility in water.

Volatility This material contains an active ingredient that will not readily enter into the air from hard surfaces or from a container of the pure substance. This material contains an active ingredient that will not readily enter into air from water.

Adsorption This material contains an active ingredient that is not likely to adsorb to soil or sediment if released directly to the environment.

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Partitioning This material contains an active pharmaceutical ingredient with octanol/water partition coefficient data that suggest that for environmental fate predictions, the active pharmaceutical ingredient will not have the tendency to distribute into fats.

Atmospheric fate According to a model of gas/particle partitioning of semivolatile organic compounds in the atmosphere, albuterol, which has an estimated vapor pressure of 8.9  10–9 mmHg at 25  C, is expected to exist solely in the particulate phase in the ambient atmosphere. Particulate-phase albuterol may be removed from the air by wet and dry deposition.

Terrestrial fate Based on a classification scheme, an estimated Koc value of 23 indicates that albuterol is expected to have very high mobility in soil. The pKa1 and pKa2 of albuterol are 9.2 and 10.7, respectively, indicating that this compound will partially exist protonated in the environment and cations generally adsorb more strongly to suspended solids and sediment than their neutral counterparts. Volatilization of albuterol from moist soil surfaces is not expected to be an important fate process.

Aquatic fate Albuterol is not expected to adsorb to suspended solids and sediment. Volatilization from water surfaces is not expected. An estimated bioconcentration factor of 3 suggests that the potential for bioconcentration in aquatic organisms is low.

Environmental persistency (degradation/speciation) Hydrolysis This material contains an active pharmaceutical ingredient that has been shown to be chemically stable in water. Hydrolysis is unlikely to be a significant depletion mechanism. Half-life, neutral: >1 years, measured.

Photolysis This material contains an active pharmaceutical ingredient that is unlikely to undergo photodegradation. Ultraviolet/visible spectrum: 225 nm.

Biodegradation This material contains an active ingredient that is not readily biodegradable (as defined by 1993 Organization for Economic Cooperation and Development Testing Guidelines). Aerobic–ready percent degradation: 1%, 28 days, modified Sturm test. Aerobic–soil percent degradation: 1.3–38.7%, 64 days.

Long-range transport Not known to be transported long-range.

Ecotoxicology Aquatic Activated sludge respiration. This material contains an active ingredient that is not toxic to activated sludge microorganisms. IC50: >830 mg L−1, 3 h.

Daphnid This material contains an active pharmaceutical ingredient that is not toxic to daphnids. EC50: 243 mg L, 48 h, Daphnia magna, static test. No observed effect concentration: 83.2 mg L, 48 h, Daphnia magna, static test.

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PubChem URL Salbutamol | C13H21NO3—PubChem (nih.gov)

References Brambilla G, Mattioli F, Robbiano L, and Martelli A (2013) Genotoxicity and carcinogenicity studies of bronchodilators and antiasthma drugs. Basic & Clinical Pharmacology & Toxicology 112: 302–313. Libretto SE (1994) A review of the toxicology of salbutamol (albuterol). Archives of Toxicology 68(4): 213–216. Spangler DL (1989) Review of side effects associated with beta antagonists. Annals of Allergy 62: 59–62. Tilley DG, Jouser SR, and Koch WJ (2023) Adrenergic agonist and antagonists. Goodman and Gilman. In: Brunton LL and Knollmann BC (eds.) The Pharmacological Basis of Therapeutics, 14th Ed, pp. 251–260. Chicago: McGraw-Hill.

Relevant website comptox.epa.gov/ :United States Environmental Protection Agency

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Alchemy PG Maxwell-Stuart, University of St Andrews, St Andrews, UK © 2014 Elsevier Inc. All rights reserved. This is a reproduction of P.G. Maxwell-Stuart, Alchemy, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 116–119, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01226-4.

Abstract Alchemy has a long history, originating in China, India, and Hellenistic Egypt from where it spread into Europe during the Middle Ages. There it established itself firmly and has been practiced ever since in all its aspects. It has three main aims: to transmute metals into genuine gold or silver, to produce medicinal elixirs, and to effect spiritual change in the person of the alchemist. Over the centuries, alchemy has attracted important scientists such as Geber, Avicenna, Paracelsus, Michael Sendivogius, Isaac Newton, and Robert Boyle, as well as charlatans and hosts of unsuccessful experimenters. Examples of alchemical gold can be seen in at least one European museum.

Keywords Alchemy; Distillation; Elixir; Gold; Mercury; Pharmacology; Philosopher’s Stone; Silver; Sulfur; Transmutation

Alchemy is a science with two faces. One looks outward to the world of matter and seeks either to manipulate aspects of it in such a way as to effect fundamental alterations in its composition, the best-known example of this being the attempt, allegedly successful in several instances, to change lead into gold, or to manufacture an elixir or pill which will preserve or provide good health and prolong a person’s life. The other face looks inward and treats the physical processes of the laboratory either as metaphors for spiritual change in the alchemist him or herself or as vehicles with whose help that spiritual change can be made to happen. The origin of alchemy is somewhat obscure. There is a Chinese tradition, whose practice goes back many centuries, an Indian tradition not quite as long but equally distinguished, and a third from Hellenistic Egypt whose Greek word khémeia (‘a pouring together’), via its Arabic version, supposedly forms the basis of our word ‘alchemy’ and suggests a derivation from the process of smelting and refining metal ore. Alchemy is sometimes regarded as a proto-chemistry on the grounds that its experiments uncovered white arsenic, silver nitrate, alcohol, ammonium carbonate, potassium sulfate, bismuth, hydrochloric acid, zinc sulfate, ferric chloride, and a host of other substances. It is also often dismissed as though it were chemistry which had taken a wrong turning, but in fact, as its history shows, it was a good deal more complex than that. Chinese alchemy was encouraged by imperial authority to manufacture gold since gold, which neither rusts nor tarnishes, was clearly not only symbolical of health and long life but also might well, when worn, pass on those desirable properties to the wearer. Metallurgical theory in China, as in the west, said that metals and minerals grow in the earth, slowly over centuries turning from one substance into another, eventually ending as gold. Hence, what the alchemist was trying to do, in theory, was to hasten that natural process and achieve in very short span what ‘nature’ took time to do. Alongside this, the Chinese alchemist experimented with both inorganic and organic substances to produce elixirs guaranteeing not only long life but also immortality, and one finds some of the great Chinese alchemists – Wei Po-Yang in the second century AD, for example, and Ge Hong and Ko Hung from the third and fourth – writing about their practical craft in combination with appeals to philosophy, astrology, magic, and pharmacology as they sought to elevate alchemy from being merely technical experiments in a laboratory into a more potent, life-changing, life-enhancing total experience. These early days of enthusiasm lasted, on and off, until the fourteenth century, assisted by intermittent imperial patronage, but thereafter interest waned under the increasing weight of public disparagement and, to some extent, Western intellectual influences and so, while alchemy continued to be practiced in China even as late as the twentieth century, public and official support was largely gone. From China, alchemy seems to have spread to Japan, Tibet, and Burma and it may be that frequent trading contact between China and India opened the way for exchange of ideas on the subject. India certainly had an alchemical tradition of her own, because Indian alchemical works were often translated into Chinese, one of which at least can be dated to the early fifth century AD, and although the high period of Indian alchemy comes much later, in the tenth century, it is clear from their treatises that Indian alchemists were pursuing the same broad aims as their Chinese counterparts: transmutation of less-developed metals into gold and transformation of human bodies into a form which would prolong good health and attain immortality. One of the principal conduits of ideas between China and India, however, was ‘Tantrism,’ an amalgam of philosophical and religious ideas which suggested that the world of matter was real, not illusory, and that there was, therefore, no barrier between the spiritual and material worlds which could not be broken down by ritual practices designed to channel divine energy into a transformation of the material world. From the fourth or fifth century, then, this Tantrism, which seemed to meld with the philosophical requirements of alchemy, gave Indian a particular coloring and perhaps encouraged the development of elixirs at the expense of transmutation of metals. Tantrism, however, either brought or animated ideas and practices based on hatha yoga on the one hand and magic on the other,

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and so the Indian alchemist’s laboratory might well be a space in which pharmacology met religious worship, distillation shook hands with sexual metaphors, and potentially poisonous substances conjoined with spiritual transcendence. But, as in China, enthusiasm for alchemy, whether gold making or elixir producing, waned somewhat after its early energetic period and although, again as in China, alchemy continues to be practiced in India nowadays, after the thirteenth or fourteenth century, interest and experiment were never quite as intense as they had been. The history of alchemy further west, however, is rather different. There, Egypt was regarded as the cradle of the science, although attempts to derive ‘alchemy’ from Arabic al- (‘the’) and Greek khem (‘black land,’ i.e., Egypt) are somewhat dubious. Apart from one source (Pliny the Elder, first century AD) telling us that the Emperor Caligula extracted gold from orpiment, and a highly uncertain tradition that the Emperor Diocletian burned Egyptian alchemists’ books in 290, evidence for the practice of alchemy in Egypt is actually rather thin, and it is not until about the second or third century AD that one finds both alchemical texts and references to earlier alchemical practitioners. Pseudo-Demokritos, possibly following Bolos of Mendes from the second century BC, provided practical recipes and mystical observations in his Physika et Mystika, as did ‘Maria the Prophetess,’ a shadowy figure often referred to by later authors as the inventor of certain pieces of alchemical apparatus. Two papyri collections from the third and fourth centuries AD (Stockholm and Leiden X) contain a mixture of recipes for coloring gemstones, manufacturing dyes, and making alloys which look like gold and silver. Then in the early fourth century came the much-quoted Zosimos, an Egyptian alchemist and mystic, who offered a pseudo-origin for alchemy in the teaching of angels, and whose references to and descriptions of practical alchemy were made to serve as allegories of a spiritual rather than a laboratorical transformation. In spite of this, however, it was the practical side of alchemy which received detailed attention in Egypt, as in the later Byzantine Empire and Arabic revival and development of the science, and a tenth-century Byzantine encyclopedist’s definition of alchemy as ‘the fabrication of silver and gold’ underlines the point. Alchemy seems to have come to the Arabs partly at least via Persia (Iran) where Christian communities had preserved both writings and knowledge of the science, and between the eighth and eleventh centuries it enjoyed a golden period, largely under the influence of three great names: Jābir ibn Hayyan (Geber), Ab
u Bakr Muhammad ibn Zakariya al- Rāzī (Rhazes), and Ab
u ‘Aliāl-Husain ibn Sīnā (Avicenna). The first two of these were practitioners, the third was not, although his negative attitude to alchemy stimulated others to come to its defense, and while Arabic-language alchemical texts depended first on their earlier Greek counterparts, they quickly took over original research. Jābir proposed that metals happen as a result of a combination of ‘mercury’ and ‘sulfur’ in certain proportions and under specific celestial influences and that one of the principal aims of the alchemist is to discover the essential characteristics of each metal and bring these into a new balance, thereby altering the composition of the metal in question. Numerological computation was an essential skill in discovering these various balances, and so the alchemist had to be at least a competent mathematician, too. Al-Rāzī, for his part, was particularly interested in pharmacological therapy, and this led him to concentrate on the manufacture of drugs and elixirs, alchemical and non-alchemical, in his laboratory. Each of these three had treatises attributed to him, so it is sometimes difficult to gauge exactly what opinions he actually had and which were fathered upon him. But it is via the corpus of works bearing their names that alchemy can be said to have passed to the West which then translated them into Latin. These translations began to appear in Spain during the twelfth century, but it was the thirteenth and fourteenth which saw Western fascination with alchemy really burgeon, encouraged by rulers’ perpetual need for more money, and one finds that major scholars of the period – Albertus Magnus, St Thomas Aquinas, Roger Bacon, Arnald of Villanova, Ramon Lull – not only expressed interest in alchemy but also had alchemical works foisted upon them, too. Others, such as ‘Ortulanus,’ author of True Alchemical Practice (1386) and the Catalan Franciscan, John of Rupescissa (died 1362), provided practical observations on laboratory processes, although these were often couched in allegorical and metaphorical terms or, as in John’s case, mixed with instructions on how to achieve transmutation of metals rendered less obscure because the coming birth of Antichrist and the subsequent arrival of the apocalypse demanded an honesty hitherto veiled in a language of secrets. Once the printing press arrived, knowledge of alchemy spread even more quickly and a readership – interested amateurs and eager professionals – for works of both practice and theory grew in proportion. Geoffrey Chaucer, author of the Canterbury Tales (late 1380s), poked astringent fun at some of the pretensions and obscurities of many alchemical writers, pretensions which lent themselves to parody and satire. His alchemist was poor, dirty, slovenly, and stank of sulfur. He was always borrowing money – hence in part people’s suspicions that alchemists merely took one’s cash and fobbed one off with metallurgical tricks and nonsense – because alchemy was an expensive occupation. Breakages were frequent and the special equipment was not easy to replace. A fifteenth-century book attributed to Jābir describes various kinds of furnace, for example, which were to be used at different stages of the alchemical process: one for calcinations, another for sublimation, a third for ‘descension’ (separation of the desired liquefied material from extraneous matter), a fourth for smelting, a fifth for ‘solution’ (conversion of dry matter into liquid), and a sixth, known as the athenor, for ‘fixation’ (a process to make sure that any desired alteration in the alchemist’s material would be stable). Then there were stills, condensers, crucibles, retorts, ‘alembics’ (a form of still), and evaporating dishes, all of which could be made of pottery or glass, and many of which had very particular shapes and therefore needed to be made or blown to order. The crowded clutter of an alchemist’s laboratory can be seen in sixteenth-century woodcuts and seventeenth-century paintings, and the remains of a sixteenth-century alchemical laboratory have been unearthed in Austria, showing that the woodcuts and paintings were accurate in every detail. Over 300 clay crucibles, especially made to resist heat and chemical assault, and shallow ceramic plates called ‘scorifiers’ have been rescued, restored, and analyzed. What neither pictures nor archaeological relics can do, of course, is convey the stench, the heat, and the noise of these places which were kept going day and night for days and weeks at a time. Satire and evidence of widespread distrust of alchemists, however, should not mislead one into thinking that alchemy was necessarily spurned or distrusted by everyone. Pope John XXII may have issued a decree against it, but Pope Leo X tolerated it, and

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monarchs and nobles were keen to support it in the hope of benefiting from the gold and silver they hoped it would produce; indeed, over the centuries there were sufficient apparently successful demonstrations of its abilities to ensure that alchemy would continue to enjoy a large measure of such support. English rulers issued licenses to individual practitioners and encouraged their researches, and the sixteenth and early seventeenth centuries saw at least one King of Denmark, two Electors, one King of Spain, one Queen of Sweden, and two Holy Roman Emperors witnessing public transmutations of base metal into gold before large and potentially skeptical audiences. The curious affair of so-called Rosicrucian pamphlets between 1604 and 1616, which suggested that a secret alchemical brotherhood was poised to reform Europe and introduce a period of peace and prosperity, was also taken seriously and made the science even more alluring. Failure, however, or discovery in the perpetration of fraud resulted in swift punishment. One such alchemist in Germany was tortured with red-hot pincers, then drawn and quartered, and his remains were hung from a public gibbet. Another was hanged on a gold-plated gallows, evidence of his ruler’s sardonic sense of humor. The Philosopher’s Stone, as the final transformative substance was usually called, and whose reaction with baser metals caused the desired transmutation, was commonly described as a reddish powder. Producing it involved a lengthy process working with apparently toxic materials and a prima materia whose exact identification alchemists kept to themselves or concealed under a variety of extraordinary names. One says ‘apparently toxic’ because the alchemists’ habit of using imagery and metaphorical language and of changing the terms they applied to their working substances means that we now find it difficult to be sure that, for example, when they spoke of ‘vitriol,’ they meant what we mean by it now. Martin Ruland’s Lexicon of Alchemy (1612) provides several entries for it, some modified by reference to color. Thus, ‘metallic vitriols are the salts of metals’; ‘red vitriol is the perfect sulfur of the philosophers at the red stage’; ‘white vitriol is white galitzen stone’; ‘Roman vitriol is green atrament’; and for ‘atrament,’ he gives 32 separate definitions including ink (made from soot) and various forms of iron and copper sulfate. To add to the confusion, modern commentators regard ‘oil of vitriol’ as referring to sulfuric acid, which would make working with it highly dangerous. Mercury, one of the three substances most commonly referred to by alchemists along with sulfur and salt, was certainly known as the material recognized by that name nowadays. It was used as a life-prolonging elixir, an ingredient in cosmetics, and was employed by at least some alchemists who sought to ‘purify’ it by cleansing it with distilled sal ammoniac or copperas or vinegar. On the other hand, developing alchemical theory proposed that just as humans are composed of spirit, soul, and body, so metals consist of mercury, sulfur, and salt. “Mercury is the spirit, sulphur is the soul, and salt is the body,” wrote the Swiss physician and alchemist Paracelsus in 1537. So when alchemical texts mention mercury, do they mean the chemical element or the metallic principle? “You must always be careful to distinguish what is generally and particularly stated concerning mercury,” warned Ruland, “as to whether it be about ordinary mercury or about our [i.e. alchemical] mercury. Do not make a mistake, otherwise the information will be useless.” Likewise, sulfur presents two general possibilities. Ruland again: “Ordinary sulphur, by whatever name we call it, remains an enemy of all metals. It consumes, blackens, and destroys. But philosophical [i.e. alchemical] sulphur is life-giving, matures and blackens, but does not destroy.” Hence it follows that ‘salt’ had more than one meaning and, indeed, was applied as a general term to various substances including borax (sal albus), sal ammoniac, niter, saltpeter, alum, mercury, urine, calcinated tartar, and common rock salt. Trying to identify the components of alchemical experiments at any given time is thus fraught with difficulty, and it is quite possible that some commentators have been overconfident in their assertions that X alchemical substance is what we know as Y. Paracelsus (1493–1541) illustrates this well. He recognized a wide variety of substances under a single alchemical principle: for example, his ‘salt’ seems to have included potassium nitrate, the vitriols of copper, iron, and magnesium, and the salts generated by mercury, arsenic, antimony, and lead. To him, alchemy was most important as an adjunct of medicine, and he was convinced that the human body acted as a kind of alchemical laboratory, separating pure substances from impure. “Everything,” he wrote in his Volumen Medicinae Paramirum (c.1520), “is perfect in itself, but both a poison and a benefit to something else,” and elsewhere, “Arsenic is the most poisonous of substances and a drachm of it will kill a horse. But fire it with salt of nitre and it is no longer a poison.” Hence if a toxic substance is introduced into the body, (while taking nourishment, for instance), its effects can be combated by the administration of another poison whose therapeutic action purifies the affected organ or organs and therefore cures the illness or at least offers some kind of palliation. In order for the curative or palliative poison to work beneficially, of course, its dosage should be very small. This was crucial. “Only the dose allows something to avoid being poisonous,” he wrote, and clearly this must have been true of his use of arcanum vitrioli (‘the secret thing enclosed in vitriol’) mixed in wine to treat epilepsy, His ‘oil of vitriol,’ on the other hand, which he employed for skin complaints of various kinds, seems to have been corrosive and therefore very painful. Once again, however, it may be difficult to elicit from his descriptions exactly what it was he would use in his recipes. “There are many kinds of vitriol,” he says, noting that “[it] offers a complete cure of jaundice, sands and stones, fevers, worms and falling-sickness . . . [as well as] for surgical diseases such as hereditary scabies, leprosy, ringworm, etc. . . .. These are vigorously attacked by vitriol, which cures them from the root” (Diseases Which Deprive People of Their Reason, written c.1528). Not surprisingly, therefore, Paracelsus appears to have had a reputation among his contemporaries for poisoning his patients, and has been given another by moderns, ‘the Father of Toxicology.’ Alchemists, then, dealt with substances which were often highly toxic but, like Paracelsus, were concerned more with the ultimate uses to which their derivatives could be put rather than the poisonous properties those substances contained. So in view of the dubious nature of many of those substances, it is not surprising to find that alchemists were convinced they had to reduce their basic ingredients by fire, liquefy them, and distil them over and over again, many hundreds of times. This rendered them ‘safe.’ Then practitioners carefully watched for distinctive changes, usually signified in rapid changes of color known as the ‘Peacock’s Tail’ before the material finally settled into the desired red. These procedures fell into three principal stages: (1) the black stage during which the basic material was broken down (‘died’) and reconstituted as something different; (2) the multiple color stage; and (3) the red stage out of which the Stone emerged, and it was during the course of these procedures that medicinal elixirs and

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tinctures were usually obtained. ‘Rectified aqueous alcohol,’ for example, was to be dripped slowly over the residue left after producing the Stone, and this would result in a golden-reddish clear liquid which, taken in wine, was said to cure any ailment. Metaphorical language – that of death, marriage, birth, and regeneration – was used to describe these various stages, as was that of sacramental theology, and so alchemical writings frequently offer a combination of chemistry and Christian mysticism. It is this which seems to account for the deep interest and unwearied experimental efforts of such people as Andreas Libavius, George Starkey (‘Eugenius Philalethes’), Robert Boyle, and Isaac Newton, and even Emmanuel Swedenborg who was hostile to alchemy pursued his chemical experiments in the light of his deeply particular and personal religious convictions. Newton, on the other hand, was deeply devoted to alchemy and doggedly pursued it for mainly religious reasons, on the grounds that if alchemy could show that there was some kind of universal spirit responsible for both creation and the workings of creation, it would provide important evidence for God’s active presence in the universe. But the eighteenth century did alchemy few favors. Charlatans continued to flourish, as did secret and not so secret societies offering pseudo-mystical experiences to their members. Worried that these might tarnish the respectability of the growing number of scientific bodies both within and outwith academic circles, experimenters began to withdraw from the wider rough and tumble and concentrate on more limited, more specialized, less controversial fields of inquiry. Even so, alchemy was still being practiced at Harvard in the early years of the century, although by the middle of the nineteenth chemists had carefully and successfully distinguished themselves from alchemists, and interested observers such as Ethan Hitchcock, an adviser to Abraham Lincoln, preferred to regard alchemy as a spiritual discipline rather than a laboratory activity. Many in the nineteenth and the twentieth centuries agreed with him, and yet that did not stop dedicated individuals from persisting with practical alchemical work. Theodore Tiffereau, for example, conducted experiments between 1854 and 1855 to transmute Mexican silver into gold using, among other materials, nitric acid, hyponitrous acid, nitrogen peroxide, and concentrated sulfuric acid, with trace amounts of gold to act as reagents. He was followed in the 1890s by Stephen Emmens who attempted much the same kind of process and persuaded the United States Mint to buy a large quantity of the gold thus produced. Between 1908 and 1920 when he published the results of his work, François Jollivet-Castelot also transmuted silver into gold by using orpiment, antimony sulfide, tellurium, nitric acid, and a little pure silica, along with gold reagents, these experiments being replicated by others, apparently with some degree of success. But while the early twentieth century saw continued efforts to produce alchemical gold, the attention of alchemists was beginning to turn toward spagyric medicine and the manufacture of health-giving elixirs. Husband and wife Richard and Isabella Ingalese, for example, while claiming to have made the Philosopher’s Stone between 1917 and 1920, made the more astonishing report that in 1917 they had succeeded in producing the white stone of the philosophers – an alchemical product preliminary to the final red Stone – and had used this to resuscitate a dead woman who then lived for a further 7 years. In England, Archibald Cockren worked on various transmuting tinctures during the 1930s, producing among others one which he called ‘philosophic gold,’ until at last he was successful in manufacturing the red Stone itself. Armand Barbault in France during the 1960s and 1970s worked with dew, earth, and plants as his basic materials, adding powdered gold at one stage in the process with a view to producing a series of transformative elixirs, and likewise Albert Riedel (better known as ‘Frater Albertus’), basing his alchemy on Paracelsus’s proposition that matter consists of three principles – spirit/mercury, sulfur/soul, and body/salt – worked mainly with plants to produce therapeutic elixirs. The twentieth century also saw a growing interest in promoting alchemy as a system of spiritual exploration and regeneration, sometimes, however, working alongside laboratory experiments. This, for Frater Albertus, was one crucial difference between alchemy and chemistry. Another was that ‘any poison can be removed alchemically from any herb or metal and its healing and curing properties set free.’ These principles, too, were important for Jean Dubuis, nuclear physicist and alchemist, who founded Les Philosophes de Nature in 1979, and for various online alchemical organizations such as the Paracelsus College and the Spagyricus Institute. But has alchemy ever genuinely succeeded in the transmutation of baser metal into gold? By way of answer, history points to three medallions in the Kunsthistorisches Museum in Vienna, one silver and two gold, which claim to be the results of transmutations successfully performed before witnesses in 1675, 1677, and 1716, and another example in the British Museum in London, dated 1814.

See also: Chemical Interactions; The history of toxicology; Antimony; Bismuth; Copper; Gold; Lead; Mercury; Metals; Silver; Sulfuric acid.

Further reading Alchemy websitewww.levity.com/alchemy/. Ball P (2006) The Devil’s Doctor: Paracelsus and the World of Renaissance Magic and Science. London: William Heinemann. Burland CA (1967) The Arts of the Alchemist. London: Weidenfeld & Nicolson. Crisciani C (2002) Il papa e l’alchimia. Viella: Roma, Italy. Dobbs BJT (1975) The Foundations of Newton’s Alchemy. Cambridge: Cambridge University Press. Greiner F (1998) Aspects de la tradition alchimique au xviie siècle. Paris–Milan: SEHA-ARCHE. Maxwell-Stuart PG (2008) The Chemical Choir: A History of Alchemy. London: Continuum. Moran BT (2005) Distilling Knowledge: Alchemy, Chemistry, and the Scientific Revolution. Cambridge, MA: Harvard University Press. Newman WR (2006) Atoms and Alchemy. Chicago and London: University of Chicago Press.

Alchemy Newman WR and Principe LM (2002) Alchemy Tried in the Fire. Chicago and London: University of Chicago Press. Nummendal T (2007) Alchemy and Authority in the Holy Roman Empire. Chicago and London: University of Chicago Press. Patai R (1994) The Jewish Alchemists. Princeton, NJ: Princeton University Press. Principe LM (1998) The Aspiring Adept: Robert Boyle and His Alchemical Quest. Princeton, NJ: Princeton University Press. Roberts G (1994) The Mirror of Alchemy. London: The British Library. Szydlo Z (1994) Water Which Does Not Wet Hands. Polish Academy of Sciences: Warsaw, Portland.

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Alcoholic beverages and health effects Parna Haghparast and Tina N Tchalikian, Department of Pharmacy Practice, West Coast University, School of Pharmacy, Los Angeles, CA, United States © 2024 Elsevier Inc. All rights reserved. This is an update of K. Shankar, H.M. Mehendale, Alcoholic Beverages and Health Effects, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 120–122, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00227-X.

Introduction Alcohol metabolism Alcohol and neurotransmitters Alcohol and effects on brain Alcohol and weight gain Effect of alcohol on gender Males Females Reproductive health Blood alcohol concentration Alcohol and Wernicke-Korsakoff syndrome Effect of alcohol on pregnancy Alcohol use disorder Alcohol withdrawal Chronic excessive alcohol use leads to chronic diseases Alcohol and nutrition Alcohol use and obesity Pharmacogenetics of alcohol Conclusion References Further reading

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Abstract World Health Organization (WHO) Classification of Diseases reported that consumption of alcohol may impact an individual’s health and increase the risk of harmful health conditions. The purpose of this chapter is to provide a general overview of the short term and long-term effects of alcohol on the body. Some of the areas that are included in this chapter are association between alcohol consumption and its effect on neurotransmitters, reproductive health, pregnancy, liver disease, cardiovascular health, nutrition, cancer, and obesity. Recent trends in treatment for alcoholism includes combination of medications, behavioral treatment, and peer support. USFDA approved drugs for treatment of alcohol dependence are disulfiram, naltrexone and acamprosate, although these treatments are not equally effective for every individual. Top investigators in this field suggest that several other factors, such as psychosocial and economic, as well as genetic variation are dependent upon interindividual variation, clinical presentation of alcohol problems and response to a given treatment. LD50 doses of ethanol are 661 mg/kg (oral) in rats, and 561 mg/kg in mouse (subcutaneous). Recent evidence regarding alcohol use disorders (AUD), fetal alcohol spectrum of disorders (FASD), and pharmacogenetic considerations are discussed in this chapter.

Keywords Alcohol; Alcohol use disorder; Alcohol withdrawal; Blood alcohol concentration; Cardiovascular; Cirrhosis; Health; Liver; Nutrition; Obesity; Pregnancy; Wine; Wernicke-Korsakoff syndrome

Key points

• • •

This chapter includes association between alcohol consumption and its effect on the body Metabolism of alcohol varies among individuals and may be impacted by multiple factors such as presence or absence of food, gender, concentration of alcohol, type of beverage and pharmacogenetic considerations Individual may build tolerance and dependence when consuming alcohol in a repetitive manner, by adapting to the neurotransmitter changes

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Chronic alcohol consumption decreases inhibitory neurotransmitters and increases excitatory neurotransmitters Binge drinking and high alcohol consumption may increase the risk of type 2 diabetes in women Heavy drinking or binge drinking (but not light to moderate drinking) may be a risk factor to obesity/weight gain Chronic consumption of excessive alcohol may adversely affect semen quality parameters in men Alcohol abuse may result in thiamine (vitamin B1) deficiency due to decreased absorption. One of the complications of thiamine deficiency is Wernicke-Korsakoff syndrome Alcohol abuse can impair nutritional status needed for the growth of developing fetus, resulting in Fetal Alcohol Spectrum Disorder (FASD) Chronic excessive alcohol use leads to chronic diseases such as liver disease and development different types of cancer

Abbreviations ADH AUD BAC BMI FAS FASD FSH LH MEOS ROS WHO

Alcohol dehydrogenase Alcohol use disorder Blood alcohol concentration Body mass index Fetal alcohol syndrome Fetal alcohol spectrum disorder Follicle stimulating hormone Luteinizing hormone Microsomal ethanol-oxidizing system Reactive oxygen species World Health Organization

Introduction Alcohol can have harmful effects on health that may lead to unintended consequences. According to the 2019 National Survey on Drug Use and Health (NSDUH), 85.6% of people aged 18 years and older reported that they drank alcohol at least once in their life, 69.5% reported that they drank within the last year, and 54.9% reported that they drank in the past month. About 14.1 million adults aged 18 years and older had alcohol use disorder in 2019 (SAMHSA, 2019). In 2018, of the 83,517 liver disease deaths among individuals ages 12 and older, 42.8% involved alcohol. Excessive alcohol use is harmful on health and impacts the U.S. economy. Loss of workplace productivity, healthcare expenses, increase in crime, and losses from motor vehicle crashes are all associated with alcohol use. Studies by Centers for Disease Control and Prevention (CDC) have found that excessive drinking cost the U.S. $223.5 billion (CDC, 2021). According to National Institute on Alcohol Abuse and Alcoholism (NIAAA), a standard drink contains roughly 14 g of pure alcohol. Different types of alcoholic beverages have different amounts of alcohol content. One standard drink containing 14 g of alcohol can be found in 12 oz of beer, 5 oz of wine, or 1.5 oz of distilled spirits (gin, rum, tequila, vodka, whiskey, etc.) (NIAAA, 2021c). NIAAA defines moderate alcohol consumption as 1 drink per day for women and 2 drinks per day for men (NIAAA, 2004). As shown in Table 1, NIAAA defines binge drinking as a drinking pattern that leads to a peak blood alcohol concentration (BAC) of
0.08% (or 0.08 g/dL). This typically means
4 drinks for women and
5 drinks for men in a 2-h period (NIAAA, 2004). Similarly, Substance Abuse and Mental Health Services Administration (SAMHSA) defines binge drinking as
4 drinks for women and
5 drinks for men on the same occasion on at least 1 day in the past month (SAMHSA, 2019). Heavy alcohol use is defined as more than 3 alcoholic drinks in a day or more than 7 a week for women and more than 4 drinks a day or 14 a week for men (NIAAA, 2004). SAMHSA, on the other hand, defines heavy drinking as binge drinking 5 or more days in the past month (SAMHSA, 2019).

Table 1

Data shows NIAAA and SAMHSA definitions of binge-drinking in relation to alcohol consumption.

Definitions

NIAAA

SAMHSA

Binge drinking

Drinking that leads to BAC
0.08 Women:
4 drinks in 2 h Men:
5 drinks in 2 h
3 alcoholic drinks in a day or
7 a week for women and
4 drinks a day or 14 a week for men

Women:
4 drinks on the same occasion on at least 1 day in 30 days Men:
5 drinks on the same occasion on at least 1 day in 30 days

Heavy drinking

Binge drinking
5 days in 30 days

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Alcohol metabolism It is important to understand the metabolism of alcohol to further understand the health effects on the body. The enzymes involved in alcohol metabolism in the liver are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Majority of alcohol is metabolized by ADH (approx. 80%), but 20% is oxidized to acetaldehyde by CYP2E1, which is then further metabolized into acetate by ALDH (Wall et al., 2016). CYP2E1 mediated oxidation of alcohol is called microsomal ethanol oxidizing system (MEOS), and this pathway produce different free radicals (such as hydroxyethyl radical) and reactive oxygen species. C2H5OH Ethanol

ADH

CH3CHO Acetaldehyde

ALDH

CH3COOH Acetate

Metabolism of alcohol varies among individuals and may be impacted by multiple factors such as presence or absence of food, gender, concentration of alcohol, and type of beverage (Lu and Cederbaum, 2018). Alcohol metabolizes faster during the fed state since there are higher levels of ADH present compared to the fasting state (Lu and Cederbaum, 2018). If there is presence of liver damage, the rate of alcohol oxidation and elimination will decrease. Alcohol consumers, specifically women, have a low first pass metabolism due to low ADH activity, increasing sensitivity to alcohol and leading to a higher BAC (Cederbaum, 2013). Subsequently, men have a higher first pass metabolism of alcohol, resulting in a lower BAC. Furthermore, variations in ADH and ALDH modifies the association between alcohol use and diabetes (Rachdaoui and Sarkar, 2013). In an in-vitro study, it was found that ethanol generates reactive oxygen species (ROS) resulting in beta cell dysfunction and reduced insulin secretion (Rachdaoui and Sarkar, 2013). Reduced levels of insulin secretion may lead to diabetes mellitus (DM) with high glucose levels (Rachdaoui and Sarkar, 2013). An observational study by Carlsson et al. showed that binge drinking, and high alcohol consumption increase the risk of type 2 diabetes in women, but not men (Carlsson et al., 2003).

Alcohol and neurotransmitters By consuming alcohol in a repetitive manner, an individual may build tolerance and dependence to alcohol by adapting to the neurotransmitter changes (Colrain et al., 2014). In normal conditions, there is a balance between both inhibitory and excitatory neurotransmitters. Acute consumption of alcohol alters the balance toward inhibition by increasing inhibitory neurotransmitters, such as GABA, glycine, and adenosine, in addition to decreasing excitatory neurotransmitters such as glutamate and aspartate. Short term effects of alcohol consumption causes depression on the brain, decreases attention, causes change in mood. However, chronic alcohol consumption attempts to restore the balance by decreasing inhibitory neurotransmitters and increasing excitatory neurotransmitters (Valenzuela, 1997). The body attempts to reach equilibrium between the two systems to compensate for the inhibitory effects of alcohol.

Alcohol and effects on brain Consumption of alcohol impacts the nervous system, brain in particular. These actions cause behavioral changes in an individual such as increased use of alcohol, development of tolerance and dependence, and alcohol seeking behavior (Abrahao et al., 2017). Alcohol consumption leads to high levels of BACs, resulting in changes such as euphoria, decreased motor skills, and sedation (Abrahao et al., 2017). Furthermore, alcohol impairs the function of excitatory and inhibitory neurotransmitters in the brain, decreasing glutamate receptors and increasing GABA and glycine receptors (Abrahao et al., 2017). By decreasing glutamate, firing of neurons are inhibited resulting in changes in brain activity (Abrahao et al., 2017). Alcohol’s effect on dopamine on locomotor skills and reward system have been extensively studied. Once alcohol is consumed, firing of the dopaminergic neurons causes more dopamine to enter the ventral tegmental area (VTA) and continue to move to downstream structures such as amygdala, prefrontal cortex, and nucleus accumbens (Morel et al., 2019; Abrahao et al., 2017; Deehan Jr et al., 2016). VTA’s dopamine neurons play a critical role in drug-related behaviors such as associative learning and reinforcement (Morel et al., 2019; Oliva and Wanat, 2016). VTA dopamine neurons encode rewards and mediate signals which promote reward seeking behaviors that may contribute to addiction related behaviors (Morel et al., 2019). Increasing evidence shows that the laterodorsal tegmentum (LDT) which regulates firing activity of dopamine neurons is also involved in drug-related behaviors. LDT, made up of acetylcholine, GABA, and glutamate neurons, send signals to the midbrain VTA dopamine system (Oliva and Wanat, 2016). In vivo studies have shown that stimulation of LDT activates an action potential which causes firing in VTA dopamine neurons (Morel et al., 2019; Oliva and Wanat, 2016). Alcohol produces injury to cells by dehydration and precipitation of the cytoplasm or protoplasm. This accounts for its bacteriocidal and antifungal action. When alcohol is injected in close proximity to nerve tissues, it produces neuritis and nerve degeneration (neurolysis). Ninety to ninety eight percent of ethanol that enters the body is completely oxidized. Ethanol is also used as a cosolvent to dissolve many insoluble drugs and to serve as a mild sedative in some medicinal formulations. Ethanol also binds to GABA, glycine, NMDA receptors and modulates their effects as described above (PUBCHEM, n.d.).

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Alcohol and weight gain Although there is conflicting evidence regarding alcohol intake and its association with obesity/weight gain, it appears that heavy drinking or binge drinking (but not light to moderate drinking) may be a risk factor to obesity/weight gain. Potential explanations of alcohol’s effect on weight include (1) its additive energy intake to other food sources (2) alcohol stimulating food intake by affecting peripheral and central neurotransmitters. It appears that the individuals do not necessarily change the amount of food intake to compensate for the added energy from alcohol when drinking before or during a meal (Yeomans, 2010). In addition to the added energy from alcohol, it plays an important role on hormones used for regulating hunger and appetite (Traversy and Chaput, 2015). As shown below, alcohol may contribute to stimulating appetite by inhibiting leptin and glucagon-like peptide-1 (GLP-1) and affecting the gamma-aminobutyric acid (GABA), opioid and the serotonergic pathway (Röjdmark et al., 2008; Raben et al., 2003). Alcohol effects on hunger and energy via peripheral signals and central neurotransmitter systems has been described by Traversy and Chaput (2015). Normally, upon appropriate peripheral signals, neurotransmitters leptin and GLP-1 are released; both suppress hunger and energy intake and show corresponding decline on intake on hormone/neurotransmitter response. In contrast, when central neurotransmitter system, such as GABA is released, it stimulates hunger/energy intake, and show agonistic effect on intake on hormone/neurotransmitter response (Traversy and Chaput, 2015).

Effect of alcohol on gender Males Alcohol, especially chronic and excessive consumption, can adversely affect the male reproductive hormones and spermatogenesis (Sansone et al., 2018). Chronic alcoholism has been associated with increase in luteinizing hormone (LH), follicle-stimulating hormone (FSH), E2 and decrease in testosterone level (Muthusami and Chinnaswamy, 2005). In one of the earlier studies in 1985, the effect of alcohol on male reproductive system was studied in 20 men with alcohol dependence syndrome (Kucheria et al., 1985). The authors found considerable decrease in testosterone level, seminal fluid volume and sperm concentration compared to controls. Similarly, in a meta-analysis looking at psycho-social risk factors for semen quality, found that alcohol consumption can adversely affect sperm quality parameters such as sperm motility and the number of morphologically normal sperm (Li et al., 2011). Consuming excessive alcohol chronically can in turn have a negative effect on fertility and can delay the pregnancy time in couples who wish to get pregnant (Condorelli et al., 2015; Hassan and Killick, 2004). That said, it appears that moderate alcohol intake may not adversely affect male fertility and that chronic consumption of alcohol may be a more influential factor than acute consumption (Taylor et al., 2009; La Vignera et al., 2013; Jensen et al., 2014; Sansone et al., 2018).

Females The female reproductive system may be affected by alcohol consumption similar to males. Prospective studies have evaluated the harmful effects alcohol has on the female reproductive system and menstrual cycling (Schliep et al., 2015; Lyngso et al., 2014; Hahn et al., 2013). Findings report that higher alcohol intake is correlated with elevated risk of heavy menses and anovulation (Schliep et al., 2015; Lyngso et al., 2014; Hahn et al., 2013). According to the World Health Organization, menses length was defined as the number of days of a bleeding episode that included two or more days of bleeding in 3 days, followed by two or more bleed free days (Schliep et al., 2015). Women who drink one drink per day on average report a higher risk of heavy menses than non-drinkers (Schliep et al., 2015). Hahn et al. reported in a cross-sectional study that heavy alcohol consumption of 14 or more drinks per week resulted in higher incidence of heavy menstruation (Hahn et al., 2013). In addition, the study reports that alcohol consumption is associated with higher estradiol levels (Schliep et al., 2015). Depending on number of drinks per day, the average estradiol levels increase by 5.82% (Schliep et al., 2015). Thus, there is a strong correlation between alcohol intake and elevated estradiol. Elevated estradiol concentrations alter the stimulation of the adrenal gland and block the conversion of testosterone into estradiol (Wozniak et al., 2019). Alcohol induced increased estradiol can affect the female reproductive system, resulting in changes in the body including menstrual cycle disturbances (Schliep et al., 2015; Lyngso et al., 2014). Excess estrogen affects hormones and sex hormone binding globulin (Hahn et al., 2013). A prospective study had observed the correlation between number of drinks and affects fertility of couples undergoing IVF treatment. Women drinking at least 4 drinks/week had 16% less odds of a live birth rate compared with those who drank fewer than four drinks per week (Rossi et al., 2011). Among those who were included in the study presenting for IVF treatment, 45–66% had reported alcohol use (Rossi et al., 2011).

Reproductive health Alcohol consumption has a strong association with sexual behaviors increasing the risk of human immunodeficiency virus (HIV) and sexually transmitted infections (STIs) (Carey et al., 2019). Correlation between alcohol use and risky behaviors were examined in a study in an STI clinic. In this study, women reported that alcohol consumption impairs thinking and focuses attention on salient cues, increasing the risk of sexual behavior (Carey et al., 2019). Women binge drinkers were found to be involved in risky behaviors in comparison to non-drinkers. Alcohol use is associated with having multiple sex partners, a risky sexual behavior because of the increased likelihood of contracting an STI (Hutton et al., 2008). Adolescents and those who drank alcohol were also

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at increased risk of unintended pregnancy. Women with an unintended pregnancy are more likely to consume alcohol and take part in risky behavior in comparison to women who plan a pregnancy. Women with an unintended pregnancy are more likely seen in alcohol consumers (Hutton et al., 2008).

Blood alcohol concentration Blood alcohol concentration (BAC), typically reported as a percentage, is the amount of alcohol in the blood after acute consumption of ethanol. Acute consumption of alcohol can lead to euphoria, loss of coordination, slurred speech, confusion, nausea, vomiting, drowsiness, memory impairment, and loss of consciousness. The effect of alcohol is primarily dependent on the blood alcohol concentration. The higher the BAC, the more significant impairments as described above. Side effects of a higher BAC include slurred speech, slowed reflexes, loss of physical coordination, and troubled memory (U.S. National Library of Medicine, 2020) (Table 2). BAC continues to increase until the rate of elimination exceeds the rate of absorption (Mitchell Jr et al., 2014). The BAC is affected by different factors such as gender, body weight, amount of alcohol consumed, how quickly the alcohol is consumed, and food intake (Dasgupta, 2017). Women tend to have a higher BAC compared to men of comparable weight after drinking same amount of alcohol Furthermore, food intake decreases rate of absorption and increases rate of ethanol elimination, leading to a decreased BAC. Some studies suggest that different concentrations of ethanol in a beverage may influence rate of absorption alcohol and consequently the BAC (Roine et al., 1993; Mellanby, 1919; Mitchell Jr et al., 2014). In a study conducted by Mitchell and colleagues, the effect of different alcoholic beverages (5.1% beer, 12.5% wine and 20% vodka/tonic) on the rate of rate of absorption and BAC in a fasting state was evaluated. The BAC was higher after vodka/tonic than after wine or beer. Beer and wine were absorbed slower than vodka/tonic, therefore BAC was the lowest after drinking beer. Thus, the bioavailability of alcohol was the least after drinking beer. The authors concluded that despite similar amount of ethanol in 12 oz of beer, 5 oz of wine and 1.5 oz of liquor, the peak BAC may be higher in individuals drinking more concentrated beverages in the absence of food (Mitchell Jr et al., 2014). Alcohol consumption effects the brain resulting in difficulty walking, blurred vision, slurred speech, slowed reaction times, and impaired memory (Alcohols Damaging Effects on the Brain, 2021). Heavy drinking can cause permanent impairment on the brain, whereas moderate drinking can cause short term impairment on the brain (Alcohols Damaging Effects on the Brain, 2021). Blackouts occur when an individual drinks large amounts of alcohol in a short period of time or on an empty stomach where they cannot recall events (Alcohols Damaging Effects on the Brain, 2021; Parsons, 1996; White, 2003). Drinkers who experience blackouts typically drink too much and too quickly, which causes their blood alcohol levels to rise very rapidly (Alcohols Damaging Effects on the Brain, 2021; Parsons, 1996; White, 2003). An average of six people die from alcohol poisoning every day in the U.S. (Alcohol Poisoning Deaths, 2021). Alcohol poisoning occurs from consuming large amounts of alcohol in a short period of time (Alcohol Poisoning Deaths, 2021). Presence of high amounts of alcohol, specifically BAC levels reaching 0.250–0.399%, in the body severely impairs parts of the brain responsible for breathing, heart rate, and body temperature leading to loss of consciousness and alcohol poisoning (Alcohol Poisoning Deaths, 2021; University of Notre Dame, 2021). Signs of alcohol poisoning include inability to wake up, vomiting, slow breathing, irregular heart rate, seizures, and hypothermia (Alcohol Poisoning Deaths, 2021).

Alcohol and Wernicke-Korsakoff syndrome Alcohol abuse may result in thiamine (vitamin B1) deficiency due to decreased absorption. One of the complications of thiamine deficiency is Wernicke-Korsakoff syndrome seen in patients with alcohol use disorder (Akhouri et al., 2020). Chronic alcohol use is the main factor associated with Wernicke-Korsakoff syndrome but may also occur in patients that are deficient in vitamin B1 for other reasons such as bariatric surgery (Akhouri et al., 2020). Wernicke-Korsakoff encompasses two different syndromes—Wernicke Table 2

Physiological effects of blood alcohol levels as reported in NIAAA/PUBCHEM.

Blood alcohol levels (BAC)

Physiological effects

0.01–0.04% (10–40 mg/dL) 0.05–0.07% (50–70 mg/dL) 0.08% (80 mg/dL) 0.08–0.12% (80–120 mg/dL) 0.12–0.15% (120–150 mg/dL) 0.15–0.2% (150–200 mg/dL) 0.2–0.3% (200–300 mg/dL) 0.3–0.4% (300–400 mg/dL) 0.4–0.5% (400–500 mg/dL) Above 0.5% (500 mg/dL)

Relaxed feeling, being social with others Mild impairment of driving skills US legal limit to drive a vehicle Moderate to significant impairment of driving skills; changes in mood depression or excitement Severe impairment of motor function, speech, and judgement; slurred speech Appears drunk, severe visual impairment Nausea, vomiting, incontinence Needs assistance walking, loss of consciousness Possible coma or respiratory failure Possible death

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encephalopathy (WE) and Korsakoff syndrome. Some scientists believe that WE and Korsakoff are two different stages of the same syndrome, where the WE represent the acute phase with reversible symptoms if treated early, but the Korsakoff syndrome is chronic/ late stage with potentially irreversible symptoms (NINDS, 2021). Wernicke encephalopathy (WE) is characterized by a confusional state, nystagmus, and ataxia. Other symptoms include hypothermia, hypotension, and coma (NINDS, 2021). In addition to the symptoms present in the WE, Korsakoff syndrome is characterized by memory loss and, confabulation. Symptoms of KP include delirium and sudden memory loss (Day et al., 2013). When patients present with the full triad of symptoms and disorder progresses, it is commonly known as Wernicke-Korsakoff syndrome (Day et al., 2013). Wernicke-Korsakoff syndrome’s development may be prevented and/or treated with administration of thiamine and abstaining from alcohol. Improvement in mental status may occur after 2–3 weeks of treatment (Martin et al., 2003; Day et al., 2013; Akhouri et al., 2020). Although treatment is recommended, full symptom recovery is uncommon.

Effect of alcohol on pregnancy Prevalence of pregnant women drinking alcohol has increased over the years. The fetus may undergo physical and neurologic impairment if alcohol is consumed during pregnancy. When nutritional status is impaired by alcohol abuse, the nutrients needed for the growth of developing fetus are not available, resulting in Fetal Alcohol Spectrum Disorder (FASD) (Sebastiani et al., 2018). A systematic review and meta-analysis estimated that the global prevalence of FASD among children is 7.7 per 1000 population of women who drank alcohol (Lange et al., 2017). Fetal Alcohol Spectrum Disorder (FASD) prevalence is associated with alcohol use and may be preventable if alcohol abstinence is attained (Wozniak et al., 2019). FASD includes conditions such as growth retardation, neurological abnormalities, cognitive and behavioral impairment, birth defects, and craniofacial anomalies (Wozniak et al., 2019). Furthermore, epilepsy may be seen in patients with FASD due to irregular development of cortical areas (Wozniak et al., 2019). FASD is a generalized term that includes four subgroups: Fetal Alcohol Syndrome (FAS), partial FAS, alcohol related neurodevelopmental disorder (ARND), and alcohol related birth defect (ARBD) (Wozniak et al., 2019). The global prevalence of FAS among the population is estimated to be 1.4 per 1000 (Sebastiani et al., 2018). Although any drink may be considered harmful during pregnancy, the risk for the fetus is the highest when the mother drinks more than 6 drinks per day (Sebastiani et al., 2018). Women with lower BMI and binge drink are not able to effectively eliminate alcohol allowing more alcohol to cross the placenta (Sebastiani et al., 2018). If fetal exposure to alcohol increases, the risk of teratogenic effects increases. Collaboration on FASD Prevalence (CoFASP) diagnostic criteria addresses four of the FASD sub-types. Diagnosis of FAS by the Institute of Medicine (IOM) criteria must include dysmorphic face, growth deficiency, brain abnormality, and cognitive or behavioral impairment (Table 3). Diagnosis of partial FAS must include dysmorphic face, cognitive or behavioral impairment, and one of the two: growth deficiency or brain abnormality (Table 3). Diagnosis of alcohol-related neurodevelopmental disorder (ARND) must include cognitive or behavioral impairment. Diagnosis of alcohol-related birth defects (ARBD) must include systemic malformation and confirmed prenatal alcohol exposure (Table 3). Prenatal alcohol exposure is described as “six or more drinks per week for two weeks or three or more drinks on two or more occasions, positive biomarker for alcohol, or evidence of maternal drinking on a screening tool” (Jauhar et al., 2014; Wozniak et al., 2019).

Alcohol use disorder Alcohol Use Disorder (AUD) is a medical condition by which the individual is unable to control their pattern of alcohol consumption (NIAAA, 2021a,b). Symptoms include but are not limited to being unable to cut down on the amount of alcohol, building a tolerance to alcohol, craving alcohol, having withdrawal symptoms when not drinking and having interpersonal problems caused by alcohol (Witkiewitz et al., 2019; Hartwell and Kranzaler, 2019). Based on a national survey in the United States, about 14 million adults (5%) and 400,000 youths (1.7%) reported AUD in 2019 (NIAAA, 2021a,b). Individuals who misuse alcohol, in other words, binge drink or drink heavily are at risk for developing AUD over time. Additionally, individuals that start Table 3

CoFASP diagnostic criteria.

Diagnosis

Confirmed prenatal alcohol exposure

Dysmorphic face

Growth deficiency

Brain abnormality

Cognitive or behavioral impairment

Other systemic malformation

FAS

Yes No Yes No

Required Required Required Required

Required Required Not Required Required if growth deficiency is not present

Required Required Required Required

Not Required Not Required Not Required

Yes Yes

Not Required N/A

Required Required Not Required Required if brain abnormality is not present Not Required N/A

Not Required N/A

Required N/A

Not Required Required

Partial FAS

ARND ARBD

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drinking before the age of 15, are five times more likely to report AUD compared to those that waited till the age of 21 (NIHAAA, 2021). There are screening tools for AUD, such as the CAGE questionnaire which asks whether the individual felt a need to cut down on drinking, was annoyed by criticism of drinking, felt guilty about drinking, or needed a drink first thing in the morning (American Psychiatric Association, 2018). “Women also displayed lower rates of binge drinking (i.e., four or more drinks on the same occasion in the past-month for women, five or more drinks for men; 20.5%), heavy drinking (i.e., binge drinking on at least 5 days in the past month; 4.2%), and alcohol use disorder (4.1%) than men (29.6%, 8.9%, 7.8%, respectively)” (McHugh et al., 2018). Pharmacological treatment used for AUD include disulfiram, acamprosate, gabapentin, naltrexone, topiramate, sertraline, and baclofen (Singal et al., 2018). Nonpharmacological treatments include alcohol abstinence and cognitive behavioral therapy (Singal et al., 2018). The American Psychiatric Association recommends naltrexone or acamprosate as the drug of choice for patients with mild to moderate alcohol use disorder achieving abstinence (American Psychiatric Association, 2018). Naltrexone has been associated with reduced cravings, reduced likelihood of return to drinking, and fewer drinking days (American Psychiatric Association, 2018). Acamprosate has been associated with reduced likelihood of return to drinking after attaining abstinence and reduced number of drinking days (American Psychiatric Association, 2018). Acamprosate is recommended once abstinence is attained and continued if patient relapses. Depending on patient specific considerations, naltrexone or acamprosate are considered appropriate initial therapy (McHugh and Weiss, 2019; Maiese, 2021; Flores-Bonilla and Richardson, 2020; Meredith et al., 2021; Patel and Balasanova, 2021).

Alcohol withdrawal Binge or heavy drinkers who abruptly discontinue or decrease use of alcohol, may experience withdrawal symptoms such as anxiety, tremor, nausea, irritability, and insomnia. Symptoms within 6–24 h post alcohol cessation may include nausea, vomiting, hypertension, tachycardia, tremor, hyperreflexia, irritability, anxiety, and headache (Singal et al., 2018). After 24 h, symptoms may include delirium tremens, generalized seizures, coma, cardiac arrest, or death (Singal et al., 2018). Individuals having a hard time quitting may need pharmacological treatment. Treatment goals for patients with alcohol withdrawal are to reduce symptoms of withdrawal, prevent or treat seizures, and prepare the patient for alcohol abstinence. The onset of alcohol withdrawal seizures may occur 24–72 h after the last alcohol intake. Seizures may last up to 5 min and may progress into delirium, increasing risk of death. Individuals experiencing such symptoms should be treated in an in-patient setting (Tiglao et al., 2021). The most common medication used to treat alcohol withdrawal is benzodiazepines. Benzodiazepines, such as chlordiazepoxide and lorazepam, are commonly used to prevent and treat acute alcohol withdrawal and work by acting on GABA-A receptors. Adjunct therapy, in addition to the primary treatment, may be used for patients who do not benefit from the primary treatment alone. Adjunct therapy for alcohol withdrawal includes alpha-agonists, such as clonidine, and beta-blockers, such as atenolol (Witkiewitz et al., 2019).

Chronic excessive alcohol use leads to chronic diseases Liver: Long term alcohol use can lead to development of alcoholic liver disease (ALD). ALD is a clinical-histological liver spectrum that ranges from asymptomatic or early ALD (hepatic steatosis) to advanced ALD (alcoholic hepatitis (AH), cirrhosis and its complications). Alcohol use disorder is one of the primary causes of preventable liver disease and death with ALD accounting for about half of the cirrhosis-associated deaths. In addition to death associated with ALD, death associated with alcohol-impaired driving is also common (accounting for 31% of driving fatalities in US). About 10–20% of heavy drinkers develop severe forms of ALD such as AH and cirrhosis. Women who drink more than 2 and men who drink more than 3 alcoholic beverages a day for over 5 years are at risk for developing ALD. Females are at risk of alcoholic liver disease at a lower daily intake of alcohol likely due to higher body fat and lower gastric ADH activity. There is a direct relationship between amount of alcohol and ALD, however, more studies are required to evaluate the impact of drinking patterns and type of alcohol on ALD. Apart from, amount of alcohol, behavioral (e.g., cigarette smoking), environmental (e.g., obesity), genetics and co-existence of chronic hepatitis (e.g., Hepatitis C) can play a role. About 90% of heavy drinkers develop hepatic steatosis. About 20–40% of patients with hepatic steatosis on liver biopsy will develop fibrosis and about 8–20% will develop cirrhosis. Alcoholic hepatitis (AH) can occur at any stage of liver disease and is associated with liver disease complications. ALD is diagnosed when there is documentation of chronic heavy alcohol use, elevation in liver enzymes, aspartate transaminase (AST), alanine aminotransferase (ALT), and exclusions of other causes (Singal et al., 2018). The Alcohol Use Disorder Inventory Test (AUDIT) is a validated tool to help identify patients with alcohol use dependence. Since breakdown of alcohol occurs in the liver, toxic products such as ADH may damage the liver. The severity of alcoholic liver disease depends on additional risk factors. The most effective therapy is abstinence, which can not only prevent progression of liver disease but also potentially reverse liver damage. Liver transplants are also options for AH in select patients. Per the American College of Gastroenterology Clinical guidelines, patients with obesity and hepatitis C, should avoid alcohol consumption and those with ALD should abstain from cigarettes (Singal et al., 2018). Cardiovascular: Alcohol use has several effects on cardiovascular (CV) health and diseases such as hypertension and stroke. Hypertension: Low to moderate amounts of alcohol does not appear to have substantial short term effect on blood pressure. However, binge drinking of more than 5 drinks a day has been associated with transient increases in blood pressure, specifically 4–7 mmHg increase for systolic and 4–6 mmHg increase for diastolic (Piano, 2017). Some studies suggest that women and men

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who drink more than 1–2 drinks/day are at increased risk of hypertension. Additionally, some of the systematic reviews suggest that alcohol consumption 30 g/d was associated with stroke (Ronksley et al., 2011; Zhang et al., 2014). Digestive problems: Alcohol induces intestinal inflammation and may result in multiple organ dysfunctions and chronic disorders associated with alcohol consumption (Bishehsari et al., 2017). Alcohol consumption disrupts the normal gut flora and promotes bacterial growth, leading to inflammation (Bishehsari et al., 2017). Additionally, individuals with AUD tend to have vitamin deficiency which in turn has effects on digestive system (Bishehsari et al., 2017; Zhong et al., 2013). Pancreas: The pancreas is susceptible to tissue injury from alcohol use (Molina and Nelson, 2018). Alcohol-induced tissue damage may include inflammation, fibrosis, oxidative stress, and apoptosis. In turn, the pancreas may undergo pathological changes and lead to pancreatitis (Molina and Nelson, 2018). Chronic consumption of alcohol contributes to the development of pancreatitis (Molina and Nelson, 2018) especially when consumed >40 g/day. Retrospective clinical studies have shown that binge alcohol drinking is associated with severe pancreatitis (Molina and Nelson, 2018; Samokhvalov et al., 2015; Tsermpini et al., 2022). Alcohol and Cancer: Chronic alcohol use is associated with cancer development, specifically in the upper aerodigestive tract, liver, colorectal, and breast. About 3.5% of all cancer-related deaths worldwide are associated with chronic alcohol drinking (Boffetta et al., 2006). The primary metabolite of ethanol (acetaldehyde), and oxidative stress, and DNA methylation may contribute to alcohol-induced cancer (Shield et al., 2016; Varela-Rey et al., 2013). Memory problems: Rehm et al. have shown in a retrospective study that alcohol use may play a critical role in developing dementia (Rehm et al., 2019; Schwarzinger et al., 2018). Alcohol and its metabolite acetaldehyde cause a neurotoxic effect causing brain damage (Rehm et al., 2019; Kruman et al., 2012; NIAAA, 2000). A systematic review has shown that daily heavy alcohol use was associated with an increased risk of developing dementia (Schwarzinger et al., 2018; Rehm et al., 2019; Hersi et al., 2017). Additionally, chronic alcohol use may cause thiamine deficiency and development of Wernicke-Korsakoff syndrome resulting in neuropsychiatric symptoms (Rehm et al., 2019; Jauhar et al., 2014; Martin et al., 2003). Dementia related cases among individuals 64 years of age or older were associated with alcohol use or previous alcohol use disorder (AUD). Furthermore, individuals with previous AUD cases among any age were associated with dementia (Schwarzinger et al., 2018; Prince et al., 2013; Arskey and O’Malley, 2007).

Alcohol and nutrition Consumption of alcohol interferes with nutrition intake and may alter normal eating or drinking habits. Individuals may likely alter their normal habits of eating or drinking, resulting in decreased levels of energy, poor nutritional status, and altered behavior (Wozniak et al., 2019). Regular consumption of alcohol may result in malnutrition, including loss of vitamins, omega-3, folic acid, and zinc. Alcohol can interfere with the uptake of essential amino-acids and vitamins, specifically B1 (thiamine), B2 (riboflavin), B6 (pyridoxine), vitamin A and C and folic acid (Sebastiani et al., 2018). Individuals who consume more than 30% of their total daily calories in the form of alcohol would be less likely to consume high amounts of protein, fats, vitamins, or carbohydrates. An unbalanced diet results in reduced nutrient intake and absorption. Combining Alcohol with Other Substances: Drinking alcohol and taking medication is contraindicated in most cases. It can be harmful and may cause more sleepiness, drowsiness, lightheadedness or many other unknown side effects (NIAAA, 2021a,b). Certain medications have a high percentage of alcohol, such as 10% alcohol. Additionally, some medications may be less effective when combined with alcohol. Some of the commonly used medication classes that interact with alcohol include painkillers (acetaminophen, aspirin), antidepressant, antidiabetic, antibiotics and antileptics (NIAAA, 2021a,b).

Alcohol use and obesity Since a gram of alcohol provides 7.1 kcal, alcohol consumption plays a huge factor in weight gain because of the high calories in alcoholic beverages (Traversy and Chaput, 2015). It is more common in individuals who are binge drinkers or heavy drinkers to experience excess body weight. Prospective studies have shown the association between men who increased frequency of alcohol and increase in body mass index (BMI) (Traversy and Chaput, 2015). The highest incidence of weight gain was seen among beer drinkers (Traversy and Chaput, 2015). Shelton and Knott reported the association between alcoholic calorie consumption and obesity in adults (Shelton and Knott, 2014). The highest risk of obesity was 70%, which was seen among individuals who had the highest alcohol intake (Traversy and Chaput, 2015; Shelton and Knott, 2014). Comparing between different beverages and risk of

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obesity, individuals consuming beer, spirits, or more than one beverage type were at a higher risk than individuals consuming wine. This is due to the different caloric intake among beverages. Individuals that consume more than the recommended daily allowance were more likely to report obesity among adults (Shelton and Knott, 2014).

Pharmacogenetics of alcohol Alcohol metabolism is predominantly handled by ADH and ALDH, although numerous “candidate genes” for alcoholism have been suggested which exhibit genetic heterogeneity. It is known for decades that ADH2 which is an atypical form of ADH, discerns a variant beta 2 subunit instead of the usual beta-1 subunit. Both are significantly different and show remarkable variations in their kinetic properties. Interestingly however, one is found more frequently among the Japanese, Chinese and other Mongoloid populations than in Caucasians and Africans. Another frequent genetic polymorphism has been observed for ALDH; livers of about 50% of Japanese and Chinese show an inactive ALDH (ALDH2 isozyme) whereas none of the Caucasian or African populations show this aberrant isozyme. Differences in the in vivo elimination rate of ethanol and acetaldehyde, may explain variable alcohol-related behaviors and its disease outcomes. Likewise, Asians who possess an atypical ALDH2 gene are more sensitive to acute responses to alcohol, tend to be discouraged from drinking alcohol, and consequently are at lower risk of developing alcohol-related ailments. Results of research in alcoholism show great variability in inter-individual and racial, and alcohol and acetaldehyde metabolism patterns (PUBCHEM, n.d.). These findings safely propel the hypothesis that the individual and racial differences in alcohol metabolism are based on the genetically determined variability of the participating enzymes, alcohol dehydrogenase and aldehyde dehydrogenase. In Orientals lacking the mitochondrial low Km aldehyde dehydrogenase, acetaldehyde accumulates and produces symptoms of intoxication (Agarwal and Goedde, 1992; Juraeva et al., 2015; Kranzler and Soyka, 2018; Purohit et al., 2018; Hartwell and Kranzaler, 2019).

Conclusion Chronic excessive alcohol use leads to chronic diseases, such as liver cirrhosis and significantly increase the risk of cardiovascular diseases, memory loss, and digestive issues. Moreover, the effect of alcohol is primarily dependent on the blood alcohol concentration. The higher the BAC, the more significant neurological impairment and physical functioning, ultimately harming host’s health. These impairments can be avoided by refraining from alcohol consumption. Since pattern of alcohol metabolism cannot be predicted in any individual group or population, no limit is a safe limit.

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Aldicarb Virginia C Moser, Independent Consultant, Apex, NC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of V.C. Moser, Aldicarb, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 123–125, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00093-2.

Chemical profile Background Uses Exposure and exposure monitoring Toxicokinetics Mechanisms of toxicity Acute and short term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Neurotoxicity Endocrine toxicity Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines References

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Abstract This chapter covers aldicarb, an N-methyl carbamate insecticide that rapidly and reversibly inhibits acetylcholinesterase. The nervous system is the most sensitive target, and there is little evidence of other forms of toxicity. Aldicarb is highly toxic to humans and wildlife. Ground water and food contamination of aldicarb, along with its sulfoxide and sulfone metabolites, have restricted its use over the years. Aldicarb is currently banned in most countries worldwide, with limited use in remaining countries.

Keywords Acetylcholinesterase; Aldicarb; Neurotoxicity; N-methyl carbamate

Chemical profile

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Name: Aldicarb IUPAC Name: [(E)-(2-methyl-2-methylsulfanylpropylidene)amino] N-methylcarbamate. Synonyms: Temik®; ENT 27093; UC21149; RCRA waste number P070; AI3-27093; OMS 771; NCI C08640; SHA 098301. CAS: 116-06-3 Molecular formula: C7H14N2O2S Chemical Structure:

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Background Aldicarb is an N-methyl carbamate insecticide that rapidly and reversibly inhibits acetylcholinesterase, the enzyme responsible for metabolizing the neurotransmitter acetylcholine at cholinergic nerve terminals. The nervous system is the most sensitive target, and there is little evidence of other forms of toxicity. Aldicarb is one of the most potent N-methyl carbamate insecticides, and has undergone extensive field, laboratory, and human studies. Ground water and food contamination of aldicarb, along with its sulfoxide and sulfone metabolites, have restricted its use over the years. Aldicarb was introduced in 1970, and within a decade its use was restricted due to high levels of water contamination and food residues. Following a special review process and assessments based on additional toxicity data, the US EPA determined that residues found in citrus and potatoes may pose unacceptable dietary risks to infants and children (US EPA, 2016). Discussions with the registrant resulted in additional risk mitigation measures and lowered application rates for remaining uses, leading to a voluntary phase-out of all uses in the US by 2018. Aldicarb is designated “extremely hazardous” by the World Health Organization, and is subject to regulation under the Rotterdam Convention, an international treaty designed to reduce trade of the most hazardous chemicals (www.pic.int). Most recently, the US EPA has allowed its use specifically on citrus in Florida (US EPA, 2021).

Uses Aldicarb is a systemic insecticide, acaricide, and nematicide used on a variety of agricultural crops, including cotton, potatoes, citrus, peanuts, sugar cane, and beets. It is commercially sold only as a granular formulation (5, 10, or 15% active ingredient), which is to be incorporated directly into the soil to provide maximum efficacy and to minimize hazard to birds and other wildlife. Aldicarb is a Restricted Use Pesticide and can be purchased and used only by certified applicators. Home and garden use is not permitted (Risher et al., 1987; US EPA, 2016).

Exposure and exposure monitoring Environmental measures of aldicarb exposure should include parent, sulfoxide, and sulfone; indeed, maximum residue levels are expressed as the sum of these. Dietary risk and ground water contamination have been the primary concerns for the use of aldicarb. Occupational exposure should be low, since handlers must use either a closed system or appropriate personal protective equipment (Baron, 1994). Aldicarb is persistent in groundwater, and studies show degradation half-lives from weeks to years (WHO, 2003). In a survey of pesticide detections in groundwater, it was one of the most often detected (Ritter, 1990). Aldicarb remains as one of the most frequently detected pesticides in a survey of groundwater in Long Island, decades after its use was discontinued (Fisher et al., 2021). The ingestion of contaminated foods and accidental exposures to workers has resulted in a number of human poisoning incidents, some quite severe. Several outbreaks of food poisoning, involving up to hundreds of affected individuals, have implicated improperly treated crops, e.g., cucumbers, watermelons, and bananas. In one summer, over 2000 cases of poisoning were reported following ingestion of tainted watermelons in the US (CDC, 1986). Some estimates of exposure from these incidents suggest aldicarb is more toxic than expected, with poisoning occurring at levels estimated as low as 0.0011 mg kg−1, well below low-effect levels in human studies (Goldman et al., 1990).

Toxicokinetics Toxicokinetic parameters are similar across species. Aldicarb is rapidly and well-absorbed and through the oral, dermal, and inhalation routes of exposure. Upon uptake, it is quickly metabolized and excreted, with half-lives of elimination being 1.1 h in rats and 1.7 h in humans. Oxidation reactions rapidly convert aldicarb to aldicarb sulfoxide, which is as toxic as the parent, with slower and lesser formation of the less-toxic sulfone. Hydrolysis pathways yield inactive oximes and nitriles (US EPA, 2007). Limited studies suggest that aldicarb is detoxified to some extent by carboxylesterases (Gupta and Dettbarn, 1993). Animal studies have indicated aldicarb and its metabolites are widely distributed to tissues, including fetal tissue. Tissue accumulation has not been reported. The major route of excretion is urinary, with at least 80% of the dose eliminated within 24 h. A minor route of biliary elimination undergoes enterohepatic recirculation, and small amounts are excreted in feces and milk (Baron, 1994; Risher et al., 1987). A physiologically based pharmacokinetic (PBPK) model has been developed and validated for aldicarb, based on parameters from both rat and human experimental data. This model predicts 9.5 greater blood and brain concentrations over time in humans compared to rats following equivalent doses (Pelekis and Emond, 2009).

Mechanisms of toxicity Aldicarb (and its sulfoxide metabolite) binds and inhibits acetylcholinesterase, via carbamylation of the serine residue at the active site of the enzyme. The resultant accumulation of synaptic acetylcholine causes overstimulation of the cholinergic pathways and produces central and peripheral toxicities. The acetylcholinesterase inhibition is readily reversible with rapid reactivation occurring through spontaneous decarbamylation, and recovery of function is evident within minutes to hours. Studies show similar

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inhibition of acetylcholinesterase in whole blood, plasma, erythrocytes, and brain of rats, mice, and dogs following acute and repeated exposures, and in plasma and erythrocytes in humans following acute exposure (Baron, 1994; Blacker et al., 2010; Risher et al., 1987). As with all N-methyl carbamates, the rapidity with which the acetylcholinesterase enzyme recovers impacts the ability to accurately measure aldicarb-induced inhibition. That is, standard assays that are typically used for organophosphate chemicals will greatly underestimate the true inhibition produced by carbamates, a consideration that must be kept in mind for both experimental studies and clinical measures (US EPA, 2007).

Acute and short term toxicity Animal Signs of acute exposure in animals are due to acetylcholinesterase inhibition and mirror effects of other N-methyl carbamates. Aldicarb is very potent (Toxicity Category I), with lethality associated with cholinesterase inhibition occurring at doses less than 1 mg kg−1 in most species. Aldicarb is not a skin irritant and does not cause dermal sensitization (Baron, 1994; US EPA, 2007).

Human Humans exposed to aldicarb show the same toxic signs and symptoms as seen with experimental animals, with rapid onset and recovery. Sublethal poisoning generally subsides by 6 h with no long-term complications. Mild cases of exposure, sufficient to produce sweating, headache, and nausea, may be confused with the flu (Risher et al., 1987).

Chronic toxicity Animal With repeated exposures, acetylcholinesterase inhibition remains the predominant form of toxicity, and there is little evidence of progressive or chronic effects due to the rapid recovery of enzyme function. Aldicarb does not inhibit neurotoxic esterase (NTE) and does not produce delayed neuropathy.

Human Data on chronic toxicity are not available. The few epidemiological studies in the literature do not show causal relationships between aldicarb exposure and long-term adverse outcomes.

Immunotoxicity Studies of individuals drinking aldicarb-contaminated water reported altered lymphocyte and T-cell counts. Laboratory animal immunotoxicity studies report contradictory results, with some finding altered humoral or functional immune status (Sharma, 2006), whereas studies submitted for regulatory purposes were negative (US EPA, 2016). Thus, immunological findings are inconclusive and it is generally concluded to not be immunotoxic.

Reproductive and developmental toxicity Several developmental studies, including multi-generational, in laboratory rats and rabbits showed no evidence of embryotoxicity, fetotoxicity, or teratogenicity at doses that are not overtly maternally toxic (Blacker et al., 2010; Risher et al., 1987; US EPA, 2016).

Genotoxicity The weight of evidence from a range of in vitro and in vivo studies of gene mutations, chromosomal aberrations, and unscheduled DNA synthesis support conclusions that aldicarb is not a mutagen (Blacker et al., 2010; US EPA, 2016).

Carcinogenicity There is inadequate evidence of carcinogenicity in 2-year studies with rats and mice, with no tumors related to aldicarb administration (US EPA, 2007, 2016). Analysis of pesticide users in the ongoing Agricultural Health Study showed a slight but significant

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increase in colorectal cancer associated with aldicarb use (Lee et al., 2007); however, interpretation is not clear due to small numbers and lack of concordance from other studies. Aldicarb is currently classified as category E: Evidence of non-carcinogenicity in humans (US EPA, 2021).

Organ toxicity Neurotoxicity This action leads to signs of cholinergic crisis, including sweating, nausea, dizziness, miosis, blurred vision, abdominal pain, vomiting, and diarrhea, progressing to tremors, convulsions, and death. Animal studies show a clear relationship between the severity of toxicity and the level of cholinesterase inhibition and subsequent cholinergic stimulation (Baron, 1994; Moser, 1995). There is evidence from comparative studies in rats that the young are more sensitive than adults to these acute effects (Moser, 1999; US EPA, 2016).

Endocrine toxicity Only a few studies have reported estrogenic/antiestrogenic effects of aldicarb, but it is sometimes listed among endocrine-disrupting pesticides (Kitamura et al., 2006; Mnif et al., 2011).

Clinical management Exposed dermal areas should be cleaned thoroughly with soap and water, and eyes should be flushed with generous amounts of clean water for at least 15 min. If the patient is not in a life-threatening condition, and is treated soon after exposure, activated charcoal may be used to reduce absorption from the gastrointestinal tract. Gastric lavage should be considered if more toxic quantities have been ingested, if possible within 1 h of exposure. Emergency management should not be delayed. Support for the airway, breathing, and circulation of the patient is most important. Patients may have excessive secretions, bronchorrhea, bronchospasm, and weakness of respiratory muscles; intubation and mechanical ventilation may be necessary. Muscarinic effects (e.g., salivation, lacrimation) may be reduced by administration of atropine, with repeated high doses until pulmonary secretions dissipate. Benzodiazepines may be used to control seizure. The use of pralidoxime is controversial, but may be useful if poisoning includes organophosphorus compounds. Some clinical reports suggest it is contraindicated for carbamate-only poisoning, whereas others have reported that it is effective in reducing morbidity and mortality in severe poisonings. Furosemide may be useful for pulmonary edema that continues after atropinization (US EPA, 2013). Plasma or erythrocyte cholinesterase measurements may be used to indicate the type of agent involved, but due to the rapid reversibility of the inhibition produced by carbamates, such laboratory assays may not be accurate and in fact could be misleading. Metabolite analysis of a urine sample may allow confirmation of the pesticide.

Environmental fate and behavior Aldicarb rapidly degrades to the sulfoxide and sulfone forms, which are moderately persistent. These chemicals are highly soluble in water and mobile in soil. The toxic residues degrade slowly in the upper soil layer, move fairly rapidly into the subsurface, and potentially persists in ground water. They have been detected at high levels in ground water and have contaminated wells and drinking water supplies. Breakdown and persistence in soil and water depends on bacteria, sunlight, moisture, and a number of other factors, leading to wide variations in estimates of environmental exposures (Baron, 1994; US EPA, 2016).

Ecotoxicology Aldicarb is extremely toxic to birds; however, due to its formulation and applications, it is not implicated in bird kills (Moore et al., 2010a). It is very toxic to fish, bees, and wildlife. Exposures of non-target species occur from contaminated water or consumption of applied granules. At typical usage rates, risks for freshwater fish and invertebrates are minimal, but maximum applications may cause high levels of concern. Bioaccumulation in the environment and bioconcentration in tissues are low (Moore et al., 2010b; US EPA, 2007).

Exposure standards and guidelines The US EPA point of departure for aldicarb is 0.013 mg kg−1, derived from benchmark dose modeling of erythrocyte cholinesterase inhibition data from a study in human volunteers exposed to single doses of aldicarb. With incorporation of an additional factor to assure safety to infants and children, the US EPA population adjusted dose becomes 0.00027 mg kg−1 (US EPA, 2016). The Joint FAO/WHO Meeting on Pesticide Residues established an acceptable daily intake value of 0.003 mg kg−1, based on cholinesterase

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data from the same human study (IPCS/INCHEM, 1992). The US EPA Maximum Contaminant Level (MCL) for aldicarb and in drinking water is 0.003 mg l−1, whereas WHO has set a limit of 0.01 mg l in drinking water (WHO, 2003). Aldicarb is currently banned in most countries worldwide, with highly restricted use in remaining countries.

References Baron RL (1994) A carbamate insecticide: A case study of aldicarb. Environmental Health Perspectives 102(suppl 11): 23–27. Blacker AM, Kelly ID, Lantz JL, Mihlan GJ, Jones RL, and Young BM (2010) Aldicarb: Toxicity, exposure and risks to humans. In: Krieger R (ed.) Hayes’ Handbook of Pesticide Toxicology, 3rd ed., pp. 1619–1632. Elsevier Academic Press: Burlington MA. CDC (Centers for Disease Control) (1986) Epidemiologic notes and reports aldicarb food poisoning from contaminated melons – California. MMWR Weekly 35: 254–258. Fisher IJ, Phillips PJ, Bayraktar BN, Chen S, McCarthy BA, and Sandstrom MW (2021) Pesticides and their degradates in groundwater reflect past use and current management strategies, Long Island, New York, USA. Science of the Total Environment 752: 141895. Goldman LR, Beller M, and Jackson RJ (1990) Aldicarb food poisonings in California, 1985-1988: Toxicity estimates for humans. Archives of Environmental Health 45: 141–147. Gupta RC and Dettbarn WD (1993) Role of carboxylesterases in the prevention and potentiation of N-methylcarbamate toxicity. Chemico-Biological Interactions 87: 295–303. IPCS (International Programme on Chemical Safety) and INCHEM (Internationally Peer Reviewed Chemical Safety Information) (1992). Aldicarb. http://www.inchem.org/documents/ jmpr/jmpmono/v92pr03.htm. Kitamura S, Sugihara K, and Fujimoto N (2006) Endocrine disruption by organophosphate and carbamate pesticides. In: Gupta RC (ed.) Toxicology of Organophosphate and Carbamate Compounds, pp. 481–494. Elsevier Academic Press: Burlington MA. Lee WJ, Sandler DP, Blair A, Samanic C, Cross AJ, and Alavanja MCR (2007) Pesticide use and colorectal cancer risk in the Agricultural Health Study. International Journal of Cancer 121: 339–346. Mnif W, Hassine AIH, Bouaziz A, Bartegi A, Thomas O, and Roig B (2011) Effect of endocrine disruptor pesticides: A review. International Journal of Environmental Research and Public Health 8: 2265–2303. Moore DR, Teed RS, Rodney SI, Thompson RP, and Fischer DL (2010a) Refined avian risk assessment for aldicarb in the United States. Integrated Environmental Assessment and Management 6: 83–101. Moore DR, Thompson RP, Rodney SI, Fischer D, Ramanarayanan T, and Hall T (2010b) Refined aquatic risk assessment for aldicarb in the United States. Integrated Environmental Assessment and Management 6: 102–118. Moser VC (1995) Comparisons of the acute effects of cholinesterase inhibitors using a neurobehavioral screening battery in rats. Neurotoxicology and Teratology 17: 617–625. Moser VC (1999) Comparison of aldicarb and methamidophos neurotoxicity at different ages in the rat: Behavioral and biochemical parameters. Toxicology and Applied Pharmacology 157: 94–106. Pelekis M and Emond C (2009) Physiological modeling and derivation of the rat to human toxicokinetic uncertainty factor for the carbamate pesticide aldicarb. Environmental Toxicology and Pharmacology 28: 179–191. Risher JF, Mink FL, and Stara JF (1987) The toxicologic effects of the carbamate insecticide aldicarb in mammals: A review. Environmental Health Perspectives 72: 267–281. Ritter WF (1990) Pesticide contamination of ground water in the United States – A review. Journal of Environmental Science and Health. Part. B 25: 1–29. Sharma RP (2006) Organophosphates, carbamates, and the immune system. In: Gupta RC (ed.) Toxicology of Organophosphate and Carbamate Compounds, pp. 495–507. Elsevier Academic Press: Burlington MA. US EPA (US Environmental Protection Agency) (2007) Reregistration Eligibility Decision for Aldicarb. https://www3.epa.gov/pesticides/chem_search/reg_actions/reregistration/red_ PC-098301_1-Sep-07.pdf. US EPA (US Environmental Protection Agency) (2013) Recognition and Management of Pesticide Poisonings, 6th edn. http://www.epa.gov/pesticide-worker-safety/recognition-andmanagement-pesticide-poisonings. US EPA (US Environmental Protection Agency) (2016) Aldicarb: Draft human health risk assessment to support registration review. https://www.regulations.gov/document/ EPA-HQ-OPP-2012-0161-0022. US EPA (US Environmental Protection Agency) (2021) Human health risk assessment in support of new uses on oranges and grapefruits in Florida. https://www.regulations.gov/ document/EPA-HQ-OPP-2020-0600-0021. WHO (World Health Organization) (2003) Aldicarb in drinking water. https://www.who.int/water_sanitation_health/dwq/chemicals/aldicarb.pdf.

Relevant websites https://inchem.org/documents/ehc/ehc/ehc121.htm :IPCS (International Programme on Chemical Safety): INCHEM (Internationally Peer Reviewed Chemical Safety Information) (1991). Environmental Health Criteria 121: Aldicarb. https://pubchem.ncbi.nlm.nih.gov/compound/Aldicarb :PubChem National Library of Medicine. https://cfpub.epa.gov/ncea/iris2/chemicalLanding.cfm?substance_nmbr¼3 :US EPA (US Environmental Protection Agency) (1993). Aldicarb: IRIS summary. https://comptox.epa.gov/dashboard/dsstoxdb/results?search¼DTXSID0039223&abbreviation¼IRIS :US EPA comptox.

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Aldrin Martin P Boland, Leidos Australia, Scoresby, VIC, Australia © 2024 Elsevier Inc. All rights reserved. This is an update of M. Honeycutt, S. Shirley, Aldrin, Editor (s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 126–129, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00094-4.

Chemical profile Uses Environmental fate and behavior Partition behavior in water, sediment, and soil Environmental persistence Long-range transport Bioaccumulation and biomagnifications Exposure and exposure monitoring Routes and pathways Human exposure Environmental exposure Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Chronic toxicity Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Freshwater–sediment organism toxicity Marine organism toxicity Terrestrial organism toxicity Other hazards Exposure standards and guidelines Miscellaneous References Further reading

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Abstract Aldrin is an organochlorine pesticide related to dieldrin. Following initial phase out was retained as an anti-termite treatment until 1987. The only difference between the structures of aldrin and dieldrin is the presence, in dieldrin, of an epoxied ring at the site of one of the carbon–carbon double bonds in aldrin. Because aldrin is rapidly metabolized to dieldrin in the body and converted to dieldrin in the environment, please see entry for dieldrin for additional information.

Keywords Aldrin; Aquatic toxicity; Banned pesticide; Bioconcentration factor; Cyclodiene; Dieldrin; GABA antagonist; Insecticide; Organochlorine pesticide; Persistent organic pollutant

Key points

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Organochlorine pesticide phased from mid-1970s (final removal 1987) Exposure can lead to convulsions, with high doses causing death Rapidly converted to dieldrin in environment or body Some evidence exposure may cause Parkinsonism

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Chemical profile

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Chemical Abstracts Service Registry Number: CAS 309-00-2 RTECS: IO2000000 Chemical Name: 1,2,3,4,10,10-Hexachloro-1,4,4a,5,8,8a-hexahydro-1,4-endo,exo-5,8-dimethanonaphthalene, abbreviated HHDN Trade Names: Aldocit, Aldrex, Aldrosol, Compound 118, Drinox, ENT 15,949, Hexachlorohexahydro-endo, exo-dimethanonaphthalene, HHDN, Kortofin, Octalene, OMS 194, Seedrin Molecular Formula: C12H8Cl6 Relative Molecular Mass: 364.91 MP 104–105.5
C Vapor pressure 7.4  10–5 mmHg at 20
C LogOW 6.50 LogOC 7.67 Water solubility 11 mg L–1 at 20
C Density 1.6 g mL–1, at 20
C

Uses The organochlorine pesticide aldrin (CAS 309-00-2) was used to control insects on crops until 1970–74, the US Department of Agriculture (USDA), Environmental Protection Agency (USEPA) and manufacturer canceled all uses except prophylactic termite mitigation (ATSDR, 2022; EPA, 1986). In 1987, the manufacturer canceled (EPA, 1989). Production in the US ended in 1974, and importation ended in 1985 (EPA, 1986).

Environmental fate and behavior Partition behavior in water, sediment, and soil Aldrin is very insoluble in water, but with a high affinity for organic materials, it readily binds to organic components of soil. However, aldrin is highly susceptible to epoxidation to the related compound dieldrin. Despite the low vapor pressure, Aldrin is prone to evaporation. These factors combine to give aldrin an estimated soil half-life in the order 53–120 days (Freedman, 1989).

Environmental persistence As noted above, Aldrin readily converts to the related compound dieldrin, which is covered in a separate chapter. Despite the expected degradation, aldrin has been found in environmental samples long after degradation would be expected, this persistence is probably to the high hydrophobicity causing the material to be sequestrated in soils and fatty tissues of fish and animals.

Long-range transport Due to the propensity to volatilization, Aldrin may be susceptible to being redistributed by air currents. The compound has been detected in non-agricultural areas in the Arctic or American and Canadian Great Lake regions, although this may be due to use in termite protection in regions closer to the discovered locations (ATSDR, 2022). Photodegradation of aldrin in the atmosphere seems to favor epoxidation to form dieldrin.

Bioaccumulation and biomagnifications Although some bioconcentration of aldrin is has been noted, the rapid conversation to dieldrin, the latter is more likely to be detected. Further information on dieldrin can be found in that chapter.

Exposure and exposure monitoring Routes and pathways Aldrin use in North America declined precipitously from the early 1970s and was terminated in 1987 (ref ). Due to the rapid degradation, air, water and soil exposures are unlikely. Some residual risk of exposure from unused stocks is possible. Dietary intake is considered possible.

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Human exposure The most recent survey data for aldrin exposure in the United States is from 2001 to 2004. Blood samples taken from 4224 individuals were below the detection threshold of 7.8 ng g−1) (CDC, 2022). Exposure in areas that have had more recent or extensive use is known.

Environmental exposure Due to the ease of conversion to dieldrin, relatively high probability of aerial distribution and low water solubility, aldrin is infrequently detected in environmental samples (CDC, 2022).

Toxicokinetics PBPK models of human absorption for aldrin are not available. There is dearth of data directly measuring direct absorption of aldrin in humans. A study of oral absorption over 2 years led to the detection of dieldrin from blood and fat samples (Hunter and Robinson, 1967; Hunter et al., 1969). Epidemiological studies suggest that absorption through dermal and/or inhalation routes is possible (Dobbs and Williams, 1983). A study of dermal absorption detected Aldrin in the urine of participants within 4 h of application to the arm (Feldmann and Maibach, 1974). As noted, aldrin is generally converted to dieldrin, hence distribution studies of this compound are limited. Metabolism of aldrin is dominated by a monooxygenase catalyzed epoxidation to create dieldrin. For further information see that chapter. However, aldrin has been detected in breast milk of women relatively recently suggesting that the conversion is not complete (Kao et al., 2019).

Mechanism of toxicity Aldrin is primary neurotoxic to mammals. Symptoms range from headaches to convulsions, potentially leading to death. Although a range of dysregulations of neurotransmitters and ions have been linked with aldrin exposure. The accepted model is that aldrin blocks the GABAA complex, preventing the cell from taking up chloride ions. The neurophysiological result of this is hyper-excitation of the neurons (ATSDR, 2022).

Acute and short-term toxicity As Aldrin is rapidly converted to dieldrin, the majority of the toxic effects are likely to be caused by that species. Only two specific LD50 studies are known for aldrin, with males occurring at 39 mg/kg compared with females at 60 mg/kg (Gaines, 1960). The second trail using only female rats showed an LD50 of 48.3 mg/kg (Treon et al., 1951).

Chronic toxicity Although prolonged exposure has been associated with episodes of headaches and similar symptoms, discontinuation of exposure led to the symptoms declining. Unlike other organochlorine compounds has not been linked with cases of Parkinson’s disease. Animal studies have shown changes in weight, particularly weight loss in dogs when chronically exposed to Aldrin (Fitzhugh et al., 1964).

Immunotoxicity A single case report of an agricultural worker suffering from immune-hemolytic anemia is linked with Aldrin (Pick et al., 1965). Very little specific immunotoxicity study has been conducted with Aldrin. Relevant studies are also discussed in the dieldrin chapter.

Reproductive toxicity Aldrin has been detected at significantly higher levels in the tissues of women who have had premature labor or miscarriages (Saxena et al., 1980).

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Animal studies of Aldrin are sparse, but a multigeneration study (six) showed decreased fertility of mice (Keplinger et al., 1970). More studies have been conducted on dieldrin.

Genotoxicity Studies of human samples for genotoxic effects have shown a single effect, sister chromatid exchange in the lymphocytes of occupationally exposed workers (Dulout et al., 1985), although a later study did not repeat the result. In contrast micronuclei, DNA single strand breaks and chromosomal aberration studies have all be negative. Studies on prokaryotic organisms have universally failed to show positive effects (ATSDR, 2022).

Carcinogenicity Results of liver cancer studies on mice show that aldrin cancer within these models, but in line with the genotoxicity results, the cause does not seem to be direct DNA damage. However, these results have lead to the USEPA to classify aldrin as a probable human carcinogen (category B2) based on the mouse results. Aldrin is considered unclassifiable (Group 3) by IARC. Epidemiological results do not suggest any high probability link between aldrin and human cancers based on studies of both manufacturers and agricultural workers. Weaknesses in these studies include relatively low numbers of subjects, assessment for numerous types of cancers, and a lack of a doses response (ATSDR, 2022).

Clinical management Ingestion should be treated by inducing vomiting and/or application of charcoal. Otherwise only treatment of symptoms and supportive care is available. Milk should not be given as this has been linked with increased effect (CAMEO, 2022).

Ecotoxicology Freshwater–sediment organism toxicity The Australian and New Zealand Guidelines for Fresh and Marine Water Quality (ANZECC and ARMCANZ, 2000) include an assessment of the toxicity of aldrin to freshwater organisms. Sixteen fish species were studies using 48–96 h timeframes yielding LC50 values in the 0.9–53 mg L−1 range, although the Saccobranchus fossilis, Oryzias latipes and Clarius batrachus,all substantially exceded this range (447–3500 mg L−1 which exceeds the solubility of the analyte). Similar results were seen for amphibians, crustaceans and mollusks, with LC50 values reaching 209 mg/L for Paratelphusa masoniana.

Marine organism toxicity ANZECC and ARMCANZ (2000) reports that marine fish, mollusks and crustaceans exhibit similar sensitivity to freshwater species with LC50 values in the range 0.32–40 mg L−1. A NOEC of 3.3 mg L−1 was determined for the Fundulus heteroclitus species, which was reported to be similar to the 96 h LC50.

Terrestrial organism toxicity As would be expected, honey bees have a rather low LD50 at 60 years) and in younger persons with preexisting illnesses such as diabetes, chronic renal disease, and hypertension with a history of transient ischemic attacks. The mouse LD50 of DA is 3.6 mg kg−1 when injected IP.

Okadaic acid Okadaic acid (okadeic acid, ocadaic acid, OA, CAS 78111-17-18, C44H68O13) and its analogues dinophysistoxin-1 (DTX-1, CAS 81720-10-7, C45H70O13) and dinophysistoxin-2 (DTX-2, CAS 139933-46-3 C44H68O13) are lipophilic, polyether toxins produced by various dinoflagellates in the genera Prorocentrum (e.g., P. lima, P. arenarium, P. hoffmannianum, P. maculosum, P. faustiae, P. levis, and P. belizeanum) and Dinophysis (e.g., D. acuta, D. acuminata, D. caudata, D. fortii, D. miles, D. norvegica, D. rapa, and D. sacculus). Okadaic acid

Toxins within the OA group are responsible for provoking DSP in humans after the consumption of shellfish that have accumulated these toxins in their digestive gland. OA was originally isolated from marine sponges of the genus Halichondria and DTX-1 and -2 were first isolated from mussels. OA and DTXs are polyketide compounds containing furane- and pyrane-type ether rings and an alpha-hydroxycarboxyl function differing only in the number or position of the methyl groups. Many derivatives of these parent toxins have been described after being found in shellfish and algae, including OA esters, okadaates, OA-diol-esters (e.g., acylated derivatives with fatty acids in the DTX-3 group, primarily with hexadecanoic acid, diol-esters formed in the unsaturated diols, and esterfication of the diol-esters with sulfated chains with or without and amide function in the DTX-4 and DTX-5a-c groups) and other compounds that comprise changes to the OA backbone (e.g., norokadanone, 19-epi-OA, and belizeanic acid). Prorocentrin is another compound that shows similarity with OA that is produced by P. lima. Of particular interest is the fact that significant portions of the toxins found in bivalves are the acylated derivatives. These derivatives may be a product of animal metabolism not produced directly by the algae and they show increased liposolubility when compared to their parent toxins. DSP was first identified in 1978 after a series of food poisonings resulting from eating contaminated mussels and scallops in the Tohoku district of Japan affected 164 individuals. DSP outbreaks have been predominantly reported in Japan, Europe, and Australia, but cultures of P. lima isolated from the gulf of California and Mexico are capable of producing toxin. Thus the problem is generally considered to be a worldwide phenomenon. Toxins within the OA group can withstand mildly acidic to strongly basic pH but degrade rapidly in strong mineral acids. However, without acid, OA group compounds are largely stable to heat and are not degraded with normal cooking procedures and contaminated foods may serve to buffer against degradation of the toxins by the gastric juices. Compounds within the OA group produce toxic effects on hydrolysis within the human digestive tract. They are inhibitors of the serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A), enzymes responsible for dephosphorylation of proteins, which are essential to metabolic processes in eukaryotic cells. Symptoms of DSP poisoning are nausea, diarrhea, vomiting, and abdominal pain starting within 3–12 h of initial consumption. Although originally the cause of the symptoms of diarrhea were thought to be caused by sodium secretion of intestinal cells, an increase in the paracellular permeability of intestinal cells by the toxins is now thought to be the likely cause of diarrhea. The Report of the Joint Food and Agriculture Organization of the United Nations, Intergovernmental Oceanographic Commission of United Nations Educational, Scientific, and Cultural Organization (UNESCO), and World Health Organization ad hoc Expert Consultation on Biotoxins in Bivalve Molluscs established a lowest observed adverse effect level (LOAEL) of 1 mg OA kg−1 bw. They established a provisional acute reference dose of 0.33 mg OA kg−1 bw. Most individuals recover within 3 days, and there have been no reported long-term effects or deaths reported due to acute DSP poisoning. However, these toxins have been shown to be tumor promoters and ancillary evidence has associated these toxins with digestive cancer. Additionally, there is evidence for cytotoxicity and potentially genotoxicity including formation of unspecific DNA adducts.

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The LD50 for mouse injected IP for OA is 0.2–0.225 mg kg−1 bw, for DTX-1 is 0.16 mg kg−1 bw, for DTX-2 is 0.35 mg kg−1 bw, and for DTX-3 is 0.2–0.5 mg kg−1 bw. However, IP doses have been shown to have little effect on the digestive tract, primarily affecting the liver. Lethal oral doses have been reported for mouse from 2 to 10 times higher than the IP dose (Dominguez et al., 2010).

Saxitoxins Saxitoxins (STXs, PSTs, CAS 35523-89-8, C10H17N7O4) are group of neurotoxic purine alkaloids that are responsible for causing PSP in humans after consumption of contaminated shellfish or other seafood, particularly lobster and puffer fish (not to be confused with poisonings caused by tetrodotoxin). STXs responsible for most reports of PSP are primarily produced by dinoflagellates of the genera Alexandrium (A. fundyense, A. catenella, A. tamarense, A. hiranoi, A. monilatum, A. minutum, A. lusitanicum, A. tamiyavanichii, A. taylori, and A. peruvianum), Gymnodinium (G. catenatum), and Pyrodinium (P. bahamense) but can also be produced by some species of cyanobacteria (see below) in the genera Anabaena (A. circinalis and A. lemmermannii), Aphanizomenon (A. gracile and A. issatschenkoi), Cylindrospermopsis (C. raciborskii), Lyngbya (L. wollei), Planktothrix, and Rivularia. There are suggestions that saxitoxins may actually have a bacterial origin, but the evidence is inconclusive at this point. Saxitoxin

Substitutions with different hydroxyl, carbamyl, and sulfate functional groups at four sites along the backbone structure have resulted in the identification of at least 24 saxitoxin-like compounds that can vary by more than three orders of magnitude in toxicity. The most toxic of these are the carbamate toxins (saxitoxin (STX), neosaxitoxin (NEO), and gonyautoxins 1–4 (GTX)) followed by the decarbamoyl toxins (dcSTX, dcNEO, dcGTX 1–4, not common) and the N-sulfocarbamoyl toxins (B1 [GTX5], B2 [GTX6], and C1–C4), respectively. A fourth group of toxins, the hydroxybenzoate toxins, are produced by G. catenatum (GC 1–3), but more research needs to be conducted to determine the extent of their toxicity. Saxitoxin is the most well-studied member of this group. The first PSP event on record occurred in 1927 in San Francisco, USA, coinciding with a bloom of the A. catenella, affecting 102 people and causing 6 deaths. However, saxitoxin was first isolated from butter clams, Saxidomus giganteus. The problem is now known to be a worldwide, having been reported in 27 locations by 1990. STXs are heat and acid stable, thus cooking the seafood does not denature the toxins. Saxitoxin acts by specifically and selectively binding to voltage-gated sodium channels on excitable cells functionally blocking sodium conductance and preventing impulse generation in peripheral nerves and skeletal muscles. STX exposure studies have been conducted on cats, chickens, dogs, guinea pigs, monkeys, mice, rats, birds, and rabbits including IP injections, intravenous injections, inhalation, and oral dosing. Saxitoxin can also block action potentials directly in skeletal muscles. Symptoms of poisoning generally occur within 30 min of consuming contaminated seafood including tingling sensations of the lips, mouth, and tongue, numbness of extremities, paresthesias, weakness, ataxia, floating/dissociative feelings, nausea, shortness of breath, dizziness, vomiting, headache, dysphagia, dysarthria, diastolic and systolic hypertension, and death. Death is caused by asphyxiation. Onset of symptoms has been reported as starting within a few minutes of seafood consumption, and death has been reported within 3–4 h of consumption. Medical treatment consists of providing respiratory support and fluid therapy. Humans typically start to recover within 12–24 h with no long-lasting effects; however, little if anything is known about the chronic effects of these toxins. Currently, STX is strictly regulated by the Organization for the Prohibition of Chemical Weapons listed as a schedule 1 chemical intoxicant. The Report of the Joint Food and Agriculture Organization of the United Nations, Intergovernmental Oceanographic Commission of UNESCO, and World Health Organization ad hoc Expert Consultation on Biotoxins in Bivalve Molluscs established an LOAEL of 2 mg STX kg−1 bw and a provisional acute reference dose of 0.7 mg STX kg−1 bw. Generally, the action limit for STX for most seafood and shellfish is 0.8 mg STX equivalents per kilogram of tissue that has been accepted by many regulatory agencies worldwide. This standard has been in place for approximately 50–60 years. Recently, the European Food Safety Authority suggested a level of 75 mg STX equivalents per kilogram of tissue. The mouse bioassay is the primary determinant of PSP toxins in shellfish for most regulatory agencies. The LD50 for mice injected peritoneally for STX is 0.008 mg kg−1 bw, injected intravenously is 8.5 mg kg−1 bw, and administered orally is 263 mg kg−1 bw.

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Acute and chronic toxicity and mechanisms of action: Cyanobacterial toxins There are at least 13 different genera of cyanobacteria that have been shown to produce toxins, often several different toxins per species. The main toxin-producing genera include Anabaena, Aphanizomenon, Cylindrospermopsis, Gloeotrichia, Hapalosiphon, Lyngbya, Microcystis, Nodularia, Nostoc, Oscillatoria, Schizothrix, Spirulina, and Synechocystis. Toxic blooms of cyanobacteria with associated animal poisonings have been reported in all continents except Antarctica. There have been frequent reports of thirsty domestic animals and wildlife consuming fresh water contaminated with toxic cyanobacterial algal blooms and dying within minutes to days from acute neurotoxicity and/or hepatotoxicity. Mammals and birds appear to be more susceptible to cyanobacterial algal toxins than aquatic invertebrates and fish, with some species variability. Prolonged morbidity and mortality have been reported in animals exposed to cyanobacterial algae in the wild. There are individual case reports of persons exposed through swimming to cyanobacterial algal blooms with skin irritation and allergic reactions (both dermatologic and respiratory) with continued positive reaction on skin testing. In particular, urticaria (hives), blistering, and even deep desquamation of skin in sensitive areas like the lips and under swimsuits have been reported, especially with Lyngbya majuscula in tropical areas. Consumption of or swimming in cyanobacterial toxin-contaminated waters has also yielded increased case reports of gastrointestinal symptoms, especially diarrhea. One severe outbreak in Brazil was associated with lethality from hepatotoxicity in dialysis patients exposed to water contaminated with microcystins; another outbreak in Australia was also associated with lethality from hepatorenal syndrome in children and adults exposed to contaminated drinking water. In addition to gastrointestinal and dermatologic symptoms, eye irritation, asthma, and ‘hay fever symptoms’ have been reported repeatedly with exposure to contaminated recreational water exposure in the United States, Canada, the United Kingdom, and Australia. The chronic effects of exposure to small quantities of cyanobacterial algal toxins are still under study. In the mid-1980s, studies were done in China, where people were drinking untreated water contaminated with cyanobacterial algal toxins. It was found that drinking contaminated pond and ditch water was associated with high rates of liver cancer. When the quality of drinking water sources was improved in these areas, the rate of liver cancer decreased. The incidence of liver cancer attributable to cyanobacterial algal toxins in the United States is unknown.

Aplysiatoxins Aplysiatoxins (CAS 52659-57-1, C32H47BrO10) and debromoaplysiatoxins are alkyl phenols produced by Lyngbya gracilis, L. majuscula, Calothrix crustacean, Nostoc muscorum, Schizothrix calcicola, and S. muscurom. Aplysiatoxin

Aplysiatoxins can cause severe skin dermatitis and are potent tumor promoters and protein kinase C activators. Aplysiatoxins and debromoaplysiatoxins from blue-green algae attached to seaweed (Gracilaria coronopifolia) are suspected of causing gastrointestinal symptoms, including diarrhea, nausea, and vomiting in a poisoning case in Hawaii in 1994. Mouse studies have shown small intestinal bleeding and large intestinal edema following toxic injections of aplysiatoxin. The mouse LC50 of aplysiatoxin is 118 mg kg−1, IP.

Anatoxins Anatoxins, are alkaloid neurotoxins, represented here by anatoxin-A (CAS 64285-06-9, C10H15NO) and anatoxin-A (S) (CAS 103170-78-1, C7H17N4O4P), produced by species of the genera Anabaena, Planktothrix, Cylindrospermum, Aphanizomenon, and Phormidium.

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Anatoxin-A and Anatoxin-A (S)

To date, only cattle, dog, and bird poisonings have been documented. Anatoxin-A acts like the neurotransmitter acetylcholine, except that it cannot be degraded by acetylcholinesterase. Anatoxin-A (S) is a natural organophosphate that binds to acetylcholinesterase enzymes, resulting in uncontrolled muscle hyperstimulation. Hypersalivation, lacrimation, and urinary incontinence, signs of parasympathetic stimulation, characterize anatoxin-A (S) poisoning. Anatoxin-A and Anatoxin-A (S) have mouse LC50 of 250 and 40 mg kg−1, IP.

Saxitoxins Saxitoxin and neosaxitoxin are both neurotoxins that may also be classified as cyanobacterial toxins. See above for details.

Microcystins Microcystins, of which there are at least 80 variants, are based on a cyclic heptapeptide structure. Toxic variants contain the unique hydrophobic amino acid, 3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca-4(E),6(E)-dienoic acid (ADDA), and are represented by the prototype compound microcystin-LR or cyanoginosin LR; CAS 101043-37-2, C49H74N10O12. Microcystins are produced by a wide variety of planktonic cyanobacteria, including Microcystis aeruginosa, M. virdis, M. ichthyoblabe, M. botrys, Planktothrix agardhii, P. rubescens, P. mougeotii, Anabaena flos-aquae, A. cirinalis, A. lemmermannii, Nostoc spp., and Snowella lacustris, as well as the benthic cyanobacteria Hapalosiphon hibernicus and Oscillatoria limosa. Microcystin-LR

Experimentally, acute high-dose administration of microcystin can lead to death from hepatoencephalopathy within hours. Chronic administration of sublethal amounts of Microcystis (a cyanobacterial algae which produces microcystin) extracts in drinking water to mice resulted in increased mortality with chronic active liver disease, even at fairly low doses and in relatively short time periods in the laboratory. Studies in mice have also shown that some cyanobacterial algal toxins cause precancerous damage to both

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the liver and the bowel. In the laboratory experimental animals, teratogenic activity has been demonstrated with oral administration of Microcystis extracts;
10% of otherwise normal neonatal mice had small brains with extensive hippocampal neuronal damage. Poisoning by microcystins can lead to visual disturbances, nausea, and vomiting. Acute exposure can lead to liver failure and death within hours to days. Microcystins inhibit protein phosphatases, particularly PP1 and PP2A, resulting in hyperphosphorylation of many cellular proteins, including the hepatocellular cytoskeleton, causing loss of cell-to-cell contact and intrahepatic hemorrhaging. Other effects include altered mitochondrial membrane permeability, generation of reactive oxygen species, and initiation of programmed cell death (apoptosis). Microcystins are also believed to cause damage to cell DNA by the activation of endonucleases and have been linked to human liver and colon cancer. Microcystin-LR has an LC50 of 60 mg kg−1 IP in mice.

Nodularins Nodularin (CAS 118399-22-7, C41H60N8O10), a cyclic pentapeptide toxin similar to microcystin, was first isolated from Nodularia spumigena. Because this species tends to inhabit brackish waters, humans generally are at low risk to exposure through drinking waters. Nodularin

ADDA is present in nodularins, as in microcystins, but other amino acids are different. For example, dehydroalanine is replaced by N-methyl-dehydrobutyrine. The presence of ADDA results in similar phosphatase inhibition and the many subsequent effects as seen in microcystins. The smaller size of nodularins prevents the molecule from binding covalently to active sites (as seen in microcystins), allowing nodularins to affect other sites in cells, and possibly explaining observed carcinogenic effects. Nodularin has a mouse LC50 of 60 mg kg−1, IP.

Cylindrospermopsin Cylindrospermopsin (CAS 143545-90-8, C15H21N5O7S), a cyclic guanidine alkaloid, with at least three variants, is produced by Cylindrospermopsis raciborskii, Aphanizomenon ovalisporum, Anabaena bergii, Umezakia natans, Raphidiopsis curvata, and other unidentified species. Cylindrospermopsin

Cylindrospermopsins are included in the cyanobacterial hepatoxin group which blocks protein synthesis. Acute exposure to cylindrospermopsin results in lipid accumulation in the liver followed by hepatocellular necrosis. Other organs are also affected with widespread necrosis of the tissues of the kidneys, bladder, ureter, and spleen. Cylindrospermopsin has a delayed toxicity, with an LC50 of 2100 mg kg−1 IP in mice at 24 h, but 200 mg kg−1 after 5 days.

Other cyanobacterial toxins A large variety of other toxins are produced by cyanobacteria but are not as well documented. These include lyngbyatoxin (dermatotoxic), endotoxins, and other substances as yet undescribed, including additional tumor promoters.

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Acute and chronic toxicity and mechanisms of action: Other toxins Numerous other algal toxins have been described. Many are believed to play important roles in prey capture and predator avoidance. These compounds tend to be ichthyotoxic, but their toxicity to humans is less certain than the toxins described above.

Cyclic imines Cyclic imines, including gymnodimine (GYM), spirolides (SPX), pinnatoxins (PnTx), prorocentrolide, and spirocentrimine, are fast-acting toxins. The presence of this group of compounds in shellfish was discovered because of their very high acute toxicity in mice upon IP injections of lipophilic extracts. All the cyclic imines for which data are available are toxic to mice after IP administration. Mouse LC50 values for GYMs range 6.5–100 mg kg−1, for SPX, 6.5–8 mg kg−1, and for PnTx, 16–45 mg kg−1. There is no evidence that any of the cyclic imines have been responsible for toxic effects in humans.

Dinophysistoxin See OA.

Golden algal toxins Blooms of algal genera within the Prymnesiophyceae, notably species of Prymnesium, Chrysochromulina, and Phaeocystis, and the Raphidophyceae, primarily species of Chattonella, Heterosigma, and Fibrocapsa, are well known for massive fish kills that have led to great economic losses, but no cases of human toxicity have been reported. Four species of Prymnesium are reported to be toxic to vertebrates or invertebrates and toxicity in two other species is suspected. Several hemolytic compounds termed ‘prymnesins’ have been described in P. parvum, but have yet to be fully characterized. These include several galactolipids and two polyoxy-polyene-polyethers (prymnesin-1 and -2). Mouse LC99 values for prymnesins-1 and prymnesins-2 were 50 and 80 mg kg−1; LC50 values for the fish Tanichthys albonubes were 8 and 9 nM. A different assemblage of ichthyotoxic polyunsaturated fatty acids and their conjugated galactoglycerolipid progenitors, consisting primarily of stearidonic acid (LC50 ¼ 21.9 mM, 10- to 14-day-old fry of the fish Pimephales promelas), and including docosahexanoeic acid (LC50 ¼ 4.7 mM), arachinodonic acid (LC50 ¼ 9.2 mM), pinolenic acid (LC50 ¼ 18.2 mM), and eicosapentaenoic acid (LC50 ¼ 23.6 mM), was identified in laboratory cultures of P. parvum. Some of these toxins were present in bloom and fish kill sites, but below toxic concentrations. Instead a different, yet-characterized, ichthyotoxic fatty acid was detected. Cytotoxicity to a human (MDA-MB-435) cancer cell line was observed for one of the fatty acids isolated from P. parvum in cultures (GAT 512A, IC50 ¼ 24.2 mM). Chrysochromulina polylepis produces two compounds, one hemolytic and one ichthyotoxic. The hemolytic compound was characterized as a galactolipid, 1-acyl-3-digalacto-glycerol. Small amounts of a polyunsaturated fatty acid, octadecapentaenoic acid, were also detected.

Karlotoxins Karlotoxins are water-soluble hemolytic, cytotoxic, and ichthyotoxic compounds produced by the dinoflagellate Karlodinium veneficum.

Pfiesteria toxins The dinoflagellate Pfiesteria spp. is believed to produce and release into the environment potent extracellular toxins, or exotoxins, referred to generally as Pfiesteria toxins (PfTx) that have been linked to mass fish mortalities and human disease in mid-Atlantic estuaries. Learning impairments have been seen as long as 10 weeks after a single acute exposure to Pfiesteria in Sprague–Dawley rats. A hydrophilic toxin (PfTx) isolated from P. piscicida cultures when applied locally to the ventral hippocampus on repeated acquisition of rats in the radial-arm maze impaired choice accuracy and early learning which was persistent across 6 weeks of testing after a single administration of the toxin. Adverse health effects, including cognitive disturbance, were found in humans following accidental exposure to P. piscicida in laboratory facilities. Cognitive deficits have also been described in people believed to be exposed to Pfiesteria through sea spray in coastal Maryland during a Pfiesteria bloom. These adverse health effects have been termed Possible Estuary-Associated Syndrome by the Centers for Disease Control and Prevention, symptoms of which include cognitive and visual contrast sensitivity deficits, pulmonary impairment, gastrointestinal disruptions, and immunologic dysfunction.

Pectenotoxins Pectenotoxins (PTXs) are a group of polyether macrolides produced by the dinoflagellates of the genus Dinophysis (D. fortii, D. acuminata, D. acuta, D. caudate, D. rotunda, D. norvegica). PTXs have also been detected in Protoperidinium divergens, P. depressum, and P. crassipes. PTXs are suspected to be DSP toxins because they are detected with the same extraction methods

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and bioassays used for OA and were first isolated from the scallop, Patinopecten yessoensis. There is no evidence of adverse acute or chronic health effects of pectenotoxins in humans. IP injection of PTXs in mice leads to liver necrosis. The mouse LC50 values (IP) for PTXs range between 250 and 770 mg kg−1 for PTX1, 2, 3, 4, 6, and 11 and greater than 5000 mg kg−1 for PTX7, 8, 9, and 2-SA. Toxicity in other PTX analogues has not been demonstrated.

Yessotoxins Yessotoxins (YTXs) are disulfated polycyclic polyethers that resemble brevetoxins, produced by the dinoflagellate Lingulodinium polyedrum. Like PTX, YTX was first isolated from the digestive glands of the scallop P. yessoensis and are suspected to be DSP toxins because they are detected with the same extraction methods and bioassays used for OA. There have been no reports of ill effects in humans attributable to YTX. IP injection of YTXs in mice leads to cardiac muscle damage. Mouse LC50 values (IP) range between 80 and 750 mg kg−1 for YTX and its analogues.

Clinical management Very little clinical research has been conducted to determine effective treatments. Medical care is primarily supportive. Medical treatment of CFP has been to a large extent symptomatic; a variety of agents, including vitamins, antihistamines, anticholinesterases, steroids, and tricyclic antidepressants, have been tried with limited results. If given within 3 days of exposure, intravenously, mannitol (1 mg kg−1 given rapidly over 1 h) has been demonstrated in a single-blinded control trial to resolve acute symptoms and prevent chronic symptoms, although repeated administrations may be necessary if symptoms return; a more recent clinical trial did not find an effect; however, this trial included subjects treated long after the initial 3-day window. Gut emptying and decontamination with charcoal have been recommended, although often the severe ongoing vomiting and diarrhea prevent this. Atropine is indicated for bradycardia and dopamine or calcium gluconate for shock. It is recommended that opiates and barbiturates be avoided since they may cause hypotension, and opiates may interact with maitotoxins. Amitriptyline (25–75 mg b.i.d.) and similar medications do seem to have some success in relieving the symptoms of chronic ciguatera such as fatigue and paresthesias. It is possible that nifedipine may be appropriate as a calcium channel blocker to counteract the effects of maitotoxins. Anecdotal food avoidance as mentioned above is also recommended. In addition, there is no immunity to these illnesses, and recurrences of actual ciguatera in the same individual appear to be worse than the initial illnesses. A rapid, accurate diagnosis and treatment of CFP within the first 72 h after exposure may be critical in preventing some of the neurologic symptoms that might otherwise become chronic and debilitating. The treatment of DSP caused by OA is symptomatic and supportive. In general, hospitalization is not necessary; fluid and electrolytes can usually be replaced orally. Supportive measures are the basis of treatment for PSP that is caused by saxitoxins, especially ventilatory support in severe cases. In animals, artificial respiration is the most effective treatment. Up to 75% of severely affected persons die within 12 h without supportive treatment. When the ingestion of contaminated food is recent, gut decontamination by the gastric lavage and administration of activated charcoal or dilute bicarbonate solution is recommended. Care must be taken concerning aspiration with the neurologically compromised patient. In general, the only treatment available for exposure to cyanobacterial algal toxins is supportive medical treatment after complete removal from exposure. If the exposure was oral, administration of activated carbon to decrease gut absorption may be efficacious if given within hours of exposure. Based on past outbreaks, monitoring of volume, electrolytes, liver, and kidney function should all be considered in the case of acute gastroenteritis associated with some of the cyanobacterial algal toxins.

Exposure standards and guidelines Global seafood safety standards have not been established. In the United States, US Food and Drug Administration (FDA) enacted the Hazard Analysis and Critical Control Points (HACCP) program of 1997. The FDA has established action levels in suspected seafood for the toxins causing some of the shellfish poisonings (see Table 1). When an action level is reached, the HACCP plan must be followed to prevent unsafe products from reaching the consumer. Table 1

US FDA action levels in seafood for natural toxins associated with shellfish and fish poisoning.

Poisoning

US FDA action level

PSP NSP DSP ASP CFP AZP

0.8 ppm (80 mg per 100 g) saxitoxin equivalents 0.8 ppm (20 mouse units per 100 g) brevetoxin-2 equivalents 0.16 ppm total OA equivalents (i.e., combined free OA, dinophysistoxins, acyl-esters of OA, and dinophysistoxins) 20 ppm domoic acid, except in the viscera of Dungeness crab, where the action level is 30 ppm 0.01 ppb P-CTX-1 equivalents for Pacific ciguatoxin and 0.1 ppb C-CTX-1 equivalent for Caribbean ciguatoxin 0.16 ppm azaspiracid equivalents

Source: US FDA. (April 2011). Fish and Fishery Products Hazards and Controls Guidance, fourth ed. Department of Health and Human Services, Public Health Service Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Food Safety.

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See also: Ciguatoxin; Okadaic acid; Saxitoxin

References Dominguez HJ, Paz B, Daranas AH, Norte M, Franco JM, and Fernández JJ (2010) Dinoflagellate polyether within the yessotoxin, pectenotoxin, and okadaic acid toxin groups: Characterization, analysis and human health implications. Toxicon 56(2): 191–217. https://doi.org/10.1016/.toxicon.2009.11.005. 19925818. Etheridge SM (2010) Paralytic shellfish poisoning: Seafood safety and human health perspectives. Toxicon 56(2): 108–122. https://doi.org/10.1016/j.toxicon.2009.013. 20035780. Kirkpatrick B, Fleming LE, Squicciarini D, et al. (2004) Literature review of Florida Red Tide: Implications for human health. Harmful Algae 3(2): 99–111. https://doi.org/10.1016/j. hal.2003.08.005. 20411030. Lange WR, Lipkin KM, and Yang GC (1989) Can ciguatera be a sexually transmitted disease? Journal of Toxicology. Clinical Toxicology 27(3): 193–197. https://doi.org/ 10.3109/15563658909038583. 2810444. Lefebvre KA and Robertson A (2010) Domoic acid and human exposure risks: A review. Toxicon 56(2): 218–230. https://doi.org/10.1016/j.toxicon.2009.05.034. 19505488. Lehane L and Lewis RJ (2000) Ciguatera: recent advances but the risk remains. International Journal of Food Microbiology 61(2–3): 91–125. https://doi.org/10.1016/s)168-1605(00) 00382-2. 11078162. Plakas SM and Dickey RW (2010) Advances in monitoring and toxicity assessment of brevetoxins in molluscan shellfish. Toxicon 56(2): 137–149. https://doi.org/10.1016/j. toxicon.2009.11.007. 19925816.

Further reading FAO/IOC/WHO (2005) Report of the Joint FAO/IOC/WHO ad hoc Expert Consultation on Biotoxins in Bivalve Molluscs. Oslo, Norway: Food and Agriculture Organization of the United Nations, Intergovernmental Oceanographic Commission of UNESCO, World Health Organization. September 26–30, 2004. Granéli E and Turner JT (eds.) (2006) Ecology of Harmful Algae. Berlin, Germany: Springer. Hallegraeff GM, Anderson DM, and Cembella AD (eds.) (2003) UNESCO Manual on Harmful Marine Algae. UNESCO/WHO: Geneva, Switzerland. Hudnell HK (ed.) (2008) Cyanobacterial Harmful Algal Blooms: State of the Science and Research Needs. New York: Springer. Hui YH, Kits D, and Stanfield PS (eds.) (2001) Seafood and Environmental Toxins. New York: Dekker.

Relevant websites http://oceanservice.noaa.gov/hazards/hab/ :Harmful Algal Blooms: Simple Plants with Toxic Implications. National Oceanic and Atmospheric Administration, National Ocean Service. http://yyy.rsmas.miami.edu/groups/ohh/ :University of Miami. Oceans and Human Health Center. http://www.whoi.edu/science/B/redtide/species/speciestable.html :US Fish, Shellfish and Wildlife Affected by Toxic or Harmful Microalgal Species. Woods Hole Oceanographic Institution, The Harmful Algae Page. http://www-cyanosite.bio.purdue.edu :A Webserver for Cyanobacterial Research. Purdue University, Department of Biological Sciences. http://www.issha.org :The International Society for the Study of Harmful Algae (ISSHA).

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Alkalis Timothy J Wiegand, University of Rochester Medical Center and Strong Memorial Hospital, Rochester, NY, USA © 2024 Elsevier Inc. All rights reserved. This is an update of V Lawana, Alkalis, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 142–143, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00228-1.

Chemical profile Background Uses Environmental fate Exposure routes and pathways Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Animals Human Chronic toxicity Animals Human Carcinogenicity Clinical management Ecotoxicology Exposure standards and guidelines References Further reading

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Abstract Alkalis are groups of chemicals with high pH. These types of chemicals are almost omnipresent and they hold many applications in both biological systems and industrial settings. Common Alkalis include aluminum hydroxide, sodium hydroxide, potassium hydroxide, ammonium hydroxide, and lithium carbonate. Among these Alkalis, ammonium hydroxide and lithium carbonate are often used to treat a wide variety of diseases and conditions. Bicarbonate-containing Alkalis are frequently used in clinical medicine to treat metabolic acidosis. Biological fluids (blood, serum, urine, saliva, vaginal fluids) or soil with pH 7.4 or higher are considered alkaline. Ingestion of Alkalis can result in esophageal and gastric injury. Endoscopy should be utilized in cases of intentional ingestion of Alkalis with subsequent staging determining further treatment and prognosis.

Keywords Alkali; Alkaline solutions; Base; Calcium carbonate; Caustic agent; Corrosive; Industrial chemical toxicity; Sodium hydroxide; Usta protocol

Key points

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Alkalis are basic salts with a pH above 7.0. Common Alkalis are sodium hydroxide, calcium carbonate, sodium bicarbonate and aluminum hydroxide although there are numerous alkaline salts available. The Poisoning Prevention Act legislation called for child resistant packaging for all caustic agents used as household cleaning products with a concentration over 10%. This was later reduced to 2% and it led to a reduction in pediatric caustic ingestions. Ingestion of alkaline agents can cause esophageal injury with subsequent stricture formation. The risk of esophageal cancer is increased after ingestion of Alkalis. Endoscopy is the preferred method of evaluation after ingestion of Alkalis although CT scan (with contrast) is also used in certain circumstances.

Encyclopedia of Toxicology 4th Edition

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Chemical profile

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Name: Alkalis Chemical Abstracts Service Registry Number: N/A (ID: D000468000) Synonyms: Base, Basic solutions, Alkaline solutions/mixtures

Background Alkalis are basic salts with a pH above 7.0. An alkali is a base that can dissolve in water. Alkalis are among the oldest forms of chemical reagents, and they are used in countless number of experimental and therapeutic settings. Their application varies from preparation of surgical equipment to manufacturing of disinfectants. Alkalis are usually an inorganic class of chemicals. Physiologically, Alkalis also play a crucial role in maintaining proper pH in body compartments (e.g., kidney-urine); however, such mechanisms are usually controlled and regulated within the body. A number of compounds with alkaline properties have tremendous therapeutic value (Table 1). Alkaline agents are used for cleaning in a variety of settings. Household cleaning products may contain alkaline agents. The Poisoning Prevention Act of 1970 mandated child resistant containers be used for products which contained caustics, including Alkalis, at concentrations over 10%. This was later reduced to 2% for household products (Hoffman et al., 2020). This led to a reduction in the number of caustic alkaline ingestions in children, but farm products were excluded from this legislation. Alkalis are used as liquid pipe cleaners on farms and a number of accidental pediatric ingestions of Alkalis from farm products are reported annually (Neidich, 1993). Ingestion of high levels of alkaline salts such as calcium carbonate or sodium bicarbonate can cause a variety of metabolic changes. Milk-alkali syndrome develops from drinking too much milk (which is high in calcium) and taking certain antacids, especially calcium carbonate or sodium bicarbonate (baking soda), over a long period of time. Calcium deposits in the kidneys and in other tissues can occur in milk-alkali syndrome, and high levels of vitamin D can worsen this condition. Although extremely rare today, milk-alkali syndrome was more commonly associated as a side effect of peptic ulcer disease treatment with antacids containing calcium. Sophisticated medications that do not contain calcium are used today for treating ulcers. Individuals taking calcium carbonate to prevent osteoporosis sometimes face this condition (even in those taking as little as 2 g of calcium per day). Patients with chronic kidney disease are at higher risk of toxicity with milk-alkali syndrome (Picolos and Orlander, 2005).

Uses Alkalis are widely used compounds. They are used in antacid preparation to treat gastritis as well as systemic acidosis. Depending on the specific alkali, they are also used in bleaches, cleaning agents, detergents, unslaked lime, and in a variety of other products. Calcium hydroxide is a common alkali used for dental and endodontic procedures. It has strong antimicrobial effects and is used during root canal and capping procedures. Pure calcium hydroxide paste has a high pH of 12.5–12.8 (Mohammadi and Dummer, 2011). Alkalis aluminum hydroxide and calcium phosphate have been used to study the antibody response to snake venom when they are used during venom toxicity tests (Olmedo et al., 2014). Ammonium nitrate is used as a fertilizer (Ortiz-Santaliesta et al., 2012).

Table 1

Useful therapeutic properties of some well-known alkaline compounds.

Name of compound

Disease treatment

Alkalis Alkalis Aluminum hydroxide Aluminum hydroxide Aluminum hydroxide Lithium carbonate Magnesium hydroxide Nickel hydroxide Potassium hydroxide

Hypercalcemia, leishmaniasis Renal insufficiency Status epilepticus Acute kidney injury Seizures Seizures Reperfusion injury; wounds and injuries; Crohn’s disease Dermatitis, irritant

http://ctdbase.org—Comparative Toxicogenomics database; Mount Desert Island Biological Lab.

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Environmental fate In cases of a solid alkali spill on soil, groundwater pollution occurs. Precipitation will dissolve some of the solid and create an aqueous solution of that alkali, which then would be able to infiltrate the soil. However, prediction of the concentration and properties of the solution produced are difficult. Ammonium nitrate fertilizer, as concentration increases, can have adverse effects on terrestrial organisms (Ortiz-Santaliesta et al., 2012).

Exposure routes and pathways Usual exposure routes include dermal exposure, oral ingestion (usually accidental although intentional ingestion of Alkalis also occurs), and via inhalation.

Toxicokinetics Toxicokinetics varies depending on the type of alkali. Alkalis are in general small molecular weight molecules and undergo weak dissociation in gastric acid pH. In general, they are readily converted into salt depending on the microenvironment.

Mechanism of toxicity Alkalis usually act by altering pH of the extracellular fluid. They exhibit their toxic effects by liquefactive necrosis, meaning that they damage the cell membrane, and thus cell integrity is ruptured. This causes cell lysis. This is why they are used in lysis buffer preparations for various biochemical assays. Intracellular alkalinization is more serious; such conditions alter intracellular redox balance and alter metabolic pathways, ultimately breaking down cellular homeostasis.

Acute and short-term toxicity Animals The toxicity of Alkalis in animals is the same as that of humans.

Human Alkalis can cause skin irritation and skin burns. Also, they cause damage to mucosal membranes and the eyes almost immediately on contact. However, the absence of burns, irritation, erythema, or other such signs in the oral or circumoral area does not necessarily indicate that esophageal injury does not exist. Inhalation of the fumes may cause pulmonary edema or pneumonitis.

Chronic toxicity Animals Toxic manifestations of Alkalis in animals are the same as that of humans.

Human Chronic exposure to any alkali is generally not beneficial and causes severe toxicity. Burns that appear to be mild at the time of injury can sometimes go on to cause opacification, vascularization, ulceration, or perforation. Direct exposure is always injurious, and prolonged alkalinization of any biological material can induce irreversible changes. Ingestion of alkaline agents can cause esophageal injury with subsequent stricture formation. The risk of esophageal cancer is increased after ingestion of Alkalis.

Carcinogenicity There is no evidence of alkali-mediated tumorigenic effects. Alkaline burns increase the risk of subsequent cancer development in esophageal tissue.

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Clinical management Exposure should be terminated as soon as possible by removing the victim to fresh air. The skin, eyes, and mouth should be washed with copious amounts of water. A 20–30 min wash may be necessary to neutralize and remove all residual traces of the contamination. Contaminated clothing and jewelry should be removed and isolated. Oral ingestion requires immediate dilution therapy with water or milk. Antidotes such as vinegar or lemon juice are absolutely contraindicated. Emesis should be avoided in case of ingestion. Endoscopy is the most common method of evaluating the esophagus and gastric mucosa for injury and staging. Extent of injury at initial evaluation has been used to predict morbidity and mortality (Kay and Wyllie, 2009). Intentional ingestions of caustics including Alkalis requires endoscopy. Prior to endoscopy the airway should be assessed and protected if any signs impairment. If endoscopy is not readily available and there is a high concern for injury, then a CT scan (with contrast). Endoscopy or CT scanning will allow for staging of injury and determination of further treatment. Grade 2B injuries are recommended to receive the “Usta protocol” which consists of methylprednisolone (1 g per 1.73 m2 of body-surface-area per day given over 3 days), ranitidine 4 mg kg−1 in children or standard adult dose given parenterally, with ceftriaxone (Hoffman et al., 2020). Injuries less than 2B (0, 2, 2A) can have a trial of clear liquids after brief NPO. More severe injuries (3A, 3B, 4) require CT scan after endoscopy and urgent surgical consultation.

Ecotoxicology These chemicals are easily degradable in environment and form salts readily. They are not known to have any profound ecotoxic impact except in exceptional circumstances such as accidental spills.

Exposure standards and guidelines The guidelines given here are for sodium hydroxide (common alkali). US Occupational Safety and Health Administration standards: permissible exposure limit: 8 h time-weighted average is 2 mg m−3. Threshold limit values: ceiling limit is 2 mg m−3. US National Institute for Occupational Safety and Health recommendations: recommended exposure limit is 2 mg m−3.

See also: Potassium; Sodium

References Hoffman RS, Burns MM, and Gosselin S (2020) Ingestion of caustic substances. The New England Journal of Medicine 382(18): 1739–1748. 348645 1.1056/NEJMra1810769. Kay M and Wyllie R (2009) Caustic ingestions in children. Current Opinion in Pediatrics 12(5): 651–654. https://doi.org/10.1097/MOP.0b013e32832e2764. PMID: 19543088. Mohammadi Z and Dummer PM (2011) Properties and applications of calcium hydroxide in endodontics and dental traumatology. International Endodontic Journal 44(8): 697–730. 21535021 https://doi.org/10.1111/j.1365-2591.2011.01886.x. Neidich G (1993) Ingestion of caustic alkali farm products. Journal of Pediatric Gastroenterology and Nutrition 16(1): 75–77. https://doi.org/10.1097/00005176-199301000-00015. PMID: 8433244. Olmedo H, Herrera M, Rojas L, Villalta M, Vargas M, Leiguez, Teixeira C, Estrada R, Gutierrez JM, Leon G, and Montero ML (2014) Comparison of the adjuvant activity of aluminum hydroxide and calcium phosphate on the antibody response towards Bothrops asper snake venom. Journal of Immunotoxicology 11(1): 44–49. 23506358 https://doi.org/10. 3109/1547691X.2013.772267. Ortiz-Santaliesta ME, Fernández-Benéitez MJ, and Marco A (2012) Density effects on ammonium nitrate toxicity on amphibians. Survival, growth and cannibalism. Aquatic Toxicology 110–111: 170–176. 22326654 https://doi.org/10.1016/j.aquatox.2012.01.010. Picolos MK and Orlander PR (2005) Calcium carbonate toxicity: The updated milk-alkali syndrome; report of 3 cases and review of the literature. Endocrine Practice 11(4): 272–280. 16006300 https://doi.org/10.4158/EP.11.4.272.

Further reading Klassen CD, Casarett LJ, and Doull J (2013) Casarett and Doull’s Toxicology: The Basic Science of Poisons, 8th edn. McGraw-Hill: New York. NIOSH (1975) Criteria Document: Sodium Hydroxide, pp. 76–105. DHEW, NIOSH.

Relevant website http://ctdbase.org/detail.go?type¼chem&acc¼D000468&view¼disease :Comparative Toxicogenomics database.

Alkyl halides Diana Migueza and Jose V Tarazonab, aWater and Environment Division, Latitud – Fundación LATU, Montevideo, Uruguay; bRisk Assessment Unit, National Centre for Environmental Health, Instituto de Salud Carlos III, Madrid, Spain © 2024 Elsevier Inc. All rights reserved. This is an update of S Kulkarni, Alkyl Halides, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 144–145, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00229-3.

Chemical profile Background Uses Banning and restrictions Physical-chemical properties Exposure routes and pathways Toxicokinetics Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Clinical management Environmental fate and concerns Conclusion References

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Abstract Alkyl halides are a group of compounds formed via substitution of a halogen for hydrogen in an alkane. Some, mostly chlorinated and brominated compounds, are of natural origin but the majority are synthetic compounds manufactured by different production processes. These compounds may be classified according to the type of halogen. They are widely used commercially as refrigerants, flames retardants, and solvents. Alkyl halides are of interest due to their widespread use and diverse beneficial and toxic impact. Due to their toxic properties and environmental impacts, the use of alkyl halides is closely regulated in many countries.

Keywords Alkyl halides; Haloalkanes; Halogenated hydrocarbon

Chemical profile

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Name: Alkyl Halides Chemical Abstracts Service Registry Number: None as a group Synonyms: haloalkanes, Halogenated hydrocarbons, Haloalkanes Individual components: The list of alkyl halides is very long, some representative compounds are Methyl bromide, Methyl chloride, Methyl iodide, Dichloromethane, Tetrachloroethane, Carbon tetrachloride, Trichloroethene, Trichloroethylene, A number of fluorinated hydrocarbons (e.g., Freons) Molecular Formula: R(X)n, where R is a hydrocarbon alkyl group and X is a halogen. One or more halogens may be present in one compound

Background Alkyl halides are a group of compounds formed via substitution of a halogen for hydrogen in an alkane. Can be classified according to the position of the halogen atom (as primary, secondary or tertiary alkyl halides), the number of halogen atoms, or according to the type of halogen. Some are naturally occurring such as bromoethane and 1-bromopropane; while most are manufactured from reaction with alcohols, alkenes, alkanes, or carboxylic acids. Due to the diversity of alkyl halides and their properties, most regulatory

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assessments are conducted on individual substances, but some group assessments have also conducted, such as the recent evaluation by Canada, covering bromoethane, chloroethane, 1-bromopropane, and trans-1,2-dichloroethene (Canada, 2022). A group of particular interest are the hydrochlorofluorocarbons (HCFCs) phased out under the Montreal Protocol due to their potential for depleting the stratospheric ozone layer. Other Alkyl Halides with ozone depletion capacity are carbon tetrachloride, 1,1,1-trichloroethane, methyl bromide, bromochloromethane, and several hydrochlorofluorocarbons (HCFCs) and hydrobromofluorocarbons.

Uses Many halogenated hydrocarbons have important commercial applications. Alkyl halides are important intermediates in synthesis, as solvents in the laboratory and industry, and as dry cleaning fluids. They also find use as anesthetics and refrigerants. For example, trichloroethene is a common dry cleaning solvent. The fluorinated hydrocarbons (Freons) are used as refrigerants, industrial solvents, fire extinguishers, local anesthetics, and glass chillers, but mainly as propellants in aerosol products. Methyl bromide, methyl chloride, and methyl iodide are used as refrigerants in chemical synthesis and as fumigants. Methyl bromide is used with carbon tetrachloride in fire extinguishers. Methyl chloroform is used as a solvent for cleaning and degreasing, and in paint removers. Dichloromethane is used in paint removers and as an industrial solvent. Tetrachloroethane is used as a solvent in industry and occurs as a contaminant in other chlorinated hydrocarbons. It is occasionally present in household cleaners. Carbon tetrachloride is used as a solvent and intermediate in many industrial processes.

Banning and restrictions The Montreal Protocol has identified those with ozone-depleting potential (ODP), additional substances have been incorporated in specific jurisdictions, such as the “new substances” under the EU Ozone Regulation, or suggested in scientific reviews (Engel et al., 2019). Halons, CFCs, carbon tetrachloride, methyl chloroform and hydrobromofluorocarbons were phased out from 1993 to 1995 (United States Environmental Protection Agency, 2022a; United States Environmental Protection Agency, 2022b). On the other hand, the Kyoto Protocol, signed in 1997 and which went into effect in 2005, under the Kigali amendment, agreed to gradually phase down the use of hydrofluorocarbons (HFCs) by 80–85% by the late 2040s (United Nations Environment Programme, 2016). These compounds are widely used in refrigeration and air conditioning systems as a replacement for CFCs and hydrochlorofluorocarbons (HCFCs), potent ozone-depleting substances. While HFCs are less harmful to the ozone layer, they are still potent greenhouse gases.

Physical-chemical properties Alkyl halides are practically insoluble in water. They are miscible in all proportions with liquid hydrocarbons and are, in general, good solvents for many organic substances. Most of the common organic halides are liquids. Like alkanes, halogen compounds are insoluble in and inert to cold concentrated sulfuric acid. In a series of alkyl halides, the boiling point rises with an increase in molecular weight due to the presence of either a heavier halogen atom or a larger alkyl group. Bromides boil at temperatures distinctly higher than the corresponding chlorides, and iodides are higher boiling than the bromides. Increase in the halogen content decreases their flammability.

Exposure routes and pathways Inhalation, dermal, and ocular contact are common routes of exposure. Considering the variability in uses and properties, the exposure levels should be estimated for each compound or commercial mixture. In contact with an open flame or very hot surface, fluorocarbons may decompose into highly irritant and toxic gases such as chlorine, hydrogen fluoride, or chloride, and even phosgene.

Toxicokinetics The information is limited for many chemicals in this group. In general, the melting and boiling points of chloro-, bromo-, and iodoalkanes are higher than the analogous alkanes; nevertheless, readily absorption after inhalation exposures is expected for bromoethane, 1-bromopropane, or trans-1,2-dichloroethene (Canada, 2022) among others. Fluorocarbon compounds are lipid soluble and, thus, generally well absorbed through the lung. Absorption after ingestion is much lower than after inhalation. Most of the fluorinated hydrocarbons are immediately absorbed via inhalation. There is a significant accumulation of fluorocarbons in the brain, liver, and lungs compared to blood levels, signifying a tissue distribution of fluorocarbons similar to that of chloroform. Fluorocarbons are concentrated in body fat where they are slowly released into blood at a concentration that should not cause any risk of cardiac sensitization. Fluorocarbons are excreted primarily by the lungs.

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Acute and short-term toxicity Animal Deliberate ocular exposure in rabbits to liquid Freon 12 produced effects related to the duration of exposure. Severe corneal damage with opacity occurred following exposure for 30 s. In dogs, inhalation of fluorinated hydrocarbon vapors causes bradycardia followed by deterioration to ventricular fibrillation in some animals.

Human Freons are very toxic when inhaled in high concentrations or for extended periods. Inhalation of fluorinated hydrocarbons such as those caused by leaking air conditioners or refrigerators usually results in transient eye, nose, and throat irritation. Palpitations and lightheadedness are also seen. Headache was a common complaint, reported in 71% of 31 workers exposed to bromotrifluoromethane in one incident. Inhalation of halides at sufficient concentrations associated with deliberate abuse, or spills or industrial use occurring in poorly ventilated areas, has been associated with ventricular arrhythmias, pulmonary edema, and sudden death. Fluorinated hydrocarbons are believed to cause arrhythmias by sensitizing the myocardium to endogenous catecholamines. Freon solvents are used as degreasers. Dermal contact with fluorinated hydrocarbons may result in defatting, irritation, contact dermatitis, or skin injury. Severe frostbite was reported as a rare effect of severe Freon exposure. Mucosal necrosis and perforation of the stomach developed in one patient after ingesting a small amount of trichlorofluoromethane. Fluorocarbons containing bromine are more toxic than the corresponding chlorine compounds. There is a significant interpatient variation following exposure to fluorocarbons and it is difficult to predict symptoms following exposure. Compounds like dibromochloropropane, in which occupational exposure has affected male fertility, have now been removed from the market. Following acute exposure to methyl bromide, chloride, or iodide, nausea and vomiting, blurred vision, vertigo, weakness or paralysis, oliguria or anuria, drowsiness, confusion, hyperactivity, coma, convulsions, and pulmonary edema are noted. Pulmonary edema and bronchial pneumonia are most often the cause of death. Skin contact causes irritation and vesiculation. Methyl chloroform and dichloromethane are central nervous system (CNS) depressants. Methyl chloroform sensitizes the myocardium to catecholamine-induced arrhythmias. Following exposure to tetrachloroethane, irritation of the eyes and nose, followed by headache and nausea, is observed. Cyanosis and CNS depression progressing to coma may appear after 1–4 h.

Chronic toxicity Animal Some of the chlorinated hydrocarbon solvents such as methylene chloride and chloroform have caused cancer in several species of experimental animals and are suspect human carcinogens.

Human A syndrome of impaired psychomotor speed, impaired memory, and impaired learning has been described in workers with chronic occupational exposure to fluorinated hydrocarbons. Skin irritation and defatting dermatitis upon prolonged or repeated contact with the skin to trichloromonofluoromethane have been reported. An excess of CNS symptoms was seen in a group of workers chronically exposed to trichloromonofluoromethane. Repeated exposure to methyl bromide, methyl chloride, and methyl iodide will cause blurring of vision, numbness of the extremities, confusion, hallucinations, somnolence, fainting attacks, and bronchospasm. Chronic toxicity has not been reported with dichloromethane. Headache, tremor, dizziness, peripheral paresthesia, and anesthesia have been reported after chronic inhalation or skin exposure to tetrachloroethane. The US National Institute of Occupational Safety and Health recommends that methyl chloride, methyl bromide, and methyl iodide be considered as potential occupational carcinogens and that methyl chloride be considered a potential occupational teratogen. Carbon tetrachloride poses significant risks to human health, as it is toxic if ingested, inhaled, or absorbed through the skin and can cause liver and kidney damage, respiratory problems, neurological effects, and other health problems. The United States Environmental Agency has classified carbon tetrachloride as a Group B2, probable human carcinogen (United Nations Environment Programme, 2016).

Clinical management This management is intended for use in the absence of a specific treatment protocol for a product or a chemical. Symptomatic and supportive care is the primary therapy. The general approach to a poisoned patient is to first assess the vital signs of the patient followed by assessing the route of administration for potential toxicity. Measures to prevent further absorption of the compound may be useful. Victims of inhalation exposure should be moved from the toxic environment and administered 100% humidified supplemental oxygen with assisted ventilation as required. Exposed individuals should have a careful and thorough medical examination performed to look for abnormalities. Patients with fluorohydrocarbon poisoning should not be given epinephrine or

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similar drugs because of the tendency of fluorohydrocarbon to induce cardiac arrhythmia, including ventricular fibrillation. Monitoring including complete blood count, urine analysis, and liver and kidney function tests is suggested for patients with significant exposure. Activated charcoal or gastric lavage may be indicated to prevent further absorption. Exposed eyes should be irrigated with copious amounts of tepid water for at least 15 min. If irritation, pain, swelling, lacrimation, or photophobia persists after 15 min of irrigation, an ophthalmologic examination should be performed.

Environmental fate and concerns Many alkyl halides are gases or volatile liquids, and their main environmental concerns are related to their ozone depletion and greenhouse potential. The depletion of the stratospheric ozone layer is associated with severe environmental hazards, linked to solar ultraviolet (UV) radiation, and more recently climate change (Neale et al., 2021). The Montreal Protocol under the United Nations signed in 1987 was the first step in international efforts to protect stratospheric ozone, covers over around 100 individual substances, and has been considered as an international success (Barnes et al., 2021). It is supported by global atmospheric-measurement networks that have been able to detect unexpected emissions rises, such as the one detected in China in 2013 (Park et al., 2021). Methyl bromide is used as fumigant and has been extensively studied in terms of environmental fate (Yates et al., 2003); and ecotoxicity, with observed high acute toxicity for birds; and similar toxicity for the different aquatic taxonomic groups (EFSA, 2011). For a majority of alkyl halides, ecotoxicological information is limited, however, in silico approaches, and particularly quantitative structure toxicity relationship (QSTR) analysis, may facilitate the assessment as the alkyl halide group is frequently included in QSAR development also for ecotoxicity (Banjare et al., 2023). The Canadian assessment concluded low environmental risk for the four substances in their Alkyl Halides Group, through the integration of several lines of evidence (Canada, 2022).

Conclusion Alkyl halide constitutes a large and heterogeneous chemical group. The nature, number and position of the halogen substitutes modifies the properties of the respective alkane, providing new uses as well as specific human and environmental concerns. The ozone depletion and greenhouse potential are very relevant for several alkyl halides. QSAR analysis offers options for completing mixing experimental information.

References Banjare P, Singh J, Papa E, and Roy PP (2023) Aquatic toxicity prediction of diverse pesticides on two algal species using QSTR modeling approach. Environmental Science and Pollution Research International 30(4): 10599–10612. https://doi.org/10.1007/s11356-022-22635-3. PMID: 36083366. Barnes PW, Bornman JF, Pandey KK, Bernhard GH, Bais AF, Neale RE, Robson TM, Neale PJ, Williamson CE, Zepp RG, Madronich S, Wilson SR, Andrady AL, Heikkilä AM, and Robinson SA (2021) The success of the Montreal Protocol in mitigating interactive effects of stratospheric ozone depletion and climate change on the environment. Global Change Biology 27(22): 5681–5683. https://doi.org/10.1111/gcb.15841. PMID: 34392574. Canada (2022) Draft Screening Assessment - Alkyl halides Group. Environment and Climate Change Canada and Health Canada, March 2022, 65 pp. Available at file:///C:/Users/ jtarazona/Downloads/draft-screening-assessment-alkyl-halides-group.pdf (Accessed on 21 April, 2023). EFSA (2011) Conclusion on the peer review of the pesticide risk assessment of the active substance methyl bromide. EFSA Journal 9(1): 1893. 32 pp https://doi.org/10.2903/j.efsa. 2011.1893. Engel A, Rigby M, Burkholder JB, Fernandez RP, Froidevaux L, Hall BD, et al. (2019) Update on Ozone-Depleting Substances (ODSs) and Other Gases of Interest to the Montreal Protocol. World Meteorological Organization. Neale RE, Barnes PW, Robson TM, Neale PJ, Williamson CE, Zepp RG, et al. (2021) Environmental effects of stratospheric ozone depletion, UV radiation, and interactions with climate change: UNEP Environmental Effects Assessment Panel, Update 2020. Photochemical & Photobiological Sciences 20(1): 1–67. Park S, Western LM, Saito T, Redington AL, Henne S, Fang X, et al. (2021) A decline in emissions of CFC-11 and related chemicals from eastern China. Nature 590(7846): 433–437. United Nations Environment Programme (2016) Available at: https://www.unep.org/news-and-stories/news/kigali-amendment-montreal-protocol-another-global-commitment-stopclimate (Retrieved 21 April, 2023). United States Environmental Protection Agency (2022a) Available at: https://www.epa.gov/sites/default/files/2016-09/documents/carbon-tetrachloride.pdf (Retrieved 21 April, 2023). United States Environmental Protection Agency (2022b) Available at: https://www.epa.gov/ods-phaseout/accelerated-phaseout-class-i-ozone-depleting-substances#:
:text¼Carbon %20tetrachloride%20was%20widely%20used,was%20discovered%20to%20be%20carcinogenic (Retrieved 21 April, 2023). Yates SR, Gan J, and Papiernik SK (2003) Environmental fate of methyl bromide as a soil fumigant. Reviews of Environmental Contamination and Toxicology 177: 45–122. https://doi. org/10.1007/0-387-21725-8_2. PMID: 12666818.

Allyl alcohol TM Shashkovaa, Ya O Mezhueva, and Aristides Tsatsakisb, aMendeleev University of Chemical Technology of Russia, Moscow, Russia; b Laboratory of Toxicology and Forensic Chemistry, Medical School, University of Crete, Heraklion, Crete, Greece © 2024 Elsevier Inc. All rights reserved. This is an update of S.P. Sawant, H.S. Parihar, H.M. Mehendale, Allyl Alcohol, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 146–148, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00230-X.

Chemical profile Introduction Uses Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Chronic toxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Exposure standards and guidelines References

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Abstract Allyl alcohol (CAS # 107-18-6) is a colorless liquid with a pungent, mustard-like odor. It is soluble in water and has a chemical structure of CH2]CHCH2OH. It is used as an industrial solvent, herbicide, and fungicide and as raw material in manufacturing of various polymers, pharmaceuticals, pesticides, and other allyl compounds. Preclinical acute toxicity studies have shown that allyl alcohol causes skin and eye irritation and kidney and liver damage in animals. The most important adverse effects of occupational exposures to allyl alcohol are upper respiratory tract irritation and burning of the eyes. It is also hazardous to aquatic organisms and plants.

Keywords Allyl alcohol; Fungicide; Hepatotoxicant; Herbicide; Skin/eye irritant

Key points

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Allyl alcohol is metabolized to highly toxic acrolein; Allyl alcohol is hepatotoxic; Allyl alcohol causes irritation of the upper respiratory tract and burning in the eyes.

Chemical profile

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Name: Allyl alcohol Chemical Abstracts Service Registry Number: 107-18-6 Synonyms: 1-Propen-3-ol, 2-Propenol, 2-Propen-1-ol, Vinyl carbinol Molecular Formula: C3H6O Chemical Structure:

H2C

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Introduction Allyl alcohol (CAS # 107-18-6) is a colorless liquid with a pungent, mustard-like odor, soluble in water, and with a chemical structure of CH2CHCH2OH. It is synthesized by the hydrolysis of allyl chloride or isomerization of propylene oxide. It is used as a raw material in manufacturing of various polymers, pharmaceuticals, pesticides, and other allyl compounds (Atzori et al., 1989; Auerbach et al., 2008; Irwin, 2006; Li et al., 2012).

Uses Industrial solvent, herbicide, fungicide and a monomer for producing polymeric materials (Atzori et al., 1989; Auerbach et al., 2008; Irwin, 2006; Li et al., 2012).

Environmental fate and behavior Allyl alcohol is a colorless water soluble liquid. The melting point, boiling point, vapor pressure, and the octanol–water partition coefficient (log Kow) are −129
C, 97
C, 26.1 mmHg at 25
C, and 0.17, respectively (Li et al., 2012). The Henry’s law constant is 4.99  10–6 atm m3 mol–1. Allyl alcohol’s production, its use as an industrial solvent and as a raw material/intermediate in the preparation of pharmaceuticals, polymers, organic chemicals, in the manufacture of glycerol and acrolein, and in the production of insecticides and herbicides, may result in its release to the environment. The vapor pressure of allyl alcohol, 26.1 mmHg at 25
C, indicates that if released in the air, it will exist mainly as a vapor in the ambient atmosphere. If released to soil, allyl alcohol is expected to have very high mobility based upon an estimated Koc of 1.3 and will be distributed mainly in the water and soil. If released into water, allyl alcohol will stay in the water and is not expected to adsorb to suspended solids and sediments. Allyl alcohol is stable in water since it lacks functional groups that hydrolyze under environmental conditions and hence hydrolysis is not expected to be an important environmental fate process. In an aerobic biodegradation study, allyl alcohol was found to readily degradable (82–86%) in 14 days. The estimated bioconcentration factor of 3.2 based on the low log Kow indicates that the potential to bioaccumulate in aquatic organisms is expected to be low.

Exposure and exposure monitoring Occupational exposure to allyl alcohol may occur through inhalation and dermal contact where allyl alcohol is produced or used (Li et al., 2012). Monitoring data indicate that the general population may be exposed to allyl alcohol via inhalation of ambient air or ingestion of food.

Toxicokinetics The metabolism of allyl alcohol involves the intermediate formation of acrolein as a result of oxidation (Atzori et al., 1989; Auerbach et al., 2008; Irwin, 2006; Li et al., 2012; Ohno, 1985; Golla et al., 2015). Also, allyl alcohol can be metabolized to glycidol oxide (Auerbach et al., 2008). The conversion of allyl alcohol to acrolein is mediated by alcohol dehydrogenase (ADH) (Atzori et al., 1989) and then may be further oxidized to acrylic acid by Nicotinamide adenine dinucleotide- or Nicotinamide adenine dinucleotide phosphate-dependent enzymes in the liver cytosol or microsomes or to glycidaldehyde by a microsomal enzyme with subsequent conversion to glyceraldehyde by epoxide hydrolase. Alternatively, acrolein may react directly both enzymatic and nonenzymatic reactions to form stable adducts with glutathione (GSH) or other low molecular weight thiol compounds prior to excretion in the urine as mercapturate (Atzori et al., 1989; Ohno, 1985).

Mechanism of toxicity Allyl alcohol is inactive per se and its toxic effect is mediated by its ADH oxidation to form acrolein, which is responsible for the hepatotoxic action (Atzori et al., 1989; Silva and O’Brien, 1989; Tukov et al., 2006). The toxicity of the alcohol (or its metabolite acrolein) is dependent on the concentration of GSH (Atzori et al., 1989; Ohno, 1985). After severe depletion of GSH, the reactive metabolite of allyl alcohol can bind to essential sulfhydryl groups in the cellular macromolecules, leading to structural and functional changes that may lead to cell death. In this case, the appearance of lipid peroxidation could be merely the consequence of cell death. In the liver, Kupffer cell (Tukov et al., 2006) activation has been implicated in playing a prominent role during progression of toxicity. The key role of GSH in the binding of acrolein, the product of biological oxidation of allyl alcohol, is indicated by the fact that the hepatotoxicity in rats with Torii-Leprfa Spontaneous Diabetes mellitus with a reduced level of GSH was significantly greater than

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in the control group. Therefore, a greater toxic effect of allyl alcohol can be expected for people with type 2 diabetes mellitus (Takahashi et al., 2019). It is noteworthy that the mechanism of detoxification of allyl alcohol and acrolein is not associated with the formation of glutathione disulfide, however the introduction of dithiothreitol had a pronounced protective effect. Therefore, it can be assumed that the decrease in the toxic effect of allyl alcohol and acrolein in the presence of GSH is associated with the addition of the thiol group of GSH to acrolein. Apparently, an important role in minimizing toxic effects belongs to the aldehyde dehydrogenase oxidation of acrolein to acrylic acid. This is supported by the increased hepatotoxicity of allyl alcohol in the presence of aldehyde dehydrogenase inhibitors such as cyanamide and disulfiram (Silva and O’Brien, 1989). The toxicity mechanism of allyl alcohol is likely very complex. Genome-wide analysis of the transcriptome of budding yeast cells treated with a sublethal dose of allyl alcohol showed the development of oxidative stress, DNA damage processes as well as disruption of the integrity of the cell wall (Golla et al., 2015).

Acute and short-term toxicity The oral LD50 values reported in rat and mice are 64, 70, and 99–105 mg kg−1 and 85 and 96 mg kg−1, respectively (Li et al., 2012). The dermal LD50 (rabbit) is 89 mg kg−1 (Li et al., 2012). The inhalation of LC50 in rat and mouse are 76 ppm per 8 h (Li et al., 2012) and 500 mg m−3 per 2 h, respectively. The LD50 values reported in rats and mice following intraperitoneal administration are 37 and 60 mg kg−1, respectively (Irwin, 2006). Studies have shown that allyl alcohol is slightly irritating to skin and irritating to the eyes in rabbits. Allyl alcohol is not a skin sensitizer in guinea pigs. Acute exposure to allyl alcohol causes liver and kidney damage in animals. Allyl alcohol is classified as a periportal hepatotoxicant since it selectively damages the periportal region of the liver. Studies have shown that in adult rats, allyl alcohol produces a moderate to marked periportal necrosis with attendant inflammation and hemorrhage, and also decreases hepatic cytochrome P450, benzphetamine N-demethylation, and ethoxyresorufin O-deethylation activities by about 30%. In immature rats, it lowered both cytochrome P450 activity (30%) and ethoxyresorufin O-deethylation (75%). Benzphetamine N-demethylation was not significantly affected in immature rats. Intraperitoneal administration of 1.5 mmol kg–1 allyl alcohol to starved Swiss albino mice causes the development of hemolysis in nearly 50% of the animals. Other toxic effects include renal necrosis, pulmonary edema, and central nervous system effects at higher dose levels. The most important adverse effects of occupational exposures to allyl alcohol are upper respiratory tract irritation (Li et al., 2012) and burning of the eyes. The substance may cause effects on the muscles, resulting in local spasms and aching. The appearance of these effects may be delayed after exposure.

Chronic toxicity Chronic exposure to allyl alcohol can cause liver and kidney damage (Atzori et al., 1989; Auerbach et al., 2008; Ohno, 1985; Silva and O’Brien, 1989; Tukov et al., 2006).

Reproductive toxicity A reproductive/developmental toxicity study was conducted using both sexes of Sprague–Dawley rats. The rats were dosed 2, 8, or 40 mg per kg bodyweight per day via oral gavage. The male rats were dosed from 14 days before mating for a total of 42 days and the female rats were dosed 14 days before mating and throughout mating and a pregnancy period to day 3 of lactation. Clinical findings reported in parental animals at 40 mg per kg bodyweight per day were salivation, lacrimation, irregular breathing, decrease in locomotor activity in both sexes, and loose stools in males. Histopathological examination at 40 mg per kg bodyweight per day revealed atrophy of the thymus and hyperplasia of luteal cells in the ovary in females. In the livers, necrosis, fibrosis, and proliferation of bile ducts, hypertrophy, brown pigmentation in perilobular hepatocytes, and diffuse clear cell changes were observed in male and female rats at 40 mg per kg bodyweight per day. Reproductive effects such as extension of estrous cycle length and increases in estrous cycle were reported in females at 40 mg per kg per bodyweight per day. In offspring, a decrease in viability index on day 4 and total litter loss (from one dam) was reported at 40 mg per kg bodyweight per day. The lowest observed adverse effect level was reported as 40 mg per kg bodyweight per day based on parental and reproductive/developmental toxicity and a no observed adverse effect level is reported as 8 mg per kg bodyweight per day.

Genotoxicity Several in vivo and in vitro genotoxicity studies have been conducted. Three out of seven in vitro genotoxicity assays including bacterial and mammalian cells gave positive results for these assays. Three in vivo genotoxicity assays, in vivo tests for chromosomal mutations (rat bone marrow and mouse erythrocyte micronuclei tests), and in vivo dominant lethal tests, produced negative results. Thus, based on the in vitro assays and in vivo tests, there is equivocal evidence that allyl alcohol is genotoxic (Irwin, 2006).

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Carcinogenicity Allyl alcohol is classified as A4 (not classifiable as human carcinogen) by the American Conference of Governmental Industrial Hygienists (Auerbach et al., 2008).

Clinical management Exposure should be terminated as soon as possible by removal of the patient to fresh air. Skin, eyes, and mouth should be washed with copious amounts of water. Contaminated clothing should be removed. A mild soap solution may be used for washing the skin, but should not be used in the eye. Dilution with water may be effective if small amounts are swallowed until additional medical attention is available.

Ecotoxicology The toxicity of allyl alcohol has been investigated in aquatic animals and plants. Several studies have shown that allyl alcohol causes acute toxicity in fish (medaka and fathead minnow), daphnia magna, polychaete, and green algae, and chronic toxicity in daphnia magna. Based on these studies, allyl alcohol is toxic to aquatic organisms and plants.

Exposure standards and guidelines The Occupational Safety and Health Administration general industry permissible exposure limit: 2 ppm, 5 mg m−3 8-h time-weighted average (TWA) (skin). The National Institute for Occupational Safety and Health recommended exposure limit: 2 ppm 10-h TWA, 4 ppm 15-min short-term exposure limit (skin). American Conference of Governmental Industrial Hygienists threshold limit value is 8-h TWA: 0.5 ppm, skin.

See also: Acrylic acid; Acrolein; Liver

References Atzori L, Dore M, and Congiu L (1989) Aspects of allyl alcohol toxicity. Drug Metabolism and Drug Interactions 7: 295–319. Auerbach SS, Mahler J, Travlos GS, and Irwin RD (2008) A comparative 90-day toxicity study of allyl acetate, allyl alcohol and acrolein. Toxicology 253(1–3): 79–88. Golla U, Bandi G, and Tomar RS (2015) Molecular cytotoxicity mechanisms of allyl alcohol (acrolein) in budding yeast. Chemical Research in Toxicology 28(6): 1246–1264. Irwin RD (2006) NTP technical report on the comparative toxicity studies of allyl acetate (CAS No. 591-87-7), allyl alcohol (CAS No. 107-18-6) and acrolein (CAS No. 107-02-8) administered by gavage to F344/N rats and B6C3F1 mice. Toxicity Report Series 48: 1–73. A1–H10. Li AA, Fowles J, Banton MI, Picut C, and Kirkpatrick DT (2012) Acute inhalation study of allyl alcohol for derivation of acute exposure guideline levels. Inhalation Toxicology 24(4): 213–226. Ohno Y (1985) Mechanism of allyl alcohol toxicity and protective effects of low-molecular-weight thiols studies with isolated rat hepatocytes. Toxicology and Applied Pharmacology 78(2): 169–179. https://doi.org/10.1016/0041-008x(85)90281-9. Silva J and O’Brien PJ (1989) Allyl alcohol- and acrolein-induced toxicity in isolated rat hepatocytes. Archives of Biochemistry and Biophysics 275(2): 551–558. Takahashi T, Matsuura C, Toyoda K, Suzuki Y, Yamada N, Kobayashi A, Sugai S, and Shimoi K (2019) Estimation of potential risk of allyl alcohol induced liver injury in diabetic patients using type 2 diabetes spontaneously diabetic Torii-Leprfa (SDT fatty) rats. The Journal of Toxicological Sciences 44(11): 759–776. Tukov FF, Maddox JF, Amacher DE, Bobrowski WF, Roth RA, and Ganey PE (2006) Modeling inflammation-drug interactions in vitro: A rat Kupffer cell-hepatocyte coculture system. Toxicology In Vitro 20(8): 1488–1499.

Relevant websites https://www.ncbi.nlm.nih.gov/books/NBK224941/ :National Center for Biotechnology Information https://www.cdc.gov/niosh/docs/81-123/pdfs/0017-rev.pdf?id¼10.26616/NIOSHPUB81123 :U.S. Department of Health and Human Services; U.S. Department of Labor https://www.atamanchemicals.com/allyl-alcohol_u25613/ :ATAMAN Chemicals https://pubchem.ncbi.nlm.nih.gov/compound/Allyl-alcohol :National Center for Biotechnology Information, PubChem https://www.nj.gov/health/eoh/rtkweb/documents/fs/0036.pdf :New Jersey Department of Health http://www.ilo.org/dyn/icsc/showcard.display?p_card_id¼0095&p_version¼2&p_lang¼en :ILO and WHO

Allyl formate Sushma Ramsinghani, University of the Incarnate Word, San Antonio, TX, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.P. Sawant and H.M. Mehendale, Allyl formate, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 152–153, ISBN 978012364550.

Chemical profile Introduction Uses/occurrence Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Toxicogenomics Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines Conclusion PubChem URL Comptox URL References

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Abstract Allyl formate (CAS#1838-59-1) is a highly flammable, clear, colorless liquid. It is used as a solvent in spray lacquers, enamels, varnishes, and latex paints and as an ingredient in paint thinners and strippers, varnish removers, and herbicides. It is also used in liquid soaps, cosmetics, industrial and household cleaners, and dry-cleaning compounds. Acute exposure to allyl formate causes liver and kidney damage. Allyl formate is classified as a periportal hepatotoxicant since it selectively damages the periportal region of the liver in rodents. The most important adverse effect of occupational exposure to allyl formate is upper respiratory tract irritation. This compound may be fatal if inhaled, ingested, or absorbed through skin.

Keywords Acrolein; Aldehyde dehydrogenase; Allyl alcohol; Allyl formate; Epoxide hydrolase; Hepatotoxicity

Key points

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Allyl formate is used in the paint industry, dry-cleaning, and in household cleaners Acute exposure to allyl formate can cause liver and kidney damage Allyl formate may be fatal if inhaled, ingested, or absorbed through skin Occupational exposure causes upper respiratory tract irritation.

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Allyl formate

Chemical profile

• • • •

Synonym: Formic acid, prop-2-enyl formate (IUPAC name), 2-propenyl ester, allyl ester, 2-propen-1-yl ester, Formic acid allyl, 2-propenyl formate, prop-2-en-1-yl formate, 2-propenyl ester of formic acid. CAS Number: 1838-59-1. Molecular Formula: C4H6O2 Chemical Structure:

O

O

Introduction Allyl formate is a clear, colorless liquid, soluble in water. It is labeled as a flammable liquid poison. Allyl formate is highly toxic by ingestion, inhalation, and skin contact. Ingestion of allyl formate can cause liver injury in animals. The most common effect in humans following occupational exposure to allyl formate is upper respiratory tract irritation.

Uses/occurrence It is used as a solvent in spray lacquers, enamels, varnishes, and latex paints and as an ingredient in paint thinners and strippers, varnish removers, and herbicides. It is also used in liquid soaps, cosmetics, industrial and household cleaners, and dry-cleaning compounds.

Exposure and exposure monitoring The principal route of exposure is through the skin. Allyl formate is harmful if inhaled or in contact with skin, and toxic if swallowed. Overexposure may cause headache, dizziness, tiredness, nausea, and vomiting. Exposure monitoring is better accomplished by biological monitoring of exposed workers than air monitoring since uptake is greatly influenced by workload.

Toxicokinetics Allyl formate is rapidly cleaved in vivo by nonspecific esterases to allyl alcohol. Allyl alcohol is metabolized via two alternative oxidative pathways leading to the formation of acrolein or the epoxide, glycidol. The epoxide may then be converted to glycerol by epoxide hydrolase. The conversion of allyl alcohol to acrolein is mediated by alcohol dehydrogenase, which may then be further oxidized to acrylic acid by nicotinamide adenine dinucleotide- or nicotinamide adenine dinucleotide phosphate-dependent enzymes in the liver cytosol or microsomes or to glycidaldehyde by a microsomal enzyme with subsequent conversion to glyceraldehyde by epoxide hydrolase. Alternatively, acrolein may react directly, both enzymatically and nonenzymatically, to form stable adducts with glutathione (GSH) or other low molecular weight thiol compounds prior to excretion in the urine as mercapturate (Serafini-Cessi, 1972; Yap et al., 2006).

Mechanism of toxicity Allyl formate is cleaved by nonspecific esterases to allyl alcohol, which is then oxidized by alcohol dehydrogenases to the reactive acrolein, which is responsible for the hepatotoxic action (Rees and Tarlow, 1967; Athersuch et al., 2006). The toxicity of allyl alcohol via its metabolite acrolein is dependent on the concentration of GSH. After depletion of GSH, the reactive metabolite of allyl alcohol can bind to essential sulfhydryl groups in the cellular macromolecules, leading to structural and functional modifications, which can be responsible for hepatic injury (Droy et al., 1989). Appearance of lipid peroxidation signals events that follow toxication mechanisms initiated by acrolein, and subsequent and continued lipid peroxidation could be merely the consequence of cell death.

In vitro toxicity data No data is available.

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Acute and short-term toxicity Animal The LD50 reported following oral administration in rats and mice is 124 and 96 mg/kg respectively. The LC50 reported following inhalation exposure in rats and mice is 980 and 610 mg/m3 respectively. Acute exposure to allyl formate causes liver damage. At high doses the hepatotoxicity caused hypercreatinemia and hypercreatinuria in rats, but it was unclear if that led to kidney damage (Clayton et al., 2004). Allyl formate also causes general depressed activity with somnolence.

Human The most important adverse effect of occupational exposures to allyl formate is upper respiratory tract irritation.

Chronic toxicity Animal Chronic exposure to allyl formate can cause liver damage.

Human Long-term exposure may lead to liver damage.

Immunotoxicity No data is available.

Reproductive and developmental toxicity No data is available.

Genotoxicity No data is available.

Carcinogenicity No data is available.

Organ toxicity Allyl formate is classified as a periportal hepatotoxicant since its metabolite, acrolein, selectively damages the periportal region of the liver in rodents. It is also a neurotoxin and can cause acute solvent syndrome – typical symptoms include hepatotoxicity, anesthesia, respiratory irritation, and dermatitis (HARD). Contact with skin may cause moderate to severe erythema and moderate edema. It may also cause serious eye injury.

Interactions Allyl formate is incompatible with oxidizing substances and spontaneously flammable products.

Toxicogenomics No data is available.

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Clinical management Exposure should be terminated as soon as possible by removal of the patient to fresh air. Skin, eyes, and mouth should be washed with copious amounts of water. Contaminated clothing should be removed. A mild soap solution may be used for washing the skin but should not be placed in the eye. Dilution with water may be effective if small amounts are swallowed before medical attention is sought.

Environmental fate and behavior No data is available.

Ecotoxicology The substance is reported to be very toxic to aquatic organisms.

Exposure standards and guidelines No data is available.

Conclusion Allyl formate is a volatile organic compound used in paints, varnishes, dry-cleaning, and certain household cleaners. It is harmful if inhaled or absorbed through skin. Allyl formate is metabolized via allyl alcohol to acrolein which causes hepatotoxicity. Data for several toxicities such as immunotoxicity, genotoxicity, and reproductive toxicity are not available.

PubChem URL Allyl formate | C4H6O2 - PubChem (nih.gov).

Comptox URL https://comptox.epa.gov/dashboard/chemical/details/DTXCID8035843. Material Safety Data Sheet https://ehslegacy.unr.edu/msdsfiles/21426.pdf.

References Athersuch TJ, Keun H, Tang H, and Nicholson JK (2006) Quantitative urinalysis of the mercapturic acid conjugates of allyl formate using high-resolution NMR spectroscopy. Journal of Pharmaceutical and Biomedical Analysis 40(2): 410–416. Clayton TA, Lindon JC, Everett JR, et al. (2004) Hepatotoxin-induced hypercreatinaemia and hypercreatinuria: Their relationship to one another, to liver damage and to weakened nutritional status. Archives of Toxicology 78(2): 86–96. Droy BF, Davis ME, and Hinton DE (1989) Mechanism of allyl formate-induced hepatotoxicity in rainbow trout. Toxicology and Applied Pharmacology 98(2): 313–324. Rees KR and Tarlow MJ (1967) The hepatotoxic action of allyl formate. Biochemical Journal 104(3): 757. Serafini-Cessi F (1972) Conversion of allyl alcohol into acrolein by rat liver. The Biochemical Journal 128(5): 1103–1107. Yap IK, Clayton TA, Tang H, et al. (2006) An integrated metabonomic approach to describe temporal metabolic disregulation induced in the rat by the model hepatotoxin allyl formate. Journal of Proteome Research 5(10): 2675–2684.

Allylamine Somayeh Salarinejada and Alireza Foroumadia,b, aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; bDrug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of NV Soucy, Allylamine, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 149–151, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01160-X.

Chemical profile Uses Exposure Toxicokinetics Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Human toxicity Lethal toxicity Nonlethal toxicity Animal toxicity Lethal toxicity Nonlethal toxicity Chronic toxicity Human toxicity Animal toxicity Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Toxicogenomics Clinical management Environmental fate and behavior Soil fate Aquatic fate Atmospheric fate Exposure standards and guidelines Preparation Polymerization PubChem URL References Further reading

310 310 310 310 310 310 310 310 310 311 311 311 311 311 311 311 311 311 312 312 312 312 312 312 312 312 312 313 313 313 313 313 313 314

Abstract Allylamine as a primary unsaturated alkylamine is a colorless volatile liquid with a very sharp odor resembling ammonia. This flammable and oxidizable substance are generally utilized in the pharmaceutical industry and vulcanization of rubber. Mainly owing to the alkaline character of allylamine, it is considered a severe irritant for the respiratory, eye, skin, and above all cardiovascular system when after human exposure, whether orally, by injection, or by inhalation at high doses. Scientific evidence reveals that the two metabolites, acrolein, and hydrogen peroxide, induce cardiac side effects. Heart failure, lung failure, or a combination can be lethal.

Keywords Allylamine; Exposure; Guideline; In vitro; Toxicity

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Chemical profile

• • • • • •

Name: Allylamine Synonyms: 2-Propen-1-amine, 2-Propenylamine, 3-Amino-1-propene, 3-Aminopropene, Monoallylamine CAS number: 107-11-9 Molecular formula: C3H7N Molecular weight: 57.1 g/mol Chemical Structure:

Uses Usage of allylamine can be classified into two categories: industrial and medical uses. In industry, it is used as a solvent and also to vulcanize rubber. Using alkylamine in the synthesis of ion-exchange resins has also been reported. Furthermore, allylamine can be used in the synthesis of water-dispersible copolymers, which can be useful in water purification, and also as flocculating agents. In industry, when steel pickling in acid, allylamine can be useful as a corrosion inhibitor. In the pharmaceutical industry, allylamine is utilized in the synthesis of some antibiotics, diuretics, and sedative agents. In medicine, allylamine is utilized as an antifungal agent in the treatment of skin and scalp ringworm in the form of pills and cream. Although the pill can cause headaches and stomach issues, the cream has no side effects. ‘Athlete’s food is the other skin disease in which allylamine is used in its treatment.

Exposure It is indicated that exposure to allylamine can be highly toxic if inhaled or ingested which may cause permanent injury or even death when exposed to small quantities for a short time. When allylamine is absorbed into the skin, it can be moderately toxic and may cause irreversible and reversible issues. In humans, the Toxic Concentration, Lowest (TClo) of allylamine in air is 5 ppm over 5 min. However, it can irritate the nose and throat at 2.5 ppm (US Environmental Protection Agency, 1998).

Toxicokinetics A toxicokinetic study in rats revealed that allylamine is rapidly absorbed into numerous organs, especially in the aorta and coronary arteries, followed by the liver and kidney. The half-life in adrenals, aorta, coronaries, heart, kidneys, and lungs was less than 1 h. Thereafter, up to 60% of a single dose was excreted into the urine within 24 h. When allylamine is administrated orally, it is metabolized to acrolein and hydrogen peroxide which is believed to be catalyzed by benzylamine oxidase. Acrolein is then conjugated with glutathione to form 3-hydroxypropyl mercapturic acid, the only metabolite in the urine. It is believed that semicarbazide as a benzylamine oxidase inhibitor can protect myocytes against myocardial damage in vitro (Boor, 1985).

Mechanism of toxicity It is proposed that the formation of acrolein and hydrogen peroxide as active metabolites is the most significant factor involved in the toxicity of allylamine. It is widely accepted that lipid peroxidation, mitochondrial membrane damage, and adjustment of cellular glutathione are caused by acrolein and hydrogen peroxide. Moreover, the metabolism of allylamine by semicarbazide-sensitive amine oxidase (SSAO) which leads to the formation of acrolein, hydrogen peroxide, and ammonia induces cardiotoxicity and results in ischemia and subendocardial necrosis (Cox et al., 1990).

In vitro toxicity data The in vitro studies for allylamine such as the embryotoxic potential of allylamine have been conducted on fertilized eggs from White Leghorn fowl named in vitro Chick Embryotoxicity Screening Test (CHEST). More information about the test has been explained in the Reproductive and Developmental Toxicity section (HSBD, n.d.).

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Acute and short-term toxicity Human toxicity Lethal toxicity There has been no reported quantitative data on the lethality of allylamine in humans. Nevertheless, some severe symptoms through allylamine inhalation such as irregular respiration, cyanosis, excitement, convulsions, and eventually, death should be considered (Zenz et al., 1994; US EPA, n.d.; ILO, n.d.).

Nonlethal toxicity

According to an unpublished source, it has been reported that “olfactory cognition” was observed among all of the volunteers who were exposed to the concentration value of 2.5 ppm. Furthermore, the odor threshold concentration at which humans can tolerate allylamine for 5 min is 58 ppm. While dose-related increases were observed in all test subjects for mild or moderate eye irritation, nose irritation, and pulmonary issues, this was not the case for Central Nervous System (CNS) effects, including headache or nausea. The irritation of the eyes, nose, and throat and pulmonary issues would be unbearable at a concentration value of 14 ppm. A case report at a chemical manufacturing company reported irritation of the mucous membranes, nausea, and disagreeable odor but not cardiovascular effects following exposure of the operators (US EPA, n.d.; ILO, n.d.).

Animal toxicity Lethal toxicity According to the performed studies, any route of administration can be toxic in multiple animal species. The oral and inhalation LD50 in the rat have been determined 106 mg kg−1 and 286 ppm, respectively. In rabbits, a concentration value of 35 mg kg−1 is reported for dermal administration (US EPA, n.d.; ILO, n.d.).

Nonlethal toxicity Multiple exposure studies were conducted on rats, mice, rabbits, and monkeys. Small evidence revealed that at the concentration value of 40 ppm for 7 h of exposure per day in rats, acute arteriole inflammation, focal muscle bundle necrosis, and EKG changes were observed during 10 days. After 20 days, healing in some areas was seen. Fragmentation of muscle bundles and edematous arterioles were the symptoms after 40 days of exposure (US EPA, n.d.; ILO, n.d.).

Chronic toxicity Human toxicity Dermatitis is resulted from prolonged contact of allylamine with skin. Furthermore, some side effects on the respiratory tract and lungs would be probable which may lead to chronic inflammation and disfunctions (ILO, n.d.).

Animal toxicity It has been demonstrated that administration of allylamine by parenteral routes leads to the induction of pathological lesions in the heart, aorta, and coronary arteries, while hepatic cellular vacuolization, thickening of the pulmonary artery, and vascular sclerosis of the kidney have resulted from intravenous administration (ILO, n.d.).

Immunotoxicity There has been no research on the human immunotoxicity potential of allylamine. It has also not been evaluated in animal studies.

Reproductive and developmental toxicity There have been no reported in vivo studies on allylamine exposure in animals. The in vitro Chick Embryotoxicity Screening Test (CHEST) has been utilized in fertilized eggs. On day 1.5, the onset of the embryotoxicity was determined between 3 and 30 mg/ embryo. The evidence did not reveal any body malformations by using 3–30 mg allylamine on 2–4-day-old embryos (until day 8). The mortality value in this study has been reported as 48%.

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Genotoxicity Studies on the genotoxicity of allylamine have been performed just on some bacterial strains, both with and without metabolic activation. The evidence has not shown any genotoxicity for allylamine.

Carcinogenicity No studies on the carcinogenicity of allylamine or its proposed metabolite, acrolein in humans were reported (HSBD, n.d.).

Organ toxicity Although allylamine can cause eye and skin irritations and respiratory effects, cardiovascular dysfunctions being of a more concern (Boor and Hysmith, 1987). In a reported study, it is demonstrated that both vascular and muscular lesions are visible. However, those lesions were restricted to the vessels of the myocardium, but not seen in the aorta or elsewhere. It should be noted that just short-term exposure to low concentrations of allylamine can create these lesions and they may be aggravated by physiologic or other stress.

Interactions In the case of exposure with a concentration value of 100 mM of both allylamine and acrolein, it is shown that mercaptoethanesulfonate, as a scavenger of reactive species, can prevent their toxicity to myocardial myocyte reaggregate cultures in serum-supplemented medium. This is not the case for cultures in a serum-free medium which means allylamine is metabolized to acrolein by an extracellular metabolism. Moreover, desferrioxamine as an iron chelator can prevent the toxicity of allylamine and also of acrolein which indicates that free radicals play an important role in myocardial myocyte reaggregate cultures. Furthermore, alpha-tocopherol succinate that inhibits lipid peroxidation can reduce allylamine toxicity of myocardial myocyte reaggregate cultures. In a study, it was shown that beta-aminopropionitrile (beta APN) can have a synergistic necrotizing toxic effect in a rat model and can induce vascular toxicity (HSBD, n.d.).

Toxicogenomics There has been no reported toxicogenomics data for allylamine.

Clinical management It is reported that direct contact with allylamine can lead to severe eye and skin irritations. Additionally, it can harm many organs such as the heart, liver, kidney, and central nervous system. Death may result from the over-exposure of the victim. Immediate first aids play a crucial role in saving the victim’s life. Adequate decontamination following supportive clinical care is required (US Environmental Protection Agency, 1998).

Environmental fate and behavior Soil fate It is widely accepted that allylamine, with a Koc of 8 has very high mobility in soil. With a pKa of 9.70, allylamine will exist in the cationic form in the environment which leads to better absorption into the soils. It suggests that allylamine is not volatilized from moist soil surfaces because of its cationic form at pH values of 5–9. However, due to its vapor pressure of 242 mmHg at 25
C, volatilization from dry soil is expected. Consequently, biodegradation of the soil is an important environmental fate process (HSBD, n.d.).

Aquatic fate Similar to the terrestrial fate, due to the cationic nature of allylamine at pH values of 5–9, it is not expected to volatilize from water surfaces. Moreover, a low value was estimated for the potential of bioconcentration in aquatic organisms. Evidence suggests that biodegradation should be considered an important fate process in water.

Allylamine Table 1

313

Summary of AEGL values for allylamine.

Classification

10 min (ppm)

30 min (ppm)

1h (ppm)

4h (ppm)

8h (ppm)

End point (reference)

AEGL-1a (nondisabling) AEGL-2 (disabling)

0.42 3.3

0.42 3.3

0.42 3.3

0.42 1.8

0.42 1.2

AEGL-3 (lethal)

150

40

18

3.5

2.3

Mild human irritation or discomfort (Hine et al., 1960) Human eye and respiratory irritation and NOAEL for severe irritation (1 h; Hine et al., 1960); NOAEL for cardiovascular lesions in rats (4 h; Hine et al., 1960) Lethality NOEL in rats (Hine et al., 1960)

Reference: Acute Exposure Guideline Levels for Selected Airborne Chemicals: Volume 6 a Odor threshold is 2.5 ppm.

Atmospheric fate Since the vapor pressure of allylamine is 242 mmHg at 25
C, it is estimated to be in the form of vapor in the atmosphere which may be degraded through a reaction with photochemically-produced hydroxyl radicals.

Exposure standards and guidelines AEGLs or acute exposure guideline levels have been created by the Federal Advisory Committee to identify and interpret the toxicologic data for acutely toxic chemicals. In Table 1. a summary of AEGL Values for Allylamine has been shown. AEGL-1 values were determined according to a study in which 35 young adult human volunteers were exposed for 5 min to 2.5, 5, or 10 ppm allylamine (Hine et al., 1960). AEGL-2 values were evaluated according to two studies. The 10, 30, and 60 min AEGLs were determined by Hine et al. (1960). AEGL-3 values were obtained from a study in which rats exposed to the chemical with a lethal concentration in 50% of the sample (LC50) through inhalation for 1, 4, or 8 h (Hine et al., 1960).

Preparation Allylamine can be produced by a reaction of allylchloride with ammonia followed by distillation. Alternatively, hydrolysis of isothiocyanate achieves a purer sample.

Polymerization Allylamine can be polymerized to produce homopolymers and co-polymers that may be utilized in reverse osmosis. One of the most well-known polymers is poly (allylamine hydrochloride) which is applicable in the biomedical field, especially in cell encapsulation.

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/Allylamine#section¼Industry-Uses

References Boor PJ (1985) Allylamine cardiovascular toxicity: V. Tissue distribution and toxicokinetics after oral administration. Toxicology 35(3): 167–177. https://doi.org/10.1016/0300483x(85)90013-7. PMID: 4012794. Boor PJ and Hysmith RM (1987) Allylamine cardiovascular toxicity. Toxicology 44(2): 129–145. https://doi.org/10.1016/0300-483x(87)90144-2. PMID: 3551196. Cox LR, Murphy SK, and Ramos K (1990) Modulation of phosphoinositide metabolism in aortic smooth muscle cells by allylamine. Experimental and Molecular Pathology ISSN: 00144800. 53(1): 52–63. Hine CH, Kodama JK, Guzman RJ, and Loquvam GS (1960) The toxicity of allylamines. Archives of Environmental Health: An International Journal 1(4): 343–352. HSBD (n.d.) Hazardous Substances Data Bank. https://pubchem.ncbi.nlm.nih.gov/source/hsdb/2065. ILO (n.d.) International Chemical Safety Cards (ICSC), Allylamine. https://www.ilo.org/dyn/icsc/showcard.display?p_version¼2&p_card_id¼0823. US Environmental Protection Agency (1998) Extremely Hazardous Substances (EHS) Chemical Profiles and Emergency First Aid Guides. Washington, DC: U.S. Government Printing Office. US EPA (n.d.) Acute Exposure Guideline Levels (AEGLs), Allylamine. https://www.epa.gov/aegl/allyl-amine-results-aegl-program. Zenz C, Dickerson OB, and Horvath EP (eds.) (1994) Occupational Medicine, p. 707. St. Louis: Mosby-Year Book.

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Further reading Allylamin 98% | Sigma-Aldrich (n.d.) Allylamin 98% | Sigma-Aldrich. http://www.sigmaaldrich.com/. Bhattacharya D (2023, April 8) Allylamine, Formula, Properties, Antifungal, MSDS, Uses. Chemistry Learner (n.d.) https://www.chemistrylearner.com/allylamine.html. Fishersci.com (2023) https://fscimage.fishersci.com/msds/87403.htm (Accessed 20 May 2023). ICSC 0823—Allylamine(n.d.) https://inchem.org/documents/icsc/icsc/eics0823.htm (Accessed 20 May 2023). National Academies Press (US) (2008) Allylamine Acute Exposure Guideline Levels. Acute Exposure Guideline Levels for Selected Airborne Chemicals—NCBI Bookshelf. https://www. ncbi.nlm.nih.gov/books/NBK207880/. NIOSH (n.d.) National Institute for Occupational Safety and Health. https://www.cdc.gov/niosh-rtecs/BA52C768.html. Registration Dossier—ECHA (n.d.) https://echa.europa.eu/registrationdossier/-/registered-dossier/5470/7/7/2. The Good Scents Company—Aromatic/Hydrocarbon/Inorganic Ingredients Catalog Information (n.d.) http://www.thegoodscentscompany.com/data/rw1216951.html.

Alpha-1 adrenergic receptor antagonists Andrew J Chambers and Kirk L Cumpston, Virginia Commonwealth Univeristy Health System, Department of Emergency Medicine, Section of Clinical Toxicology, Richmond, VA, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.M. Miller, K.L. Cumpston, Alpha Blockers, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 154–155, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00714-4.

Introduction Exposure routes and pathways Toxicokinetics Mechanism of toxicity Acute and short-term toxicity (or exposure) Human Reproductive toxicity Clinical management Conclusion/summary/outlook Further reading

315 316 316 316 317 317 317 317 317 317

Abstract Alpha receptor adrenergic antagonists can be selective or non-selective for alpha-1 and alpha-2 adrenergic receptors in peripheral smooth muscle. This article will focus on the toxicity of selective alpha-1 adrenergic antagonists, not nonselective alpha-blockers or combination of alpha and beta-blockers. In clinical therapeutics, alpha-1 selective adrenergic antagonists are used in the treatment of primary hypertension, urinary dysfunction secondary to benign prostatic hyperplasia, and post-traumatic stress disorder associated nightmares. The most significant complications from toxicity are hemodynamic changes such as tachycardia and hypotension. Treatment is centered on fluid resuscitation and pressor agents that counteract the peripheral vasodilation.

Keywords Alpha blockers; Alpha receptor adrenergic antagonists; Antihypertensive; Benign prostatic hyperplasia; Doxazosin; Orthostatic hypotension; Prazosin; Reflex tachycardia; Tamsulosin; Terazosin

Key points

• • • •

Alpha receptor adrenergic antagonists can be selective or nonselective for alpha-1 and 2 receptors in peripheral smooth muscle. Alpha-1 adrenergic antagonism leads to excessive vasodilation from smooth muscle relaxation, subsequently causing hypotension and a reflex tachycardia. Treatment is supportive and typically responds well to intravenous fluids without need for vasopressor support. If vasopressor support is needed, norepinephrine and vasopressin are first line agents.

Introduction Alpha receptor adrenergic antagonists can be selective or nonselective for post-synaptic alpha-1 and 2 adrenergic receptors in peripheral smooth muscle. Selective alpha-1 adrenergic antagonists such as prazosin, terazosin, and doxazosin will be the focus of this article. These are used for several different modalities including treatment for urinary dysfunction secondary to benign prostatic hyperplasia (BPH), primary hypertension, and sleep disturbances secondary to post-traumatic stress disorder. Prazosin has also been studied in the treatment of alcohol dependence, pheochromocytomas, Raynaud phenomenon, and scorpion envenomation.

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Exposure routes and pathways Ingestion and injection are the most common routes of accidental and intentional toxicity.

Toxicokinetics Prazosin has a bioavailability of around 60%, hepatic metabolism, 97% protein binding, volume of distribution of 0.5 L kg−1, and a half-life of approximately 2–3 h. Terazosin is approximately 90% protein bound, extensively metabolized by the liver and achieves peak concentration around 1 h after oral ingestion. The volume of distribution for prazosin is 25–30 L, and it has an elimination half-life of 9–12 h. Doxazosin has a bioavailability of 65%, with 98% protein binding, and it achieves a peak concentration 2–3 h after immediate-release forms are taken orally. There is an extensive hepatic metabolism of doxazosin and it has an elimination half-life of approximately 22 h. Tamsulosin has more than 90% bioavailability and achieves peak concentrations within 4–8 h after oral use. Tamsulosin undergoes extensive hepatic metabolism, has 76% renal excretion, and has an elimination half-life of 9–13 h.

Mechanism of toxicity Alpha-1 adrenergic receptors are post-synaptic receptors primarily found in vascular smooth muscle but they also located in the genitourinary tract and in the central nervous system (CNS). Under normal circumstances, stimulation of these receptors results in vasoconstriction. The alpha adrenergic antagonists produce arterial smooth muscle relaxation, vasodilation, and a reduction of the blood pressure. Excessive vasodilation causes hypotension and can lead to reflex tachycardia. Activation of alpha-1 adrenergic receptors in the CNS can sometimes result in somnolence.

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Acute and short-term toxicity (or exposure) Human Alpha-1 adrenergic receptor antagonists can produce symptoms of postural hypotension, including light-headedness, syncope, or palpitations, particularly after the first dose, or if the dosing is rapidly increased. Hypotension, leading to a reflex tachycardia, is most often seen, but cases of bradycardia have been reported and are thought to be associated with a sympatholytic effect seen with the alpha adrenergic antagonists in some patients. CNS depression ranging from lethargy to coma have been reported in overdose. Priapism can also occur as a side effect. Other common adverse effects includes weakness, nausea, and headache.

Reproductive toxicity Based on experimental animal studies, therapy with alpha-adrenergic antagonists during pregnancy is not expected to increase the risk of congenital abnormalities however prescribers should weigh the potential benefits of drug treatment against potential risks before prescribing these drugs during pregnancy. Alpha-adrenergic antagonists are present in breast milk and caution should be exercised when they are administered during nursing and lactation.

Clinical management Patients generally do well with supportive care if toxicity or overdose. Patients who are awake after a significant ingestion can be treated with activated charcoal. Basic and advanced life-support measures should be implemented, if needed, to normalize vital signs. Airway management is not generally required but patients should be monitored for potential decreases in mental status. For hypotension, patients should receive intravenous fluid boluses. A vasopressor with alpha-1 adrenergic receptor agonism properties like norepinephrine or phenylephrine is the first choice for individuals who are unresponsive to fluids. Alpha-1 agonists counteract the alpha-1 adrenergic receptor antagonism, by increasing the blood pressure. Interestingly, a case was reported to be recalcitrant to fluids, norepinephrine, and epinephrine but improved with vasopressin, possibly by its ability to bypass the direct alpha blockade itself.

Conclusion/summary/outlook Alpha receptor adrenergic antagonists can be selective or non-selective for alpha-1 and alpha-2 receptors predominately in peripheral smooth muscle. Selective alpha-1 adrenergic antagonists such as prazosin, terazosin, tamsulosin and doxazosin are primarily used in the treatment of primary hypertension, urinary dysfunction secondary to benign prostatic hyperplasia, and post-traumatic stress disorder associated nightmares or night terrors. The most significant complications from toxicity are hemodynamic changes such as hypotension and reflex tachycardia. Treatment is centered on fluid resuscitation and pressor agents that counteract the peripheral vasodilation. If hypotension is unresponsive to fluid administration, norepinephrine, phenylephrine and vasopressin are appropriate choices because of their vasoconstrictive properties.

Further reading Anderson C, Lynch T, Gupta R, and Lim RK (2018) Refractory hypotension caused by prazosin overdose combined with acetaminophen and naproxen toxicity: A case report and review of the literature. The Journal of Emergency Medicine 55(6): e141–e145. Cubeddu LX (1988) New alpha 1-adrenergic receptor adrenergic antagonists for the treatment of hypertension: Role of vascular alpha receptors in the control of peripheral resistance. American Heart Journal 116: 133–162. Froehlich JC, Hausauer BJ, Federoff DL, Fischer SM, and Rasmussen DD (2013) Prazosin reduces alcohol drinking throughout prolonged treatment and blocks the initiation of drinking in rats selectively bred for high alcohol intake. Alcoholism, Clinical and Experimental Research 37(9): 1552–1560. Kim AK and Souza-Formigoni ML (2013) Alpha 1-adrenergic drugs affect the development and expression of ethanol-induced behavioral sensitization. Behavioural Brain Research 256C: 646–654. Lenz K, Druml W, and Kleinberger G (1985) Acute intoxication with prazosin: Case report. Human Toxicology 4: 53–56. Lip GYH and Ferner RE (1995) Poisoning with anti-hypertensive drugs: Alpha adrenoreceptor adrenergic antagonists. Journal of Human Hypertension 9: 523–526. Robbins DN, Crawford ED, and Lackner LH (1983) Priapism secondary to prazosin overdose. The Journal of Urology 130: 975. Satar S, Sebe A, Avci A, et al. (2005) Acute intoxication with doxazosin. Human & Experimental Toxicology 24(6): 337–339. Seak CJ and Lin CC (2008) Acute intoxication with terazosin. The American Journal of Emergency Medicine 26(1): 117–126.

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Aluminosilicate fibers Mark J Utella, Joseph J Kucherab,c, and Paul Boymeld,e, aProfessor Emeritus of Medicine and Environmental Medicine, Pulmonary and Critical Care Division, University of Rochester Medical Center, Rochester, NY, United States; bProduct Stewardship Consultant, Alkegen, Tonawanda, NY, United States; cFormer Vice President of Product Stewardship, Alkegen, Tonawanda, NY, United States; dIndependent Consultant—Technology Management and Material Science, Naples, FL, United States; eWorldwide Vice President, Technology, Unifrax I LLC Information, Tonawanda, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of L.D. Maxim, M.J. Utell, Aluminosilicate Fibers, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 156–160, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01161-1.

Chemical profile Background Uses Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity Acute and short-term toxicity (animal/human) Chronic toxicity (animal/human) Clinical management lmmunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Ecotoxicity Other hazards Exposure standards and guidelines Conclusion References Further reading

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Abstract Aluminosilicate fibers (142844-00-6) or aluminosilicate wool (ASW) are synthetic fibers made by melting a combination of alumina and silica and then either spinning or blowing the heated melt to produce fibers. ASW can be used directly as bulk fiber for some applications or (more typically) is converted into a variety of other forms. ASW is used chiefly in industrial applications as a high temperature insulation material. As produced or used, some of these fibers are respirable. Chronic animal studies indicate that the fiber has the potential to cause interstitial fibrosis, lung cancer, and mesothelioma. Ongoing epidemiological studies have shown that occupationally exposed cohorts have not developed interstitial fibrosis, incremental lung cancer or mesothelioma but have reported respiratory symptoms, developed pleural plaques, and experienced statistically significant but clinically insignificant decrements in certain measures of lung function. Exposure data indicate that weighted average exposures range from approximately 0.2–0.3 fibers/mL (individual time weighted average values have substantial variability and depend upon specific jobs and tasks), beneath most recommended or mandated occupational exposure limits.

Keywords Aluminosilicate fiber; Biopersistence; Fibrosis; Insulating refractory; Lung cancer; Mesothelioma; Occupational exposure limits; Respirable fiber

Abbreviations ASW HTIW MMMF MMVF

Aluminosilicate wool High temperature insulating wool Man-made mineral fiber Man-made vitreous fiber

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RCF REACH SVF

Refractory ceramic fiber Registration, Evaluation, Authorisation and Restriction of Chemicals Synthetic vitreous fiber

Chemical profile

• • • •

•

Name: Aluminosilicate fiber Chemical Abstracts Service Registry Number: 142844-00-6 Synonyms: Refractory ceramic fiber (RCF), aluminosilicate wool (ASW). Molecular Formula: This fiber is produced by melting a mixture of alumina (Al2O3) and silica (SiO2) in approximately equal proportions. Other inorganic oxides, such as ZrO2, Cr2O3, B2O3, and TiO2, are sometimes added to alter the properties (e.g., the maximum end-use temperature) of the resulting product. The International Agency for Research on Cancer (IARC) and NIOSH provide illustrative composition ranges for aluminosilicate fibers. Compositions vary by manufacturer and intended end use; detailed data can be found in manufacturer’s technical and safety data sheets. Standard fibers range in reported composition (percentage by weight) from 40% to 55% alumina and 45% to 60% silica (exclusive of minor ingredients). Higher-temperature fiber compositions range in composition from 28% to 40% alumina, 43% to 56% silica, and 14% to 18% zirconia. These fibers are referred to as zirconia aluminosilicate or AZS fibers. Chemical Structure: Amorphous fibers of variable dimensions.

Background Aluminosilicate fibers, historically called refractory ceramic fibers (RCFs) and now more commonly referred to as Aluminosilicate wools (ASWs) are amorphous fibers belonging to a class of materials termed synthetic vitreous fibers (SVFs), also termed man-made mineral fibers (MMMF) or man-made vitreous fibers (MMVF). This class of materials also includes glass wool, rock (stone) wool, slag wool, mineral wool, and special- purpose glass fibers. Fibers can be classified in (changed font) various ways, such as natural vs synthetic, organic vs inorganic, and crystalline vs amorphous. Several fiber taxonomies have been proposed; Fig. 1 shows the taxonomy of SVFs used by IARC (2002). Aluminosilicate wools were first invented in the early 1940s and commercialized in the 1950s in the United States and somewhat later in other countries. Substantial energy price increases beginning in the 1970s increased the economic benefits of energy conservation and the market for these fibers. ASWs are SVFs produced by melting (at
1925  C) alumina, silica, and other inorganic oxides, and then blowing or spinning these melts into fibers. These fibers can also be produced by melting blends of calcined kaolin, alumina, and silica. The bulk fibers produced by this process can be used directly for some applications, but are more commonly converted into other physical forms,

Synthetic Vitreous Fibers (SVFs)

Wools

Filaments

Glass Wool

Insulation Wool

Rock (Stone) Wool

Slag Wool

Special Purpose Wool

Fig. 1 Taxonomy of synthetic vitreous fibers (SVFs). Source: Modified from IARC, 2002.

Aluminosilicate Wool & Zirconia Aluminosilicate Wool

Other Fibers (e.g., Alkaline Earth Silicate Wool)

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Fig. 2 Photomicrograph of aluminosilicate wool produced by spinning.

including blanket, modules (folded blanket capable of being installed rapidly in industrial furnaces), paper, felt, board, vacuum formed parts, textiles, and putties or pastes (Utell and Maxim, 2010; Maxim and Utell, 2018). Conversion to various physical forms takes place at locations where aluminosilicate fibers are produced, facilities operated by converters (producers of intermediate goods) or end users. Primary manufacturing facilities for aluminosilicate fibers are located in North and South America, Europe, and Asia. Conversion facilities and end users are distributed throughout the industrialized world. Fig. 2 shows a photomicrograph of RCF produced by spinning.

Uses ASWs have several desirable properties for use as high- temperature insulating materials, including low thermal conductivity, low heat storage (low volumetric heat capacity), thermal shock resistance, lightweight, good corrosion resistance, and ease of installation. Depending upon the fiber composition, the maximum end-use temperature for ASWs can be as high as 1430  C (2600  F) (NIOSH, 2006; ATSDR, 2004). Because of this capability, these fibers are also included in the class of high-temperature insulation wools (HTIWs). Benefits of the use of ASW insulation include reduced energy costs and reduced greenhouse gas emissions (Utell and Maxim, 2010). The energy savings can be substantial when compared to conventional high-temperature insulation such as insulating firebrick. Applications and markets for ASWs are principally industrial and vary by product form and country including furnace linings and components in the cement, ceramic, chemical, fertilizer, forging, foundry, glass, heat treating, nonferrous metals, petrochemical, power generation (cogeneration), and steel industries. ASWs are used for passive fire protection applications where thin, lightweight materials are needed to prevent flame penetration. ASWs have also been used in automotive applications such as heat shield insulation, and emission control applications as catalytic converter support mat, and filtration media for air bag inflators (Maxim and Utell, 2018). Though sometimes referred to in the literature as a substitute for asbestos, aluminosilicate fibers are not typically used in asbestos applications. Aluminosilicate fibers are priced substantially higher than various types of asbestos and have maximum end-use temperatures substantially greater than those for asbestos which vary depending upon the product but are typically 850  C. Over the past 10 years, in applications where the use temperatures are 850–1100  C, such as passive fire protection and some automotive applications the use of ASW has largely been replaced with products produced from Alkaline Earth Silicate Wools (AES) (Utell and Maxim, 2010).

Environmental fate and behavior Aluminosilicate fibers are white fibrous solids, soluble to a degree in human lung fluid (see below). The usual physicochemical parameters relevant to fate and transport (e.g., solubility, vapor pressure, octanol-water partition coefficient, and Henry’s law constant) are not applicable or relevant; vapor pressure, octanol-water partition coefficient, and Henry’s law constant are exceedingly low and not measurable. Fibers are capable of being transported in the air and are removed by gravitational settling. Fiber products are treated as “chemicals” for the purpose of regulatory risk management. The two major regulatory models are Europe’s “Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) and the U.S. Environmental Protection Agency standard,” the “Toxic Substances Control Act” (TSCA). REACH has classified RCF as a substance of very high concern (SVHC) based on animal studies (REACH Registration Dossier—ECHA (europa.eu)). The EPA views RCF exposures as a workplace risk management issue and has not regulated RCF.

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Exposure and exposure monitoring Possible pathways for human exposure include ingestion, inhalation, and dermal contact. There are only limited data on ASW concentrations in the environment, typically in the vicinity (fence line) of manufacturing facilities and at one landfill. Arithmetic mean fence line boundary concentrations range from beneath the detection limit to 0.02 fibers per milli-liter (f/mL). The greatest exposure to aluminosilicate (and other) SVFs occurs from inhalation by workers who manufacture, convert, use, or remove these fibers. It is estimated that approximately 30,000 workers in the United States are occupationally exposed to ASWs (NIOSH, 2006) and a similar number in Europe. As of this writing, no consistent estimates are available for the size of the exposed population in Asia. The ASW industry has developed and maintained a comprehensive (now nearly worldwide) product stewardship program (PSP) to identify, manage, and mitigate the risks associated with production or use of these fibers. The goal of the product stewardship is to minimize the health, safety, and environmental impacts of using products through their life cycle from cradle to grave. In the United States, this voluntary program is overseen by the Occupational Safety and Health Administration. In Europe the program is referred to as the “CARE” program (Control And Reduce Exposure) and has been shared with the Health & Safety communities in each of the EU’s member states. The program along with other scientific work on ASWs has been used in establishing the regulatory guidance for ASW in Europe. Two components of the PSP are directed to measuring and controlling occupational exposures to aluminosilicate and related HTIWs. These efforts are well documented. As part of the PSP, industrial hygienists from ASW producers monitor exposures (including 8-h time-weighted average (TWA) respirable fiber concentrations) at plants operated by producers and also at customer facilities. Data collected by this program include TWA fiber concentrations, jobs (partitioned into eight functional job categories (FJCs)), tasks within jobs, exposure controls, and use of personal protective equipment (PPE). Key results of the exposure monitoring program include the following: (1) exposures vary by FJC and task within FJC; exposures are the highest for removal of after-service insulation, (2) for comparable jobs exposures are slightly lower at plants operated by producers, but the gap has narrowed over the years, and (3) weighted (by number of workers in each FJC) fiber concentrations have decreased over the years and now average between 0.2 and 0.3 f/mL, absent any correction for protection associated with the use of PPE. However, there is substantial variability in TWA fiber concentrations—even within a specific FJC and plant the coefficient of variation s/m is typically >1.0 (Utell and Maxim, 2010; Maxim et al., 2008). Fig. 3 shows weighted arithmetic mean time trends in TWA fiber concentrations in the United States. Another component of the PSP established for ASW includes research and development of alternative High Temperature Insulation Wools (HTIWs) with reduced biopersistence. As described further below, a fibers biopersistence correlates well with both short-term animal lung clearance studies and lifetime animal inhalation studies for cancer endpoints. The biopersistence of fibrous materials is mostly a function of its chemical composition, though the interactions and contributions of different chemical compositions is not straight forward. Since ASW are typically used at very high temperatures and in demanding chemical and mechanical environments, developing materials that are acceptable alternatives while being less biologically active is a challenging goal. Over the past decade new compositions in the alkali earth silicate (AES) family of compositions (typically consisting of calcium and/or magnesium silicates with limited Al2O3) have successfully met and replaced ASW in applications 20 mm long from inhalation studies, is approximately 53 days, which shows that ASW is more biopersistent than many SVFs, but comparable to those for other SVFs including one type of rock wool, E-glass, and 475 glass (Zoitos et al., 1997; Hesterberg et al., 1998). The biopersistence of aluminosilicate fibers is substantially lower than that for crocidolite asbestos (
800 days), which suggests that the toxic effects of aluminosilicate fibers are likely to be markedly less than those for this form of asbestos (Utell and Maxim, 2010).

Mechanism of toxicity Numerous in vitro and in vivo studies have been conducted on both natural and synthetic fibers to try to understand and measure cytotoxicity, mutagenicity, and genotoxicity. Many of these studies have proven inconclusive, so mechanism(s) of action are still unclear (Oberdörster, 2000; Utell and Maxim, 2010; Greim et al., 2014). Other studies have indicated that aluminosilicate fibers are less active biologically than various forms of asbestos (Zoitos et al., 1997; Hesterberg, et al., 1998).

Acute and short-term toxicity (animal/human) Aluminosilicate fibers have a recognized potential to cause mild mechanical irritation to the respiratory tract (nose, throat, and lungs), eyes, and skin of exposed individuals, a property typically listed on safety data sheets (Lockey et al., 2007).

Chronic toxicity (animal/human) Aluminosilicate fibers have been evaluated in several chronic animal studies with various routes of exposure (inhalation, intratracheal instillation, intrapleural injection, and intraperitoneal injection). Of greatest potential relevance are the results of chronic inhalation studies, which indicated that rats and hamsters exposed to aluminosilicate fibers developed fibrosis and tumors (Mast et al., 2000). Interpretation of the results is made more difficult by the fact that the experiments employed an aluminosilicate fiber with a high and nonrepresentative content of particulate material, believed to be an artifact of the test sample preparation method, resulting in lung overload. Acknowledging this complication, IARC nonetheless concluded that there was sufficient evidence for carcinogenicity of aluminosilicate fibers in experimental animals, resulting in a cancer classification of Group 2B. Other regulatory or advisory agencies have reached similar conclusions. As noted above, ASW has been classified as an SVHC in Europe. We are not aware of any new chronic animal bioassays conducted since the earlier publication.

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The aluminosilicate fiber industry has sponsored several epidemiological studies (both morbidity and mortality) on workers exposed to these fibers (McKay et al., 2011; LeMasters et al., 2017). The studies evaluated/measured symptoms, X-rays, pulmonary function, and mortality. Collectively, these previous studies indicated that exposed workers (1) exhibited symptoms (e.g., dyspnea) similar to those reported in other dust-exposed populations, (2) developed statistically significant, but not clinically significant, deficits in certain measures of pulmonary function in a cross-sectional study, but no excessive decline in a later longitudinal study, and (3) a dose-related increase in pleural plaques, but no interstitial fibrosis (Maxim and Utell, 2018). The results of the updated epidemiology studies (2017) confirmed that occupational exposure to RCF was associated with development of pleural plaques and minor decrements in lung function. After 30 years of follow-up, LeMasters et al. (2017) reported no excess lung cancers in the mortality study and no significant association of x-ray findings of interstitial fibrosis were found in the workers. There was one reported but pathologically unconfirmed mesothelioma case in an RCF worker with asbestos exposure. In 2022, LeMasters and colleagues updated the RCF epidemiology study and again found no excess lung cancers. They identified one former employee currently living with a pathologically confirmed mesothelioma. Of note, the standardized mortality rate (SMR) was non-significant even when both the mesothelioma from the deceased and living workers were included in the analyses (LeMasters et al., 2022). The association of these two mesothelioma cases with RCF exposure alone was unclear because of past significant asbestos exposure for each of the workers. In addition, urinary and bladder cancer mortality were increased compared to background populations among the higher RCF-exposed workers. However, the number of individuals with these cancers was quite small (n ¼ 9). Evidence supporting a finding that urinary tumors were associated with RCF exposure remained but is confounded by smoking and occupational exposure to solvents and other chemical carcinogens. Six of the nine individuals had a history of smoking, which is an important risk factor for urinary tumors. An elevated SMR for leukemia was found, but was absent in the highly exposed group, there was no consistent leukemia cell type, and leukemia has not been observed in studies of other mineral fibers. The mortality study is ongoing and deaths from all causes are being monitored (LeMasters et al., 2022). A recent cross-sectional study among Chinese workers evaluated the association between occupational exposure to RCFs and respiratory health (Gu et al., 2020). Several serum biomarkers were measured and RCF workers showed higher levels of transforming growth factor b1 (TGF-b1) and 8-hydroxy-2-deoxyguanosine (8-OHdG) and lower level of Clara cell protein 16 (CC16) compared to controls. However, RCF workers were also exposed to high levels of dust and the absence of a non-RCF dust exposed only control, lack of a dose-response relationship, and the non-specific nature of the biomarkers limit interpretation of the findings.

Clinical management The results of the ongoing epidemiology studies confirm that occupational exposure to RCF is associated with the development of pleural plaques and minor decrements in pulmonary function, but no interstitial fibrosis or incremental lung tumors (LeMasters et al., 2017). Pleural plaques are a marker of fiber exposure. With the absence of other nonmalignant pleural disease, pleural plaques are not associated with respiratory symptoms (in most individuals) or clinically significant impairments of pulmonary function. In cohorts occupationally exposed to asbestos, there is a correlation between the presence of pleural plaques and malignant effects (lung cancer and mesothelioma). However, the evidence indicates that this relationship is a consequence of the degree of exposure to asbestos and that the presence of pleural plaques does not, of itself, independently affect risk levels (Maxim et al., 2015). Although pleural plaques are detected on imaging studies as evidence of fiber exposure, they are not pre-malignant. There is no specific medical treatment for pleural plaques. Clinical management of workers in the RCF industry, with or without pleural plaques, should include a medical surveillance program that includes completion of a respiratory questionnaire, chest x-rays, and spirometry. NIOSH (2006) initially recommended periodic exams every 5 years for workers with less than 10 years of RCF exposure and then every 2 years for workers with more than 10 years of exposure. However, the University of Cincinnati studies have demonstrated from their 30-year epidemiological study that surveillance every 3 years would be reasonable (LeMasters et al., 2017). Although the epidemiology has generally been limited to the finding of pleural plaques, there remains a need for ongoing follow-up of the RCF-exposed cohort to better understand possible linkages between RCF exposure and renal and/or bladder disease, leukemia, and mesothelioma given it’s long latency and relative infrequency in the population. The U.S. RCF industry will continue the product stewardship program including the mortality study at least through 2025.

lmmunotoxicity There are limited reports indicating that aluminosilicate fibers are immunotoxic (ATSDR, 2004). Additionally, depending upon the end-use temperature and duration, the hot face of aluminosilicate fiber insulation that has reached the end of its service life may form crystalline quartz, cristobalite, or tridymite. Respirable crystalline silica (RCS) is known to be immunotoxic (Parks et al., 1999).

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Reproductive and developmental toxicity Reproductive toxicity screening was conducted as part of the REACH submittal for these fibers. Results were negative: at what was regarded as the maximum usable dose there were no adverse reproductive effects seen with RCF administered by gavage. Exposure to reproductive organs is extremely unlikely (REACH, 2011—Registration Dossier—ECHA (europa.eu)).

Genotoxicity For the purposes of compiling a complete REACH registration dossier for RCF/ASW, Covance (an independent quality- assured toxicology testing facility) conducted studies to determine the genotoxic potential of ASW. The studies concluded that in standard regulatory mutagenicity tests using five different histidine-requiring strains of Salmonella typhimurium (TA98, TAl00, TA1535, TA1537, and TA102), ASW at concentrations up to 5000 mg per plate did not induce gene mutations (Harrison et al., 2015; REACH, 2011—Registration Dossier—ECHA (europa.eu)). And, in a standard regulatory in vitro cytogenicity assay using duplicate cultures of Chinese hamster ovary cells, ASW up to and including a maximum practicable concentration of 1000 mg/mL did not induce micronuclei in either pulse or continuous exposure regimes (Harrison et al., 2015; REACH, 2011—Registration Dossier—ECHA (europa.eu)). Study authors concluded that ASW has no mutagenic activity in standard test systems for this endpoint (Note: In the absence of positive in vitro mutagenicity results, in vivo genotoxicity testing is not normally required and was not conducted). The genotoxicity of aluminosilicate fibers has been evaluated by two agencies (Health Council of the Netherlands and The Scientific Committee on Occupational Exposure Limit Values {SCOEL, 2011}) that identified and reviewed the available literature (Greim et al., 2014). These agencies concluded that results of the applicable studies and the information that inflammation is the underlying mechanism of fiber carcinogenicity strongly indicate that the genotoxic effects observed in some (but not other) studies of aluminosilicate were secondary. This conclusion may lead to the possibility of a dose threshold for observed carcinogenic effects (see below).

Carcinogenicity Exposure to aluminosilicate fibers has been shown to cause interstitial fibrosis, lung cancer, and mesothelioma in laboratory animals (hamsters and rats) exposed by various routes, including nose-only inhalation. As noted above, the ongoing epidemiology studies of occupationally exposed cohorts have not resulted in any interstitial fibrosis, incremental lung cancer, or mesothelioma. In addition, a brief report from France (Gérazime et al., 2015) examined the relationship between occupational exposure to RCF and the risk of head and neck cancer and lung cancer by analyzing data from a case-control study population, Icarus Study. The risk of cancer of the lung or head and neck was not higher among those exposed to both asbestos and RCF than in subjects exposed to asbestos only. Although a moderate increase in risk cannot be excluded, the results did not support an association between RCF exposure and occurrence of head and neck cancer or lung cancer. Aluminosilicate fibers have been classified variously as (1) possibly carcinogenic to humans (Group 2B) by lARC (2) reasonably anticipated to be a human carcinogen by the National Toxicology Program, (3) according to Regulation (EC) No 1272/2008 under the CLP Regulation (classification, labeling, and packaging of substances and mixtures), ASW has been classified as a 1B carcinogen (“presumed to have carcinogenic potential for humans, classification is largely based on animal evidence”), and (4) classification according to directive 67/548/EEC aluminosilicate fibers have been classified as a Category 2 carcinogen (“substances which should be regarded as if they are carcinogenic to man”).

Ecotoxicity Aluminosilicate fibers are inorganic, inert, stable, and not soluble in water (solubility 100 mg l−1. Among nonexposed individuals, the upper reference limit is 16 mg l−1. Several factors have suggested a correlation between blood levels of aluminum and the onset of Alzheimer’s disease; however, thus far this has not been satisfactorily demonstrated. Occupational exposure to aluminum powder has resulted in pulmonary fibrosis. While cancer and coronary heart disease have been observed among aluminum production workers, it is unlikely that aluminum alone is the causative agent.

Keywords Alum; Aluminum compounds; Aluminum powder; Deferoxamine; Hans Christian Øersted; Metals; Neurotoxic

Chemical profile

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Valence States: +1, +2, +3 Name: Aluminum Synonyms: Aluminum, Molten, Metana, Aluminum powder, Pyrophoric Chemical Abstracts Service Registry Number: 7429-90-5 Molecular Formula: Al Chemical Structure:

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Background Although aluminum was one of the last metals to be commercialized, it has been recognized for centuries. Aluminum was first recognized by the Romans as an astringent substance, and they called it ‘alum.’ By the Middle Ages it was manufactured as ‘alum stone,’ a subsulfate of alumina and potash. In 1825, Hans C. Øersted was able to isolate a few drops of the raw material, and by 1886 it had patents from both Charles Martin Hall of the United States and Paul-Louis-Toussaint Heroult of France. Aluminum was commercialized in industry by the end of the nineteenth century.

Uses/occurrence Aluminum can be used in several different ways, either alone, as an alloy component, or compounded, and in a variety of forms, including powder. Aluminum is frequently used in food packaging, and also in utensils and electrical conductors. Aluminum compounds are widely used in industry, in the form of alums in water treatment and alumina in abrasives and furnace linings. However, aluminum is used alone very rarely since it is such a soft metal. It is often combined with other metals to create a stronger, more durable aluminum alloy. Aluminum alloys are used extensively in aircraft. Aluminum and aluminum salts can also be found in many consumer products such as antiperspirants, food additives, antacids, astringents, and buffered aspirins. Powdered aluminum is used to make explosives and fireworks. There has been concern about the exposures resulting from leaching of aluminum from cookware and beverage cans; however, aluminum beverage cans are usually coated with a polymer to minimize such leaching. Leaching from aluminum cookware becomes potentially significant only when cooking highly basic or acidic foods, for example, in one study, tomato sauce cooked in aluminum pans was found to accumulate 3–6 mg aluminum per 100 g serving. Aluminum is absorbed from the soil by many plants that humans consume. The amount that a person would inhale depends on where they reside, and aluminum levels are much higher in industrial and urban areas. Another route of exposure is through skin contact with soil, water, and with aluminum metal. Aluminum is a good conductor of both heat and electricity. These properties make it suitable for industrial purposes. Aluminum is used in alloys with copper, zinc, manganese, and magnesium.

Exposure Aluminum is the most abundant metal, and the third most abundant element in the Earth’s crust. Human exposure to this metal is common and unavoidable. However, intake is relatively low because aluminum is highly insoluble in many of its naturally occurring forms. Humans are always exposed to some form of aluminum by eating food, drinking water, ingesting aluminum-containing medicinal products, or just breathing air. The average human intake is estimated to be 30–50 mg day−1. This intake comes primarily from foods, drinking water, and pharmaceuticals. Food additives can contain aluminum; due to certain additives, processed cheese and cornbread are two major contributors to high aluminum exposures in the American diet. Some common over-the-counter medications such as antacids and buffered aspirin contain aluminum and can increase intake significantly.

Toxicokinetics (ADME) Less than 1% of that taken into the body orally is absorbed from the gastrointestinal tract. Aluminum can increase the absorption of other chemicals such as fluoride, calcium, iron, and phosphates. Most of the aluminum absorbed into the body will eventually end up in the bones or lungs. Aluminum that is not absorbed by the bones or lungs and is excreted by the kidneys. Dermal absorption of aluminum may be problematic.

Mechanism of toxicity Aluminum binds diatomic phosphates and possibly depletes phosphate, which can lead to osteomalacia. High aluminum serum values and high aluminum concentration in the bone interfere with the function of vitamin D. The incorporation of aluminum in the bone may interfere with deposition of calcium; the subsequent increase of calcium in the blood may inhibit release of parathyroid hormones by the parathyroid gland. The mechanism by which aluminum concentrates in the brain is not known; it may interfere with the blood brain barrier.

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In vitro toxicity data Aluminum has not been reported as genotoxic or mutagenic, nor is it notably cytotoxic.

Acute and short-term toxicity Animal Acutely, aluminum itself has minimal systemic toxicity. Overall, animals become weaker and less active due to exposure (Table 1).

Human Aluminum has not been shown to alter the immune system in humans exposed by the oral or inhalation routes. Skin sensitization may occur.

Chronic toxicity Animal Cats and rabbits are aluminum sensitive and have showed neurotoxic effects from aluminum. Toxicity of aluminum in animals differs from humans because animals are much more sensitive to high exposures. Monkeys on a low calcium, high aluminum diet showed neurological disease similar to those of amyotrophic lateral sclerosis and Parkinsonism. Rats and hamsters showed signs of lung damage after breathing large amounts of aluminum dust. Death often occurred after the inhalation of air highly concentrated with the chemical (Table 2).

Human Fibrosis of the lung may occur through inhalation of aluminum dust particles. Aluminum has been associated with encephalopathy, bone disease, and anemia related to dialysis. It has also been thought that aluminum may be a cofactor in the etiopathogenesis of some neurodegenerative diseases, including Alzheimer’s disease. Direct evidence, however, cannot link the two together. Aluminum toxicity has been well recognized in patients with renal failure. Also, an increased concentration of aluminum in infant formulas and in solutions for home parenteral nutrition has been associated with neurological consequences and metabolic bone loss.

Immunotoxicity There are insufficient data to support immunotoxicity effects of aluminum.

Reproductive and developmental toxicity There is no evidence that aluminum has adverse effects on reproduction or development in mammals or humans. When AlCl3 was orally administered in various species such as rat, guinea pigs, and rabbits, maximum doses of 27 mg Al kg−1 for 20–30 days resulted in ‘slight’ gonadal toxicity. Oral doses of 100–200 mg Al kg−1 given for 6 months resulted in decreased numbers and motility of spermatozoa and proliferation of testicular interstitial cells at highest doses. The highest doses (100 and 200 mg Al kg−1 for >4 weeks) via intraperitoneal injection of soluble aluminum nitrate nonahydrate in male rats before mating decreased pregnancy rates and weight loss in males. A dose of 200 mg kg−1 significantly decreased epididymal spermatocytes and testicular

Table 1

Acute in vivo toxicological effects of aluminum.

Assay

Compound

Species

Route

Exposure

Effect

References

Acute Toxicity

Fumed alumina Aluminum flakes

Rat Rat

Oral Inhalation

Single Single; 4 h

LD50 > 15,900 mg/kg NOAEC ¼ 10 mg/m3 LOAEC 200 mg/m3 Multifocal microgranulomas in terminal airways and alveolar septae

ECHA for Aluminum (2022)

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Aluminum

Table 2 Assay Repeat Dose Toxicity

Chronic in vivo toxicological effects of aluminum. Compound Aluminum Aluminum flakes

Species Human Rat

Aluminum chlorohydrate Hamster

Route

Inhalation

Aluminum chlorohydrate Hamster

Inhalation

Aluminum oxide

Rat

Inhalation

Aluminum chlorohydrate Rat

Inhalation

Aluminum chlorohydrate Guinea Pig

Inhalation

Aluminum powder

Inhalation

Effect

References 3

ATSDR (2008) LOAEC ¼ 4.6 mg/m NOAEC ¼ 10 mg/m3 LOAEC ¼ 50 mg/m3 Increased inflammatory mediators in BAL fluid 3 days; 4–6 h/day NOAEC ¼ 3 mg/m3 LOAEC ¼ 7 mg/m3 Increased lung weight LOAEC ¼ 33 mg/m3 Alveolar wall thickening; inflammatory response 6 months; 5 days/week; NOAEC ¼ 0.061 mg/m3 6 h/day LOAEC ¼ 0.61 mg/m3 Increase in alveolar macrophage; granuloma formation in lungs 5 or 6 weeks; LOAEC ¼ 10 mg/m3 5 days/week; 6 h/day Alveolar thickening and cellular inflammatory response 86 weeks; 5 days/week; NOAEC ¼ 2.45 mg/m3 6 h/day 12–24 months; NOAEC ¼ 0.65 mg/m3 5 days/week; 6 h/day LOAEC ¼ 5.4 mg/m3 Significant increase in relative lung weight; decreased body weight 12–21 months; LOAEC ¼ 0.065 mg/m3 5 days/week; 6 h/day Increase in relative lung weight 12 months; 6 h/day, LOAEC ¼ 50 mg/m3 ECHA for 5 days/week Alveolar proteinosis Aluminum (2022)

Inhalation 12 years (occupational) Inhalation 5 days; 4 h/day

Aluminum chlorohydrate Rat; guinea pig Inhalation

Rat

Exposure

spermatid, and decreased organ weight as well. Considering the use, the forms of aluminum, and the amount encountered in daily life, the capacity should appear to be considerable in the safe margins between unacceptable reproductive events and daily conditions of usage or ingested aluminum.

Genotoxicity Several studies indicate that various species of aluminum compounds or complexes are capable of interaction with DNA contained in chromosomes, which might lead to abnormalities in their configuration and replication. In vitro experiments indicate that aluminum-induced DNA binding and cross-linking resulted in clastogenic effects, led to configuration changes, and altered sister chromatid exchange, ineffective or reduced DNA replication, and circular dichroism of DNA. It is also suggested that accumulation of aluminum in cell nuclei potentially alters protein–DNA interactions and calmodulin biosynthesis. Theoretically, the altered configuration of calmodulin can affect calcium modulation of the second messenger system that is activated by neurotransmitters. Other in vitro effects suggest aluminum-induced chromatid alteration. The alternation and generalization of transformed cells to normal, untreated cells are unknown.

Carcinogenicity Aluminum is not classifiable as a human carcinogen; the American Conference of Governmental Industrial Hygienists (ACGIH) classifies it as group A4. Most animal studies have failed to demonstrate carcinogenicity attributable to aluminum powder or several aluminum compounds. In 1987, the International Agency for Research on Cancer (IARC) concluded that there is sufficient evidence that certain exposures occurring during aluminum production cause cancer of the lung and bladder.

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Organ toxicity Aluminum can have adverse effects on the respiratory system and gastrointestinal system. Prolonged exposure during dialysis can cause neurologic effects such as encephalopathy. High doses of absorbed aluminum ca inhibit bone recovery.

Interactions Aluminum can cause a decrease in the absorption of amprenavir, doxycycline, Methylene blue, and tetracycline, resulting in reduced serum concentration and potentially a decrease in efficacy. The Serum concentration of Digoxin and Magnesium can be decreased when it is combined with aluminum. Aluminum can cause an increase in the absorption of Raltegravir resulting in an increased serum concentration and potentially a worsening of adverse effects.

Clinical management Aluminum overload has very few treatment options. Besides symptomatic treatment, deferoxamine is used as a chelating agent to lower systemic (blood) levels.

Environmental fate and behavior

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•

Routes and pathways relevant physicochemical properties Melting point ¼ 660  C. Boiling point ¼ 2450  C. Specific gravity ¼ 2.708 g cm−1. Solubility: insoluble in H2O, HNO3. Soluble in HCl, H2SO4. Partition behavior in water, sediment, and soil

The contribution of aluminum from drinking water is about 100 mg day−1. Air aluminum concentrations vary between 20 and 500 ng m−3 in rural settings and 1000 and 6000 ng m−3 in urban areas. Humans exposed to ambient aluminum concentrations of 2000 ng m−3 and particle size 5000 mg kg − 1 of aluminum. Lycopodium, some fern species, and members of genera Symplocos or Orites may contain high levels of aluminum. It does not appear to accumulate to any significant degree in cow’s milk or beef tissue, and it is therefore not expected to undergo biomagnification in terrestrial food chains.

Ecotoxicology Aluminum occurs naturally in soil, water, and air. It is redistributed or moved by natural and human activities. High levels in the environment can be caused by the mining and processing of its ores and by the production of aluminum metal, alloys, and compounds. Small amounts of aluminum are released into the environment from coal-fired power plants and incinerators. Virtually all food, water, and air contain some aluminum, which nature is well adapted to handle.

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Exposure standards and guidelines The United States Pharmacopeia (USP) considers aluminum to be a Class IV metal (metal impurities recognized as micronutrients with a high upper intake limit or no upper intake limit) and recommends the limit for oral and parenteral routes be set at 50,000 mg/ day and 500 mg/day respectively (USP, 2009). The ACGIH and the Occupational Safety and Health Administration (OSHA) in the United States have the following airborne exposure limit:

• •

ACGIH threshold limit value: Aluminum oxide: 10 mg m−3 (time-weighted average (TWA)) inhalable (total) particulate matter containing no asbestos and < 1% crystalline silica, A4. Soluble salts as Al: 2 mg m−3 (TWA). OSHA permissible exposure limit: Alpha alumina (aluminum oxide): 15 mg m−3 total dust, 5 mg m−3 respirable fraction. Aluminum as metal: 15 mg m−3 total dust, 5 mg m−3 respirable fraction.

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/5359268.

See also: Metals

References ATSDR (Agency for Toxic Substances & Disease Registry) (2008) Toxicological Profile for Aluminum. Available online at: Toxicological Profile for Aluminum (cdc.gov). USP (2009) Digest of comments received on the stimuli article “General Chapter on Inorganic Impurities: Heavy Metals” Pharmacopeial Forum 34(5) Available online at: https://www. usp.org/sites/default/files/usp/document/our-work/chemical-medicines/key-issues/2009-04-22MetalImpuritiesCommentDigest.pdf. ECHA for Aluminum (2022) https://echa.europa.eu.Registration. Accessed 10 January 2022. Dossier- Aluminum.

Further reading Ae N, Arihara J, Okada K, and Srinivasan A (eds.) (2011) Plant Nutrient Acquisition: New Perspectives: The Role of the Root Cell Wall in Aluminum Toxicity, pp. 201–226. Tokyo Japan: Springer Verlag. Becaria A, Campbell A, and Bondy SC (2002) Aluminum as a toxicant. Toxicology and Industrial Health 18(7): 309–320. Bingham E and Cohrssen B (eds.) (2012) Patty’s Toxicology. 6th edn, In: vol. 2, pp. 354–406. New York: Wiley. Mailloux RJ, Lemire J, and Appanna VD (2011) Hepatic response to aluminum toxicity: Dyslipidemia and liver diseases. Experimental Cell Research 317(6): 2231–2239. Nordberg GF, Fowler BA, and Nordberg M (2015) Handbook on the Toxicology of Metals, 4th edn. London: Academic Press, pp. 549–564. Wier HA (2012) Aluminum toxicity in neonatal parenteral nutrition: What can we do? The Annals of Pharmacotherapy 46(1): 137–140.

Relevant websites http://www.inchem.org :Aluminum (Environmental Health Criteria). http://www.aluminum.org :The (US) Aluminum Association Website. http://www.atsdr.cdc.gov :Agency for Toxic Substances and Disease Registry. Toxicological Profile for Aluminum.

Aluminum phosphide Mahshid Ataeia, Omid Mehrpourb,c, and Mohammad Abdollahia,d, aToxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; bData Science Institute, Southern Methodist University, Dallas, TX, USA; cMichigan Poison & Drug Information Center, Wayne State University School of Medicine, Detroit, MI, United States; d Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of M. Abdollahi, O. Mehrpour, Aluminum Phosphide, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 164–166, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00467-X.

Chemical profile Background Uses Exposure routes and pathways Epidemiology Toxicokinetics Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Clinical management Environmental fate Ecotoxicology Artificial intelligence view Exposure standards and guidelines Prognostic factors and methods Other hazards Zinc phosphide References

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Abstract Aluminum phosphide (AlP) is a highly effective outdoor and indoor insecticide and rodenticide. Moisture in the air mixes with phosphide grains and sets off phosphine (hydrogen phosphide, phosphorus trihydride, PH3), which is the active form of AlP. Exposure occurs mainly in cases of acute poisoning with suicidal intent. AlP inhibits cytochrome oxidase, interferes with cellular respiration, and induces severe oxidative stress. The main manifestations of AlP poisoning are severe metabolic acidosis and severe and refractory cardiogenic shock. There is no antidote available, and the treatment is mainly supportive. However, new and novel treatment potentials are proposed. The mortality rate in AlP-poisoning cases is 30–100%. Therefore, prognostic factors to better indicate the risk of mortality have been studied. Ecotoxicity is another crucial issue affecting environmental health, and novel AlP detection approaches like artificial intelligence are addressed.

Keywords Aluminum phosphide; Phosphine gas; Toxicity

Chemical profile

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Name: Aluminum phosphide Chemical Abstracts Service (CAS) Registry Number: 20859-73-8 Synonyms (Various Brand Names): Celphos, Alphos, Quickphos, Phosfume, Phostoxin, Talunex, Degesch, Synfume, Chemfume, Phostek, Delicia, rice tablets Molecular Formula: AlP Chemical Structure:

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Background Aluminum phosphide (AlP) is one of the most lethal toxic components. Several epidemiological studies showed that AlP is responsible for a high death rate among different kinds of poisons. Therefore, it is widely used by the young generation as a suicide agent. Unfortunately, no antidote is available for AlP. Consequently, physicians should consider that AlP, which is highly toxic, is widely known and has no actual cure. The toxicity caused by this substance should be treated immediately with the best supportive care.

Uses The primary use for AlP is as a fumigant to control insects and rodents in both food and non-food crops in indoor environments. It is also used to control rodents outdoors via application to their burrows or in grain storage areas. Due to the widespread use of this compound for protecting rice, it is also called rice tablets in some countries (Abdollahi and Mehrpour, 2014). Rice tablets are used for suicide, which increased the rate of toxicity and mortality (Alinejad et al., 2017). AlP is formulated in solid form and is available as a tablet, pellet, or dust. It is marketed as dark gray 3-g tablets consisting of AlP (56%) and carbamate (44%), under brand names such as Celphos, Alphos, Quickphos, Phosfume, Phostoxin, Talunex, Degesch, Synfume, Chemfume, Phostek, and Delicia in porous hags or blister packs (Abdollahi and Mehrpour, 2014).

Exposure routes and pathways AlP is usually formulated as dark gray or dark yellow crystals, similar to decaying fish or garlic. Most lethal exposures to AlP occur via the oral route with suicidal intent. Because it is a solid material, dermal absorption of AlP is unlikely. AlP is highly reactive with water, such that any contact with moisture results in decomposition to phosphine gas. Phosphine gas is colorless, flammable, and explosive at room temperature. Therefore, the primary exposure route is via inhalation and absorption by the lungs. Exposure is also possible through ingestion of commodities, such as grains and nutmeats, treated with AlP; these foods may contain residues of phosphine gas. Residues of phosphine gas in treated commodities are expected to be C, and m.1494C>T (McDermott et al., 2021; Selimoglu, 2007). Unlike traditional genetic variations where a diplotype exists, only one genetic variant exists for mitochondrial DNA, thus the presence of any of these mutations increases the

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susceptibility to aminoglycoside toxicity. If known to carry an MT-RNR1 variant of interest, the Clinical Pharmacogenetics Implementation Consortium strongly recommends that aminoglycoside antibiotics should be considered relatively contraindicated unless there is a lack of safer alternatives (McDermott et al., 2021).

Clinical management Management of toxic doses or an overdose of an aminoglycoside should begin with prompt assessment of respiratory and cardiovascular function and support when necessary. This may be critical if the patient experiences neuromuscular blockade leading to respiratory paralysis. For patients who have received an intravenous injection of these agents, managing renal function is also important. Proper hydration should help to reduce the potential for nephrotoxicity in patients with normal hydration status and kidney function, and routine analysis of fluid balance, serum creatinine, and serum trough values should be performed (MacDougall, 2017). In patients with serum trough levels that are prolonged, hemodialysis may be considered as aminoglycosides are dialyzable (Nayak-Rao, 2010). If patients experience ototoxicity, discontinuation of the aminoglycoside is the only way to reduce the potential for irreversible damage. Risks and benefits must be weighed carefully with this approach. Systemic toxicity with inhaled aminoglycosides is rare; however, large doses, when inhaled, may cause severe bronchospasm in patients (Childs-Kean et al., 2019). These patients should be managed with inhaled b-2 adrenergic agonists, such as albuterol, and other respiratory supportive care.

Environmental fate and behavior Aminoglycosides are water-soluble compounds that are naturally occurring products of bacteria found in the soil (MacDougall, 2017). They are highly polar compounds and under acidic conditions may become positively charged by protonation. This positive charge may lead to adsorption to clay and soil materials, which under normal parameters are negatively charged. They are readily degraded in the environment with relatively minor persistence and accumulation.

Ecotoxicology As products that are or were originally derived from bacteria found commonly in the soil, their potential for ecotoxicity exists only if major releases of large quantities occurred. Given their relatively low molecular weight (2.0 mg/kg/day, the animals showed salivation and muscular weakness and brain weights were slightly decreased. No changes were observed in blood, urinalyses, or in histopathological examinations (Aminopyridines | US EPA archive document, n.d.).

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Human There are several cases of acute toxicity of 4-AP due to accidental or intentional exposure. There are, however, no data available to report long-term effects of low-dose exposure to 4-AP.

Immunotoxicity No studies could be found which examine the potential effects of acetamide on the immune system.

Toxicity reproductive and developmental toxicity No adverse reproductive outcomes were noted in a study in quail, but otherwise there are no data available.

Genotoxicity 4-AP was negative in several mutagenicity assays.

Carcinogenicity No data were available to assess the carcinogenic potential of 4- aminopyridine.

Clinical management Pancuronium is a pharmacologic antidote and is recommended in severely poisoned human patients. Propranolol appears to block some of the cardiac toxicity of 4-AP. Seizures can be treated with diazepam. In severe cases, phenobarbital or phenytoin can be given if there is no response to diazepam. General symptomatic and supportive treatment is rewarding. Bicarbonate should be added to the fluid therapy to treat acidosis. Exposed persons should be removed to fresh air. If not breathing, then give artificial respiration. Medical attention should be sought if the cough or other symptoms still appear. If swallowed, wash out mouth with water provided person is conscious. Do not induce vomiting. Medical attention is needed if large amounts are ingested. For skin contact, the exposed area should be washed with soap and water for 15 min; Remove contaminated clothing. Medical attention should be sought if irritation develops or if the allergic reaction happens. For eye contact, Rinse thoroughly with large quantities of water for at least 15 min while lifting the lower and upper eyelids occasionally; immediate medical attention should be sought if the symptoms persist (4-aminopyridine—Jubilant Ingrevia, 2012).

Accidental release measures Wear personal protective equipment. Keep people without personal protective equipment away. Avoid raising and breathing dust and provide adequate ventilation. Do not allow material to reach groundwater, waterway, and sewer system. Place in appropriate containers for disposal.

Ecotoxicology As an avicide, the toxicity of 4-aminopyridine to the environment has been studied extensively.

Bird 4-Aminopyridine induces mortality in many non-target avian species. 4-Aminopyridine, or its hydrochloride salt, were highly toxic to 39 species of birds, including doves, parakeets, magpies, grackles, ducks, jays, robins, finches, sparrows, pheasants, quails, starlings, blackbirds, bobwhites, queleas, chicken, pigeons, and mannikins. The oral LD50 ranged from 2.4 to 20 mg/kg. The 8-day dietary LC50 is 447 ppm in Japanese quail, 316 ppm in mourning doves, and 722 ppm in mallard ducks. Avian reproduction studies

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suggest ingestion of sublethal amounts of 4-aminopyridine is unlikely to cause negative effects on birds’ reproductive systems. There is a large potential for exposure of non-target, particularly grain-feeding birds. Migratory birds, finches, and other small seed-feeding birds may ingest lethal doses that are applied to corn and sunflower fields (Aminopyridines | US EPA archive document, n.d.).

Fish EPA has characterized 4-aminopyridine as moderately toxic to warm water fish. Fish become increasingly sensitive with increased exposure. The LC50 ranges from 4 mg/L (in soft water) to 2.43 mg/L (in hard water) in channel catfish. The LC50 in is 3.40 mg/L (in soft water) to 3.20 mg/L (hard water) in bluegill fish (Aminopyridines | US EPA archive document, n.d.).

Others Endangered species may be adversely affected by 4-aminopyridine. There is low or nonexistent potential for secondary poisoning in animals such as cats, dogs, or birds of prey that may feed upon birds killed by Avitrol (4-aminopyridine—Extoxnet Pip, 1996).

Exposure standards and guidelines OSHA has currently set exposure limits for 4-AP of 0.5 ppm (2 mg/m3) for an 8-h TWA. In addition, due to 4-AP exposure, the American Conference of Governmental Industrial Hygienists (ACGIH) set its threshold limit value, at 0.5 ppm (2 mg/m3) for an 8-h TWA. In order to protect others from adverse health effects Short Term Exposure Limit (STEL) of 2 ppm is also recommended. The National Institute for Occupational Safety & Health (NIOSH) permissible exposure limit is 0.5 ppm for an 8-h TWA and 5 ppm as IDLH. No observed effect level of 4-AP is reported to be 200 ppm in dogs and 3 ppm in rats. Acceptable daily intake is reported to be 0.0015 mg/kg/day (4-aminopyridine—Jubilant Ingrevia, 2012).

See also: Pesticides and its toxicity

References Aminopyridines | US EPA archive document. (n.d.). Retrieved March 30, 2023, from https://archive.epa.gov/osa/hsrb/web/pdf/nci-2006.pdf Chin LS, Park CC, Zitnay KM, Sinha M, DiPatri AJ Jr., Perillán P, and Simard JM (1997) 4-Aminopyridine causes apoptosis and blocks an outward rectifier K+ channel in malignant astrocytoma cell lines. Journal of Neuroscience Research 48(2): 122–127. PMID: 9130140. Extoxnet Pip. (1996). 4-Aminopyridine. Retrieved June 31, 1996, from http://extoxnet.orst.edu/pips/4-aminop.htm. Hayes KC (2006) The use of 4-aminopyridine (fampridine) in demyelinating disorders. CNS Drug Reviews 10(4): 295–316. https://doi.org/10.1111/j.1527-3458.2004.tb00029.x. Kostadinova I and Danchev N (2019) 4-aminopyridine—The new old drug for the treatment of Neurodegenerative Diseases. Pharmacia 66(2): 67–74. https://doi.org/10.3897/ pharmacia.66.e35976. Jubilant Ingrevia. (2012). 4-Aminopyridine. Retrieved March 30, 2023, from https://jubilantingrevia.com/uploads/files/39msds_0078GjGhs09Div.3sds4-Aminopyridine.pdf. Sedehizadeh S, Keogh M, and Maddison P (2012) The use of aminopyridines in neurological disorders. Clinical Neuropharmacology 35(4): 191–200. https://doi.org/10.1097/ wnf.0b013e31825a68c5.

Further reading Johnson NC and Morgan MW (2006) An unusual case of 4-aminopyridine toxicity. The Journal of Emergency Medicine 30: 175–177. King AM, Menke NB, Katz KD, and Pizon AF (2012) 4-aminopyridine toxicity: A case report and review of the literature. Journal of Medical Toxicology 8(3): 314–321. https://doi.org/ 10.1007/s13181-012-0248-9. Pickett TA and Enns R (1996) Atypical presentation of 4-aminopyridine overdose. Annals of Emergency Medicine 3: 382–385. US Environmental Protection Agency (1980) Pesticide Registration Standard: 4- Aminopyridine: Avitrol. Washington, DC: Office of Pesticides and Toxic Substances.

Relevant websites https://pharmacia.pensoft.net/article/35976/ :Kostadinova IND (2019) 4-aminopyridine—The new old drug for the treatment of neurodegenerative diseases. Pharmacia. http://extoxnet.orst.edu/pips/4-aminop.htm :EXTOXNET PIP—4-AMINOPYRIDINE (1996) Extension Toxicology Network. Prepared for NCI by Technical Resources International, Inc. to support chemical nomination under contract no. N02-CB-07007 (06/03;rev. 11/05; 3/06). http://sitem.herts.ac.uk/aeru/ppdb/en/Reports/1348.html :University of Hertfordshire. (1982). 4-aminopyridine (Ref: PRC-1237). Toxicology.

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Amiodarone Jeanna M Marraffa, Department of Emergency Medicine, Upstate NY Poison Center, Upstate Medical University, Syracuse, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of J.M. Marraffa, Amiodarone, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 197–199, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00691-6.

Chemical profile Uses Exposure routes and pathways Pharmacokinetics/toxicokinetics Mechanism of action Mechanism of toxicity Acute and short-term toxicity (or exposure) Animal Human Chronic toxicity (or exposure) Animal Human In vitro toxicity data Clinical management References

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Abstract Amiodarone is an antiarrhythmic drug used for the management of acute and chronic ventricular and supraventricular arrhythmias. Although amiodarone is a class III antiarrhythmic agent it has effects at all four of the Vaughn Williams classifications. It is a sodium channel blocker with relatively fast on-off kinetics; it has nonselective beta-adrenergic antagonist activity; blocks potassium channels and has a small degree of calcium channel antagonist activity. Acute toxicity from amiodarone generally includes symptoms of nausea, vomiting, bradycardia and hypotension. Chronic toxicity can affect the thyroid and 5% of patients treated chronically with amiodarone can develop pulmonary toxicity including fibrosis.

Keywords Amiodarone; Antiarrhythmic drugs; Atrial fibrillation; Atrial flutter; Class III antiarrhythmic agent; Paroxysmal reentrant supraventricular tachycardia; Pulmonary toxicity; Ventricular fibrillation; Ventricular tachycardia

Key points

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Amiodarone is still used worldwide as an antiarrhythmic drug. It is used in the management of both acute and chronic ventricular and supraventricular arrhythmias. It can be used intravenously or orally. Chronic use is associated with numerous adverse events including pulmonary fibrosis, which can be fatal. Amiodarone is associated with multiple drug interactions.

Chemical profile

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Uses Despite its high incidence of side effects after chronic use, amiodarone is still used worldwide as an antiarrhythmic drug. It is used in the management of both acute and chronic ventricular and supraventricular arrhythmias. Amiodarone is used in life-threatening, recurrent, ventricular tachycardia or fibrillation when other interventions, such as epinephrine, have failed. It is also used in the management of supraventricular tachyarrhythmias including atrial fibrillation, atrial flutter and paroxysmal reentrant supraventricular tachycardia. Amiodarone is included in Advanced Cardiac Life Support (ACLS) Algorithms for numerous unstable rhythms. For unstable rhythms, amiodarone is used in the parenteral (intravenous) form. Oral amiodarone is used for chronic therapy once a patient is stabilized (Hamilton et al., 2020).

Exposure routes and pathways Accidental and intentional overdoses of amiodarone are not prevalent and only a few cases are reported in the literature. If overdose does occur, however, ingestion is the most common route of exposure.

Pharmacokinetics/toxicokinetics Following oral administration, amiodarone is slowly and variably absorbed from the gastrointestinal (GI) tract. The oral bioavailability varies greatly with a range of 22–86% (average of 50%). The reason for the variable bioavailability is not known but postulated to be: metabolism of drug in the gut lumen; first pass metabolism in the liver; and/or poor dissolution characteristics of the drug. Food, in particular, those high in fat content, results in an increase in the rate and extent of absorption (Freedman and Somberg, 1991). The peak plasma concentrations occur within 3–7 h (range: 2–12 h) after oral administration. The onset of action, however, is delayed for 2–3 days and up to 1–3 weeks post drug initiation. Although not clearly established, the maximal antiarrhythmic effect occurs within 1–5 months after initiation of oral therapy. The antiarrhythmic effect generally persists for 10–150 days after withdrawal of therapy (Freedman and Somberg, 1991). Amiodarone is extensively metabolized to an active metabolite, N-desethylamiodarone. The apparent volume of distribution is 65.8 L/kg (range: 18.3–147.7 L/kg). After chronic administration, amiodarone and its metabolite are extensively distributed to adipose tissue and many other organs including the liver, lung, spleen, and skeletal muscle. Amiodarone is extensively bound to plasma proteins, mainly albumin. The drug and its metabolite cross the placenta and is distributed into breast milk. The metabolism of amiodarone is not fully elucidated but appears to be at least biphasic. After a single IV dose of amiodarone, the terminal elimination half-life is on average, 25 days (range: 9–47 days). The elimination half-life of N-desethylamiodarone is equal to or longer than the parent drug. The half-life appears to be much more prolonged following multiple doses rather single doses. After chronic oral administration, the drug appears to be eliminated biphasically with an initial elimination half-life of about 2.5–10 days which is followed by a terminal elimination half-life averaging 53 days (range: 26–107 days). The exact metabolism of amiodarone has not been fully described but the drug appears to be extensively metabolized in the liver by N-deethylation to N-desethylamiodarone. The excretion of amiodarone and its metabolite are not fully described though it appears that it is nearly completely excreted in the feces as unchanged drug and N-desethylamiodarone presumably via biliary elimination (Hamilton et al., 2020; Papiris et al., 2010). Amiodarone and its major metabolite are not amenable to hemodialysis for drug removal.

Mechanism of action Amiodarone displays electrophysiologic characteristics of all four classes within the Vaughan-Williams classification scheme. It is a sodium channel blocker with relatively fast on-off kinetics; it has nonselective beta-adrenergic antagonist activity; blocks potassium channels and has a small degree of calcium channel antagonist activity. Its most prominent activity is potassium channel blockade and therefore, is classified as a Class III antiarrhythmic agent. It delays repolarization via prolongation of the action potential duration and effective refractory period; decreases AV conduction; depresses sinus node and junctional automaticity; acts as a noncompetitive alpha- and beta- receptor inhibitor and slows automaticity of Purkinje fibers (Freedman and Somberg, 1991).

Mechanism of toxicity Despite its popularity and frequency of use, amiodarone is a complex drug and displays unusual pharmacologic effects and pharmacokinetics. Even more peculiar is the multi-system organ adverse events attributed to amiodarone. The exact mechanisms

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of these adverse events remain unclear. Fatal complications of amiodarone include ARDS, pulmonary fibrosis, cirrhosis and bradycardia leading to cardiac arrest. Amiodarone pulmonary toxicity is probably related to a combination of different mechanisms including: a cytotoxic effect on pneumocytes; an immune mediated mechanism in genetically predisposed patients and possibly an effect on the angiotensin enzyme system. This results in a disruption of lysosomal membranes by amiodarone and a release of toxic oxygen radicals leading to apoptosis of lung epithelial cells (Hamilton et al., 2020; Papiris et al., 2010; Wiley Online Library, n.d.; Ruzieh et al., 2019).

Acute and short-term toxicity (or exposure) Animal The oral LD50 dose in dogs is more than 3 g/kg.

Human There is no published/established oral toxic dose of amiodarone. The determination of toxicity is based on observation and clinical effects. Symptoms include: nausea; vomiting; bradycardia; hypotension; heart block; QT prolongation with torsades de Pointes being a rare effect; tremor and ataxia.

Chronic toxicity (or exposure) Animal Chronic studies in rats demonstrate an increase carcinogenicity risk for thyroid tumors. The effects are dose related and have been described at doses as low as 5 mg/kg. Daily doses of 90 mg/kg in pregnant rats showed reduced fertility. Daily doses in rabbits of 25 mg/kg showed no change in fertility or effects on the fetus. However, higher daily doses of 75 mg/kg resulted in an increased rate of spontaneous abortion in these rabbits.

Human Acute administration of amiodarone is generally well-tolerated by patients however, chronic administration can result in severe and devastating adverse effects. The exact mechanisms of these adverse effects remain postulated and not clearly elucidated. Severe bradycardia has been reported to occur. Hypo- and hyper-thyroid have been reported with an unclear mechanism of toxicity. Pulmonary toxicity has been reported to occur in up to 5% of treated patients. The development of lung toxicity appears to be associated with older age, duration of treatment, cumulative dose, high levels of the metabolite, history of cardiothoracic surgery, use of iodinated contract media and probably pre-existing lung disease. The devastating pulmonary toxicity may develop as early as the first few days of treatment to several years later. The onset of this toxicity can be either insidious or rapidly progressive. Pulmonary fibrosis has resulted in death (Hamilton et al., 2020; Wiley Online Library, n.d.). Other side effects include: elevated liver function tests; uncommonly fulminant hepatitis; fatigue; tremor; dizziness; ataxia; corneal microdeposits (which usually do not affect vision); optic neuropathy/neuritis which can lead to blindness; blue-gray skin discoloration especially in areas exposed to the sun; skin necrosis rarely has occurred (Atıcı et al., 2019).

In vitro toxicity data Amiodarone has not been shown to be mutagenic in Ames, micronucleus and lysogenic tests.

Clinical management In patients with intentional overdose of amiodarone, treatment is mainly supportive. Amiodarone is adsorbed to activated charcoal and it should be considered in patients with large, intentional ingestions. There is little experience in managing patients with intentional overdoses of amiodarone because this is not commonly reported. In such cases, treatment is largely based on symptoms. Vasopressors should be considered for blood pressure support. Atropine should be considered if symptomatic bradycardia occurs. Hemodialysis to remove amiodarone has little benefit. In patients with toxicity secondary to therapeutic, chronic use of amiodarone should be managed supportively as well. Patients on chronic amiodarone therapy should be routinely followed and have continued monitoring including: liver function tests, thyroid function tests, eye exams, chest radiographs and pulmonary function tests. In patients demonstrating adverse effects, a risk vs. benefit assessment should be performed to determine whether amiodarone therapy should be continued. In patients that

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develop pulmonary toxicity, discontinuation of therapy should be strongly considered. In patients with severe pulmonary toxicity and ARDS, discontinuation of therapy is necessary and consideration of corticosteroids should be employed.

See also: Iodine

References _l (2019) Amiodarone-related blue–gray skin discoloration. Anatolian Journal of Cardiology 21: E11–E12. https://doi.org/10.14744/ Atıcı A, Aso
glu R, Barman HA, and Sahin ¸ AnatolJCardiol.2019.84890. Freedman MD and Somberg JC (1991) Pharmacology and pharmacokinetics of amiodarone. Journal of Clinical Pharmacology 31: 1061–1069. https://doi.org/10.1002/j.15524604.1991.tb03673.x. Hamilton D, Nandkeolyar S, Lan H, Desai P, Evans J, Hauschild C, Choksi D, Abudayyeh I, Contractor T, and Hilliard A (2020) Amiodarone: A comprehensive guide for clinicians. American Journal of Cardiovascular Drugs 20: 549–558. https://doi.org/10.1007/s40256-020-00401-5. Papiris SA, Triantafillidou C, Kolilekas L, Markoulaki D, and Manali ED (2010) Amiodarone. Drug Safety 33: 539–558. https://doi.org/10.2165/11532320-000000000-00000. Ruzieh M, Moroi MK, Aboujamous NM, Ghahramani M, Naccarelli GV, Mandrola J, and Foy AJ (2019) Meta-analysis comparing the relative risk of adverse events for amiodarone versus placebo. The American Journal of Cardiology 124: 1889–1893. https://doi.org/10.1016/j.amjcard.2019.09.008. Wiley Online Library (n.d.) Population-level incidence and monitoring of adverse drug reactions with long-term amiodarone therapy—Rankin—2017—Cardiovascular Therapeutics—Wiley Online Library. https://onlinelibrary.wiley.com/doi/full/10.1111/1755-5922.12258 (accessed 1.27.22).

Amitraz Virginia C Moser, Independent Consultant, Apex, NC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of V.C. Moser, Amitraz, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 200-202, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00097-X.

Chemical profile Background Uses Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity Acute and short term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Neurotoxicity Liver and kidney Endocrine toxicity Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines References

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Abstract This chapter covers the pesticide amitraz, a formamidine acaricide that is widely used in agriculture and veterinary medicine. The actions of amitraz on a2-adrenoreceptors leads to neurotoxicity, which is the primary toxic endpoint of concern. Amitraz also has reproductive, developmental, and endocrine effects with longer-term exposures.

Keywords Adrenergic system; Amitraz; Developmental toxicity; Endocrine toxicity; Formamidine; Neurotoxicity; Reproductive toxicity

Chemical profile

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Common name: Amitraz IUPAC Name: N,N0 -[(methylimino)dimethylidyne]di-2,4-xylidine Synonyms: BTS 27419, BAAM®, Preventic®, Apivar®, Zamitraz® CAS Number: 33089-61-1 Molecular Formula: C19H23N3 Chemical Structure:

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Background Amitraz is a formamidine insecticide and acaricide used on crops, livestock, and pets. Neurotoxicity is the primary endpoint of concern for short-term exposures, mostly through its actions on the adrenergic nervous system. Amitraz also has reproductive, developmental, and endocrine effects with longer-term exposures.

Uses Amitraz is a contact insecticide and acaricide that is widely used in agriculture and veterinary medicine. It can control psylla, whiteflies, and mites on cotton and pear crops and mites, lice, and ticks on livestock, wildlife, and pets. It is applied via spray-dip machines, low-pressure ground sprayers, dip or hand spray, or in impregnated dog collars. In the US, current uses are in dog collars for ticks, and impregnated strips for mites on honeybees (US EPA, 2021). In other countries, amitraz is also widely used on crops and in livestock dips.

Exposure and exposure monitoring Humans may be exposed to amitraz residues in foods, particularly honey. The highest potential for exposures, both residential and occupational, involves its use as a tick dip and in pet collars resulting in dermal or incidental oral ingestion (US EPA, 2021).

Toxicokinetics Amitraz is rapidly and well-absorbed via oral exposure. After a single dose, onset of effects is rapid (within hours of dosing). A major metabolite, N0 -(2,4-dimethylphenyl)-N-methylformamidine (BTS 27271), has been shown to have the same biological activity and perhaps greater potency compared to amitraz. Several subsequent metabolites include hydrolysis and conjugated products that are excreted primarily in urine, and to lesser extent in feces. Metabolism is similar across species, including humans (Knowles and Benezet, 1981). Kinetic studies of single, low doses indicate that amitraz is eliminated within hours after a single dose, but behavioral and pharmacological studies suggest dose-dependency. Following a low dose (5 mg kg−1), greater than half of a dose is eliminated within 24 h, whereas higher doses (50–75 mg kg−1) require several days. Similarly, behavioral studies show that effects of lower doses are reversible within hours to days, whereas higher doses produced effects that were still evident after 8 days (Knowles and Benezet, 1981). The BTS 27271 metabolite reaches higher concentrations and is more persistent in the brain, and may be responsible for prolonged toxicity (Hu et al., 2019).

Mechanism of toxicity Formamidines produce behavioral changes in target pests, including altered feeding and mating behaviors, representing a novel mode of pesticidal action. These chemicals stimulate the light organ of the firefly, causing it to glow, confirming their actions as octopamine receptor agonists in insects (Hollingworth and Murdock, 1980). Similarity between the target insect octopamine receptors and mammalian a2-adrenoreceptors give support for the mechanism of toxicity produced by amitraz, and most adverse effects are ascribed to activation of these receptors (reviewed in Costa, 2020; Evans and Gee, 1980; Hollingworth, 1976). Amitraz-induced bradycardia, mydriasis, sedation, intestinal stasis, hyperglycemia, and even lethality can be blocked using pharmacological antagonists of the a2-adrenoreceptors (e.g., yohimbine, piperoxan, atipamezole), but not by blockers of a1-adrenoreceptors or of other neurotransmitter receptors. This relative selectivity has been confirmed both in vivo and in vitro using receptor binding assays (Costa, 2020). Monoamine oxidase (MAO) inhibition is another known action of formamidine chemicals (Costa, 2020). MAO inhibition ex vivo was only measured at highly toxic doses of amitraz (Moser and MacPhail, 1989), whereas dysregulation of neurotransmitter systems, possibly through altered estradiol levels, has been suggested as a mechanism underlying altered levels of monoamine neurotransmitters (Del Pino et al., 2017). In addition to these neurological actions, amitraz and other formamidines are anti-inflammatory and antipyretic by means of blocking prostaglandin E2 synthesis (Costa, 2020). It also decreases insulin release, and increases glucagon secretion, leading to hyperglycemia (Abu-Basha et al., 1999).

Acute and short term toxicity Animal Amitraz is moderately toxic by dermal exposure, and slightly toxic by oral exposure or inhalation. It is non-irritating and does not cause skin sensitization. Dogs and baboons are the most sensitive, with oral LD50 values around 100–250 mg kg−1, with rats

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somewhat less sensitive (LD50 values around 500–800 mg kg−1), and mice the least sensitive with LD50 values >1600 mg kg−1 (Costa, 2020; US EPA, 2021).

Human Studies, in human volunteers as well as poisonings, report nervous system effects, including excitation followed by sedation, hypothermia, bradycardia, and hypotension. These signs mostly mirror those observed in laboratory animal studies (Avsaro gullari et al., 2006), and are similar across species, sexes, and routes of administration. Some signs in poisoning cases, such as vomiting, that are not reported in animal studies may be due to solvents and adjuvants in amitraz formulations (Dhooria and Agarwal, 2016). Humans appear to be the most sensitive species, and in one human study the No Observable Adverse Effects Level (NOAEL) was 0.125 mg kg−1 (US EPA, 2021).

Chronic toxicity Animal Neurotoxic effects were evident in subchronic and chronic studies in rodents and non-rodents. The effects at low doses do not appear to become progressively worse with repeated exposure. Repeated doses in several species also produce decreased weight gain, hyperglycemia, and liver and kidney toxicity (IPCS/INCHEM, 1998).

Human Little is known regarding chronic effects of amitraz in humans.

Immunotoxicity A guideline immunotoxicity study in rats submitted for regulatory purposes showed no adverse effects at the highest dose tested (US EPA, 2021). On the other hand, another study using higher doses reported increased adrenal weight, decreased splenic plaque-forming cells, and attenuated delayed-type hypersensitization reaction in rats (Institoris et al., 2007).

Reproductive and developmental toxicity In rat and rabbit developmental studies, embryotoxicity (increased fetal death, decreased size) and teratogenicity (fetal visceral and skeletal abnormalities) were observed at doses lower than or equal to those producing maternal toxicity (clinical signs, decreased weight gain) (Kim et al., 2007; US EPA, 2021). A rat multi-generational study showed reduced litter size and pup survival in all three generations; this was also observed in a guideline extended one-generation study (US EPA, 2021). Decreased sperm motility and male fertility, increased resorptions, and changes in reproductive organ weights were indicative of adverse effects on fertility and reproductive systems in both rats and mice (Al-Thani et al., 2003; Lim et al., 2010; Omoja et al., 2016a). Prenatal exposure of rats resulted in altered ages of physical developmental landmarks (vaginal opening, fur development, incisor eruption, righting reflex), and changes in open-field behavior in the offspring for up to 1 month of age (Palermo-Neto et al., 1997). Pre- and postnatal exposures also resulted in long-term changes in noradrenergic, serotonin, and dopamine neurochemistry (Del Pino et al., 2011), as well as brain morphometric changes and decreased thyroxine levels in the offspring (US EPA, 2021).

Genotoxicity Amitraz has shown to produce DNA damage (Comet assay) but was negative in bacterial assays (Ames test) (Padula et al., 2012; Radakovic et al., 2013; Tudek et al., 1988). Despite these contradictory data, it is generally accepted that amitraz does not have genotoxic potential (US EPA, 2021).

Carcinogenicity Mouse studies have reported lymphoreticular, lung, and liver tumors at the highest dose tested, which produced considerable toxicity, in females only. No findings of carcinogenicity have been reported in rats. The data are generally non-compelling, and the US EPA has classified amitraz as “suggestive evidence of carcinogenicity” (US EPA, 2021).

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Organ toxicity Neurotoxicity The actions of amitraz on a2-adrenoreceptors leads to signs of neurotoxicity that are sympathomimetic in nature. Acute neurotoxic signs include hypotension, bradycardia, hypothermia, periods of excitability and aggression, ataxia, and decreased spontaneous activity. High doses produce tremors, convulsions, signs of hypothalamic depression (Moser, 1991). Other pharmacological actions include slowed gastrointestinal transit, which can exacerbate colic in horses, and decreased insulin release (Costa, 2020). In addition to laboratory animal studies, there are a number of reports of neurotoxic effects following amitraz use in dogs and horses, which appear to be very sensitive.

Liver and kidney There are reports of changes in liver enzymes and kidney markers as well as hepatic vacuolar and generalized tubular degeneration; however, most of these studies use the formulation that includes xylene as a solvent or DMSO as a vehicle (e.g., Filazi et al., 2003; Omoja et al., 2016b). The liver and/or kidney toxicity of amitraz itself is not well-documented.

Endocrine toxicity Amitraz-induced altered hormone levels (e.g., 17b-estradiol, testosterone) may result from altered metabolism and decreased hormone release (Chou et al., 2008; Del Pino et al., 2017; Omoja et al., 2016a). Special reproductive studies have indicated altered hormonal cycling in female rats and mice (US EPA, 2021); weak antiestrogenic activity have also been reported in vitro (Ueng et al., 2004). While the US EPA Toxcast program reported significant effects on the androgen receptor assays, and a few estrogen receptor effects, amitraz has not yet been subject to that Agency’s full Endocrine Disruptor Screening Program (EDSP) (US EPA, 2021). An extended one-generation reproduction toxicity study in rats reported decreases in thyroxine (T4) levels in the F1 adult females at relatively low doses (US EPA, 2021).

Clinical management Poisoning with amitraz can occur via oral, inhalation, or dermal routes. Ingestion of sufficient amounts leads to depressed respiration, hypotension, bradycardia, gastric stasis, hyperglycemia, and coma. These signs and symptoms are somewhat similar to cholinesterase inhibitor poisoning and have sometimes been misdiagnosed as such (Bhartiva et al., 2019). While the usefulness of gastric lavage or activated charcoal is unclear, symptomatic treatments and supportive care (intubation and assisted ventilation) are usually effective and morbidity and mortality are low (Avsaro gullari et al., 2006; Dhooria and Agarwal, 2016). Although a2-antagonists (e.g., yohimbine, atipamezole) have been shown to block amitraz effects in animal studies, this treatment has not been explored in clinical cases (Proudfoot, 2003).

Environmental fate and behavior Amitraz is rapidly degraded (aerobic soil t1/2 of about 1 day) to several transformation products, and contamination of ground or surface waters is not a concern. On the other hand, the degradates are moderately persistent in aquatic and terrestrial environments, and are relatively immobile in soil (US EPA, 2021).

Ecotoxicology The parent amitraz is only slightly toxic to several avian species (8-day LD50 values >1000 ppm in diet), but causes reproductive toxicity (eggshell cracking, decreased viability of embryos and chicks). On the other hand, amitraz is highly toxic to fish (96-h LC50 values 1–10 ppm) and aquatic invertebrates. The degradates of amitraz, which are more environmentally stable, tend to have the reverse toxicity pattern. Amitraz is practically nontoxic to bees (US EPA, 2021).

Exposure standards and guidelines The US EPA has established an acute RfD of 0.0125 mg kg−1, based on a no-effect level from a study of neurotoxicity in human volunteers. The chronic RfD of 0.0017 mg kg−1 day−1 is based on the no-effect level for thyroid toxicity from the recent extended one-generation developmental study in rats; this is expected to be protective of all other effects, including human outcomes (US EPA, 2021). The Joint FAO/WHO Meeting on Pesticide Residues established an acute RfD of 0.01 mg kg−1, based on the human volunteer study, as well as an ADI value of 0.01 mg kg−1 day−1, based on the no-effect level of 1.3 mg kg−1 day−1 for developmental effects in rats (IPCS/INCHEM, 1998).

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References Abu-Basha EA, Yibchok-Anun S, Hopper DL, and Hsu WH (1999) Effects of the pesticide amitraz and its metabolite BTS 27271 on insulin and glucagon secretion from the perfused rat pancreas: Involvement of a2D-adrenergic receptors. Metabolism 48: 1461–1469. Al-Thani RK, Al-Thani AS, Elbetieha A, and Darmani H (2003) Assessment of reproductive and fertility effects of amitraz pesticide in male mice. Toxicology Letters 138: 254–260. Avsarogullari L, Ikizceli I, Sungur M, Sözüer E, Akdur O, and Yücei M (2006) Acute amitraz poisoning in adults: Clinical features, laboratory findings, and management. Clinical Toxicology 44: 19–23. Bhartiva M, Hans B, Sundaray S, and Sagar A (2019) Amitraz poisoning: The not so (un)common poisoning. Cureus 11: e5438. Chou C-P, Lu S-Y, and Ueng T-H (2008) Modulation of serum concentrations and hepatic metabolism of 17b-estradiol and testosterone by amitraz in rats. Archives of Toxicology 82: 729–737. Costa LG (2020) Neurotoxicity of amitraz, a formamidine pesticide. In: Aschner M and Costa LG (eds.) Advances in Neurotoxicology. vol. 4, pp. 255–276. Cambridge, MA: Elsevier Inc. Del Pino J, Martínez MA, Castellano VJ, Ramos E, Martínez-Larrañaga MR, and Anadón A (2011) Effects of prenatal and postnatal exposure to amitraz on norepinephrine, serotonin and dopamine levels in brain regions of male and female rats. Toxicology 287: 145–152. Del Pino J, Moyano P, Ruiz M, Anadón MJ, Díaz MJ, García JM, Labajo-González E, and Frejo MT (2017) Amitraz changes NE, DA and 5-HT biosynthesis and metabolism mediated by alterations in estradiol content in CNS of male rats. Chemosphere 181: 518–529. Dhooria S and Agarwal R (2016) Amitraz, an underrecognized poison: A systematic review. The Indian Journal of Medical Research 144: 348–358. Evans PD and Gee JD (1980) Action of formamidine pesticides on octopamine receptors. Nature 287: 60–62. Filazi A, Sireli M, and Kalkan F (2003) The influence of amitraz on biochemical parameters in mice. Human & Experimental Toxicology 22: 99–101. Hollingworth RM (1976) Chemistry, biological activity, and uses of formamidine pesticides. Environmental Health Perspectives 14: 57–69. Hollingworth RM and Murdock LL (1980) Formamidine pesticides: Octopamine-like actions in a firefly. Science 208: 74–76. Hu SX, Benner CP, White JA, Martin RA, and Feenstra KL (2019) Pharmacokinetics and brain distribution of amitraz and its metabolites in rats. Environmental Toxicology and Pharmacology 65: 40–45. Institoris L, Banfi H, Lengyel Z, Papp A, and Nagymajtenyi L (2007) A study on immunotoxicological effects of subacute amitraz exposure in rats. Human & Experimental Toxicology 26: 441–445. IPCS (International Programme on Chemical Safety) and INCHEM (Internationally Peer Reviewed Chemical Safety Information) (1998) Amitraz. http://www.inchem.org/documents/jmpr/ jmpmono/v098pr02.htm. Kim JC, Shin JY, Yang YS, Shin DH, Moon CJ, Kim SH, Park SC, Kim YB, Kim HC, and Chung MK (2007) Evaluation of developmental toxicity of amitraz in Sprague-Dawley rats. Archives of Environmental Contamination and Toxicology 52: 137–144. Knowles CO and Benezet HJ (1981) Excretion balance, metabolic fate and tissue residues following treatment of rats with amitraz and N’-(2,4-dimethylphenyl)-N-methylformamidine. Journal of Environmental Science and Health. Part. B 16: 547–555. Lim J-H, Kim S-H, Kim K-H, Park N-H, Shin I-S, Moon C, Park S-H, Kim S-H, and Kim J-C (2010) Reproductive and developmental toxicity of amitraz in Sprague-Dawley rats. Toxicology Research 26: 67–74. Moser VC (1991) Investigations of amitraz neurotoxicity in rats: IV. Assessment of toxicity syndrome using a functional observational battery. Fundamental and Applied Toxicology 17: 7–16. Moser VC and MacPhail RC (1989) Investigations of amitraz neurotoxicity in rats. III. Effects on motor activity and inhibition of monoamine oxidase. Fundamental and Applied Toxicology 12: 12–22. Omoja VU, Anika SM, and Asuzu IU (2016a) The effects of sub-chronic administration of sub-lethal doses of amitraz/xylene on selected reproductive parameters of male Wistar rats. Iranian Journal of Veterinary Research 17: 277–280. Omoja VU, Asuzu IU, and Anika SM (2016b) Assessment of the hepatic and renal effects of sub-chronic administration of sub-lethal doses of amitraz/xylene in albino Wistar rats. Comparative Clinical Pathology 25: 203–209. Padula G, Ponzinibbio MW, Picco S, and Seoane A (2012) Assessment of the adverse effects of the acaricide amitraz: In vitro evaluation of genotoxicity. Toxicology Mechanisms and Methods 22: 657–661. Palermo-Neto J, Sákaté M, and Flório JC (1997) Developmental and behavioral effects of postnatal amitraz exposure in rats. Brazilian Journal of Medical and Biological Research 30: 989–997. Proudfoot AT (2003) Poisoning with amitraz. Toxicological Reviews 22: 71–74. Radakovic M, Stevanovic J, Djelic N, Lakic N, Knezevic-Vukcevic J, Vukovic-Gacic B, and Stanimirovic Z (2013) Evaluation of the DNA damaging effects of amitraz on human lymphocytes in the Comet assay. Journal of Biosciences 38: 53–62. Tudek B, Gajewska J, Szczypka M, Rahden-Staron I, and Szymczyk T (1988) Screening for genotoxic activity of amitraz with short-term bacterial assays. Mutation Research 204: 585–591. Ueng T-H, Hung C-C, Wang H-W, and Chan P-K (2004) Effects of amitraz on cytochrome P450-dependent monooxygenases and estrogenic activity in MCF-7 human breast cancer cells and immature female rats. Food and Chemical Toxicology 42: 1785–1794. US EPA (US Environmental Protection Agency) (2021) Amitraz. Revised Draft Human Health Risk Assessment for Registration Review. https://www.regulations.gov/docket/ EPA-HQ-OPP-2009-1015/document.

Relevant websites http://www.cdpr.ca.gov/docs/risk/rcd/amitraz.pdf :CDPR (Department of Pesticide Regulation, California Environmental Protection Agency). (1995). Amitraz Risk Characterization Document https://pubchem.ncbi.nlm.nih.gov/compound/Amitraz :PubChem National Library of Medicine https://comptox.epa.gov/dashboard/dsstoxdb/results?search¼DTXSID5023871#details :US EPA (US Environmental Protection Agency) Comptox

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Amitrole Atoosa Karimi Babaahmadi and Maryam Armandeh, Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of R. Sellamuthu, Amitrole, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 203–205, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01163-5.

Chemical profile Background Uses Environmental fate and behavior Exposure and exposure monitoring Toxicokinetic Mechanism of toxicity Acute and short-term toxicity Chronic toxicity Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Interaction Clinical management Ecotoxicology Exposure standards and guidelines References Further reading

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Abstract Amitrole (3-amino-1,2,4-triazole) is a colorless, crystalline powder primarily synthesized by reacting aminoguanidine with formic acid. This nonselective herbicide is used to control annual grasses and aquatic weeds. Due to the restricted use of amitrole in non-food products, the risk of amitrole exposure in human is low. Nevertheless, it is a possible human carcinogen that mainly affects the thyroid gland in human and other species.

Keywords Amitrole; Carcinogenicity; Herbicide; Pesticide; Thyroid tumor; Triazole chemical

Chemical profile

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Name: Amitrole. Chem. Abstr. Serv. Reg. No.: 61-82-5 Molecular Formula: C2H4N4 (NIH, Pozdnyakov et al., 2018) Synonyms: Aminotriazole; 2-amino-1,3,4-triazole; 3-aminotriazole; 3-amino-1,2,4-triazole; 3-amino-1H-1,2,4-triazole; 5-amino-1,2,4-triazole; 5-amino-1H-1,2,4-triazole; AT; 3,A-T; ATA; ENT 25445 (NIH, Ilager et al., 2020). IUPAC Systematic Name: 3-Amino-s-triazole; 1H-1,2,4-triazol-3-ylamine (NIH, Pozdnyakov et al., 2018). Chemical Abstracts Service Registry Number: 61-82-5, 65312-61-0, 65312-62-1 (NIH). Chemical Structure: H N N N NH2

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Background Amitrole is a colorless, odorless crystalline, aromatic amine with a bitter taste (PubChem). It was primarily synthesized in 1898 by reacting aminoguanidine with formic acid, and it was first commercially manufactured in the late 1940s in the United States. Amitrole was recognized as an herbicide in the United States in 1948. In the late 1950s, there were reports of amitrole misuse and residue detection in cranberry crops known as the Cranberry Crisis of 1959. Following these events, the Delaney Clause law was enacted, prohibiting the addition of any potentially carcinogenic substances to food. Then in 1996, new rules were introduced in the Food Quality Protection Act that changed the way pesticides are regulated in foods. Meanwhile, the US Environmental Protection Agency revoked the amitrole of food products and restricted its use to non-food products (EPA, PubChem).

Uses The triazole chemical, amitrole, is used as a nonselective broad-spectrum herbicide (NIH) and plant growth regulator (Ilager et al., 2020). It is primarily used on outdoor areas, specifically nonagricultural areas on orchids and roadsides, to control annual grasses, deep-rooting weeds (annual and perennial broad leaf ), grasses, aquatic weeds in drainages and marsh land, and poison ivy (Siswana et al., 2008). It is mixed with other herbicides and used as a total herbicide for weed control in postharvest applications and before certain crops’ annual sowing. Amitrole is also used on non-cropland soils and inter row weed control in vineyards, but not animal grazing land or water sources. It is available in soluble concentrates, wettable powders, and water-soluble granules. Water-soluble granules containing 86% amitrole are most commonly used worldwide (Fernández et al., 2016).

Environmental fate and behavior Amitrole is a non-volatile crystalline compound that dissolves readily in water (280 g/L at 25
C) and polar solvents including ethanol, methanol, and chloroform. Amitrole levels have been estimated in various environmental compartments. However, water and soil are the only compartments that contribute to the ecological fate of amitrole. Amitrole does not enter the atmosphere because of its low vapor pressure. Excessive application of amitrole above the recommended level on soil leads to soil contamination and leaching, depending on the soil type. The leached-out amitrole contaminates the surface as well as groundwater. Leaching behavior is affected by dissolved organic matter despite the high water solubility (Howard, 2017). Under aerobic circumstances, amitrole found in the soil is easily degraded by microorganisms (mineralization), the primary breakdown pathway. Other routes like abiotic mechanisms contribute less to the degradation process. Laboratory studies indicated that amitrole is moderately persistent with an aerobic half-life of 26 days in soil and 57 days in water. However, it is more persistent in water under anaerobic condition with more than a 1-year half-life. Studies also demonstrated that photodegradation of amitrole is slow in soil but stable in water. There will be no bioaccumulation or biomagnification of amitrole expected due to its high water solubility, a very low log Kow, and non-persistence in animals (Li et al., 2009).

Exposure and exposure monitoring Amitrole is not discharged into the surroundings from production sites when used as recommended. Processing methods in manufacturing plants, such as dry crushing and bagging, release significant amounts of amitrole into the environment, with concentrations as high as 100 mg m−3, posing a danger of inhalation exposure to employees. In addition, herbicide spraying in the atmosphere is likely to cause inhalation exposure in living areas (Li et al., 2009). The risk of amitrole exposure in humans is minimal due to its limited use in non-food products. However, its exposure has been found in the workplace, particularly among factory workers and those spraying herbicide to agricultural areas. Inhalation, unintentional ingestion, dermal and eye contact are all possible routes of occupational amitrole exposure (HSDB, 2009). Human exposure data are limited; only a few occupational exposure surveys from the United States, Sweden, and Finland reported that workers were exposed to amitrole during handling processes. Thyroid function tests are routinely performed to monitor workers exposed to amitrole. Irritation of eyes and skin, skin dryness, dyspnea, muscle spasms, ataxia, lassitude, anorexia, salivation, increased body temperature, lethargy, depression due to thyroid function suppression are the symptoms of amitrole exposure (Li et al., 2009).

Toxicokinetic Amitrole is quickly and virtually fully absorbed from the gastrointestinal system and lungs. Minimal metabolic transformation occurs in mammals. Humans excrete the chemical through their kidneys and urine in less than 24 h, with no alteration (Cocco, 2002). A woman who swallowed approximately 20 mg kg−1 body weight of the herbicide had unaltered amitrole excretion in her urine at a concentration of 1 g/L. The concentration of amitrole in the blood was high (13 mg L−1) 12 h after consuming a pesticide

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comprising a combination of amitrole and ammonium thiocyanate, which eventually led to the victim’s death (WHO/FAO, 1993). Amitrole is rapidly absorbed from the gastrointestinal system after consumption in mice, rats, and dogs. It is absorbed up to 30% after cutaneous application. Based on the distribution studies, radiolabeled amitrole given to mice intravenously or intragastrically accumulated in the bone marrow, spleen, thymus, liver, and gut mucosa (WHO, 1994). Amitrole is metabolized in the liver, and 50% is metabolized within 3 h in a dose-dependent manner. In rodents, the unchanged form of amitrole is eliminated in the urine, and blood levels are drastically reduced within 24 h Metabolic studies in rats detected three metabolites in urine. Concentrations of eliminated metabolites were higher after 24 h post-dosing with low levels of amitrole (PubChem). The metabolism of amitrole to mutagenic intermediates occurs through peroxidases, such as prostaglandin synthase and lactoperoxidase (Cocco, 2002).

Mechanism of toxicity As a thyroid-disrupting chemical, amitrole suppresses the thyroid peroxidase enzyme, which produces the thyroxine hormone (T4) (Singh et al., 2020). Animal studies showed amitrole had a substantial inhibitory effect on T3 and T4 secretion in rats (NIH). Based on the In vitro studies, the reactive metabolites from amitrole are generated by thyroid peroxidase (TPO) and prostaglandin H-synthase, which bind to thyroid peroxidases irreversibly and inactivate them. As a result, T4 production in the blood decreases. T4 deficiency serves as a negative feedback mechanism on the pituitary gland, causing it to secrete more thyrotropin (TSH) (Roelfsema and Veldhuis, 2013). TSH stimulates the thyroid gland, and the lack of negative feedback regulation of TSH by T4 promotes thyroid gland hypertrophy and hyperplasia, depletion of colloid, and enhanced vascularity. This mechanism is responsible for thyroid tumors, especially follicular cell adenomas and carcinomas in rats exposed to amitrole for the long term (Mattioli et al., 1994).

Acute and short-term toxicity Acute and chronic toxicity studies of pesticides in groundwater are conducted in experimental animals and data from cases of accidental ingestion, clinical exposures, and occupational exposures (NIH). When examined in numerous species and by various routes of administration, amitrole exhibits low acute toxicity (LD50s always more than 2500 mg kg−1 body weight (WHO/FAO, 1993). So, it is classified as a substance with slightly toxic properties. In humans, amitrole may cause different symptoms depending on its dose, including dry cough and severe respiratory symptoms (after a 19% amitrole exposure) and inhibition of thyroid iodide uptake (exposure to 10–100 mg of amitrole (NIH). Skin rashes, vomiting, diarrhea, and nosebleeding are common symptoms. The LD50/LC50 values of amitrole and attributed toxicities for laboratory animals are documented in Table 1. In rabbit studies, amitrole was classified as slightly toxic and practically non-toxic for ocular irritation and cutaneous toxicity, respectively. In rabbits and rats, amitrole caused moderate erythema when applied topically. In rabbits (through the conjunctival sac route), eye irritation was observed, unlike in rats (inhalation exposure) (PubChem).

Chronic toxicity Amitrole does not pose a severe risk to workers if they are sufficiently protected. However, there have been reports of goiter, tumors of the thyroid, liver, and pituitary (Cocco, 2002). There have been no reported signs of amitrole toxicity in humans, but symptoms reported in laboratory animal studies include dyspnea, muscle spasm, ataxia, anorexia, salivation, skin dryness, and reduction in thyroid functions (PubChem). The enlarged thyroid gland decreased T3 and T4 levels, follicular hyperplasia, pituitary hypertrophy, follicular hyperplasia, and mild anemia were observed in dogs fed high concentrations of amitrole for more than 1 year (13 and 32 mg kg−1 body weight per day). In both sexes of rats (during 3 months) and mice (18 months), but not in golden hamsters, continuous feeding of 100 mg kg−1 amitrole resulted in goiter. Amitrole toxicity was not observed in hamsters or rabbits in general. The difference in sensitivity to amitrole was evident in these studies (WHO/FAO, 1993). Also, following oral and subcutaneous injection of amitrole, mice and rats developed thyroid and liver cancers. However, the liver tumors were caused by large or even toxic dosages (Crouse et al., 2015). Table 1

Amitrole LD50/LC50 values and relative toxicity in laboratory animals.

Species

Dose LD50/LC50

Relative toxicity

References

Rat Rat Rat Mice Rabbit

LD50 dermal: >2500 mg kg−1 Inhalation LC50 oral: >439 mg/cu m/4 h LD50 oral: >4000–25,000 mg kg−1 LD50 oral: 11000–14,700 mg kg−1 LD50 dermal: >10,000 mg kg−1

Slightly toxic Slightly toxic Slightly toxic Slightly toxic Slightly toxic

Gaines et al. (1973) PubChem PubChem PubChem PubChem

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Immunotoxicity A human case report of allergic contact dermatitis after 6 months of exposure to amitrole documented a positive patch test and allergic contact dermatitis (NIH). In guinea pigs, dermal application of amitrole produced a sensitizing effect (WHO/FAO, 1993). Pulmonary lesions have also been observed in some cases (APVMA, 2010).

Reproductive and developmental toxicity Amitrole’s reproductive and developmental effects on humans are unknown. In pregnant rats exposed to amitrole, circulating thyroxine (T4) concentrations decreased and caused abnormal brain development in the offspring. The offspring’s brain had a congenital abnormality, with enormous clusters of ectopic neurons in the corpus callosum, a condition known as periventricular heterotopia. Amitrole inhibits the enzyme TPO, thereby altering the migration of neurons and inducing heterotopia around the ventricle (Ramhøj et al., 2022). At low doses of dietary amitrole (1.25 mg or 5 mg kg−1 for 1 day), it has no adverse effects on reproduction in rats. However, high doses of dietary amitrole had a teratogenic effect in rats during pregnancy. High amounts of amitrole fed to infant mice (36 or 75 mg kg per day) caused growth retardation and thyroid hyperplasia. In addition to increased mortality after weaning without affecting parental fertility, this substance can lead to the thymus and splenic atrophy in infants (PubChem).

Genotoxicity Amitrole’s genotoxicity has been investigated in several systems. The majority of In vivo and In vitro studies showed negative results. Amitrole has not been found to produce genotoxicity in humans (Pfuhler et al., 2021). In the lymphocytes of Chinese hamster, amitrole has chromosomal abnormalities (Pfuhler et al., 2021). However, human lymphocytes have not shown chromosomal abnormalities due to amitrole exposure (Kaya et al., 2000).

Carcinogenicity Based on sufficient evidence and various studies on laboratory animals, amitrole has been classified as a possible human carcinogen, Group B2, probably human carcinogen. This classification was based on thyroid gland and liver tumors in an animal study of both sexes of mice exposed to amitrole for 7 days (IARC). It also caused tumors of the liver and thyroid gland by subcutaneous injection in rats. In Syrian hamster embryo fibroblasts, cells known for their ability to metabolize other carcinogens, amitrole induces gene mutations and morphological transformations (Krauss and Eling, 1987). However, the data available from epidemiological studies are inadequate to evaluate the relationship between human cancer and exposure specifically to amitrole. A small cohort study of Swedish railroad workers who had sprayed herbicides found a statistically significant excess of all cancers among those exposed to both amitrole and chlorophenoxy herbicides, but not among those exposed mainly to amitrole (Axelson and Sundell, 1974).

Interaction Amitrole interferes with proteins such as protein 1TH4 (crystal structure of NADPH depleted bovine liver catalase complexed with 3-amino-1,2,4-triazole). It also interferes with 3SXV protein (Crystal structure of the complex of goat lactoperoxidase with amitrole at 2.1 A resolution) (PubChem).

Clinical management General Emergency Management In oral, dermal, or respiratory exposure is recommended by the US National Institute for Occupational Safety and Health (NIOSH) (NIH).

Ecotoxicology Amitrole has low toxicity in mammals and is unlikely to bioaccumulate. It has moderate toxicity to most terrestrial and aquatic species. Residues of amitrole above its environmental quality standard (0.1 mg/L) are regularly found in the rivers of Europe (Sánchez-Bayo et al., 2013). LD50 values of amitrole for some non-mammalian species are listed in Table 2.

Amitrole Table 2

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Amitrole LD50/LC50 values and relative toxicity in other species.

Species Mallard duck Quail Honeybee Trout (96 h) Soil microflora

Dose LD50/LC50 −1

LD50 2000 mg kg LD50 >2150 mg kg−1 LD50 >10 mg bee LC50 Oral: >1000 mg−1 At 10 normal application rate

Relative toxicity

References

Slightly toxic Practically nontoxic Practically nontoxic Practically nontoxic No adverse effect

WHO/FAO (1993) WHO/FAO (1993) WHO/FAO (1993) WHO/FAO (1993) WHO/FAO (1993)

Exposure standards and guidelines Amitrole was first considered by the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) in 1974 and given a conditional acceptable daily intake (ADI) of 0–0.00003 mg kg−1 body weight (Krauss and Eling, 1987). In the United States, the following three agencies have imposed restrictions on the use of amitrole in the workplace: 1. Occupational Safety and Health Administration (OSHA) permissible exposure limit 8 h TWA: 0.2 mg m−3. 2. NIOSH recommended exposure limit 8 h TWA: 0.2 mg m−3. 3. American Conference of State and Industrial Health Professionals (ACGIH): 0.2 mg m−3 as a TWA for 8 h during a 40-h workweek. OSHA and NIOSH limits are based on the risk of cancer associated with exposure to amitrole, and the ACGIH limits are based on the risk of systemic and reproductive effects associated with exposure to amitrole (NIH).

References APVMA (2010) Australian Pesticides and Veterinary Medicines Authority in Encyclopedia of Nanoscience and Society. 2455 Teller Road, Thousand Oaks California 91320. United States: SAGE Publications, Inc.https://doi.org/10.4135/9781412972093.n22. Axelson O and Sundell L (1974) Herbicide exposure, mortality and tumor incidence. An epidemiological investigation on Swedish railroad workers. Work, Environment, Health 11: 21–28. Cocco P (2002) On the rumors about the silent spring. Review of the scientific evidence linking occupational and environmental pesticide exposure to endocrine disruption health effects. Cadernos de saúde pública/Ministério da Saúde, Fundação Oswaldo Cruz, Escola Nacional de Saúde Pública 18(2): 379–402. https://doi.org/10.1590/S0102311X2002000200003. Crouse LCB, Lent EM, and Leach GJ (2015) Oral toxicity of 3-nitro-1,2,4-triazol-5-one in rats. International Journal of Toxicology 34(1): 55–66. https://doi.org/ 10.1177/1091581814567177. Fernández P, et al. (2016) Underlying resistance mechanisms in the Cynosurus echinatus biotype to acetyl CoA carboxylase-inhibiting herbicides. Frontiers in Plant Science 7(APR2016): 1–10. https://doi.org/10.3389/fpls.2016.00449. Gaines TB, Kimbrough RD, and Linder RE (1973) The toxicity of amitrole in the rat. Toxicology and Applied Pharmacology 26: 118–129. Howard P (2017) In: Howard PH, et al. (ed.) Handbook of Environmental Fate and Exposure Data. Routledgehttps://doi.org/10.1201/9780203719305. HSDB (2009) Hazardous Substances Data Bank. National Library of Medicine.http://toxnet.nlm.nih.gov/. Ilager D, et al. (2020) Electrocatalytic detection of herbicide, amitrole at WO30.33H2O modified carbon paste electrode for environmental applications. Science of the Total Environment 743: , 140691. https://doi.org/10.1016/j.scitotenv.2020.140691. Kaya B, et al. (2000) Genotoxicity testing of five herbicides in the Drosophila wing spot test. Mutation Research, Genetic Toxicology and Environmental Mutagenesis 465(1–2): 77–84. https://doi.org/10.1016/S1383-5718(99)00214-4. Krauss RS and Eling TE (1987) Macromolecular binding of the thyroid carcinogen 3-amino-l,2,4-triazole (amitrole) catalyzed by prostaglandin H synthase, lactoperoxidase and thyroid peroxidase. Carcinogenesis 8(5): 659–664. https://doi.org/10.1093/carcin/8.5.659. Li W, et al. (2009) Changes of thyroid hormone levels and related gene expression in Chinese rare minnow (Gobiocypris rarus) during 3-amino-1,2,4-triazole exposure and recovery. Aquatic Toxicology 92(1): 50–57. https://doi.org/10.1016/j.aquatox.2009.01.006. Mattioli F, et al. (1994) Studies on the mechanism of the carcinogenic activity of amitrole. Toxicological Sciences 23(1): 101–106. https://doi.org/10.1093/toxsci/23.1.101. Pfuhler S, et al. (2021) Validation of the 3D reconstructed human skin Comet assay, an animal-free alternative for following-up positive results from standard in vitro genotoxicity assays. Mutagenesis 36(1): 19–35. https://doi.org/10.1093/mutage/geaa009. Pozdnyakov IP, et al. (2018) Photooxidation of herbicide amitrole in the presence of fulvic acid. Environmental Science and Pollution Research International 25(21): 20320–20327. https://doi.org/10.1007/s11356-017-8580-x. Ramhøj L, et al. (2022) Perinatal exposure to the thyroperoxidase inhibitors methimazole and amitrole perturbs thyroid hormone system signaling and alters motor activity in rat offspring. Toxicology Letters 354: 44–55. https://doi.org/10.1016/j.toxlet.2021.10.010. Roelfsema F and Veldhuis JD (2013) Thyrotropin secretion patterns in health and disease. Endocrine Reviews 34(5): 619–657. https://doi.org/10.1210/er.2012-1076. Sánchez-Bayo F, et al. (2013) Calibration and field application of ChemcatcherW passive samplers for detecting amitrole residues in agricultural drain waters. Bulletin of Environmental Contamination and Toxicology 90(6): 635–639. https://doi.org/10.1007/s00128-013-0983-x. Singh S, et al. (2020) Protective effect of a polyherbal bioactive fraction in propylthiouracil-induced thyroid toxicity in ratsby modulation of the hypothalamic–pituitary–thyroid and hypothalamic–pituitary–adrenal axes. Toxicology Reports 7: 730–742. https://doi.org/10.1016/j.toxrep.2020.06.002. Siswana M, Ozoemena K, and Nyokong T (2008) Electrocatalytic detection of amitrole on the multi-walled carbon nanotube—Iron (II) tetra-aminophthalocyanine Platform. Sensors 8(8): 5096–5105. https://doi.org/10.3390/s8085096. WHO (1994) Assessing Human Health Risks of Chemicals: Derivation of Guidance Values for Health-Based Exposure Limits/Published Under the Joint Sponsorship of the United Nations Environment Programme. The International Labour Organisation, and the World Health Organization.https://apps.who.int/iris/handle/10665/40675. WHO/FAO (1993) Pesticide Residues in Food. https://apps.who.int/iris/handle/10665/39146.

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Further reading EPA. n.d. Environmental Protection Agency Office of Pesticide Programs Reregistration Eligibility Decision (no date) AMITROLE, List A, Case 0095. IARC. Monographs Amitrole, vol. 79, https://publications.iarc.fr. NIHhttps://www.ncbi.nlm.nih.gov/books/NBK396276/. PubChemhttps://pubchem.ncbi.nlm.nih.gov/compound/Amitrole#section¼InChI.

Ammonia Carolin Bischoff, Regulatory Toxicology, BASF SE, Ludwigshafen am Rhein, Germany © 2024 Elsevier Inc. All rights reserved. This is an update of RJ Parod, Ammonia, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 206–208, ISBN9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00468-1.

Chemical profile Background Uses/occurrence Exposure Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines Other References Further reading

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Abstract Ammonia is a colorless gas naturally produced by the decay of plant and animal matter. Being one of the most commonly produced chemicals, ammonia serves as a precursor for several endproducts, the most commonly known are fertilizers. The toxicity of ammonia in humans is mainly exhibited by its corrosive nature. Burns of the skin and mucous membranes (eyes, respiratory tract) are the most prominent effects observed after exposure to the gas and aqueous solutions thereof. In the environment, animal husbandry represents the most common source of ammonia exposure. Elevated amounts of ammonia in the atmosphere can lead to the deposition of ammonia in ecosystems. In soil, microbial transformation to nitrites and nitrates via nitrification processes leads to fertilization; an unfavorable condition in ecosystems which are dependent on poor soils. Ammonia is toxic for fish and aquatic invertebrates.

Keywords Ammonia toxicity; Environmental toxicants; Respiratory tract toxicology

Key points

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Ammonia is a naturally occurring gas involved in several metabolic pathways in the human body. As one of the chemicals with highest production volumes around the globe, it is mainly used in the production of fertilizers. The most prominent toxic effects of ammonia are burns of the skin (mainly caused by aqueous solutions) and mucous membranes. In the environment, animal husbandry represents the most common source of ammonia exposure. Elevated amounts of ammonia in the atmosphere can lead to its deposition in ecosystems, with the possibility of subsequent eutrophication.

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Chemical profile

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Synonyms: Ammonia gas; Liquid Ammonia; Spirit of hartshorn; Anhydrous ammonia CAS Number: 7664-41-7 Molecular Formula: NH3

•

Chemical Structure:

Background Ammonia is a naturally occurring colorless gas with a pungent odor. It is a key component of the global nitrogen cycle and of all living organisms. It is part of several metabolic pathways, most importantly, it is used in the synthesis of nucleic acids and proteins. In nature, it is generated primarily by the decay of plant and animal matter. Ammonia is one of the most commonly produced chemicals in the world and serves as a precursor for several other nitrogen compounds.

Uses/occurrence About 90% of the commercially produced ammonia is used in fertilizers with the remainder used in a variety of applications such as animal feed, household and industrial cleaners, plastics, synthetic fibers and resins, pharmaceuticals, explosives and refrigeration.

Exposure Ammonia is a gas under normal environmental conditions; thus, human exposures typically occur via inhalation. Oral exposures are also possible as ammonia is readily soluble in water. Dermal absorption is considered to be low. Background concentrations of ammonia in air (1 ppb), water (30 ppb), and soil (3 ppm) are low but can increase by orders of magnitude in areas of fertilizer production and application, sewage treatment, and animal confinement buildings. Another important source of exposure are cleaning agents, which can contain up to 25% of dissolved ammonia.

Toxicokinetics Unionized ammonia freely diffuses through tissue cells and exists in equilibrium with ammonium cations upon contact with tissue water; the ratio of NH3/NH4+ is dependent on the pH. Under normal physiological conditions (pH 7.4), ammonium ion is the predominant form in blood, as predicted by pH partitioning for a weak base (pKa ¼ 9.25) (ATSDR, 2004). During short term (2 min) human exposures to 500 ppm ammonia, most of the inspired ammonia is retained within the upper airways due to its high water solubility. This absorption process may be adaptive or saturable because most of the ammonia inspired during longer exposures (10–27 min) is exhaled with only 4–30% being retained within the upper airways and available for systemic absorption. This scrubbing of the inspired air by the upper respiratory tract protects the deeper lung from damage by ammonia. Ammonia or ammonium ion is well-absorbed by the gastrointestinal tract, and almost 100% of the ammonia produced endogenously in the human digestive tract by gut microbiota (60 mg kg−1 day−1) is absorbed and metabolized in the liver by the urea cycle to urea and glutamine. Astrocytes in the brain can also convert ammonia to glutamine. Due to first-pass metabolism in the liver, little ammonia from the gut reaches the systemic circulation, and toxicologically significant amounts of ammonia in blood (>1 mg mL−1) probably occur only in severe disease states where the metabolism of ammonia by the liver and the excretion of metabolites by the kidney are compromised. It is unlikely that a significant amount of the ammonia contacting the skin is absorbed. Absorption of aqueous ammonia however can be enhanced due to destruction of the skin barrier. Ammonia that reaches the circulation is distributed throughout the body where it can be used in protein synthesis or as a buffer. Ammonia is able to cross the blood brain barrier where it primarily enters astrocytes. Most of the absorbed ammonia is excreted in the urine as urea, with minimal amounts excreted in the feces or expired air.

Mechanism of toxicity The primary acute effects of ammonia exposure are manifested by burns to the skin, eyes, and respiratory tract. Ammonia readily dissolves in tissue water where hydroxide ions are formed that break down cellular proteins, saponify cell membrane lipids resulting

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in cell disruption and death, and initiate an inflammatory response that further damages surrounding tissues. Under pathological conditions (e.g., cirrhosis) that impair the metabolic capacity of the liver, high ammonia levels can reach the bloodstream and subsequently enter the brain, overwhelm the limited capacity of astrocytes to metabolize ammonia, and result in a potentially fatal disorder called hepatic encephalopathy.

Acute and short-term toxicity Animal The 1 h inhalation lethal concentration 50% (LC50) values in mice and rats receiving whole-body exposures were 4200 and 14,000 ppm, respectively. The rat oral lethal dose 50% (LD50) of aqueous ammonia is 350 mg kg−1, while that to ammonium salts is 2000 mg kg−1. A 12% solution of aqueous ammonia was corrosive to rabbit skin, while a 10% solution was not. Ammonium salts do not exhibit skin sensitizing properties (ECHA, 2022).

Human Ammonia has an odor threshold ranging from 1 to 5 ppm. Acute changes in respiratory function are not seen in workers exposed to 9 ppm ammonia for 8 h. Exposures between 20 and 25 ppm can cause complaints and discomfort in some workers unaccustomed to ammonia exposure but have little effect on pulmonary function or odor sensitivity. Concentrations of 100 ppm caused definite irritation of the respiratory tract and eyes, and exposures at 250 ppm ammonia are bearable for 30–60 min. Severe irritation of the respiratory tract, skin, and eyes has been observed following ammonia exposures ranging from 400 to 700 ppm. Exposure to 2500–4500 ppm ammonia can be fatal within 30 min. Immediate fatalities appear to be the result of airway obstruction, particularly laryngeal edema, and glottic spasm, while infections and other secondary complications appear to be the cause of fatality among those who survive for several days to weeks (NRC, 1979).

Chronic toxicity Animal Ammonia is considered as an irritant gas that generally causes severe local effects at the point of entry in the absence of systemic toxicity. While there are no guideline studies on ammonia itself, some information can be obtained from non-guideline studies on surrogate ammonium salts since once absorbed the ammonium ion and ammonia exist in equilibrium. When male and female rats were exposed to ammonium sulfate in the diet at doses up to 1975 mg kg−1 day−1 for 13 weeks, neither body weight, food consumption, or hematological and clinical parameters were affected. Although kidney and liver weight changes were noted, these effects occurred only at the highest dose and in the absence of histopathological changes. Diarrhea was seen in males at the highest dose while females were unaffected. The no observable adverse effect levels (NOAELs) were 886 mg kg−1 day−1 (m) and 1975 mg kg−1 day−1 (f ). In a combined repeated-dose/reproduction/developmental study performed similar to an OECD (Organization for Economic Cooperation and Development) test guideline no. 422 protocol, rats were exposed by gavage to ammonium phosphate at doses up to 1500 mg kg−1 day−1. At the two highest doses, alkaline phosphatase was increased and total blood protein was decreased resulting in a NOAEL of 250 mg kg−1 day−1 (ECHA, 2022).

Human No health effects were observed in volunteers exposed to ammonia (25–100 ppm) for 6 h day−1 over a period of 6 weeks; after 2–3 weeks of exposure, volunteers were inured to the eye, nose, and throat irritation noted at these concentrations (Ferguson et al., 1977). There were no differences in pulmonary function or respiratory and cutaneous symptoms between soda ash workers exposed for an average of 12 years to a mean timeweighted average (TWA) of 9.2 ppm ammonia and those exposed to a mean of 0.3 ppm ammonia (Holness et al., 1989). The NOAEL obtained in this worker population was used by the US Environmental Protection Agency under its Integrated Risk Information System program to establish a reference concentration of 0.7 ppm for the general population chronically exposed to ammonia.

Immunotoxicity While studies in animals have shown that exposures to ammonia (25–100 ppm) can decrease resistance to bacterial infection, there is no evidence that the secondary infections accompanying the dermal and respiratory lesions seen after human exposures to ammonia are due to an effect on immune function.

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Reproductive and developmental toxicity There are no data on the effect of ammonia in animals or humans. However, data on related ammonium compounds in animals suggest that ammonia does not affect either reproduction or development. For example, histological lesions were not seen in the reproductive organs of rats exposed to ammonium sulfate (up to 1975 mg kg−1 day−1) via the diet for 13 weeks (European Chemicals Agency (ECHA), 2022). Similarly, reproductive and developmental parameters in a screening study similar to an OECD 422 protocol were not affected when male and female rats were exposed to diammonium phosphate (up to 1500 mg kg−1 day−1) via gavage (European Chemicals Agency (ECHA), 2022).

Genotoxicity Data from in vitro and in vivo mutagenicity and clastogenicity tests of ammonia, aqueous ammonia, and ammonium salts following guideline protocols are negative. Positive and negative results have been reported in nonguideline studies, the former sometimes occurring only at cytotoxic doses (European Chemicals Agency (ECHA), 2022).

Carcinogenicity There are no data in animals or humans on the carcinogenic potential of ammonia following either inhalation or dermal exposures. In a 2-year drinking water study in mice given 193 mg ammonia kg−1 day−1 as liquid ammonia, carcinogenic effects were not observed in mice and the spontaneous development of breast cancer commonly seen in C4H mice was not affected (European Chemicals Agency (ECHA), 2022). In a study of the role of ammonia generated by Helicobacter pylori on the development of gastric lesions, 0.01% ammonia in drinking water (42 mg ammonia kg−1 day−1) increased the incidence and severity of gastric tumors induced in rats that had been pretreated with N-methyl-N0 -nitro-N-nitrosoguanidine, a known stomach carcinogen in experimental animals (Tsujii et al., 1992). This dose is more than 100-fold greater than the daily dose of ammonia ingested by humans in food and water. To study the carcinogenicity of acidogenic or alkalogenic diet in rats, ammonium chloride was tested in a study similar to OECD guideline No. 451 protocol. Doses up to 1105 mg NH4Cl kg−1 day−1 did not cause any carcinogenic effects. Moreover, an eminent adaptive capacity of the rats has been observed (European Chemicals Agency (ECHA), 2022). There are no data in humans on the carcinogenic effects of ammonia or ammonium compounds following oral exposure.

Organ toxicity The main target organs of ammonia are the respiratory tract and the eyes. There, local findings like eye and throat irritation are the first effects to be observed. If detoxifying mechanisms are disturbed (e.g., in the course of chronic liver disease), hyperammonemia can lead to significant brain damage. Aqueous ammonia solutions cause burns at the first site of contact.

Clinical management Exposures by inhalation should be monitored for respiratory tract irritation, upper airway obstruction, bronchitis, or pneumonitis. Humidified supplemental 100% oxygen should be administered to help soothe bronchial irritation. Oxygen, in combination with intubation and mechanical ventilation, may be required in severe cases. Exposed eyes and skin should be irrigated immediately with copious amounts of water; eyes should be washed for at least 30 min or until the eye reached neutral pH as tested in the conjunctival sac. If eye irritation, pain, swelling, lacrimation, or photophobia persist, the patient should be seen in a health care facility. After ingestion of aqueous ammonia solutions in accidental or suicidal attempt, patients should be monitored for oral and esophageal burns. There is a risk of gastrointestinal bleeding and perforation of the esophagus or stomach, which might occur late. Therefore, abdominal examination should be repeated more frequently. Ingested ammonia should be diluted by oral application of water; induction of vomiting must be omitted.

Environmental fate and behavior The major emission source of ammonia to the environment is agriculture: about 70% of emissions stem from animal husbandry only. With a vapor pressure of 8611 hPa at 20  C (European Chemicals Agency (ECHA), 2022), ammonia is a gas under normal environmental conditions. In the atmosphere, ammonia is estimated to have a half-life of several days. The primary fate process is the reaction of ammonia with acid air pollutants like sulfur dioxide and removal of the resulting ammonium salts by dry or wet deposition. Rain washout and reaction with photochemically produced hydroxyl radicals also contribute to the atmospheric fate of vapor-phase ammonia.

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In water, ammonia acting as a weak base (pKa ¼ 9.25) will exist in equilibrium with the ammonium ion. Ammonia will volatilize to the atmosphere due to its high vapor pressure in water (2878 hPa at 25  C) while the ammonium ion will be removed via uptake by aquatic plants, adsorption to sediments, and microbial transformation to nitrites and nitrates by nitrification. In soil, the same general processes will occur. As a result of binding of ammonium ions to soil particles and uptake by plants or microorganisms, ammonia does not readily leach through soil. However, nitrate can leach through soil due to its high water solubility and can then be present in ground water. In drinking water, nitrate levels are tightly regulated, as high concentrations can lead to methemoglobinemia in infants. Overfertilization of soil leads to a higher leakage of nitrates as well as to eutrophication and subsequent changes in susceptible ecosystems. Due to the multiple physical and biological transformation processes that exist in nature, ammonia is not expected to accumulate in the environment or living organisms.

Ecotoxicology The toxicity of ammonia is commonly evaluated using ammonium salts that dissociate in water to ammonia and ammonium cation. Unionized ammonia is considered the more toxic of the two moieties. As predicted by pH partitioning, the toxicity of ammonium salts increases with increasing. pH due to the higher fraction of unionized ammonia. Unionized ammonia levels are lower at high ionic strengths which can impact the toxicity of ammonia in estuarine and marine environments. The amount of unionized ammonia also increases with increasing temperature (US EPA, 2013). The 96-h LC50 values for ammonium compounds in a variety of fish species range between 6.9 and 175 mg total ammonia per liter depending on pH and temperature; the unionized ammonia levels under these conditions typically range between 0.16 and 3.4 mg L−1. The 48-h effective concentration 50% (EC50) values for a variety of aquatic invertebrates exhibit similar ranges. While data on the toxicity of ammonia to aquatic plants are limited, available data indicate they are more tolerant to ammonia than fish or invertebrates (European Chemicals Agency (ECHA), 2022). Algae, for example, use ammonia as a nitrogen source for growth.

Exposure standards and guidelines International occupational exposure limits (OELs) generally range between 20 and 25 ppm as an 8-h TWA (time-weighted average). The American Conference of Governmental Industrial Hygienists has established an 8-h TWA OEL for ammonia of 25 ppm with a 15-min excursion limit of 35 ppm. National Institute of Occupational Safety and Health recommends a 10-h TWA OEL of 25 ppm with a 15-min excursion limit of 35 ppm and indicates that 300 ppm ammonia is immediately dangerous to life or health. OSHA has a permissible exposure limit of 50 ppm for an 8-h TWA (Occupational Safety and Health Administration (OSHA), 2022).

Other Ammonia has lower and upper explosive limits of 15% and 28% by volume in air, respectively (Andrew, 2012).

References Andrew W (2012) Ammonia. In: Pohanish RP (ed.) Sittig’s Handbook of Toxic and Hazardous Chemical Carcinogens. 6th edn. vol. 1. Waltham, MA: Elsevier. European Chemicals Agency (ECHA) (2022) Registered Substances: Ammonia, Anhydrous. https://echa.europa.eu/de/substance-information/-/substanceinfo/100.028.760. Ferguson WS, et al. (1977) Human physiological response and adaptation to ammonia. Journal of Occupational Medicine 19(5): 319–326. Holness DG, et al. (1989) Acute and chronic respiratory effects of occupational exposure to ammonia. American Industrial Hygiene Association Journal 50(12): 646–650. National Research Council (U.S.) (NRC), Subcommittee on Ammonia (1979) Ammonia, Committee on Medical and Biologic Effects of Environmental Pollutants, Division of Medical Sciences, Assembly of Life Sciences, National Research Council. Baltimore: University Park Press. Occupational Safety and Health Administration (OSHA) (2022) Occupational Chemical Database: Ammonia. Available at https://www.osha.gov/chemicaldata/623. Tsujii M, et al. (1992) Ammonia: A possible promotor in Helicobacter pylori-related gastric carcinogenesis. Cancer Letters 65(1): 15–18. US EPA (2013) Aquatic Life Ambient Water Quality Criteria for Ammonia—Freshwater. EPA-822-R-13-001.

Further reading ATSDR (2004) Toxicological Profile for Ammonia. Available at: http://www.atsdr.cdc.gov/toxprofiles/tp126.pdf WHO (1986) Environmental Health Criteria No. 54: Ammonia. Available at: http://www.inchem.org/documents/ehc/ehc/ehc54.htm Organization for Economic Cooperation and Development (OECD) (n.d.) Existing Chemicals Database. SIDS Initial Assessment Report on Ammmonia. https://hpvchemicals.oecd.org/UI/ handler.axd?id¼7a66b9ff-c0f6-4191-b90b-ea6e1f16b5e8

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Relevant websites https://pubchem.ncbi.nlm.nih.gov/compound/222 :Pubchem. Ammonia Profile. https://comptox.epa.gov/dashboard/dsstoxdb/results?search¼DTXSID0023872 :Comptox. Compound Summary Ammonia. http://www.atsdr.cdc.gov :Agency for Toxic Substances and Disease Registry. http://www.cdc.gov/niosh/topics/ammonia/ :National Institute for Occupational Safety and Health (NIOSH).

Ammonium nitrate Sofia Angela P Federicoa, Amelia B Hizon-Fradejasa, Jeb Reece H Grabatoa, and Elmer-Rico E Mojicab, aInstitute of Chemistry, University of the Philippines Los Baños, College, Laguna, Philippines; bDepartment of Chemistry and Physical Sciences, Pace University, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of P.S. Rao, Ammonium Nitrate, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 209–211, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00235-9.

Chemical profile Introduction Beirut blast Recent incidents Uses Toxicokinetics Mechanisms of action In vitro toxicity data Acute and short-term toxicity Animal Humans Chronic toxicity (or exposure) Animal Human Genotoxicity Reproductive and developmental toxicity Clinical management Ecotoxicology Exposure standards and guidelines References Further reading

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Abstract Ammonium nitrate with the chemical formula H4NO3 is a colorless or white to gray crystalline compound solid at room temperature and standard pressure. Among its common uses include a high-nitrogen fertilizer in agriculture, oxidizing agent in explosives, component in pyro techniques, and nutrient for yeast and antibiotics. It is proven to have acute and short-term, and chronic toxicity in animals and humans, in addition to potential exploitation in chemical terrorism, thus the imposition of restrictions and standards on its use such as the regulation of the Department of Homeland Security in the United States of America.

Keywords Ammonium nitrate; Explosives; Fertilizers; Nitrate; Toxicity

Chemical profile

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Name: Ammonium nitrate Chemical Abstracts Service Registry Number: 6484-52-2; 893,438–76-1; 95,255–40-6 Chemical profile: ○ Colorless, odorless, crystalline solid; hygroscopic; strong oxidant. ○ Decomposes at 200–260
C; boiling point 2101
C (with NO evolution); melting point 170
C; density 1.72 g/cm3; 80 g/mol. ○ Highly soluble in water (213 g/100 g water at 25
C), soluble in alcohol and alkalies, slightly soluble in methanol

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Ammonium nitrate

Synonyms: Ammonium nitrate; Ammonium nitricum; Ammonium saltpeter; Ammonium(I) nitrate (1:1); Caswell No. 045; EINECS 229-347-8; EPA Pesticide Chemical Code 076101; German saltpeter; HSDB 475; Herco prills; Merco prills; Nitram; Nitrate d’ammonium; Nitrate d’ammonium (French); Nitrate of ammonia; Nitrato amonico; Nitrato amonico (Spanish); Nitric acid, ammonium salt; Norway saltpeter; UNII-T8YA51M7Y6; Varioform I Molecular Formula: H4N2O3 Chemical Structure:

Introduction Ammonium nitrate is found as colorless or white to gray crystals or odorless beads with molecular weight of 80.06 g/mol and a specific gravity of 1.725 g cm−1. It has high solubility in water and has a melting point of 169.51
C and boiling point of 2101
C with evolution of nitrous oxide. Ammonium nitrate is produced by neutralizing nitric acid (HNO3) with ammonia (NH3). The substance decomposes at temperature greater than 200C and produces toxic fumes such as nitrogen oxides upon heating. Violent combustion might also happen when a heat source is sequestered in a closed container of ammonium nitrate. The compound is incompatible or reactive with some organic and inorganic compounds including acetic acid, acetic anhydride, hexamethylene tetramine acetate, and nitric acid mixture; ammonia; aluminum, calcium nitrate, and formamide mixture; metals; alkali metals; and combustible agents. Explosive reaction can be produced when it is mixed with powdered metals such as zinc, copper, lead, and aluminum. Ammonium nitrate is also a strong oxidant.

Beirut blast One of the most devastating disasters that is caused by the explosion of ammonium nitrate was the Beirut Blast that happened on August 4, 2020, in the Port of Beirut, Lebanon (El Sayed, 2022). The detonation formed a 140-m-wide crater and a 3.3 magnitude earthquake, killing an estimate of 220 individuals and injuring more than 6500, while leaving nearly 300,000 people homeless (Al-Hajj et al., 2021). The catastrophe resulted in an economic burden that exceeded 6.7 billion US dollars. The ammonium nitrate ignition emitted irritating white and brown fumes. The process by which this emission occurs begins as white ammonia nitrate mist (NH3), nitric acid (HNO3), nitrous oxide (N2O), and water vapor are formed in the gas phase. The decomposition of ammonium nitrate allows for the reaction of these four gases to form water vapor, nitrogen and toxic brown fumes mainly consisting of nitric oxides (NOx). The most harmful Nox are nitric oxide (NO) and nitrogen dioxide (NO2) which can cause severe toxicity, primarily at the lower respiratory tract. Macrophage and immune functions are potentially altered by NO2. This was pointed out as the reason for the increased risk of COVID-19 infection in the affected communities, given that the blast happened during the pandemic. Fares et al. (2021) reported an increase in the positivity rate following the explosion. Prior to August 10, 2020, the rate was 2.7%. However, a rate of 6.4% was recorded following the explosion incident (p < 0.001). Furthermore, there was an increase in hospitalized patients from 139 (on July 27, 2020) to 266 (on August 23, 2020). The patients in the Intensive Care Unit increased from 36 to 75 in the same period. The blast also produced volumes of particulate matter that remained suspended in the air for days, prolonging impacts particularly on upper respiratory health. Released particulate matter with a diameter of around 10 mm or less (PM10) comprised the blast demolition dust.

Recent incidents Several ammonium nitrate-related incidents were recorded in different parts of the world in the past 20 years with various contributing factors and consequences. The most recent event before the Beirut Blast took place in West, Texas in 2013 as ammonium nitrate fertilizers were set on fire leading to an explosion that ensued 15 fatalities and 200 injured individuals (Laboureur et al., 2016). It was well-known that Texas was the place of among the worst industrial tragedies in 1947 where 2300 tons of ammonium nitrate exploded (Stephens, 1993). In 2007, a truck containing almost 25 tons of ammonium nitrate crashed in Coahuila Mexico (Baraza et al., 2022). In the Azote de France fertilizer factory in Toulouse, France, 300 tons of ammonium nitrate were contaminated with chloride. A blast wave struck windows up to 3 km away from the factory in 2001 (Dechy et al., 2004).

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Uses One of the most common uses of ammonium nitrate is in agriculture as fertilizer. The neutralization between its nitric acid and ammonia usually gives 80% to 90% concentration and contains between 33% to 34% N in which half of it (as nitrate) is readily available. It is marketed as prills which is made by evaporating the ammonium nitrate solution to an anhydrous melt and then solidified. It can also be mixed with urea. Ammonium nitrate can also be combined with calcium carbonate in a 3:1 ratio as the additional calcium is good for fruit and vegetable crops. Ammonium nitrate is also found as a component in pyro techniques, herbicides, insecticides, freezing mixtures, rocket propellants, and nutrients for yeast and antibiotics. Coupled with fuel oil, known as ANFO, it is used as a chemical explosive for blasting rocks, mining, quarrying, and civil construction. In addition, ammonium nitrate has therapeutic uses. It is used to compensate for alkalosis induced by diuretics. It can also be used in veterinary purposes as expectorant and urinary acidifier.

Toxicokinetics Ingested nitrate goes into the stomach and is absorbed completely in the intestines. Nitrate that moves into the kidney are recirculated in the blood, and a portion is excreted into the urine. About 20% of the nitrate is converted into nitrate by commensal bacterial through reduction, while the acid in the stomach protonates nitrate and then quickly decomposes to biologically active nitrogen oxides. Nitrates can react with low-molecular weight amines in the stomach to form N-nitroso compounds which are potential carcinogens. Nitrite can oxidize hemoglobin to methemoglobin, leading to cyanosis.

Mechanisms of action The formation of ammonium nitrate from ammonia and alkali metal is described by the following chemical equations: i) NH4NO3 + NaOH ! NH3 + H2O + NaNO3 ii) NH4NO3 + KOH ! NH3 + H2O + KNO3 iii) It can also be formed in the atmosphere from emissions of NO, SO2, and NH3. Hydrochloric acid reacts with ammonium nitrate to form ammonium chloride (NH4Cl) and nitric acid (HNO3). Upon heating, ammonium nitrate can produce nitrous oxide (N2O).

In vitro toxicity data Ammonium nitrate is not genotoxic in vitro in bacterial or mammalian cell systems.

Acute and short-term toxicity Animal LD50 Rat oral: 2085 mg kg−1 LD50 Rat dermal: >5000 mg kg−1 LD50 Rat inhalation: >88.8 mg l−1 A recent study by Lee et al., (2019) evaluated the sub-chronic toxicity of ammonium nitrate in rats. The mortality, clinical signs, body weight, and food consumption were observed using relevant techniques. Their findings suggested that ammonium nitrate induces reversible salivation, increases BUN levels, stimulates acidic diuresis with decrease in sodium, potassium, and chloride levels, and causes ZG hypertrophy. The most susceptible species to nitrate toxicity are ruminants. The primary acute toxic effect of oral exposure to this compound is methemoglobinemia, which can lead to anoxia and death in extreme cases. Several factors contribute to the toxicity such as conversion of nitrate to nitrite, ability to enzymatically reduce methemoglobin, levels of vitamins A, C, D, and E in the diet, and nutritional state of the animal. Level of methemoglobin is at peak approximately 8 h after cattle are fed once a day. When cattle are fed twice daily, methemoglobin is highest 4 to 5 h after feeding. Acute poisoning in cattle is characterized by increased heart rate, muscle tremors, vomiting, weakness, blue-gray mucous membranes, excess saliva and tear production, depression, labored or violent breathing, staggering gait, frequent urination, low body temperature, disorientation, and an inability to get up. In most cases, animals die before any signs of toxicity is observed. The effect on amphibians was also documented. In 2017, Garriga et al. revealed the negative impact of ammonium nitrate by examining the impacts on the survival and growth rate of Alytes obstetricans tadpoles. They found out that growth rate of larvae decreased at 22.5 mg/L ammonia.

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Humans Ammonium nitrate causes irritation to the eyes, nose, throat, and mucous membranes. When inhaled, this can lead to severe lung congestion, coughing, difficulty in breathing, and increased acid urine. Systemic acidosis and abnormal hemoglobin can be a result of exposure to enormous amounts of this compound. Nitrates are converted to nitrites in the body that can convert hemoglobin to methemoglobin. Nitrates also cause unconsciousness, dizziness, fatigue, shortness of breath, nausea, and vomiting. Vasodilation can be an extreme body reaction to nitrates. When transferred through the breast milk it can also cause methemoglobinemia in infants who are more predisposed to nitrate-related toxicity than adults.

Chronic toxicity (or exposure) Animal In animals, excessive consumption of nitrates can result in poisoning (toxicosis) especially to ruminants (Hall, 2018). Nitrate ions can be reduced to nitrite leading to the formation of methemoglobin that inhibit oxygen transport. Ammonium nitrate orally administered to rats at subchronic level (0, 100, 300 and 1000 mg/kg/day) were found to induce reversible salivation, increase BUN levels, induce acidic diuresis with decreases in sodium, potassium, and chloride levels, and induce ZG hypertrophy (Lee et al., 2019). Male albino Albinos wistar rats treated with increasing concentrations (200, 400 and 600 mg/kg of body weight) of ammonium nitrate for 3 weeks show variation in biochemical and biological parameters: an increase in the hepato-somatic ratio and an increase in serum glucose, cholesterol, creatinine, lactate dehydrogenase, and transaminases (GOT, GPT) (Boukerche et al., 2007).

Human In a study conducted by Bae et al. (2020), it was determined that various sperm motion parameters were significantly altered after inhalation of ammonium nitrate, particularly to a range of motion kinematic parameters and to capacitation status. It also affected protein kinase A (PKA) activity and tyrosine phosphorylation regardless of capacitation. Thus, it was concluded that inhalation of ammonium nitrate may cause adverse impact on male fertility such as sperm motility, motion kinematics, and capacitation status via unusual tyrosine phosphorylation by abnormal PKA activity. The results of another study in which 45 workers of the storage and distribution of agricultural manures were exposed to nitrate derivatives indicate no poisoning has occurred, despite the observation of kidney inflammation in 50% of the population (Boukerche et al., 2007). Common findings associated with nitrate poisoning include unconsciousness, dizziness and fatigue, shortness of breath, nausea, vomiting, coma, cyanosis, dyspnea, and pallor.

Genotoxicity Ammonium nitrate is not genotoxic in vitro in bacterial or mammalian cell systems (SIDS initial assessment profile, 2008).

 OECD 473 (Test No. 473: In vitro Mammalian Chromosome Aberration Test): Negative (Yara Chemical Compliance, 2021)  OECD 471 (Test No. 471: Bacterial Reverse Mutation Test): Negative (Yara Chemical Compliance, 2021). No genotoxic effect was reported when ammonium nitrate as part of the fertilizer: pesticides mixture was fed to rats and mice in different studies (Yang, 1993).

Reproductive and developmental toxicity Teratology studies using Sprague-Dawley rats and a continuous breeding assay using CD-1 Swiss mice (Heindel et al., 1994; Yang, 1993) showed no adverse effects was observed when ammonium nitrate in pesticide/fertilizer mixtures in drinking waters at concentrations as high as 100 times the media concentrations of the individual chemicals determined by ground water surveys are fed with water to these test organisms.

Clinical management Hypotension might occur after nitrate overdose. It is advised to administer patient 10 to 20 mL/kg 0.9% saline. In patients who are comatose or at risk of convulsing, 4–8 oz of milk or water or by gastric lavage should prevent absorption. In cases of methemoglobinemia, treatment with methylene blue is used if patient is symptomatic. The dose of methylene blue is at 1–2 mg/ kg body

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weight (0.1–0.2 mL/ kg body weight of a 1% solution in saline) over a 5- to 10-min period. If there is no response within 15 min in serious cases or 30–60 min in moderate cases, a second dose of 0.1 mL/ kgbody weight can be given. For neonates, the dosage is from 0.3 to 1 mg/kg. In patients with glucose-6-phosphate dehydrogenase (G-6-PD) deficiency, methylene blue is not advisable because of potential development of severe hemolytic anemia. Treatment options include exchange transfusion and hyperbaric oxygen therapy. To regulate seizures, diazepam, anticonvulsant, or antiarrhythmic therapy. Can be ordered. If recurrent, it can be controlled by phenytoin and phenobarbital. If there is massive hemolysis, blood transfusion might be required. When inhaled, the patient must be moved to fresh air. If there is difficulty in breathing, oxygen must be administered. Bronchospasm must be treated with an inhaled beta2-adregenic agonist. Decontamination for eye exposure include irrigation with copious amounts of room temperature 0.9% saline of water for at least 15 min. When exposed dermally, areas must be washed with soap and water for 10 to 15 min with gentle sponging.

Ecotoxicology One study reported (Camargo et al., 2005) that long term exposure to nitrate concentration of 10 mg NO3-N/l can adversely affect the following organisms: freshwater invertebrates (E. toletanus, E. echinosetosus, Cheumatopsyche pettiti, Hydropsyche occidentalis), fishes (Oncorhynchus mykiss, Oncorhynchus tshawytscha, Salmo clarki), and amphibians (Pseudacris triseriata, Rana pipiens, Rana temporaria, Bufo bufo). Another study looked on the effect of ammonium nitrate and sodium nitrate on the growth of tadpoles of Alytes obsterricans. Results showed that ammonium ion showed adverse effects at lower concentration than nitrate ions (Garriga et al., 2017). Upon decomposition, ammonium nitrate will dissociate into ammonium and nitrate ions. The nitrate ion is mobile while ammonium is adsorbed by soil. Ammonia poses toxicity hazard to fishes, but not listed as a marine pollutant.

Exposure standards and guidelines As per the standards of the US Environmental Protection Agency (EPA, 2023), the maximum contaminant level for nitrates is 10 ppm in drinking water.

See also: Nitrous oxide

References Al-Hajj S, Dhaini HR, Mondello S, Kaafarani H, Kobeissy F, and DePalma RG (2021) Beirut ammonium nitrate blast: Analysis, review, and recommendations. Frontiers in Public Health 9: 657996. Bae JW, Kwon HJ, Kim SH, Ma L, Im H, Kim E, Kim MO, and Kwon WS (2020) Inhalation of ammonium sulfate and ammonium nitrate adversely affect sperm function. Reproductive Toxicology 96: 424–431. Baraza X, Giménez J, Pey A, and Rubiales M (2022) Lessons learned from the Barracas accident: Ammonium nitrate explosion during road transport. Process Safety Progress 41(3): 519–530. https://doi.org/10.1002/prs.12396. Boukerche S, Aouacheri W, and Saka S (2007) Toxicological effects of nitrate: Biological study in human and animal. Annales de Biologie Clinique (Paris) 65(4): 385–391. Camargo JA, Alonso A, and Salamanca A (2005) Nitrate toxicity to aquatic animals: A review with new data for freshwater invertebrates. Chemosphere 58(9): 1255–1267. Dechy N, Bourdeaux T, Ayrault N, Kordek MA, and Le Coze JC (2004) First lessons of the Toulouse ammonium nitrate disaster, 21st September 2001, AZF plant, France. Journal of Hazardous Materials 111(1–3): 131–138. https://doi.org/10.1016/j.jhazmat.2004.02.039. El Sayed MJ (2022) Beirut ammonium nitrate explosion: A man-made disaster in times of the COVID-19 pandemic. Disaster Medicine and Public Health Preparedness 16(3): 1203–1207. https://doi.org/10.1017/dmp.2020.451. Epub 2020 Nov 18. Fares MY, Musharrafieh U, and Bizri AR (2021) The impact of the Beirut blast on the COVID-19 situation in Lebanon. Journal of Public Health 1–7. Garriga N, Montori A, and Llorente GA (2017) Impact of ammonium nitrate and sodium nitrate on tadpoles of Alytes obstetricans. Ecotoxicology 26: 667–674. Hall JO (2018) Nitrate- and nitrite-accumulating plants. In: Gupta RC (ed.) Veterinary Toxicology, 3rd edn, pp. 941–946. Academic Press. Heindel JJ, Chapin RE, Gulati DK, et al. (1994) Assessment of the reproductive and developmental toxicity of pesticide/fertilizer mixtures based on confirmed pesticide contamination in California and Iowa groundwater. Fundamental and Applied Toxicology 22: 605–621. Laboureur DM, Han Z, Harding BZ, Pineda A, Pittman WC, Rosas C, Jiang J, and Mannan MS (2016) Case study and lessons learned from the ammonium nitrate explosion at the West Fertilizer facility. Journal of Hazardous Materials 308: 164–172. https://doi.org/10.1016/j.jhazmat.2016.01.039 Epub 2016 Jan 18 PMID: 26812084. Lee MJ, Chung YH, Choi HY, and Cha HG (2019) Evaluation of sub-chronic repeated administration toxicity of ammonium nitrate in rats. Toxicology Research 36(2): 115–122. SIDS Initial Assessment Profile (2008) https://hpvchemicals.oecd.org/ui/handler.axd?id¼c15c1d7b-3c0a-4334-bd74-58a251f0d83e. Stephens HW (1993) The Texas city disaster: A re-examination. Industrial & Environmental Crisis Quarterly 7(3): 189–204. http://www.jstor.org/stable/26162551. USEPA (2023) https://www.epa.gov/ground-water-and-drinking-water/national-primary-drinking-water-regulations. Accessed 31 January 2023. Yang R (1993) NTP technical report on the toxicity studies of pesticide/fertilizer mixtures administered in drinking water to F344/N rats and B6C3F1 mice. Toxicity Report Series 36: 1–G3. Yara Chemical Compliance (2021) Safety Data Sheet. Grimsby, North East Lincolnshire, UK: Yara UK Limited.

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Further reading Harper C, Keith S, Todd GD, Williams M, Wohlers D, Diamond GL, Coley C, and Citra MJ (2017) Toxicological profile for nitrate and nitrite. Agency for Toxic Substances and Disease Registry. Available at: https://wwwn.cdc.gov/TSP/ToxProfiles/ToxProfiles.aspx?id¼1452&tid¼258. Speight JG (2017) Industrial inorganic chemistry. chapter 3 In: Speight JG (ed.) Environmental Inorganic Chemistry for Engineers, pp. 111–169. Butterworth-Heinemann. ISBN: 9780128498910. https://doi.org/10.1016/B978-0-12-849891-0.00003-5. National Academy of Sciences (1981) The Health Effects of Nitrite, Nitrate and N-nitroso Compounds. Washington: National Academy Press. US Environmental Protection Agency (1987) Nitrate/Nitrite Health Advisory. Washington: US Environmental Protection Agency, Office of Drinking Water. US Environmental Protection Agency (1990) National Pesticide Survey: Summary Results of Pesticides in Drinking Water Wells. Washington: US Environmental Protection Agency, Office of Pesticides and Toxic Substances. Yeruham I, Shlosberg A, Hanji V, Bellaiche M, Marcus M, and Liberboim M (1997) Nitrate toxicosis in beef and dairy cattle herds due to contamination of drinking water and whey. Veterinary and Human Toxicology 39(5): 296–298. PMID: 9311087.

Relevant websites https://wwwn.cdc.gov/TSP/ToxProfiles/ToxProfiles.aspx?id¼1452&tid¼258 :(Accessed February 20, 2023): Agency for Toxic Substances and Disease Registry (ATSDR). 2017. Toxicological profile for Nitrate and Nitrite. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service. https://www.epa.gov/sites/default/files/2015-06/documents/an_advisory_6-5-15.pdf :USEPA (2015) Chemical Advisory: Safe Storage, Handling, and Management of Solid Ammonium Nitrate Prills, prepared by United States Environmental Protection Agency, Occupational Safety and Health Administration, and Bureau of Alcohol, Tobacco, Firearms and Explosives Health Administration and Explosives, EPA 550-F-15-001 June 2015. https://pubchem.ncbi.nlm.nih.gov/compound/Ammonium-nitrate :Ammonium Nitrate, Compound Summary. PubChem [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004. PubChem Compound Summary for CID 22985, Ammonium nitrate; [cited 2023 Feb. 20]. https://www.cas.org/resources/cas-insights/safety/ammonium-nitrate-making-it-safer-today-better-tomorrow :CAS, a division of American Chemical Society (ACS) – Ammonium Nitrate information resource page). http://www.dhs.gov/ammonium-nitrate-security-program :US Dept. of Homeland Security - Ammonium Nitrate Security Program. http://www.neochim.bg/files/SDS_NPK_en.pdf :Neochim Plc - Safety Data Sheet NPK AN based, compound fertilizer.

Amphetamines Timothy J Wiegand, University of Rochester Medical Center and Strong Memorial Hospital, Rochester, NY, USA © 2024 Elsevier Inc. All rights reserved. This is an update of E.M. Pallasch, M. Wahl, Amphetamines, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 212–216, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00692-8.

Chemical profile History Uses Environmental fate and behavior Physico-chemical properties Exposure routes and pathways Toxicokinetics Mechanism of toxicity Acute and short-term toxicity (or exposure) Animal Human Chronic toxicity (or exposure) Animal Human In vitro toxicity data Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Clinical management and suggested diagnostic tests Exposure standards and guidelines Miscellaneous Drug screening References Further reading

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Abstract Amphetamines (CAS 300-62-9) are a class of drug used for narcolepsy, attention deficit disorder and obesity. They are also a popular drug of abuse. Therapeutic use, abuse and overdose may lead to sympathomimetic effects such as hypertension tachycardia and seizures. Aggressive treatment for overdose may include benzodiazepines for agitation and seizures, vasodilators for hypertension, external cooling and aggressive hydration for hyperthermia.

Keywords Amphetamines; Benzodiazepines; Catecholamines; Cathinones; Mephedrone; Methamphetamine; Methylenedioxymethamphetamine; Phenethylamine; Psychostimulant; Stimulant

Key points

• • • • •

Amphetamines are a class of drug available through prescription and illicitly (e.g., methamphetamine) that are classified as stimulants. Use and abuse can lead to sympathomimetic effects including anxiety, agitation, tachycardia, hypertension and seizures. Use of illicit methamphetamine has increased in recent years, in particular with concomitant fentanyl. Some of the amphetamines, in particular the beta-keto amphetamines, or cathinones, have been sold or misrepresented in products sold as “bath salts,” “plant food,” and other products, “not for human consumption.” Benzodiazepines, cooling, and rehydration are standard treatments for patients with signs of toxicity from amphetamines. Hyperthermia and acidosis are signs of severe toxicity and should be aggressively treated. Stimulant users, in particular users of illicit methamphetamine, are at increased risk of trauma, psychiatric complications and complications from risky behaviors including sexually transmitted infections.

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Abbreviations CERCLA GC-MS MDMA MPDV

Comprehensive Environmental Response Compensation and Liability Act Gas chromatography-mass spectrometry Methylenedioxymethamphetamine Methylenedioxyprovalerone

Chemical profile

• • • • •

Chemical Abstracts Service Registry Number: CAS 300-62-9 Synonyms: Phenethylamine; racemic b-phenylisopropylamine. Phenethylamines are a large group of structurally similar agents that includes the amphetamines, hallucinogenic tryptamines and the cathinones. Amphetamines and cathinones have similar activities, but are technically subsets of phenethylamines. Slang terms for this group of stimulants include: uppers, meth, speed, ice, dexies, and crank Chemical/Pharmaceutical/Other Class: Central nervous system stimulant Chemical Formula: C9H13N Chemical Structure (see Fig. 1):

Fig. 1 Chemical structure of amphetamine.

History Amphetamine was originally synthesized in 1887 by Edeleano, a Romanian chemist working in Germany. He originally named it phenylisopropylamine. Amphetamines first became available commercially in the form of an inhaler for use as a nasal decongestant under the name Benzedrine in the 1930s. Amphetamine was used for a variety of conditions in the 20th century. It was marketed for the treatment of narcolepsy and appetite suppression and was also used off-label for schizophrenia, morphine addiction, alcoholism and behavioral issues in children. Methamphetamine was discovered in 1893. In the 1940s methamphetamine and amphetamine were used by soldiers during World War II to fight combat fatigue and amphetamine is currently permitted for use to promote wakefulness in battle. Amphetamines were readily available over the counter and by prescription until the dangers of use, abuse and addiction were recognized. Amphetamine was classified as a schedule II substance under the federal Controlled Substances Act in 1970. Amphetamines, in particular methamphetamine, has been a popular drug of abuse. This has led to illegal production and criminal activity. Motorcycle gangs would conceal methamphetamine in the crank cases of their bikes, leading to the popular slang term “crank.” Methamphetamine abuse became rampant in certain parts of the United States in the 1980s and 1990s as clandestine synthesis of methamphetamine increased. Synthetic analogues of methamphetamine, such as methylenedioxymethamphetamine (MDMA), became available. These drugs were popularized in clubs and “rave” parties. In an effort to curb illicit methamphetamine product The Methamphetamine Control Act of 1996 mandated registration of persons trading in list 1 chemicals from the DEA list of chemicals. These included phenylpropanolamine and pseudoephedrine products. In 2005 a federal law was enacted to regulate the sale of the precursors of methamphetamine (pseudoephedrine, ephedrine) at the retail level. The law limited the purchaser to a maximum of 3.6 g of pseudoephedrine per day and required identification and the signature of the purchaser at the pharmacy counter. Newer designer drugs known as “bath salts,” comprised of amphetamine-like chemicals, cathinone derivatives, (e.g., methylenedioxyprovalerone (MPDV) and mephedrone) are analogues of amphetamines and have similar clinical and toxicologic effects (Prosser and Nelson, 2012).

Uses Amphetamines are used in a wide variety of conditions but are medically approved for the treatment of attention-deficit hyperactivity disorder, narcolepsy and weight loss. Amphetamines are also popular drugs of abuse available in several forms for different routes of administration and abuse. Amphetamine can be ingested, smoked, insufflated, sniffed, and used rectally. Methamphetamine can be ingested, smoked, insufflated, sniffed and used rectally. When it is smoked methamphetamine is often called “ice” due to the crystalline appearance.

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Prescription amphetamines are available as immediate release (IR) formulations, sustained release (SR) formulations, and even as pro-ducts (e.g., lysdexamfetamine). Levomethamphetamine is an active ingredient in some over-the-counter nasal decongestants. It does not cross the blood brain barrier and thus lacks the rewarding effects of methamphetamine. Use of illicit methamphetamine has increased in recent years, in particular with concomitant fentanyl. In one study looking at the presence of methamphetamine in oral fluid toxicology tests from outpatients in a large healthcare setting in the Northeast of the United States the presence of methamphetamine increased from 0.9% to 5.1% from 2014 to 2019 (Wakeman et al., 2021).

Environmental fate and behavior Physico-chemical properties Amphetamine is a clear to colorless liquid in freebase or white crystalline substance as a salt. As a liquid it slowly volatilizes and has a characteristic amine odor. Amphetamine base is slightly soluble in water, soluble in alcohol and ether. The melting point of amphetamine is 300  C with some decomposition occurring. Methamphetamine has a melting point of 170  C (338 F), and a boiling point of 212  C (414 F) at 760 mmHg.

Exposure routes and pathways Most exposure to amphetamines are through ingestion or through other routes of common use (insufflation, intranasal, intravenous or smoked). Exposure to methamphetamine can occur through environmental contamination as well and children and other individuals on-site at a methamphetamine production center (including a “mom-and-pop” lab in a house) can be exposed through the environment at a clandestine lab. 15% of children simply around individuals who use methamphetamine will test positive for the drug and nearly 100% of children in a clandestine lab environment will have methamphetamine detectable during toxicologic analysis (e.g., urine drug testing). Therapeutic dosing for amphetamine ranges from 5 to 60 mg per day in adults and 5–40 mg per day in children 6 years of age and older. Peak plasma concentrations are dependent on the route of exposure. Absorption from the GI tract is rapid producing peak concentrations in approximately 2–3 h, vs. 30 min when used intravenously or intramuscularly. Delayed release preparations will take longer to reach peak concentrations. Absorption into the lungs via smoking reaches the brain within 7 s. More than 50% of a dose undergoes hepatic metabolism, and about 30% is excreted unchanged in urine. The amount of non-metabolized drug recovered in urine is greater with acidic urine. The half-life ranges from 8 to 30 h. Some of the amphetamines, in particular the beta-keto amphetamines, or cathinones, have been sold or misrepresented in products sold as “bath salts,” “plant food,” and other products, “not for human consumption.” Exposure to products marked as such but containing cathinones or other types of drugs could result in clinical effects (German et al., 2014).

Toxicokinetics Amphetamines are generally well absorbed from the gastrointestinal tract in therapeutic doses. Several commercially available amphetamines are formulated as sustained or delayed release products. Peak steady state serum levels are expected within 30 min after intravenous injection and within 2–3 h after ingestion of immediate release products. In overdose and with exposure to sustained release products, delays in absorption are expected. Amphetamines have a volume of distribution of approximately 3–5 L/kg with low protein binding. Amphetamines are extensively metabolized thru hepatic and renal pathways via cytochrome P450 enzymes. Cytochrome CYP2D6 interactions may be responsible for some amphetamine-related drug toxicity. Substrate competition or inhibition at this metabolic site may increase the half-life of amphetamines. Many metabolites have amphetamine-like activity (Kraemer and Maurer, 2002). Methamphetamine has an oral bioavailability of 67%, 79% intranasally, 67–90% via inhalation (smoking) and 100% intravenously. Peak plasma concentrations of methamphetamine occur 3.13–6.3 h post ingestion. Methamphetamine is highly lipophilic and it rapidly crosses the blood-brain barrier after use, faster than other stimulants. Amphetamine, as a metabolite of methamphetamine, peaks at 10–24 h after methamphetamine use. The elimination half-life of methamphetamines ranges from 5 to 30 h with average of 9–12 in most studies. There is substantial interindividual variability in methamphetamine half-life of elimination. The primary metabolites of methamphetamine are amphetamine and 4-hydroxymethamphetamine.

Mechanism of toxicity Amphetamines are indirect acting sympathomimetics, producing their effects by inhibiting the transporters of dopamine, norepinephrine and serotonin at the presynaptic nerve terminal (Fig. 2). This increases the release of norepinephrine, dopamine and serotonin and increased norepinephrine levels at central synapses, which further stimulates alpha and beta receptors. Some

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5

VMAT-2

DA MA DA DA DA

3

MA

DA DA 2

DAT

DA

1

4

MA

DAT

MA

[1] MA competitively inhibits reuptake of DA by DAT; [2] MA causes phosphorylation of DAT resulting in internalization; [3] MA inhibits DA synaptosomal uptake by VMAT-2; [4] Intracellular uptake of MA causes reverse transport of DA via DAT into synapse; [5] MA diffuses into synaptosomes impairing storage of DA

Fig. 2 Mechanism of action of methamphetamine on dopamine neurotransmission.

amphetamines also inhibit monoamine oxidase, preventing the breakdown of catecholamines. These mechanisms combine to produce the sympathomimetic and central nervous system (CNS) effects seen with amphetamine use and intoxication. An example of this is 4-methylamphetamine (4-MA). 4-MA was initially developed as an appetite suppressant but development was stopped due to adverse effects. 4-MA resurfaced as a Novel Psychoactive Substance in Europe in 2011–2012 where amphetamine samples and toxicologic analysis after fatalities from methamphetamine use showed presence of 4-MA. 4-MA has a combination of typical amphetamine effects, MAO-inhibition, and serotonergic activity. These mechanisms increase the toxicity associated with 4-MA use. Other substituted amphetamines and cathinones share some of these mechanisms, toxicity and clinical effects (Blanckaert et al., 2013).

Acute and short-term toxicity (or exposure) Animal Amphetamine toxicity in animals manifests itself in a similar way as humans. Expected signs and symptoms include hypertension, tachycardia, seizures, coma and hyperthermia. Rhabdomyolysis may also occur if agitation and hyperthermia persist. This can lead to renal failure if not treated aggressively.

Human Toxicity in humans will follow the expected sympathomimetic toxidrome. Toxicity from amphetamines can effect multiple organ systems the most serious involve the CNS and cardiovascular system. CNS effects include hypervigilance, agitation, restlessness, decreased appetite, irritability, stereotyped repetitive behavior, and insomnia with low doses. Patients may develop psychosis due to dopaminergic effects. With larger exposures, confusion, panic reactions, aggressive behavior, hallucinations, seizures, delirium, coma, and death can occur. Intracranial bleeding can result from untreated hypertension. Trauma is common secondary to behavior changes and impaired judgment. Frequent use results in fatigue, paranoia, and depression. Cardiovascular effects include tachycardia, hypertension, chest pain, myocardial ischemia or infarction, dysrhythmias, cardiovascular collapse and death. Other effects include rhabdomyolysis, increased respiratory rate, flushing, diaphoresis and dilated pupils. Hyperthermia may lead to multi system organ failure. Serotonin syndrome is also possible in overdose of certain amphetamines alone or in combination with other serotonergic agents. Symptoms include altered mental status, hyperthermia, rigidity and autonomic instability. Rising mortality related to stimulants, in particular methamphetamine, often involves concomitant use of synthetic opioids such as fentanyl (Ciccarone and Shoptaw, 2022). In this respect the toxicity is often related to the synthetic opioid use not just the methamphetamine. Methamphetamine and other amphetamines have been shown to damage brain monoaminergic cells directly. The potential for damage varies by drug, frequency and amount of use, and other factors (McCann and Ricaurte, 2004).

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Chronic toxicity (or exposure) Animal Animal models describe changes in behavior with toxicity and withdrawal. Chronic dosing of amphetamines in animals leads to stereotypic, compulsive behaviors of searching and examining in higher animals, sniffing and biting movements in lower animals. There is no increased carcinogenic activity in rats and mice fed varying doses of amphetamine over studies as long as 2 years.

Human Chronic use may result in paranoia, psychosis, bruxism, compulsive behavior and cardiomyopathy. Acute withdrawal may lead to headache, anxiety and depression. Chronic methamphetamine use can lead to neurotoxicity and neurodegeneration, cognitive impairment, and psychiatric decompensation along with psychomotor abnormalities (Ciccarone and Shoptaw, 2022).

In vitro toxicity data Several amphetamines have been shown to have monaminergic neurotoxic properties. Recent studies of PC12 dopaminergic cells have shown increased activity of capsase-3 and mitochondrial cytochrome c release. These findings suggest that amphetamines (particularly substituted amphetamines) may induce apoptosis, possibly via a mitochondrial pathway.

Reproductive toxicity Amphetamines do not appear to cause congenital abnormalities when taken during pregnancy however intrauterine growth retardation, premature delivery and maternal and fetal morbidity is significantly increased when abuse of amphetamines occurs during pregnancy. A mild withdrawal syndrome has also been reported after delivery with amphetamine use during pregnancy. Most studies of long-term follow-up of children exposed to amphetamines during pregnancy have found no significant chronic behavioral changes. Adverse effects in-utero to the vasoconstrictive effects of amphetamines have been reported including cerebral injury. Poor outcomes occurring during amphetamine exposure during pregnancy may also be due to factors other than the drug itself including multiple drug use, poor maternal health, socioeconomic factors and other life-style variables.

Genotoxicity Amphetamines are not thought to be mutagenic.

Carcinogenicity Amphetamines are not carcinogenic in humans. Some amphetamines have been shown to have beneficial effects in the treatment of certain cancers, in particular hematologic malignancies.

Clinical management After assessment of airway, breathing, and circulation with necessary supportive care, decontamination of the gastrointestinal tract should be undertaken for substantial recent ingestions. If patients present within an hour of ingestion or have taken a modified release product, consider activated charcoal; a 10:1 ratio of activated charcoal per gram of ingested substance may be administered to patients that are awake and alert and can protect their airway. Determination of specific toxic doses is difficult in chronic users of amphetamines due to the development of tolerance. Oxygen and benzodiazepines should be administered as needed for agitation, shortness of breath, or chest pain. Increased blood pressure can be managed with benzodiazepines. Although vasodilators such as nitroprusside have been recommended, reflex tachycardia is a common result. Beta blockers are not recommended for use in overdose due to possible unopposed alpha-adrenergic effects, which may lead to exacerbation of symptoms, i.e., worsening hypertension. Benzodiazepines may be necessary for agitated or combative patients. Benzodiazepines, cooling, and rehydration are standard treatments for patients with increased temperature and rhabdomyolysis. Hyperthermia is a poor prognostic sign and should be aggressively treated. Antipsychotics should be used when the primary symptoms are psychosis, in particular after prolonged use and periods of sleep deprivation. Benzodiazepines are first-line; however, for sympathomimetic symptoms. Hazards associated with methamphetamine labs include chemical and thermal burns, chemical exposures, blast injuries and inhalational injuries. Children and bystanders may be at risk to exposures, injuries, and toxicity from methamphetamine, precursor chemicals and solves, and from drug use paraphernalia as well. Response to a methamphetamine lab should include a concentrated

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team of law enforcement, emergency medical services and public health experts with experience in hazards of clandestine methamphetamine production (Vearrier et al., 2012). Stimulant users, in particular users of illicit methamphetamine, are at increased risk of trauma, psychiatric complications and complications from risky behaviors including sexually transmitted infections. Appropriate screening and education can reduce harms in certain populations when harm reduction activities are implemented (Ciccarone, 2011).

Clinical management and suggested diagnostic tests Electrolytes, urinalysis, complete blood count, urine toxicology screen, creatine kinase and cardiac enzymes are helpful laboratory tests for evaluation. In patients with severe toxicity monitor ABG, liver function tests, coagulation studies and disseminated intravascular coagulation panel.

Exposure standards and guidelines Amphetamine is a CERCLA hazardous substance and subject to the release reporting requirement of CERCLA section 103, 40 CFR parts 302 and 355. It is an Extremely Hazardous Substance (EHS) and subject to reporting requirements when stored in amounts of 1000 lbs. or greater (the threshold planning quantity).

Miscellaneous Drug screening Qualitative tests such as immunoassays may have false positive results for products containing ephedrine and pseudoephedrine. Other substances, such as bupropion, labetalol and ranitidine, may cross-react with anti-amphetamine antibodies, giving a false positive test result as well. Selegiline, a selective monoamine oxidase inhibitor type B, is partially metabolized to amphetamine and thus will give a positive result by most analytical methods. Additionally some newer designer drugs such as methylenedioxymethamphetamine (MDMA) may not react with anti-amphetamine antibodies and will result in a negative test. Limitations such as these may make clinical interpretations more difficult and imprecise. A confirmatory test, such as gas chromatography–mass spectrometry (GC–MS) offers greater specificity and sensitivity and may be the best choice in avoiding false positives and false negatives.

See also: Drugs of abuse; Methylenedioxymethamphetamine; Poisoning emergencies in humans

References Blanckaert P, van Amsterdam J, Brunt T, van den Berg J, Van Durme F, Maudens K, and Van Bussel J (2013) 4-Methyl-amphetamine: a health threat for recreational amphetamine users. Journal of Psychopharmacology 27(9): 817–822. https://doi.org/10.1177/0269881113487950. PMID: 23784740. Ciccarone D (2011) Stimulant abuse: pharmacology, cocaine, methamphetamine, treatment, attempts at pharmacotherapy. Primary Care 38(1): 41–58. https://doi.org/10.1016/j. pop.2010.11.004. PMID: 21356420. Ciccarone D and Shoptaw S (2022) Understanding stimulant use and use disorders in a new era. The Medical Clinics of North America 106(1): 81–97. https://doi.org/10.1016/j. mcna.2021.08.010. PMID: 34823736. German CL, Fleckenstein AE, and Hanson GR (2014) Bath salts and synthetic cathinones: An emerging designer drug phenomenon. Life Sciences 97(1): 2–8. https://doi.org/10.1016/ j.lfs.2013.07.023. PMID: 23911668. Kraemer T and Maurer HH (2002) Toxicokinetics of amphetamines: Metabolism and toxicokinetic data of designer drugs, amphetamine, methamphetamine and their N-alkyl derivatives. Therapeutic Drug Monitoring 24(2): 277–289. https://doi.org/10.1097/00007691-200204000-00009. PMID: 11897973. McCann UD and Ricaurte GA (2004) Amphetamine neurotoxicity: Accomplishments and remaining challenges. Neuroscience & Biobehavioral Reviews 27: 821–826. https://doi.org/ 10.1016/j.neubiorev.2003.11.003. PMID: 15019431. Prosser JM and Nelson LS (2012) The toxicology of bath salts: A review of synthetic cathinones. Journal of. Medical Toxicology 8(1): 33–42. https://doi.org/10.1007/s13181-0110193-z. PMID: 22108839. Vearrier D, Greenberg MI, Ney Miller S, Okaneku J, and Haggerty DA (2012) Methamphetamine: History, pathophysiology, adverse health effects, current trends, and hazards associated with the clandestine manufacture of methamphetamine. Disease-a-Month 58(2): 38–89. https://doi.org/10.1016/j.disamonth.2011.09.004. PMID: 22251899. Wakeman S, Flood J, and Ciccarone D (2021) Rise in presence of methamphetamine in oral fluid toxicology tests among outpatients in a large healthcare setting in the northeast. Journal of Addiction Medicine 15(1): 85–87. https://doi.org/10.1097/ADM.0000000000000695.

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Further reading Baselt RC (2002) Disposition of Toxic Drugs and Chemicals in Man, 6th edn. Foster City, CA: Biomedical Publications pp. 64–66. Biaggioni I and Robertson D (2009) Adrenoceptor agonists & sympathomimetic drugs (Chapter 9). In: Katzung B and Trevor SBMAJ (eds.) Basic and Clinical Pharmacology, 11th ed. McGraw-Hill (Access Medicine). Chiang W (2010) Amphetamines. In: Flomenbaum , et al. (ed.) Goldfrank’s Toxicologic Emergencies, 9th ed., pp. 1078–1090. New York, NY: McGraw-Hill. POISINDEXW System: Amphetamines. Klasco RK (ed.) (2012) POISINDEXW System. Greenwood Village: Thomson Reuters. Colorado Edition expires [04/2012].

Relevant websites www.erowid.org/ :Online resource for intoxicating plants and drugs –information and resources including journal articles, timelines, media, prohibition sites, harm reduction, subjective user reports and chemical information. http://health.utah.gov/meth/html/Healthconcerns/Children.html#Environmentchild :Methamphetamine facts through Utah Department of Health Website. http://emedicine.medscape.com/article/812518-overview :Amphetamine toxicity—Medscape reference an online resource on diagnosis, mechanism of toxicity, clinical management and treatment of amphetamine toxicity. http://www.aiha.org/aihce06/handouts/po118vandyke.pdf :National Jewish Medical Center power point resource on methamphetamine particle size and persistence after methamphetamine cook. http://www.dtsc.ca.gov/SiteCleanup/ERP/upload/OEHHA_Memo-Nov2007.pdf :Office of Environmental Health Hazard Assessment for children’s exposure to methamphetamine surface residue in use and clandestine laboratory environment. http://www.drugabuse.gov/sites/default/files/drugfacts_bath_salts_final_0_1.pdf :National Institute on Drug Abuse (NIDA) information on bath salts (cathinones). Https://www.ncbi.nlm.nih.gov/books/NBK546597// :Sympathomimetics—StatPearls [Internet] Horowitz AJ, Frey D., Denault D., and Smith T. 2022.

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Amyl nitrite A Kubic and M Wahl, Illinois Poison Center, Chicago, IL, United States © 2024 Elsevier Inc. All rights reserved. This is an update of A Kubic, M Wahl, Amyl nitrite, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 217–219, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00693-X.

Chemical profile Background/uses Environmental fate and behavior Routes Stability Physicochemical properties Disposition in body Toxicokinetics Mechanism of toxicity Acute and short-term exposure Human Animal Chronic toxicity Human Animal Immunotoxicity Reproductive toxicity Carcinogenicity Clinical management Exposure standards and guidelines Miscellaneous References Further reading

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Abstract Amyl nitrite (CAS: 110-46-3) is a volatile liquid supplied in ampoules historically used recreationally as ‘poppers.’ When amyl nitrite is used this way, it is usually inhaled or ‘huffed.’ Its toxicity is primarily through its oxidative effects and presents as methemoglobinemia. It also acts as a vasodilator. Treatment with methylene blue may be indicated for significant methemoglobinemia. Amyl nitrite is flammable, reactive, and an explosion hazard in large quantities or industrial settings.

Keywords Amyl nitrite; Cyanide; Methemoglobinemia; Nitrites; Poppers; Toxicity; Vasodilator

Chemical profile

• • • • •

Name: Amyl Nitrite Chemical Abstracts Service Registry Number: 110-46-3 Synonyms: 3-methylbutyl nitrite; 3-methylbutyl ester; Isopentyl alcohol nitrite; Aspiral; Nitramyl; Isoamyl nitrite; 3-methyl-1-nitrosoxybutane; Nitrous acid, Poppers (colloquial, street slang) Molecular Formula: C5H11NO2 Chemical Structure:

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Background/uses Amyl nitrite had been used clinically as early as 1867, when the Scottish physician Sir Thomas Brunton used it as a vasodilator to treat angina pectoris in his patients. In the late 1880s, a protective effect on canine cyanide toxicity was noted when amyl nitrite was inhaled postexposure. Amyl nitrite had been used clinically as an initial step in multicomponent cyanide antidote kits. However, it has been removed in favor of dual sodium nitrite/sodium thiosulfate kits or hydroxocobalamin. It is also a recreational drug of abuse (‘poppers’) (Lefevre et al., 2018).

Environmental fate and behavior Routes A volatile liquid, amyl nitrite is slightly soluble in water. It was at one time commonly supplied in ampoules that were broken and administered by inhalation for medical purposes. It is now sold in small bottles through internet sites or retail outlets and may be marketed as room deodorizers or various types of cleaners or polish. Inhalation is the most common route of exposure, though reports of ingestion of the liquid itself have been seen. Amyl nitrite may also be absorbed through the skin.

Stability Amyl nitrite is an unstable compound. It is air and light-sensitive and flammable. Amyl nitrite forms explosive mixtures with air or oxygen, which is incompatible with oxidizing and reducing agents.

Physicochemical properties Clear to yellowish liquid with a fruity odor. Molar mass 117.15 g mol−1. Density 0.872 g cm−3 liquid (25
C). Boiling point 99
C, 372 K, 210
F. Solubility is slightly soluble to insoluble in water. Flash point of 50
F (10
C). Refractive index 1.3871. Partition coefficient (log P (octanol/water)), 2.8. Amyl nitrite is highly flammable and reactive. It is an explosion and fire hazard.

Disposition in body Amyl nitrite is absorbed rapidly from mucous membranes after inhalation but rapidly inactivated by hydrolysis. Amyl nitrite undergoes hydrolysis in the gastrointestinal tract. However, ingestion can cause significant methemoglobinemia.

Toxicokinetics Amyl nitrite is rapidly absorbed from the lungs and hydrolyzed to nitrite ions and their corresponding alcohol. The body metabolizes approximately two-thirds of the ion, while the remaining one-third is excreted unchanged in the urine. When amyl nitrite is ingested, it undergoes hydrolysis in the stomach.

Mechanism of toxicity The primary mechanism of toxicity develops from the powerful oxidative effects of nitrites on hemoglobin. Methemoglobinemia develops when the iron atom in hemoglobin loses one electron to an oxidant, causing a change from the ferrous (2+) state to the ferric (3+) state. The ferric iron (3+ state) cannot bind and transport oxygen, leading to functional anemia. When methemoglobin levels exceed 10–15%, cyanosis may be evident. Nitrites also cause vasodilation by direct action on smooth muscle. Physical effects include decreases in blood pressure, headache, flushing of the face, increased heart rate, dizziness, and relaxation of involuntary muscles, especially of the blood vessels and the anal sphincter (Long, 2019). Amyl nitrite may be irritating to the lungs and throat when inhaled. With skin exposure, amyl nitrite has irritant properties and may cause dermatitis. It can also be readily absorbed, causing systemic effects through dermal absorption.

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Acute and short-term exposure Human Breathing amyl nitrite can irritate the lungs and throat, with coughing and shortness of breath symptoms. Exposure generally occurs either through inhalation or through contact with the skin. Nausea, vomiting, central nervous system (CNS) depression, and dizziness can occur shortly after exposure. Amyl nitrite may cause symptoms due to methemoglobinemia and/or vasodilation (Edwards and Ujma, 1995). Previously healthy patients who develop symptoms of methemoglobinemia may present initially with cyanosis, progressing to dizziness, fatigue, nausea, vomiting, and headache. As the methemoglobin level rises, dyspnea, severe lethargy, and confusion may develop, culminating in a possible coma, seizures, and death as the levels approach or exceed 70% (POISONDEX, 2021). A hallmark sign of methemoglobinemia is the chocolate-brown color of the blood apparent in a blood sample. Hemolysis and hemolytic anemia may be noted in patients with G6PD deficiency (Neuberger et al., 2002). Vasodilation may cause throbbing headaches and hypotension. Amyl nitrite may increase cerebral and intraocular pressure with acute exposure. It is contraindicated in patients with cerebral hemorrhage and glaucoma.

Animal Symptoms are usually consistent with methemoglobinemia, including tachypnea, weakness, and cyanotic or chocolate brown mucous membranes in equine or porcine species. Increases in intraocular pressure have been reported in some animals.

Chronic toxicity Human Chronic exposure to amyl nitrite can cause tolerance and reduced effect on the vasodilating properties of this chemical. Visual changes in chronic ‘poppers’ users have been reported, including pain, transient increased intraocular pressure, and central bilateral vision loss. Disruption in the outer segments of the fovea is reported as the mechanism in these patients. Vision returns with cessation of recreational nitrite use (Audo et al., 2011).

Animal Chronic exposure in equine populations is suggested to cause infertility, abortion, stunted growth, and animal immunosuppression. Studies in mice have shown that amyl nitrite reacts with amines to form highly carcinogenic nitrosamines.

Immunotoxicity In vitro tests show volatile nitrites, including isobutyl and amyl nitrite, to have significant immunosuppressive effects on human lymphocytes (Newell et al., 1984).

Reproductive toxicity Nitrites are excreted in breast milk and may cause symptoms of methemoglobinemia in infants. Infants may be at greater risk for nitrite toxicity due to the lack of complete NADH methemoglobin reductase enzyme system development until approximately 6 months. The use of amyl nitrite in pregnancy can cause significant harm to the fetus because maternal blood flow is significantly reduced through reductions in systemic vascular resistance, which results in less blood flow through the placenta. Amyl nitrite is pregnancy category C.

Carcinogenicity While not known to be carcinogenic in humans, the metabolized byproducts of many nitrites, when metabolized further into N-nitroso compounds, are highly carcinogenic in some animal species (Newell et al., 1984).

Clinical management The most common route of exposure will be via inhalation. Since the onset of effects is within 30 s when inhaled and lasts 3–5 min, effects due to vasodilation and decreases in systemic vascular resistance should be transient and self-limited. In patients with orthostatic hypotension, their heads should be of low posture, and movement of their extremities and raising their extremities will

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hasten recovery. If amyl nitrite is ingested, nausea and vomiting may occur and should be treated with antiemetic agents. Patients with CNS depression should be evaluated for head injury from falling and support as needed with attention to the airway and breathing. Vomiting after amyl nitrite ingestion may lead to aspiration. A chest X-ray is indicated in these patients. Patients with concerns about methemoglobinemia will need further care and monitoring. A complete blood count, arterial blood gas, and methemoglobin levels should be determined in all symptomatic patients, along with close monitoring of vital signs and electrocardiogram. Treatment with methylene blue may be considered in patients with severe methemoglobinemia (Modari et al., 2002). The use of methylene blue in patients with known G6PD deficiency is relatively contraindicated, as methylene blue can lead to hemolysis in these patients (Pranav et al., 2011). If seizures develop, initial control should be with a benzodiazepine with the addition of phenobarbital if necessary for recurrent or refractory symptoms. Hypotension may be treated initially with intravenous fluid boluses. This effect is usually short-lived and dissipates quickly as the half-life of amyl nitrite is only minutes.

Exposure standards and guidelines The US Environmental Protection Agency considers nitrites’ maximum contaminant level goal in drinking water to be less than 1 mg L−1. World Health Organization guidelines for nitrites recommend levels less than 3 mg L–1 short term and, provisionally, levels less than 0.2 mg L−1 long term. No occupational exposure limits have been set for amyl nitrite.

Miscellaneous Amyl nitrite is typically supplied in 0.3 g in 0.3 mL capsules or ampoules. Online, it may be found marketed ostensibly as solvents or deodorizers and come in 10–30 mL containers with monikers such as “Jungle Juice” or “Super Rush”. With capsules, the liquid is covered by a special mesh that allows for crushing between the fingers and subsequent inhalation by patients with angina pectoris. Amyl nitrite is highly flammable and should be kept away from flames.

See also: Butyl nitrite; Cyanide; Poisoning emergencies in humans

References Audo I, El Sanharawi M, Vignal-Clement C, Villa A, Morin A, Conrath J, Fompeydie D, Sahel JA, Gocho-Nakashima K, Goureau D, and Paques M (2011) Foveal damage in habitual popper users. Archives of Ophthalmology 129(6): 703–708. Edwards RJ and Ujma J (1995) Extreme methemoglobinemia secondary to recreational use of amyl nitrite. Journal of Accident & Emergency Medicine 12: 138–142. Lefevre T, Nuzzo A, and Megarbane B (2018) Poppers-induced life-threatening methemoglobinemia. American Journal of Respiratory and Critical Care Medicine 198(12). Long H (2019) Inhalants. In: Nelson LS, Howland M, Lewin NA, Smith SW, Goldfrank LR, and Hoffman RS (eds.) Goldfrank’s Toxicologic Emergencies, 11th edn. McGraw-Hill. https:// accesspharmacy.mhmedical.com/content.aspx?bookid¼2569§ionid¼210260247. Modari B, Kapadia YK, Kerins M, and Terris J (2002) Methylene blue: A treatment for severe methaemoglobinaemia secondary to misuse of amyl nitrite. Emergency Medicine Journal 19: 270. Neuberger A, Fishman S, and Golik A (2002) Hemolytic anemia in a G6PD deficient man after inhalation of amyl nitrite (“poppers”). The Israel Medical Association Journal 11: 1085–1086. Newell GR, Adams SC, Mansell PW, and Hersh EM (1984) Toxicity, immunosuppressive effects and carcinogenic potential of volatile nitrites: possible relation to Kaposi’s sarcoma. Pharmacotherapy 4(5): 284–291. POISONDEX (2021) POISONDEXW System (electronic version). Greenwood Village, CO: IBM Watson Health. Available at: https://www.micromedexsolutions.com/ (cited June 06, 2021). Pranav S, Bindra VK, Kapoor S, Jain V, and Saxena KK (2011) Blue cures blue but be cautious. Journal of Pharmacy & Bioallied Sciences 3(4): 543–545.

Further reading McDonagh EM, Bautista JM, Youngster I, Altman RB, and Klein TE (2013) PharmGKB summary: methylene blue pathway. Pharmacogenetics and Genomics 23(9): 498–508. https:// doi.org/10.1097/FPC.0b013e32836498f4. Reggad A, Ficko C, Andriamanantena D, Flateau C, and Rapp C (2012) Acute hemolytic anemia in an HIV patient after inhalation of amyl nitrite. Médecine et Maladies Infectieuses 42(12): 619–620.

Relevant websites www.epa.gov :United States Environmental Protection Agency-For EPA information on amyl nitrite. http://www.erowid.org/chemicals/inhalants/inhalants_info1.shtml :Vault of Erowid content on inhalants-amyl nitrite. www.who.int/en/ :World Health Organization.

Anabolic steroids Mahwish Qureshi and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.P. Sawant, H.S. Parihar, H.M. Mehendale, Anabolic Steroids, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 220-222, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00236-0.

Introduction Mechanisms of action Clinical applications Human toxicity and adverse effects Reproductive Cardiovascular Liver Musculoskeletal Psychological Kidney Control of anabolic steroids Clinical management Conclusion References Further reading

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Abstract Anabolic steroids, also known as, anabolic androgenic steroids (AAS) are the endogenously or exogenously synthesized derivatives of the male sex hormones (androgens/testosterone) that enhance the anabolic effects of androgens and decrease or eliminate the androgenic effects. AASs are used widely by athletes and adolescents for aesthetic purposes and performance enhancers to increase lean body mass and muscle growth (Torrisi et al., 2020). The actions of anabolic steroids can be classified under two categories, androgenic (associated with masculinization such as growth of the testes, external genitalia, and the male accessory reproductive glands, deepening of the voice, hair growth, acne, and behavioral effects such as increased aggressiveness) and anabolic (associated with protein building in skeletal muscle and bone). Clinically anabolic steroids use has been indicated for treating renal failure, bone marrow failure, anemia, delayed puberty, and children with reduced growth. Testosterone preparations are used in male hypogonadism, hormone replacement therapy and as male contraception (Giannoulis et al., 2012). There are several adverse effects associated with the use of anabolic steroids. Anabolic steroids in males have been reported to reduce sperm production, cause testicular atrophy, impotence, temporary infertility, and irreversible gynecomastia. In women, anabolic steroids use is reported to cause changes in or cessation of menstrual cycle, increase in body hair, clitoral enlargement, decrease in breast size and body fat, deepening of voice, and decrease in scalp hair (baldness). Long term use of anabolic steroids has been reported to affect the cardiovascular and hepatic system (Golestani et al., 2012; Cittadini et al., 2022). These agents can increase bad cholesterol, induce high blood pressure, and enhance fluid retention. Anabolic steroids are controlled substances in several countries, including Australia, Argentina, Brazil, Canada, the United Kingdom, and the United States. In 1999, various governments, intergovernmental organizations, and other public and private bodies fighting against doping in human sport in concert with International Olympic Committee started the World Anti-Doping Agency (WADA). All the rules and detailed technical documents concerning anabolic steroids (and other drugs) are constantly evolving under WADA and up to date information can be found at the WADA web site (http: //www.wada-ama.org/en/).

Keywords Anabolic Steroids; Androgens; Drug Abuse; Steroids; Testosterone

Key points

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Anabolic steroids are man-made derivatives of the male sex hormone testosterone, aka anabolic-androgenic steroids. “Anabolic” refers to muscle building, and “androgenic” refers to increased male sex characteristics. Steroids are occasionally prescribed to treat hormonal issues, such as delayed puberty, and many other conditions, such as, muscle loss, cancer and AIDS (Salvador et al., 2013). Frequent misuse by athletes and bodybuilders in an attempt to boost performance or improve their physical appearance is common.

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Anabolic steroids normally have long-term effects on the brain unlike the drugs of abuse that exhibit short-term effects. Misuse of anabolic steroids might lead to negative mental effects, such as: paranoid (extreme, unreasonable) jealousy, extreme irritability, and aggression (“roid rage”), delusions—false beliefs or ideas, impaired judgment, severe acne, and mania. Long-Term Effects are kidney problems or failure, liver injury/dysfunction and tumors, cardiomegaly, high blood pressure, and changes in blood cholesterol (increased risk of stroke and heart attack), and increased blood clots. Testicular shrinkage, declining sperm count, baldness, breast enlargement, greater risk for prostate cancer are seen in males. In females, facial hair and/or excess body hair growth, decrease breast size, male-pattern baldness, menstrual cycle dysregulation, clitoris enlargement, and voice changes are common. Teenagers exposed to steroids show stunted growth (when high hormone levels from steroids signal to the body to stop bone growth too early), many physical changes (shrinking sex organs in men and mood disorders. Steroids are not addictive, but they can lead to ‘steroid use disorder’, which can lead to: fatigue, restlessness, loss of appetite, sleep problems, decreased sex drive, steroid cravings (Nyberg and Hallberg, 2012). Studies have shown people who take steroids via injection increase their risk of contracting or transmitting HIV/AIDS or hepatitis or other infections. Even though anabolic steroids do not cause the same high as other drugs, they can lead to addiction. Although there is no specific treatment for anabolic steroid addiction, use of behavioral therapy and medications have shown beneficial outcomes. Medicines can help treat symptoms of withdrawal in some instances.

Abbreviations AAS AIDS GABA IOC NIDA WADA

Anabolic Androgenic Steroids Acquired Immunodeficiency Syndrome Gamma-amino butyric acid International Olympic Committee National Institute of Drug Abuse World Anti-Doping Agency

Introduction Anabolic steroids, also known as, anabolic androgenic steroids (AAS) or performance enhancing drugs are the endogenously or exogenously synthesized derivatives of the male sex hormones (androgens), particularly testosterone, that enhance the anabolic effects of androgens and decrease or eliminate the androgenic effects. Some of the examples of exogenous anabolic steroids are Nandrolone, Oxandrolone, Trenbolone and endogenous anabolic steroids are testosterone, androstenediol, androstenedione, and dehydroepiandrosterone. These agents show both beneficial and harmful effects depending on the exposure levels. Many organs and systems of the body including reproductive system, muscle, bone, hair follicles in the skin, liver, kidneys, the hematopoietic, and central nervous systems are affected by the action of androgens. The actions of anabolic steroids can be classified under two categories namely, androgenic, and anabolic. The androgenic effects of these steroids can be generally considered as those associated with masculinization such as growth of the testes, external genitalia and male accessory reproductive glands (prostate, seminal vesicles and bulbourethral), deepening of the voice due to enlargement of the larynx, hair growth (in the pubic, limb, chest and facial region), an increase in sebaceous glands activity which may result in acne, and neuropsychiatric or behavioral effects such as increased aggressiveness. The anabolic effects are associated with protein building in skeletal muscle and bone which may increase physical strength and body mass. In males, androgens are necessary for sustaining reproductive function, and development and maintenance of skeletal muscle and bone, and cognitive function. Testosterone has been modified structurally with an intention to afford synthetic anabolic steroids that enhance the anabolic effects of androgens and decrease or eliminate the androgenic effects. Anabolic steroids were first developed in the late 1930s in an effort to treat hypogonadism and chronic wasting. During World War II they were given to German soldiers to enhance their aggressiveness. Their use rapidly spread and after World War II athletes were openly using anabolic steroids for performance enhancement. Tightly controlled scientific studies have shown that testosterone and its derivative, nandrolone decanoate and enanthate increases athletic performance by building muscle mass and strength. Upon exposure to steroids, they diffuse everywhere including different organs and muscles inside the body. Steroids are unique in their mechanism of action because they affect individual cells and induce them to make different proteins. These proteins are sometimes needed but occasionally not so useful to the body. The liver, for example, can get the signal from the steroids and grow tumors and develop cancer. Frequent steroid abusers may sometimes develop a rare condition called peliosis hepatis in which blood-filled cysts crop up on the liver, but unfortunately, the tumors and cysts can rupture and cause internal bleeding.

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The effects of anabolic steroids are thought to increase protein synthesis and nitrogen fixation which lead to increased muscle mass and strength. In its unmodified state, testosterone must be administered intramuscularly, sublingually, or transcutaneously and is metabolized rapidly. Anabolic steroids are synthetic compounds structurally related to testosterone, binds to androgen receptors to exert its effects. Anabolic steroids are available in sublingual, parenteral, topical and oral formulations (Kicman, 2008; Niedfeldt, 2018). Representative oral forms of AS may include: Fluoxymesterone (Android-F; Halotestin), or “Halo”, Mesterolone (Proviron), Methandienone (Dianabol), or “Dbol”, Methyltestosterone (Android 5, Android 10 etc.), Oxandrolone (Oxandrin), Oxymetholone (Anadrol-50), Stanozolol (Winstrol). Examples of injectable forms of AS may include: Boldenone undecylenate (Equipoise), Methenolone enanthate (Primobolan), Nandrolone decanoate (Deca Durabolin), Nandrolone phenpropionate (Durabolin), Testosterone cypionate (Depotest), Testosterone enanthate (Andro-Estro), Testosterone propionate (Testex), and Trenbolone acetate (Finajet).

Mechanisms of action Anabolic steroids are reported to exert their actions by genomic and non-genomic mechanisms. (1) Anabolic steroids bind to androgen receptors intracellularly to form the steroid-receptor complex, and this complex translocates to the nucleus. In the nucleus, the complex interacts with the steroid response elements resulting in gene activation and transcription resulting in mRNA synthesis and consequently translation resulting in protein synthesis. The resultant proteins cause alteration in cell function, growth, or differentiation. (2) Modulation of androgen receptor activation due to intracellular metabolism of anabolic steroids. Certain anabolic steroids, particularly derivatives of testosterone, are designed to improve their protein anabolic effects and reduce the androgenic effects. For example, Nandrolone is reported to be converted by 5a- reductase enzyme to a metabolite that binds with weaker affinity to the androgen receptor in androgenic tissues and thus reducing androgen receptor mediated response of Nandrolone in androgenic tissues. In the skeletal muscle, since the 5a- reductase enzyme activity is negligible, the parent compound, Nandrolone, binds with stronger affinity to the androgen receptor thus mediating the anabolic effects. (3) An anticatabolic effect mediated by interference with glucocorticoid receptor expression; and. (4) Affecting the genome-dependent genome-independent pathways in brain such as modulation of the gamma-amino butyric acid (GABA) receptor function in the central nervous system resulting in behavioral effects. AASs increase tolerance to exercise by allowing the muscles more capable to overload. Androgens bind to the nuclear androgen receptor (AR) in the cytoplasm and translocate into the nucleus. Skeletal muscle is the main target for anabolic effects which are mediated by ARs which are up-regulated and increase in number after exposure to AAs. AASs increase tolerance to exercise by allowing the muscles more capable to overload (Frati et al., 2015).

Clinical applications Many anabolic steroids are used as therapeutic agents. In general, anabolic steroids have been indicated for treating renal failure, bone marrow failure, anemia, delayed puberty, and children with reduced growth. Testosterone preparations are used in male hypogonadism, hormone replacement therapy and as male contraception. Males with decreased circulating testosterone associated with chronic diseases or conditions, for example, those with severe burn injuries or acquired immunodeficiency syndrome (AIDS)-associated wasting may benefit from the use of anabolic steroids. The anabolic steroid, Nandrolone decanoate, is reported to be effective in treating sarcopenia (age related, involuntary loss of skeletal muscle mass and strength) in patients receiving dialysis. Oxandrolone is indicated to stimulate muscle protein anabolism in older women.

Human toxicity and adverse effects There are several reversible and irreversible adverse/side effects associated with the use of anabolic steroids. Non-medical use of anabolic steroids such as increasing physical strength, improving body composition has been associated with several adverse effects. These adverse effects are function of steroids used, the dose, duration of use and route of administration. Some of the minor side effects associated with anabolic steroids use include headache, gastrointestinal irritation, stomach pain, and oily skin (Christou et al., 2017; Esposito et al., 2021).

Reproductive In men, anabolic steroids have been reported to reduce sperm production, cause testicular atrophy (shrinking of testicles), impotence, temporary infertility, and irreversible gynecomastia. Other effects include increased frequency and duration of erection, premature sexual development, development of breast, and difficulty or pain while urinating. Anabolic steroids are also associated with priapism, prostatic carcinoma, and prostatic hypertrophy in males (Frati et al., 2015). In women, anabolic steroids use is reported to cause changes in or cessation of menstrual cycle, increase in body hair, clitoral enlargement, decrease in breast size and

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body fat, deepening of voice, and decrease in scalp hair (baldness). Anabolic steroids cause clitoral hypertrophy, uterine and atrophy in females (Frati et al., 2015).

Cardiovascular The cardiovascular effects of anabolic steroids are dose dependent. Long term use of anabolic steroids has been reported to cause hypertension, stroke, heart attack, cardiac arrhythmias, and sudden death due to cardiac failure. Anabolic steroid use had also been reported to cause premature and permanent termination of growth among adolescents. Long term use of anabolic steroids has also been associated with liver injury, hepatitis, development of fatal cysts, hepatocellular carcinoma and hepatic angiosarcoma. Anabolic steroids cause growth of sebaceous glands and increased secretion of natural oil sebum resulting in oily skin and acne (Golestani et al., 2012). Anabolic steroids use is also associated with psychiatric and behavioral effects. AS are reported to increase aggressiveness particularly when high doses are taken. Other effects reported are depression, mood swings, fatigue, restlessness, loss of appetite, and mania (Oberlander and Henderson, 2012). Long-term use of anabolic steroid use has been correlated with an increased risk of future atherosclerotic artery disease caused by an increase in LDL levels. High doses of oral anabolic steroids cause an increase in triglyceride levels as well. These effects on cholesterol and lipoproteins are reversible however and can last for several weeks (Niedfeldt, 2018). Atherogenesis can occur by AS-induced hyperhomocysteinemia. Homocysteine is a by-product of methionine metabolism and is an independent risk factor for atherosclerosis. At high levels, homocysteine inhibits vascular reendothelialization further promoting vascular inflammation causing vascular injury (Seara et al., 2020). Cardiac remodeling has been reported with high dose anabolic steroid users. There is evidence of mitochondrial injury and cardiomyocyte hypertrophy (Seara et al., 2020). Anabolic steroids are associated with myocardial apoptosis. Animal studies have shown that ventricular myocytes were isolated from adult male rabbits treated in vitro for 20 h with Stanozolol, Testosterone Enanthate, and Testosterone (Liu and Wu, 2019). Sudden cardiac death with AASs is associated with increased risk of ventricular arrhythmias, coronary flow reserve and neurohumoral activation (Torrisi et al., 2020). AAS use increases blood pressure, alters serum lipoproteins and causes direct myocardial toxicity.

Liver The effects of anabolic steroids are thought to increase liver transaminases, risk of hepatic tumors and toxicant-associated fatty liver disease. There has been an increase in case reports of liver injury due to bodybuilding supplements with oral steroids. Overall contamination rates of supplements are between 12% and 58%. Many bodybuilders who use steroids for longer and stronger exercise with great intensity show elevations of AST and ALT levels. Hepatic tumors is another complication of anabolic steroid use either benign hepatocellular adenoma (HCA) or malignant hepatocellular carcinoma (HCC). The liver is a hormone-sensitive organ to both estrogen and androgen receptors which causes HCA and HCC through the use of synthetic steroid intake as well as oral contraceptives. Tumors are most commonly discovered after long-term use, however, has been reported with short term use as well (Niedfeldt, 2018). Two main chemical substitutions to testosterone occur in anabolic steroid formulations. Esterification of the 17-b-hydroxyl group makes the molecule more longer lasting and hydrophobic. Second, 17-a-alkylation takes place which reduces hepatic metabolism allowing for anabolic steroids to be available orally. Oral steroids are resistant to immediate degradation however, slower clearance from the liver makes them potentially more hepatotoxic (Niedfeldt, 2018) Anabolic steroids increase the expression of adhesion molecules in the vascular endothelial layer, which causes the infiltration of immune cells and LDL particles into blood vessel walls. This effect was observed after 6–12 weeks use of stanozolol, nandrolone and testosterone esters (Marshall-Gradisnik et al., 2009; Seara et al., 2020).

Musculoskeletal Anabolic steroid use causes an increase rate of muscle strains/ruptures and increased risk of musculotendinous (Frati et al., 2015).

Psychological Anabolic steroid use leads to mood swings, aggressive behavior, psychosis, and addiction (Frati et al., 2015).

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Kidney Anabolic steroids cause elevated BUN and creatinine levels. It also causes acute renal failure, focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis and Wilm’s tumor (Frati et al., 2015). A lower collagen-to-smooth muscle and an increased bladder-to-body mass ratio and ratio was observed in the bladder of rats treated with testosterone (Filho et al., 2020). Parenteral formulations of AAS are associated with hypercalcemia and vitamin D overload. Hypercalcemia further leads to nephrocalcinosis, prerenal AKI and nephrolithiasis. Glomerular and interstitial damage occur via direct renal toxicity (Filho et al., 2020). Lastly, use of anabolic steroids has been indicated to develop dependency due to feeling of well-being associated with its use. Also, abusers taking his dose of anabolic steroids are also at risk to develop dependency due to withdrawal symptoms such as depression and fatigue when they stop taking anabolic steroids (Scarth et al., 2012).

Control of anabolic steroids Anabolic steroids are controlled substances in several countries, including Australia, Argentina, Brazil, Canada, the United Kingdom, and the United States. The US congress passed the Anabolic Steroid Enforcement Act in 1990 which declared anabolic steroids as controlled substance (Schedule III, Controlled Substance Act). In 2003, the Controlled Substance Act was amended to include prohormones (steroid precursors) since they may potentially act as steroid hormones. The Anabolic Steroids Control Act 2004 was introduced which took effect in January 2005, that reclassified prohormones as controlled substances by amending sections of the Controlled Substance Act and the Anabolic Steroids Enforcement Act 1990. The International Olympic Committee (IOC) Medical Commission introduced anabolic steroids as a banned class in 1974 in order to control doping in human sport. In 1999, various governments, intergovernmental organizations, and other public and private bodies fighting against doping in human sport in concert with IOC started the World Anti-Doping Agency (WADA). All the rules and detailed technical documents concerning anabolic steroids (and other drugs) are constantly evolving under WADA and up to date information can be found at the WADA web site (Rane and Ekström, 2012; http: //www.wada-ama.org/en/). Recently, in the history of sports, professional bicyclist Lance Armstrong faced accusations of using and distributing performance-enhancing drugs (steroids). Between 1999 and 2005, Lance won the Tour de France a record seven consecutive times presumably under the influence of various forms of steroids. Based on these charges, in 2012, he was expunged of all his seven Tour de France titles and banned indefinitely from cycling by the United States Anti-Doping Agency (USADA). Further, he was found to be involved in the “most sophisticated, professionalized and successful doping program that world sport has ever witnessed”. This incident has raised worldwide concern of sophisticatedly using performance-enhancing drugs by athletes to succeed in high profile competitions. Most nations are now implementing plans and procedures to screen such performers and reduce this pandemic problem (Strano-Rossi et al., 2011).

Clinical management Current knowledge is based largely on the case studies and reports from physicians who have worked with patients undergoing steroid withdrawal. These reports indicate that supportive therapy is sufficient in some cases. Patients are educated about what they may experience during withdrawal and are evaluated for suicidal thoughts. If symptoms are severe or prolonged, medications or hospitalization may be needed. Some medications restore the disrupted hormonal system due to steroid abuse. Other medications target specific withdrawal symptoms such as, antidepressants to treat depression, and analgesics for headaches, muscle, and joint pains. Some patients exhibiting psychiatric and behavioral effects require assistance beyond simple treatment of withdrawal symptoms and are treated with behavioral/psychiatric therapies.

Conclusion Anabolic steroids (AS) are synthetic drugs that promote masculinizing effects of the male sex hormone, testosterone. AS are often referred to as a ‘performance and image enhancers’ although they have legitimate medical uses. Very small % of population develop muscle mania and end up misusing AS. Improper use can cause a variety of long-term side effects which may include cardiovascular complications, liver disease, reproductive organ injury and extreme mood swings. Support from healthcare professionals is available for those individuals who have a strong will to change their dependence on these man-made drugs. Treatment options for drug dependence or addiction may include behavioral counselling, detoxification, individual counselling, and group therapy (Table 1).

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Commonly abused anabolic steroids.

Oral agents Methandrostenolone Methyltestosterone Oxandrolone Oxymetholone Stanozolol Ethylestrenol Fluoxymesterone Danazol

Injectable agents C20H2O2 C20H30O2 C19H30O3 C21H32O3 C21H32N2O C20H32O C20H29FO3 C22H27NO2

Testosterone Nandrolone Boldenone Methenolone Trenbolone Stanozolol Dromostanolone

C19H28O2 C18H26O2 C19H26O2 C20H30O2 C18H22O2 C21H32N2O C20H32O2

See also: Androgens; Behavioral toxicology; Dietary supplements

References Christou MA, Christou PA, Markozannes G, et al. (2017) Effects of Anabolic Androgenic Steroids on the Reproductive System of Athletes and Recreational Users: A Systematic Review and Meta-Analysis. Sports Medicine 47(9): 1869–1883. https://doi.org/10.1007/s40279-017-0709-z. PMID: 28258581. Cittadini A, Isidori AM, and Salzano A (2022) Testosterone therapy and cardiovascular diseases. Cardiovascular Research 118(9): 2039–2057. https://doi.org/10.1093/cvr/cvab241. Esposito M, et al. (2021) Forensic Post-Mortem Investigation in AAS Abusers: Investigative Diagnostic Protocol. A Systematic Review. Diagnostics (Basel) 11(8): 1307. https://doi.org/ 10.3390/diagnostics11081307. Filho SLA, et al. (2020) Kidney disease associated with androgenic–anabolic steroids and Vitamin Supplements Abuse: BE AWARE!: Nefrologí a. Nefrología (English Edition). Frati P, et al. (2015) Anabolic androgenic steroid (AAS) related deaths: Autoptic, histopathological and toxicological findings. Current Neuropharmacology 13(1): 146–159. https://doi. org/10.2174/1570159X13666141210225414. PMID: 26074749. Giannoulis MG, et al. (2012) Hormone replacement therapy and physical function in healthy older men. Time to talk hormones? Endocrine Reviews 33(3): 314–377. Golestani R, Slart RH, Dullaart RP, et al. (2012) Adverse cardiovascular effects of anabolic steroids: pathophysiology imaging. European Journal of Clinical Investigation 42(7): 795–803. Kicman AT (2008) Pharmacology of anabolic steroids. British Journal of Pharmacology 154: 502–521. https://doi.org/10.1038/bjp.2008.165. Liu J-D and Wu Y-Q (2019) Anabolic-androgenic steroids and cardiovascular risk. Chinese Medical Journal. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797160/. Accessed 16 November 2022. U.S. National Library of Medicine. Marshall-Gradisnik, et al. (2009) Anabolic androgenic steroids effects on the immune system: A review. Central European Journal of Biology 4(1): 19–23. Niedfeldt MW and Medical College of Wisconsin Private Practice (2018) Anabolic steroid effect on the liver. Current Sports Medicine Reports 17(3): 97–102. Nyberg F and Hallberg M (2012) Interactions between opioids and anabolic androgenic steroids: Implications for the development of addictive behavior. International Review of Neurobiology 102: 189–206. Oberlander JG and Henderson LP (2012) The Sturm und Drang of anabolic steroid use: Angst, anxiety, and aggression. Trends in Neurosciences 35(6): 382–392. Rane A and Ekström L (2012) Androgens and doping tests: Genetic variation and pit-falls. British Journal of Clinical Pharmacology 74(1): 3–15. Salvador JAR, et al. (2013) Anticancer steroids: Linking natural and semi-synthetic compounds. Natural Products Report 2013. https://doi.org/10.1039/C2NP20082A. Scarth JP, Kay J, and Teale P (2012) A review of analytical strategies for the detection of ’endogenous’ steroid abuse in food production. Drug Testing Analysis 4(Suppl 1): 40–49. Seara FAC, Olivares EL, and Nascimento JHM (2020) Anabolic steroid excess and myocardial infarction: From ischemia to reperfusion injury. Steroids. Strano-Rossi S, Fiore C, Chiarotti M, and Centini F (2011) Analytical techniques in androgen anabolic steroids (AASs) analysis for antidoping and forensic purposes. Mini Reviews in Medicinal Chemistry 11(5): 451–458. Torrisi M, et al. (2020) Sudden cardiac death in anabolic-androgenic steroid users: A literature review. Medicina (Kaunas, Lithuania) 56(11): 587. https://doi.org/10.3390/ medicina56110587.

Further reading Amsterdam JV, Opperhuizen A, and Hartgens F (2010) Adverse health effects of anabolic-androgenic steroids. Regulatory Toxicology and Pharmacology 57: 117–123. Fitch K (2020) Proscribed drugs at the Olympic Games: Permitted use and misuse (doping) by athletes. Clinical Medicine 12(3): 257–260. Kersey RD, Elliot DL, Goldberg L, et al. (2012) National Athletic Trainers’ Association position statement: anabolic-androgenic steroids. Journal of Athletic Training 47(5): 567–588. National Institute of Drug Abuse (n.d.) Fact sheets on anabolic steroids can be ordered free, by calling NIDA Infofax at 1-888-NIH-NIDA (1-888-644-6432) or, for those with hearing impairment, 1-888-TTY-NIDA (1-888-889-6432). Steroid Abuse (n.d.) Information on steroid abuse also can be accessed through the NIDA Steroid Abuse Web Site (http://www.steroidabuse.org/). Stohs SJ and Ray SD (2012) A review and evaluation of the efficacy and safety of Cissus quadrangularis extracts. Phytotherapy Research. https://doi.org/10.1002/ptr.4846.

Relevant websites http://www.drugabuse.gov/drugs-abuse/steroids-anabolic :NIDA http://teens.drugabuse.gov/facts/facts_ster1.php :NIDA Teens http://www.medicinenet.com/anabolic_steroid_abuse/article.htm :MedicineNet.com http://www.vanderbilt.edu/AnS/psychology/health_psychology/anabolic_steroids.htm :Vanderbilt University

Analytical toxicology Shayne C Gad, Gad Consulting Services, Raleigh, NC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.C. Gad, Analytical Toxicology, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 223–225, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00812-5.

Introduction Analysis of common toxic substances Gases Volatile substances Corrosives Metals Toxic anions and nonmetals Nonvolatile organic substances Analytical chemistry in environmental toxicology Miscellaneous Analytical techniques Conclusion References Further reading

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Abstract Analytical toxicology is the use of qualitative and quantitative chemical, immunological, and/or physical techniques in sample preparation, separation, assay calibration, detection and identification, and quantification for toxicological research and testing. The diagnosis and treatment of health problems induced by chemical substances and the closely allied field of therapeutic drug monitoring rely on analytical toxicology, and advances in the field have added power and problems to toxicology, dual gifts of increases in sensitivity and specificity. For both compounds of primary interest and an ever-expanding range of accompanying molecules. Other applications of analytical toxicology occur frequently during the course of experimental studies. The regulatory interest in leachable and extractable compounds has generated expanded interest for this aspect of analytical toxicology. As a principal approach to screening for and evaluating the potential risks of impurities and degradants to patients has mostly increased interest in (and depends on) evaluating toxic risks.

Keywords Anions and nonmetals; Chromatography; Corrosives; Gases; Immunoassay; Leachables and extractables; Metals; Nonvolatile organic substances; Spectroscopy; Volatile substances

Introduction Analytical toxicology is the use of qualitative and quantitative chemical, immunological, and physical techniques in sample preparation, separation, assay calibration, detection and identification, and quantification for toxicological research and testing. Examples of the objectives of such analysis include:

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Determining the levels of exposure to potential toxicants via air, water, or food. Verifying exposure levels to doses for animals in experimental studies. Determining levels of xenobiotics and their metabolites in animal studies. Screening blood and urine for the presence of illicit drugs or their metabolites. Measuring levels of endogenous compounds and molecules to evaluate organ function and damage (clinical chemistry). Identifying metabolites and macromolecular adjuncts to identify mechanisms of action. Evaluating and quantitating the formation and distribution of antibodies. Identifying and quantifying unintended molecules that appear (leachables and extractables).

The diagnosis and treatment of health problems induced by chemical substances and the closely allied field of therapeutic drug monitoring rely on analytical toxicology, and advances in the field have added both power and problems to toxicology, largely due to the dual gifts of increases in sensitivity and specificity (Brandenberger and Maes, 2015). Although the analytes are present in matrices similar to those seen in forensic toxicology, the results must be reported rapidly to be of use to clinicians in treating patients.

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This requirement of a rapid turnaround time limits the number of chemicals that can be measured because methods, equipment, and personnel must all be available for an instant response to toxicological emergencies. Investigations for an ‘unknown’ drug or poison are usually carried out on specimens of urine (30 mL for qualitative tests) and blood (10 mL for quantitative tests). No preservatives should be added to urine specimens and blood samples should be heparinized. (Baselt, 2018; Maurer, 2010). Occupational and regulatory toxicology require strict analytic procedures for implementation or monitoring. In occupational toxicology, the analytical methods used to monitor threshold limit values and other means of estimating the exposure of workers to toxic hazards may utilize simple, nonspecific, but economical screening devices. However, to determine the actual exposure of a worker, it is necessary to analyze blood, urine, breath, or another specimen by employing methods similar to those used in clinical or forensic toxicology. For regulatory purposes, a variety of matrices (e.g., food, water, and air) must be examined for extremely small quantities of analytes. Frequently, this requires the use of sophisticated methodology with extreme sensitivity. Both of these applications of analytical toxicology impinge on forensic toxicology because an injury or occupational disease in a worker can result in a legal proceeding. Other applications of analytical toxicology occur frequently during the course of experimental studies. Confirmation of the concentration of dosing solutions and monitoring of their stability often can be accomplished with the use of simple analytical techniques. The bioavailability of a dose may vary with the route of administration and the vehicle used. (Flanagan et al., 2020) Blood concentrations can be monitored as a means of establishing this important parameter. In addition, an important feature in the study of any toxic substance is the characterization of its metabolites as well as the distribution of the parent drug, together with its metabolites, to various tissues. This requires sensitive, specific, and valid analytical procedures. Similar analytical studies can be conducted within a temporal framework to gain an understanding of the dynamics of the absorption, distribution, metabolism, and excretion of toxic chemicals.

Analysis of common toxic substances Analytical toxicology is intimately involved in many aspects of experimental and applied toxicology. Because toxic substances include all chemical and biological types of entities and their measurement may require the examination of biological or nonbiological matrices, the scope of analytical toxicology is broad. Nevertheless, a systematic approach and a reliance on the practical experience of generations of forensic toxicologists can be used in conjunction with the sophisticated tools of analytical chemistry to provide the data needed to understand the hazards of toxic substances more completely. As Paracelsus states: ‘All substances are poisons: There is none which is not a poison.’ Analytical toxicology potentially encompasses all chemical substances. (Caldwell et al., 2008) Forensic toxicologists learned long ago that when the nature of a suspected poison is unknown, a systematic, standardized approach must be used to identify the presence of most common toxic substances. An approach that has stood the test of time was first suggested by Chapuis in 1873 in Elements de Toxicologie. It is based on the origin or nature of the toxic agent. Such a categorization can be characterized as follows: 1. 2. 3. 4. 5. 6. 7.

gases, volatile substances, corrosives, metals, anions and nonmetals, nonvolatile organic substances, and miscellaneous.

In addition to considering the descriptive classification of the substance, one must determine the method for separating a toxic agent from the matrix in which it is collected and embedded. The matrix is generally a biological specimen such as a body fluid or a solid tissue. The agent of interest may exist in the matrix in a simple solution or may be bound to protein and other cellular constituents. The challenge here is to separate the toxic agent in sufficient purity and quantity to permit it to be characterized and quantified. At times, the parent compound is no longer present in large enough amounts to be separated. In such cases, known metabolites may indirectly provide measure of the parent substance. With other substances, interaction of the poison with tissue components may require the isolation or characterization of a protein adduct. Methods for separation have long provided a great challenge to analytical toxicologists. Only recently have methods become available which permit direct measurement of some analytes without prior separation from the matrix. The following sections provide a closer look at analytical toxicological issues related to substance class.

Gases Gases are most simply measured by means of gas chromatography. Some gases are extremely labile, and the specimen must be collected and preserved at temperatures as low as that of liquid nitrogen. Generally, the gas is carefully liberated by incubating the specimen at a predetermined temperature in a closed container. The gas, freed from the matrix, collects over the specimen’s

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‘headspace,’ where it can be sampled and injected into the gas chromatograph. Other gases, such as carbon monoxide, interact with protein, or the adduct can be measured independently, as in the case of carboxyhemoglobin.

Volatile substances Volatile substances are generally liquids of a variety of chemical types. Gas–liquid chromatography is the simplest approach for simultaneous separation and quantitation in many cases. The simple alcohols can be measured by injecting a diluted body fluid directly onto the column of the chromatograph. A more common approach is to use the headspace technique, as is done for gases, after incubating the specimen at an elevated temperature.

Corrosives Corrosives include mineral acids and bases. Many corrosives consist of ions that are normal tissue constituents. Clinical chemical techniques can be applied to detect these ions when they are in great excess over normal concentrations. Because these ions are normal constituents, the corrosive effects at the site of contact of the chemical, together with other changes in blood chemistry values, can confirm the ingestion of a corrosive substance.

Metals Metals are encountered frequently as occupational and environmental hazards. Elegant analytical methods are available for most metals even when they are present at extremely low concentrations. Classical separation procedures involve destruction of the organic matrix by chemical or thermal oxidation. This leaves the metal to be identified and quantified in the inorganic residue. Unfortunately, this prevents a determination of the metal in the oxidation state or in combination with other elements, as it existed when the metal compound was absorbed. For example, the toxic effects of metallic mercury, mercurous ion, mercuric ion, and dimethyl mercury are all different. Analytical methods must be selected which determine the relative amount of each form present to yield optimal analytical results.

Toxic anions and nonmetals Toxic anions and nonmetals are a difficult group for analysis. Some anions can be trapped in combination with a stable cation, after which the organic matrix can be destroyed, as with metals. Others can be separated from the bulk of the matrix by dialysis, after they are detected by colorimetric or chromatopathic procedures. Still others are detected and measured by ion-specific electrodes. There are no standard approaches for this group, and other than phosphorus, they are rarely encountered in an uncombined form.

Nonvolatile organic substances Nonvolatile organic substances constitute the largest group of substances that must be considered by analytical toxicologists. This group includes drugs, both prescribed and illegal, pesticides, natural products, pollutants, and industrial compounds. These substances are solids of liquids with high boiling points. Thus, separation procedures generally rely on differential extractions, either liquid–liquid or solid–solid in nature. These extractions often are not efficient, and recovery of the toxic substance from the sample matrix may be poor. When the nature of the toxic substance is known, immunoassay procedures are useful because they allow a toxicologist to avoid using separation procedures. Such compounds can be classified as organic strong acids, organic weak acids, organic bases, organic neutral compounds, or organic amphoteric compounds.

Analytical chemistry in environmental toxicology There are many cases in which toxic agents are either present in an environment (or the life forms in an environment) or that they may be. This may pose special problems to an analytical chemist: 1. Accurately measuring exposure levels above ambient background levels. 2. Detection and accurate measurement of moieties in complex and natural matrices (i.e., seawater, soils, aquatic, and terrestrial organisms). 3. Identification and quantification of complex mixtures of chemical moieties at low levels.

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Miscellaneous Finally, a miscellaneous category must be included to cover the large number of toxic agents that cannot be detected by the routine application of the methods described previously. Venoms and other toxic mixtures of proteins or uncharacterized constituents fall into this class. Frequently, if antibodies can be grown against the active constituent, immunoassay may be the most practical means of detecting and measuring these highly potent and difficult to isolate substances. Unfortunately, unless highly specific monoclonal antibodies are used, the analytical procedure may not be acceptable for forensic purposes. Frequently, specific analytical procedures must be developed for each analyte of this type. At times, biological endpoints are utilized to semiquantify the concentration of the isolated product.

Analytical techniques Due to increased levels of sensitivity of analytical techniques and a range of legal requirements (including Good Laboratory Practices and issues in potential litigation), particular care must be taken in collecting and handling samples to both avoid contamination and maintain a chain of custody of samples and sample records (Rifai et al., 2018; Simmons, 2013; Caldwell et al., 2008). There are a vast variety of techniques now employed in analysis:

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Chromatography Thin layer, gas, high-performance liquid chromatography Mass spectrometry Photometry/spectroscopy Spectrometry (ultraviolet, infrared, and visible light) Flame photometry, atomic absorption, nuclear magnetic resonance spectroscopy, electron spin resonance spectrophotometry, Raman spectroscopy Immunoassays Radioimmunoassay, enzyme immunoassay, fluorescent immunoassay Isotopic labeling Positron emission tomography Magnetic resonance imaging

Newer and more complex material analysis techniques are:

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Accelerator mass spectrophotometry Atomic absorption spectroscopy Auger electron spectroscopy Controlled potential coulometry Crystallographic texture measurement Electrogravimetry Electrometric titration Electron probe X-ray microanalysis Elemental and functional group analysis Extended X-ray absorption fine structure Ferromagnetic resonance Field ion microscopy High temperature combustion Image analysis Inert gas fusion Inductively coupled plasma Ion chromatography Low energy electron diffraction Low energy ion scattering spectroscopy Mass spectrometry Molecular fluorescence spectrometry Mossbauer spectroscopy Neutron activation analysis Neutron diffraction Optical emission spectroscopy Optical metallography membrane electrodes Particle induced X-ray emission potentiometric Radial distribution function analysis

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Radio analysis Rutherford backscattering spectroscopy Scanning electron microscopy Secondary ion mass spectroscopy Single crystal X-ray diffraction Small angle X-ray and neutron scattering Spark source mass spectrometry Transmission electron microscopy Voltammetry Wet analytical chemistry X-ray diffraction residual stress techniques X-ray photoelectron spectroscopy X-ray powder diffraction X-ray spectrometry X-ray topography

Conclusion Analytical Chemistry seeks to identify and quantitate chemical and biologic entities in specified matrices. Originally dependent on wet chemistry methods which had limited capabilities as to both specificity and sensitivity. Available technologies are continually improving going to instrumental methods and in immunologically based methods, as well as a range of specially modification using energy-based methods such as X-rays, voltammetry along with methodologies to validate methods and results. In toxicology, details depend on the requirements of objectives, matrices, and regulatory requirements. These needs continue to change and become increasingly more complex.

References Baselt RC (2018) The Disposition of Toxic Drugs and Chemicals in Man, 11th edn. Seal Beach, CA: Biomedical Publications 1900. Brandenberger H and Maes RAA (2015) Analytical toxicology for clinical, forensic and pharmaceutical chemists. Clinical Biochemistry. vol. 5. Berlin: Walter de Gruyter Inc. Caldwell WS, Byrd GD, deEthizy JD, and Warthen GD (2008) Modern instrumental methods for studying mechanisms of toxicity. In: Hayes AW (ed.) Principles and Methods of Toxicology, 5th edn., pp. 2041–2112. Boca Raton, FL: CRC Press. Flanagan RJ, et al. (2020) Fundamentals of Analytical Toxicology, 2nd edn. Chichester, England: John Wiley and Sons. Maurer HH (2010) Analytical toxicology. EXS 100: 317–337. Rifai N, Horvath AR, and Witter CT (2018) Tietz Fundamentals of Clinical Chemistry and Molecular Diagnostics, 8th edn. Elsevier. Simmons IL (2013) Methods in Radioimmunoassay, Toxicology, and Related Areas. Springer-Verlag, 2013.

Further reading Flanagan RJ, Cuypers E, Maurer HH, and Whelpton R (2020a) Fundamentals of Analytical Toxicology, 2nd edn. Hoboken, NJ: Wiley. Stahr HM (1991) Analytical Methods in Toxicology. New York: Wiley, pp. 328.

Relevant website http://www.jatox.com :Journal of Analytical Toxicology. Preston Publications, Niles, IL.

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Ancient warfare and toxicology A Mayor, Stanford University, Palo Alto, CA, United States © 2024 Elsevier Inc. All rights reserved. This is an update of A. Mayor, Ancient Warfare and Toxicology, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 226-229, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00996-9.

Introduction Toxic weapons in mythology Historical uses of natural toxins for weapons Plant poisons Snake venom in biological warfare Poisoning water and food Stinging insects and biting snakes in biological warfare Contagions as biological warfare agents Toxic aerosols and incendiaries as chemical weapons Practical and ethical dilemmas, ancient and modern Conclusion References Further reading

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Abstract In antiquity, a wide range of natural toxins, from poisonous plants and venoms to poisonous aerosols, were exploited to wage the earliest forms of biological and chemical warfare. Evidence for the concept and practice of toxic warfare can be traced back thousands of years in written sources. Such practices arose from centuries of observation and experimentation with easily accessible toxic materials. Many modern biochemical weapons were anticipated in ancient Greece, Rome, the Middle East, and Asia. Strategies based on attacking an enemy’s biological vulnerabilities with toxic agents were advantageous while the facing troops were superior in number, bravery, skill, or technology. Yet the practical dangers of self-injury and the ethical issues posed by biochemical warfare were recognized by most ancient cultures that employed toxic weapons.

Keywords Antiquity; Biological weapons; Chemical warfare; Greece; Poison; Rome; Venom; Warfare

Key points

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Toxic substances were weaponized by ancient cultures to wage biological and chemical warfare Written texts and modern archeology record a variety of toxic weapons and tactics around the ancient world Examples of poison weapons are found in ancient Greece and Rome, the Near East, India, China, and the Americas Practical and ethical drawbacks of toxic weapons were recognized in antiquity Concepts underlying modern biochemical armaments were foreshadowed in poison weapons devised in antiquity

Introduction In antiquity, natural toxins were exploited to devise poison weapons for waging the earliest forms of biological and chemical warfare. A wide range of substances, from toxic plants and venomous insects and reptiles to infectious agents and noxious chemicals, were weaponized in ancient Europe, the Mediterranean, North Africa, the Middle East, Central Asia, India, China, and in the Americas. Evidence for the concept and practice of toxic warfare can be traced back thousands of years. For example, cuneiform tablets from about 1200 BCE record that the Hittites of Asia Minor deliberately drove plague victims into enemy territory (Faraone, 1992). Such practices did not require a scientific understanding of toxicology, epidemiology, and chemistry, or depend on advanced technology, but were based on centuries of observation and experimentation with easily available toxic materials. Strategies based on insidiously attacking an opponent’s biological vulnerabilities with poisonous agents could be advantageous when facing troops were superior in number, courage, skill, or technology. Yet the use of toxic weapons also entailed practical and ethical dilemmas in antiquity, and this was recognized in archaic Greek mythology and in ancient historical accounts.

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Toxic weapons in mythology The earliest poison projectiles were probably devised for hunting and then turned toward war. The bow and arrow proved to be a highly effective delivery system for toxins at an early date, since a mere scratch from a treated point could be fatal. The concept of poisoned projectiles is embedded in the ancient Greek language. The word for “poison,” toxicon, is derived from toxon, the word for “arrow.” Greek mythology offers further evidence of the antiquity of the concept. The great hero of myth, Heracles, for example, invented biological weaponry when he dipped his arrows in the venom of the Hydra monster, a many-headed serpent. Homer’s Iliad, an oral epic first written down in 750–650 BCE, contains indirect allusions to the use of toxic projectiles in the legendary Trojan War. Homer’s descriptions of black blood oozing from wounds, battlefield doctors sucking out poisons, and never-healing wounds are all hallmarks of snake venom poisoning. In the Odyssey, Homer describes the Greek hero Odysseus smearing lethal plant juices on arrows intended for enemies. According to myth, Odysseus himself died from a wound inflicted by a spear tipped with the toxic spine of a marbled stingray, a common species in the Mediterranean. The epic poem recounting the legendary history of Rome, the Aeneid by Virgil, also refers to poisoned spears wielded by the early Romans. Weapons delivering poisons also appear in the mythological epic of India, the Rigveda. Myth and legend likely reflect the early invention of biological arms in various cultures and they also offered models for the actual practice of biological warfare (Mayor, 2022, chapters 1 and 3).

Historical uses of natural toxins for weapons Plant poisons About two dozen toxic Eurasian plant species, often employed as medicines in tiny dosages, were collected to make arrow poisons or other biological weapons used in historical battles. One of the most popular plant drugs was hellebore, identified by the ancients as black hellebore (probably the Christmas rose of the buttercup Ranunculaceae family, Helleborus niger) and white hellebore (the lily family, Liliaceae). The unrelated plants are each laden with powerful chemicals that cause severe vomiting and diarrhea, muscle cramps, delirium, convulsions, asphyxia, and heart attack. Hellebore was one of the arrow drugs used by the Gauls, among other groups, and it was also used to poison wells. Another favorite biowar toxin was aconite or monkshood (also called wolfsbane). Aconitum (buttercup family) contains the alkaloid aconitine, a violent poison, which in high doses causes vomiting and paralyzes the nervous system, resulting in death. Aconite was employed by the archers of ancient Greece and India, and its use in warfare continued into modernity. For example, during the war between the Spanish and the Moors in 1483, Arab archers wrapped aconite-soaked cotton around their arrowheads. Nepalese Gurkhas poisoned wells with aconite in the 19th century, and during World War II, Nazi scientists created aconitine-treated bullets. Henbane (Hyoscyamus niger), a sticky, bad-smelling weed containing the powerful narcotics hyoscyamine and scopolamine, was also collected as arrow poison in antiquity. Henbane causes violent seizures, psychosis, and death. Other plant juices used on projectiles included hemlock (Conium maculatum), yew (Taxus baccata), rhododendron, and several species of deadly nightshade or belladonna, which causes vertigo, extreme agitation, coma, and death. The fact that the Latin word for deadly nightshade was dorycnion, ‘spear drug,’ suggests that it was smeared on weapons at a very early date, as noted by Pliny the Elder (21.177–79), a natural historian of the 1st century CE (Mayor, 2022, chapters 1, 2, 3).

Snake venom in biological warfare Snake venom was another well-known arrow poison. Most snake venom is digestible, making it safe for hunting because the venom did not make game meat harmful to eat. But introducing venom into the bloodstream of an enemy brought a painful death or a never-healing wound. Numerous poisonous snakes exist around the Mediterranean and in Africa and Asia. According to the Greek and Roman writers, archers who steeped their arrows in serpents’ venom included the Gauls, the Dacians and Dalmatians (of the Balkans), the Sarmatians of Persia (now Iran), the Getae of Thrace, Slavs, Armenians, Parthians between the Indus and Euphrates, Indians, North Africans, and the Scythian nomads around the Black Sea. According to the ancient Greek geographer Strabo, the arrow poison concocted by the Soanes of the Caucasus was so noxious that its mere odor was injurious, foreshadowing modern stench weapons. Strabo (16.4.10) also reported that people of what is now Kenya dipped their arrows “in the gall of serpents.” The Roman historian Silius Italicus (Punica 1.320–415, 3.265–74) described the snake venom arrows used by the archers of Libya, Morocco, Egypt, and Sudan. Ancient Chinese sources show that arrow poisons were also in use in China at early dates (Mayor, 2022, chapter 2). In the Americas, Indigenous peoples used a wide variety of snake, frog, and plant poisons on projectiles for hunting and warfare (Jones, 2007). Complex recipes for envenomed arrows similar to those devised in the Americas are recorded in Greek and Latin texts. One of the most dreaded arrow drugs was concocted by the Scythians, who combined snake venom and bacteriological agents from rotting dung, human blood, and putrefying viper carcasses bloated with feces. Even in the case of a superficial arrow wound, such toxins

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would begin taking effect within an hour. Envenomation accompanied by shock, necrosis, and suppuration of the wound would be followed by gangrene and tetanus and an agonizing death. Several snake species contributed the venom used by the Scythians, including the steppe viper Vipera ursinii renardi, the Caucasus viper Vipera kaznakovi, the European adder Vipera berus, and the long-nosed or sand viper Vipera ammodytes transcaucasiana. In ancient India, one of the most feared poisons was derived from the rotting flesh and venom of the white-headed Purple Snake, described by the natural historian Aelian (3rd century CE). His detailed description suggests that the Purple Snake was the rare, white-headed viper discovered by modern herpetologists in the late 1880s, Azemiops feae. Different snake venoms tipped the arrows encountered by the army of Alexander the Great in his conquest of India in 327–325 BCE. According to the historians Quintus Curtius, Diodorus of Sicily, and others, the defenders of Harmatelia (Mansura, Pakistan) had smeared their arrows and swords with an unknown snake poison. Most modern historians assume cobra poison, but the ancient historians’ detailed description of the gruesome deaths suffered by Alexander’s men points to the deadly Russell’s viper. Even the slightly wounded went immediately numb and experienced stabbing pain and wracking convulsions. Their skin became cold and livid and they vomited bile. Black froth exuded from the wounds and then purple-green gangrene spread rapidly, followed by death. Death from cobra venom is relatively painless, from respiratory paralysis, but the Russell’s viper causes numbness, vomiting, severe pain, black blood, gangrene, and death – as described by Alexander’s historians (Mayor, 2022, chapter 2).

Poisoning water and food Tainting water and food was another ancient biological tactic. A legendary Greek account set in about 1000 BCE tells how King Cnopus conquered Erythrae (in what is now Turkey) by drugging a bull and tricking the enemy into eating the poisoned meat (Mayor, 2022, chapter 5). The earliest historically documented case of poisoning drinking water occurred in Greece in about 590 BCE, during the First Sacred War. Athens and allied city-states made war on the strongly fortified city of Kirrha, which controlled the road to Delphi, the site of the famous Oracle of Apollo. According to several ancient Greek historians, Kirrha had offended Apollo and was therefore to be totally destroyed. During the siege, the league of allies gathered a great quantity of hellebore and placed it in the water pipes supplying Kirrha. The soldiers guarding Kirrha’s walls – and the entire population – fell violently ill. The allies easily overran the city and slaughtered combatants and civilians alike. After the war, Athens and her allies had second thoughts and agreed among themselves not to interfere with water supplies should they ever find themselves at war with each other (Mayor, 2022, chapter 3). Roman commanders also poisoned wells. Manius Aquillius, for example, ended a long-drawn-out war to quell insurrections in the Roman province of Asia Minor in 129 BCE, by pouring poison into the springs supplying the rebelling cities. According to the Roman historian Florus (1.35.5–7), however, his victory was dishonorable because of the resort to underhanded biological tactics. The Carthaginian generals Himilco and Maharbal overcame enemies in North Africa by tainting wine with mandrake, a heavily narcotic root of the deadly nightshade. In North America, Native Americans poisoned enemy’s drinking water with rotting animal skins. In ancient India, numerous recipes for poisoning enemy food and water are given in the Arthashastra, a warfare manual dating to the 4th century BCE, attributed to Kautilya, the advisor of King Chandragupta (Mayor, 2022, chapter 3; Jones, 2007; Kautilya, 1992). In 65 BCE, naturally occurring toxic honey was used against the army of the Roman general Pompey during the war against King Mithradates VI of Pontus on the southern shore of the Black Sea. Mithradates’ allies set out tempting honeycombs along the Romans’ route and hid. The honey was made by wild bees that gathered nectar from abundant rhododendron blossoms, which contain devastating neurotoxins. As the legionnaires succumbed to the sweet treat, collapsing with vertigo, vomiting, and diarrhea, the enemy ambushed and slaughtered about 1000 of Pompey’s men (Mayor, 2022, chapter 5).

Stinging insects and biting snakes in biological warfare Stinging insects and arachnids such as wasps and scorpions and deadly vipers could also be drafted for war. Hives filled with furious bees were thrown at enemies, who were driven into chaos by the painful stings; later, catapults were used to hurl beehives. The ancient Maya of Central Mexico created ingenious booby traps to repel besiegers on their fortress walls, consisting of dummy warriors whose gourd heads were filled with hornets. In the 2nd century BCE, the Carthaginian general Hannibal devised a plan of filling clay pots with live vipers during a naval battle against Pergamum, in which he was outnumbered. The enemy sailors were panicked and easily defeated when the catapulted pots smashed on their ships’ decks, releasing masses of snakes. At the fortified city of Hatra (Iraq), in 198–199 CE, besieging Roman legions led by Emperor Septimius Severus were forced to retreat after the Hatreni defended their walls with insect bombs. They had packed clay pots with scorpions (arthropods), assassin bugs, and other poisonous insects from the surrounding desert. The historian Herodian (3.9.3–8) wrote that as the insects rained down on the Romans scaling the walls, they “fell into the men’s eyes and exposed parts of their bodies, digging in, biting, and stinging the soldiers, causing severe injuries.” The terror effect would be impressive, no matter how many men were actually stung. Scorpion stings inject a complex combination of toxins, causing intense pain, thirst, great agitation, muscle spasms, convulsions, slow pulse, irregular breathing, and torturous death. Assassin bugs, predatory, bloodsucking insects with sharp beaks, inflict an extremely painful bite and inject a lethal nerve poison that liquefies tissues. It is possible that Paederus beetles were also collected by

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the Hatreni. Pederin, the virulent poison secreted by predatory Staphylinidae (rove) beetles was well-known in ancient India and China. One of the most powerful animal toxins in the world, pederin is a blistering agent on the skin and eyes, and in the bloodstream its toxicity is more potent than cobra venom (Mayor, 2022, chapter 6).

Contagions as biological warfare agents An empirical understanding of contagion developed at a surprisingly early date. In Mesopotamia in 1770 BCE, for example, cuneiform tablets warned that disease could be spread by fomites, infectious pathogens on clothing, bedding, and other items. Legends about King Solomon suggested that he concealed plague in sealed jars in the Temple of Jerusalem to infect Babylonian and Roman invaders. During the Peloponnesian War (4th century BCE), the Athenians suspected that the Spartans had spread plague by poisoning their wells. In ancient India, Kautilya’s Arthashastra (1992) suggested ways of infecting enemies with illnesses such as fevers, wasting lung disease, and rabies. In Roman times, historians such as Seneca and Dio Cassius deplored “man-made pestilence,” the malicious transmission of plagues by saboteurs who pricked victims with infected needles during the reigns of Domitian and Commodus in the first and second centuries CE. The Great Plague of 165–180 CE, probably smallpox, spread from Babylon (modern Iraq) to Syria, Italy, and Germany, transmitted by Roman soldiers returning from the war to control Mesopotamia. According to historians of the era, the epidemic began when some Roman soldiers looted a treasure chest in an enemy temple in Babylonia. The implication of the historical accounts, that the chest was booby trapped with plague-laden items, is plausible although unprovable. The local population would have had some immunity to the epidemic, while the invading Roman army would have been vulnerable. At the very least, the reports demonstrate that the notion of deliberately spreading epidemics among the enemy was widely contemplated by that time (Mayor, 2022, chapter 4).

Toxic aerosols and incendiaries as chemical weapons Asphyxiating clouds of smoke, dust, and gases were effective chemical weapons in antiquity. One of the earliest documented examples of toxic aerosols occurred during the Peloponnesian War in 429 BCE, when Sparta besieged the city of Plataea. As reported by the historian Thucydides, the Spartans created a massive fire next to Plataea’s city walls and fueled the conflagration with liberal quantities of resinous pine tree sap and sulfur. The combination of pitch and sulfur created clouds of toxic sulfur dioxide gas, fumes that can be fatal when inhaled in large amounts. A few years later, in 424 BCE, the Spartans’ allies the Boiotians invented a “flame-throwing” machine to propel similar sulfur dioxide smoke from charcoal, resin, and sulfur used against the walled city of Delium. The Greek strategist Aeneas the Tactician, writing in 360 BCE, suggested the use of incendiaries made with pitch, hemp, and sulfur. In 189 CE, the defenders of Ambracia repelled the besieging Romans by burning chicken feathers and propelled the fumes by bellows into tunnels. Feathers are composed of keratins containing cysteine, a sulfuric amino acid: combustion releases poisonous sulfur dioxide, the same kind of gas created by the Spartans at Plateia (Mayor, 2022, chapter 7). Archeologists have discovered physical evidence to show that in 256 CE, the Sassanians attacking Roman-held Dura-Europos (on the modern border of Iraq and Syria) created a similar incendiary mixture that resulted in a deadly gas enveloping a siege tunnel, killing 19 Romans and 1 Sassanian (James, 2011). In 80 BCE, the Roman general Sertorius deployed choking clouds of dust to defeat the Characitani of Spain, who had taken refuge in inaccessible caves. The fine white soil in the area consisted of limestone and gypsum. Sertorius ordered his soldiers to pile great heaps of the powder in front of the caves. When the wind was right, the Romans stirred up the dust and raised great clouds of caustic lime powder, a severe irritant to the eyes and lungs. The Characitani surrendered (Mayor, 2022, chapter 7). A similar dust was used in China to quell a revolt in 178 CE, employing bellows to blow limestone powder at opponents (Temple, 1991). The military treatise by the Byzantine emperor Leo advised a similar tactic. Powdered lime interacts with the moist membranes of the eyes, nose, and throat with corrosive, burning effect, blinding and suffocating the victims. In the Middle East, where petroleum is abundant, naphtha (the volatile and toxic light fraction of oil) was ignited and poured on attackers. The ancient Indians and Chinese added “fire chemicals” to their incendiaries, explosive saltpeter or nitrite salts, a key ingredient of gunpowder, and they also mixed a great variety of plant, animal, and mineral poisons, such as arsenic and lead, in smoke and fire bombs (Sawyer, 2007). In the Americas and in India, seeds of toxic plants and hot peppers were burned to rout attackers (Jones, 2007). Notably, in 2006, archeologists discovered evidence of chemical incendiaries used against Alexander the Great and his Macedonian army during his campaigns in India (Ali et al., 2006).

Practical and ethical dilemmas, ancient and modern The toxicity of plants, venoms, and other poisons used in armaments posed perils to those who wielded them, and the mythology and the history of poison weapons is rife with examples of accidental self-injury and unintended collateral damage. The use of windborne toxins also involved blowback problems, as acknowledged by Kautilya in his Arthashastra (1992). He advised that protective salves and other remedies be applied before deploying poisonous smokes. Toxic weapons are notoriously difficult to control and often resulted in the destruction of noncombatants as well as soldiers, especially in siege situations.

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The use of poisons in warfare led to a search for antidotes. Ancient sources list hundreds of substances that are believed to counteract specific weaponized poisons, from rust filings to poultices made from medicinal plants. It was also believed that one could become invulnerable to toxins by ingesting minute amounts of various poisons over time. Mithradates VI of Pontus (d. 63 BCE) was an early experimenter in creating a “universal antidote” later known as mithridatium and ingested by Roman emperors such as Nero and Marcus Aurelius, and later by European royalty, to gain immunity to poisoning (Mayor 2010, chapter 11). The use of toxic weapons was surrounded by ambivalence in antiquity, though there were few rules of war governing their use. Weapons that delivered hidden poisons to make an enemy defenseless or experience excessive suffering aroused moral criticism in many cultures, even as their use was rationalized in numerous recorded instances. Ancient Greeks considered poisoned projectiles a cowardly weapon, for example, yet their mythic heroes, Heracles and Odysseus, resorted to such arms, and well poisoning and toxic aerosols were used in historical Greek conflicts. Poisoned arrows and tainting water and food supplies were deplored by many Romans, yet their generals occasionally turned to such strategies (Mayor, 2022, Introduction). The Hindu Laws of Manu (dating to 500 BCE) recommended spoiling the enemy’s food and water but forbade the use of poisoned arrows. In the same era, the Arthashastra extolled the advantages of poisoning projectiles, food, and water and asphyxiating foes with chemical and disease-laden clouds of smoke. Notably, Kautilya stressed the deterrent effect of publicizing the horrid ingredients of one’s toxic arsenal, a strategy also embraced by the Scythians and others in broadcasting their recipes for poison arrows. Sun Tzu’s Art of War (500 BCE) praised deceptive terror strategies based on fire and Chinese treatises give recipes for toxic aerosols and incendiaries. On the other hand, humanitarian codes of war in China (450–200 BCE) forbade ruses of war and harming noncombatants (Kautilya, 1992; Sawyer, 2007). Self-defense was often a rationale for the use of toxic weapons. Besieged cities and desperate populations overcome by overwhelming invaders turned to biological weapons as a last resort. Some commanders used poisons in frustration to break stalemates or long sieges. Other situations, such as holy wars, quelling rebellions, and fighting people considered “less than human” encouraged the indiscriminate use of bioweapons against entire population. The threat of horrifying toxic weapons could discourage would-be attackers or bring quick capitulation. Some commanders had no compunctions about using any weapons at hand, and in some cultures poison arrows were the customary weapons in both hunting and warfare (Mayor, 2022, Introduction). The scope of human ingenuity in weaponizing natural forces in antiquity is impressive, and many of the ancient examples anticipated, in substance or principle, many basic forms of biological and chemical weapon known today, from spreading plague to poisoning water. For example, asphyxiating smokes were precursors of mustard and other toxic gases first used in World War I. Red hot sand catapulted onto Alexander the Great’s men in the 4th century BCE is analogous to modern thermite bombs of World War II. The burning, adhering effects of ancient petroleum incendiaries, which evolved to produce Greek Fire in the Byzantine era, are recapitulated in the modern invention of napalm so notorious during the Vietnam War. Even the advanced stench and noise weapons, the so-called calmatives in mists or water supplies, and top secret insect and animal-based weapons developed by modern military scientists have antecedents in the ancient world (Mayor, 2022, chapter 7). Nor are the dangers of self-injury and disposing of poison weapons anything new. The ancient myth of the Hydra with its ever-proliferating heads is a fitting symbol of the dilemmas off creating toxic arms. Faced with the problem of disposing of the immortal central head of the Hydra, Heracles buried it deep in the ground and placed a huge boulder as a marker over the spot. A similar geological solution is used today to dispose of toxic and nuclear weapons material, with burial deep underground. The practice necessitates the creation of nuclear semiotics–urgent warnings to future generations about the perils of biochemical agents. A model for avoiding the proliferation of toxic weaponry is also found in Greek myth. The archer who inherited Heracles’ Hydra-venom arrows had experienced grievous injury from the arrows himself, before he deployed them against the Trojans. After the Trojan War, he dedicated the poisonous arrows to a temple of Apollo, the god of healing, rather than passing them on to the next generation of warriors (Mayor, 2022, chapter 1 and Afterword).

Conclusion There was no time in history when biological and chemical weapons were unthinkable, and such tactics were used much earlier than previously recognized. First imagined in mythology, and then put into practice in historical times, people of antiquity weaponized naturally occurring toxins and employed chemical accelerants in fires to create noxious and lethal weapons, thus carrying out what may be considered the earliest forms of biological and chemical warfare. Literary and archeological evidence for the concept and practice of toxic warfare can be traced back thousands of years. As we have seen, a great variety of substances, including baneful plants, stinging insects, venomous reptiles, infectious agents, incendiaries, and poisonous gases, were exploited in unconventional warfare tactics, in Europe, around the Mediterranean, in North Africa, the Middle East, Central Asia, India, China, and in the Americas. The concepts underlying many of today’s most advanced biochemical armaments were foreshadowed in the unconventional weaponry that exploited biological vulnerabilities in antiquity. Notably, in each ancient culture that left writings about such weapons, similar justifications for their use were voiced, even as doubts about practical and ethical issues were also expressed.

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See also: Animals, poisonous and venomous; Chemical warfare delivery systems; Chemical warfare; Plants, poisonous (humans); Plants, poisonous (animals); Toxicology in the arts, culture, and imagination

References Ali T, et al. (2006) Southern Asia’s oldest incendiary missile? Archaeometry 48: 641–655. Faraone C (1992) Talismans and Trojan Horses. Oxford: Oxford University Press. James S (2011) Stratagems, combat, and “chemical warfare” in the siege mines of Dura-Europos. American Journal of Archaeology 115: 69–101. Jones D (2007) Poison arrows: North American Indian hunting and warfare. Austin, TX: University of Texas Press. Kautilya (1992) (4th Century BC/1992) The Arthashastra. (Edited, Rearranged, Translated and Introduced by L. N. Rangarajan) New Delhi and New York: Penguin Books. Mayor A (2022) Greek Fire, Poison Arrows and Scorpion Bombs: Unconventional Warfare in the Ancient World. Princeton, NJ: Princeton University Press. Sawyer R (2007) The Tao of Deception: Unorthodox Warfare in Historic and Modern China. New York, NY: Basic Books. Temple R (1991) The Genius of China: 3,000 Years of Science, Discovery, and Invention. London: Prion.

Further reading Bilkadi, Z. (1995) The oil weapons—ancient oil industries.” Aramco World, January–February, 22–27. Crosby AW (2002) Throwing fire: Projectile technology through history. Cambridge, UK: Cambridge University Press. Harris R and Paxman J (2002) A higher form of killing: The secret story of chemical and biological warfare, rev ed Arrow: Harmondsworth, UK. Lockwood J (2009) Six-legged soldiers: Using insects as weapons of war. Oxford, UK: Oxford University Press. Partington JR (1999) A history of Greek Fire and gunpowder. Baltimore, MD: Johns Hopkins University Press.

Androgens Eva Israilova, Davidmierhi Pinkhasov, and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of P.S. Rao, Androgens, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 230–233, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00237-2.

Chemical profile Background Uses Acute and short-term toxicity Animal Human Chronic toxicity (or exposure) animal Human Chronic poisoning Toxicokinetics Clinical management Environmental fate Ecotoxicology Other hazards Conclusion References

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Abstract Androgen, also called androgenic hormone, is the generic term for any natural or synthetic compound, usually a steroid hormone that stimulates or controls the development and maintenance of male characteristics in vertebrates by binding to androgen receptors. This includes the activity of the accessory male sex organs and development of male secondary sex characteristics. Androgens were first discovered in 1936. Androgens are also the original anabolic steroids and the precursor of all estrogens, the female sex hormones. The primary and most well-known androgen is testosterone; other less important androgens are dihydrotestosterone and androstenedione.

Keywords 5-Alpha-reductase; Anabolic steroids; Androgen; Androstenedione; Cholesterol; Dihydrotestosterone; Estradiol; Female sex hormone; Male sex hormone; Testosterone

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Androgens are a steroid hormone which controls the development and maintenance of male characteristics. Androgens play a direct role in inducing early benign prostatic hypertrophy in baboons and similar effects were seen in humans. The International Agency for Research on Cancer (IARC) has concluded that it is reasonable to regard testosterone as if it presented a carcinogenic risk to humans. Chronic exposure has led to hepatic and cardiac damage.

Chemical profile

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Name: CAS RN : D00072800. Synonym : Male sex hormones. Structure: Testosterone (left upper panel) is a potent androgen; most androgens and estrogens are produced from biotransformation of cholesterol (right upper panel) Fig. 1.

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CH2

H 2C H

CH2 CH2

CH2

H H

H HO

CH3 CH3

OH

H H

H

O OH

H H

H

HO

Fig. 1 Testosterone is converted to its more potent form Dihydrotestosterone with the enzyme 5-alpha-reductase. Testosterone can also be converted to Estrogen with the enzyme Aromatase.

Background A subset of androgens, adrenal androgens, includes any of the 19-carbon steroids synthesized by the adrenal cortex, the outer portion of the adrenal gland (zonula reticularis—innermost region of the adrenal cortex), that function as weak steroids or steroid precursors, including dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), and androstenedione. Besides testosterone, other androgens include: Dehydroepiandrosterone (DHEA): a steroid hormone produced in the adrenal cortex from cholesterol. It is the primary precursor of natural estrogens. DHEA is also called dehydroisoandrosterone or dehydroandrosterone. Androstenedione (Andro): an androgenic steroid produced by the testes, adrenal cortex, and ovaries. While androstenediones are converted metabolically to testosterone and other androgens, they are also the parent structure of estrone. Androstenediol: the steroid metabolite that is thought to act as the main regulator of gonadotropin secretion. Androsterone: a chemical by-product created during the breakdown of androgens, or derived from progesterone, that also exerts minor masculinizing effects, but with one-seventh the intensity of testosterone. It is found in approximately equal amounts in the plasma and urine of both males and females. Dihydrotestosterone (DHT): a metabolite of testosterone, and a more potent androgen than testosterone in that it binds more strongly to androgen receptors. It is produced in the adrenal cortex (Rao, 2014).

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Uses Androgens/systemic testosterone are primarily indicated in males as replacement therapy when congenital or acquired endogenous androgen absence or deficiency is associated with primary or secondary hypogonadism. Primary hypogonadism includes conditions such as: testicular failure due to cryptorchidism, bilateral torsion, orchitis, or vanishing testis syndrome; orchidectomy, Klinefelter’s syndrome, chemotherapy, or toxic damage from alcohol or heavy metals. Hypogonadotropic hypogonadism (secondary hypogonadism) conditions include idiopathic gonadotropin or LHRH deficiency or pituitary-hypothalamic injury as a result of tumors, trauma, or radiation and are the most common forms of hypogonadism seen in older adults. Recently, androgen therapy has become the mainstay of treatment for female to male transgender individuals. It increases testosterone levels, suppresses estrogens, and treats gender dysphoria (Dimakopoulou et al., 2022). Androgen therapy also has an effect on female to male transgender patients as it lowers their voice frequency and increases their hair growth. Androgens also increase their hematocrit as well as hemoglobin levels to adult male reference ranges (Pubchem-1, 2022; Pubchem-2, 2022; Dimakopoulou et al., 2022).

Acute and short-term toxicity Animal Six adult male baboons received weekly intramuscular injections of 200 mg testosterone enanthate (equivalent to 8 mg kg−1 body weight) for up to 28 weeks, while two control animals received weekly injections of the vehicle only. Quantitative increases in the weight and volume of both prostatic lobes were seen after 15 weeks of treatment, and by week 28 there was an increase in stromal tissue with papillary ingrowth or invagination of glandular epithelium in the caudal lobe of the prostate. The serum concentrations of testosterone and dihydrotestosterone were significantly elevated, from 10 and 2–3 ng/mL to 30–40 and 5–6 ng/mL, respectively. The androstenedione concentrations were increased by three to four times and that of estradiol from 20 to 80–90 pg/mL. From this study, it was concluded that these steroids play a direct role in inducing early benign prostate hypertrophy in baboons and that their observations were similar to those in human benign prostate hypertrophy (Rao, 2014).

Human Androgens may have a virilizing effect in women. The undesirable manifestations include acne, growth of facial hair, and coarsening of the voice. Profound virilization and serious disturbances in the growth and osseous development can occur when androgens are given to children. The capacity of androgens to enhance epiphyseal closure in children may persist for several months after discontinuation of the drug. All androgens should be used with great care in children. Androgens should not be used during pregnancy since they cross the placenta and cause masculinization of the female fetus. Feminizing effects, particularly gynecomastia, can occur in men who receive androgens. The feminizing effects are particularly severe in children and men with liver disease (Rao, 2014). Water retention due to sodium chloride (salt) is a common manifestation that leads to weight gain. Edema is also found in patients with cardiac heart failure, renal insufficiency, liver cirrhosis, and hypo proteinemia. When large doses are used to treat neoplastic diseases, compounds with 17-alkyl substitutions can cause cholestatic hepatitis; at high doses, jaundice is the most common clinical feature with accumulation of bile in the bile capillaries. Jaundice usually develops after 2–5 months of therapy. It can be detected by increases in plasma aspartate aminotransferase and alkaline phosphatase (Higgins et al., 2012). Obstructive sleep apnea (OSA) causes a mild lowering of blood testosterone concentrations that is rectified by effective continuous positive airway pressure (CPAP) treatment. Although testosterone treatment has precipitated OSA and has potential adverse effects on sleep in older men, the prevalence of OSA precipitated by testosterone treatment remains unclear. It appears to be a rare idiosyncratic reaction among younger hypogonadal men, but the risk may be higher among older men as the background prevalence of OSA rises steeply with age. Hence, screening for OSA by asking about daytime sleepiness and partner reports of loud and irregular snoring, especially among overweight men with large collar size, is wise for older men starting testosterone treatment although not routinely required for young men with classical hypogonadism.

Chronic toxicity (or exposure) animal The effects of subcutaneously injected or implanted testosterone and its esters have been reviewed extensively. The working group convened by the International Agency for Research on Cancer (IARC) concluded that: “there is sufficient evidence for the carcinogenicity of testosterone in experimental animals. In the absence of adequate data in humans, it is reasonable, for practical purposes, to regard testosterone as if it presented a carcinogenic risk to humans.” The relevance of animal models to human prostate disorders has been reviewed. Besides humans, dogs are the only animals that develop prostatic cancer and benign prostatic

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hyperplasia at a high frequency. In this model, long-term treatment with androgens and estrogens is required to produce hyperplasia, although such synergism is not observed in other species. ACI rats spontaneously develop histologically evident prostatic cancer, which does not progress to clinically relevant disease when pharmacologically relevant amounts of exogenous androgen are administered. Prostate cancer has been induced only in the Noble and Lobund–Wistar strains of rat (Rao, 2014). The role of hormones, including androgens, in the development of mammary neoplasia in rodents and their relevance to human risk assessment has been reviewed. Endogenous androgens are necessary for mammary development in rodents, and it was noted that rodent models mimic some but not all the complex external and endogenous factors involved in initiation, promotion, and progression of carcinogenesis. Tumor type and incidence are influenced by the age, reproductive history, and the endocrine milieu of the host at the time of exposure. The spontaneous incidence of tumors differs in different strains of rats and mice. In rats, most spontaneous neoplasias, with the exception of leukemia, occur in endocrine organs or organs under endocrine control (Wood et al., 2012). Russo and Russo concluded that mechanism-based toxicology is not yet sufficient for human risk assessment and the approach should be coupled to and validated by traditional long-term bioassays. Fischer 344 rats were given 3,20-dimethyl-4aminobiphenyl (a prostate carcinogen) at 50 mg/kg body weight 10 times at 2-week intervals, and then, from week 20, testosterone propionate and/or diethylstilbestrol by subcutaneous silastic implant for 40 weeks, as seven cycles of 30-day treatment and 10-day withdrawal. Intermittent administration of testosterone resulted in suppression of the development of ventral prostate adenocarcinomas and slight (nonsignificant) increases in the incidences of invasive carcinomas of the lateral prostate and seminal vesicles. Diethylstilbestrol completely suppressed tumorigenesis, and the combination with testosterone propionate inhibited prostate tumor development. Hydroxyprogesterone caproate was given intramuscularly every other week at an average dose of 13 mg to 19 female rabbits, and testosterone ethanoate was given intramuscularly every other week at an average dose of 15 mg to 21 animals; both treatments were given for up to 763 days. Rabbits treated with progesterone developed numerous endometrial cysts, sometimes associated with atypical hyperplasia; active mammary secretion was also seen. Treatment with testosterone induced two adenomatous polyps of the endometrium in one animal, but no other noteworthy endometrial changes were found, and one control animal developed similar polyps. Neither significantly altered other tissues such as the ovary, adrenal, thyroid, or pituitary gland. No precancerous endometrial changes or cancers were found (Rao, 2014).

Human With prolonged treatment, as in long-term use of androgens in mammary carcinoma, male pattern baldness, excessive body hair, prominent musculature, and hypertrophy of the clitoris may develop and may be irreversible. Patients receiving the 17a-alkyl substituted androgens may develop hepatic adeno carcinoma, the complications may be more common in people with Fanconi’s anemia. Erythrocytosis is the most common adverse effect seen with testosterone treatment especially in the elderly population. Hct levels have been seen to increase greater than 50% which can lead to complications such as hyper-viscosity (Khera, 2016). Anabolic steroids have also shown a correlation to reversible cardiomyopathy in young, otherwise healthy athletes (Doleeb et al., 2019). One case of a 53-year-old bodybuilder who presented with 3 months of exertional dyspnoea. He was otherwise healthy but was taking anabolic steroids. Echocardiogram showed a left ventricular ejection fraction of 15%. After discontinuation of the anabolic steroids and appropriate guideline directed heart failure therapy, his ejection fraction normalized at 6 months (Doleeb et al., 2019).

Chronic poisoning Ingestion: Hepatic damage, manifest as derangement of biochemical tests of liver function and sometimes severe enough to cause jaundice. Newer reports, however, show that jaundice due to liver toxicity have been attributed to the orally administered alkylated forms of testosterone. Although it has been shown that oral testosterone may cause jaundice, topical gel formulations have been shown to be safe and efficacious in cirrhotic patients. (Osterberg et al., 2014). The reason for this is that topical gel formulations of testosterone avoid first pass metabolism; they are therefore a better option for patients that have chronic liver cirrhosis. Other studies though, have shown that Testosterone replacement therapy may improve hepatic function in patients who have end stage liver disease. Because of these mixed results, clinicians should use their best judgment in such scenarios (Osterberg et al., 2014). Virilization in women; prostatic hypertrophy, impotence, and azoospermia in men; acne, abnormal lipids, premature cardiovascular disease (including stroke and myocardial infarction), abnormal glucose tolerance, and muscular hypertrophy in both sexes; psychiatric disturbances can occur during or after prolonged treatment. Parenteral exposure: Virilization in women; prostatic hypertrophy, impotence, and azoospermia in men; acne, abnormal lipids, premature cardiovascular disease (including stroke and myocardial infarction), abnormal glucose tolerance, and muscular hypertrophy in both sexes. Psychiatric disturbances can occur during or after prolonged treatment. Hepatic damage is not expected from parenteral preparations. Course, prognosis, cause of death: Patients with symptoms of acute poisoning are expected to recover rapidly. Patients who persistently abuse high doses of anabolic steroids are at risk of death from premature heart disease or cancer, especially prostatic cancer. Non-fatal but long-lasting effects include voice changes in women and fusion of the epiphyses in children. Other effects are reversible over weeks or months (Rao, 2014). Genotoxicity: Testosterone and its esters are not mutagenic in bacteria. This also holds for methylnortestosterone (MENT) (Organon, unpublished data). The anabolics trenbolone, fluoxymesterone and oxymetholone were found negative in bacterial

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and mammalian cell systems with equivocal results at highly toxic concentrations for trenbolone in the mouse lymphoma L5178Y TK system (Dhillon et al., 1995). Carcinogenicity: See Chronic Toxicity (Animal) section above. Carcinogenicity is a major cause of imbalance of hormones as well as hormone replacement therapy. It develops when there is some form of DNA damage. The metabolism of estrogens may in some cases lead to DNA damage. According to the pharmacological report, Endogenous estrogens-breast cancer and chemoprevention, increased levels of estrogens can lead to a disbalance and lead to damage. It states that an increased level of estrogen quinones and depurinating adducts can happen when the production of estrogen is excessive causing an unbalance. The overexpression of estrogen can result in an unbalanced metabolism and lead to a deficiency of deactivating enzymes (Starek- Swiechowicz et al., 2021). Androgens and estrogen are both hormones that have a critical role in the initiation and the development of breast cancer (Starek- Swiechowicz et al., 2021). In a case control-study post-menopausal women that had breast cancer had higher levels of serum estradiol and estrone (E1) as compared to the controls (Pubchem-3, 2022; Starek- Swiechowicz et al., 2021).

Toxicokinetics Injected as oil, androgens are so quickly absorbed, metabolized, and excreted that the effect is very small. Esters of testosterone are more slowly absorbed and are more effective. The majority of the androgens is inactivated primarily in the liver and involves oxidation of the hydroxy groups and reduction of the steroid ring. Alkylation at the 17-position retards hepatic metabolism and hence is effective orally. About 90% of the androgens are excreted in the urine; 6% appear in the feces after undergoing enterohepatic circulation. Small amounts are also excreted as soluble glucuronide and sulfate conjugates. Many of the synthetic androgens have a longer half-life. Unaltered compounds are excreted in the urine and feces (Rao, 2014).

Clinical management Edema due to salt retention is generally treated with diuretics targeted at increased sodium excretion.

Environmental fate Hormones excreted in animal waste have been measured in surface and groundwater associated with manure that is applied to the land surface. Limited studies have been done on the fate and transport of androgenic hormones in soils. There were weak correlations of sorption with soil particle size, organic matter, and specific surface area. Testosterone was the dominant compound present in the soil column effluents, although it was found that testosterone degraded more readily than 17ß-estradiol, it appeared to have a greater potential to migrate in the soil because it was not strongly absorbed (Rao, 2014).

Ecotoxicology The EDMAR program investigated evidence of changes associated with endocrine disruption in marine life and, if so, the possible causes and potential impacts. It followed on from work that demonstrated that flounder in some UK estuaries had changes consistent with endocrine disruption. Male flounder from some industrialized estuaries showed strong vitellogenin induction. Caught sand gobies exhibited no vitellogenin induction or intersex, but feminization of secondary sexual characteristics was observed in male gobies in some estuaries. Viviparous blennies in some estuaries showed induction of vitellogenin, and incidence of intersex. Toxicity identification and evaluation (TIE) procedures deployed on the Tyne and Tees estuaries identified three natural (steroidal) and two industrial (surfactant and phthalate) estrogenic compounds as possible causes of the observed effects. A study utilizing fathead minnows was conducted to study the differences in the reproductive biology between groups of minnows from a stream directly below the effluent outfall from a feedlot, from a stream that receives runoff from an agricultural field with disbursed cattle, and from uncontaminated areas upstream from the two previous sample areas. The size, sex hormone levels and gonads of the sampled fish were tested for the effects of trenbolone-b, an active synthetic anabolic steroid. The female fish near the contaminated areas were found to have higher levels of androgens in their systems and smaller distances between internal organs than those from upstream. Similarly, male minnows had smaller testicles and closer internal organs than those from uncontaminated waters. No pathology was apparent in the ovaries or testicles of the fish collected in the contaminated water (Rao, 2014).

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Other hazards For men and women, the use of male steroids (androgens)—either the hormone testosterone or the synthetic anabolic steroids—may also increase the risk of coronary artery disease. Although it has been studied that high levels of steroid use may increase the risk of coronary artery disease a recent article states that this evidence is not clear. It shows that there is an inverse relationship between the risk of CAD and testosterone levels. The article suggests that taking supplements such as testosterone may decrease the severity of Coronary Artery Disease (Osterberg et al., 2014). These drugs lower high-density lipoprotein (HDL) (the good) cholesterol levels, increase low-density lipoprotein (LDL) (the bad) cholesterol levels, and cause high blood pressure. All of these effects may contribute to having a heart attack at an early age or to having a stroke (Rao, 2014). What effects the use of anabolic steroids early in life has later in life are unclear. Although mind-altering drugs typically are those that have potential for abuse, several other drugs that do not alter the mind (or do so only occasionally) are often taken without medical need, even when doing so endangers the quality of life or health and safety of the user. Using a drug this way is considered drug abuse. People who stop abusing any of these drugs do not experience withdrawal symptoms, but they may experience medical problems when the drug is discontinued abruptly (problems that are usually preventable if discontinuation is supervised by a doctor). Anabolic steroids are very similar to the hormone testosterone. They have many physical effects on the body, including muscle growth and increased strength as well as increased energy level. Thus, anabolic steroids are often abused to gain a competitive edge in sports. Users are often athletes, typically football players, wrestlers, or weightlifters, and almost all users are male. Very high doses of anabolic steroids may cause erratic mood swings, irrational behavior, and increased aggressiveness (often called steroid rage). Increases in androgens at puberty is a major factor that contributes to male athletic advantage. In this day in age, the international Olympic Committee has determined specific requirements that transgender woman must abide by in order to compete in the female category. Testosterone levels must be suppressed below 10 nmol/L for a minimum of 12 months prior to and during the competition. Although this rule has been implicated, whether or not this removes the male performance advantage has not been proven (Hilton and Lundberg, 2021). Anabolic steroids can damage the liver and cause jaundice. Regular use of any amount also tends to increase body hair. Acne commonly gets worse with anabolic steroid use and is one of the few side effects for which an adolescent may visit a doctor. Laboratory tests can measure anabolic steroid breakdown products in the urine. Up to 6% of boys in high school, including a number of non-athletes, have used anabolic steroids at least once. A particular problem with anabolic steroid use in adolescents is early closure of the growth plates at the ends of bones, resulting in permanent short stature. Other side effects are common to both adolescents and adults (Kersey et al., 2012).

Conclusion An Androgen is term used for both natural or synthetic compounds, which is usually a steroid that stimulates the development and maintenance of male characteristics. The primary purpose of this hormone is used as a replacement therapy when there is a deficiency of endogenous androgens. While it may be beneficial for hormonal replacement therapy, misuse has led to many different toxic effects such as cancer, hepatotoxicity, as well as cardiac toxicity in humans.

References Dhillon VS, Singh J, Singh H, and Kler RS (1995) In vitro and in vivo genotoxicity of hormonal drugs. VI. Fluoxymesterone. Mutation Research 342: 103–111. Dimakopoulou A, Millar OD, Moschonas D, and Jayasena CN (2022) The role of androgens in transgender medicine. Best Practice & Research Clinical Endocrinology & Metabolism 101617. https://doi.org/10.1016/j.beem.2022.101617. Epub ahead of print PMID: 35120800. Doleeb S, Kratz A, Salter M, and Thohan V (2019) Strong muscles, weak heart: Testosterone-induced cardiomyopathy. ESC Heart Failure 6(5): 1000–1004. https://pubmed.ncbi.nlm. nih.gov/31287235/. Higgins JP, Heshmat A, and Higgins CL (2012) Androgen abuse and increased cardiac risk. Southern Medical Journal 105(12): 670–674. Hilton EN and Lundberg TR (2021) Transgender women in the female category of sport: Perspectives on testosterone suppression and performance advantage. Sports Medicine 51(2): 199–214. https://pubmed.ncbi.nlm.nih.gov/33289906/. Kersey RD, Elliot DL, Goldberg L, et al. (2012) National Athletic Trainers’ Association position statement: Anabolic-androgenicsteroids. Journal of Athletic Training 47(5): 567–588. Khera M (2016) Testosterone therapies. The Urologic Clinics of North America 43(2): 185–193. https://doi.org/10.1016/j.ucl.2016.01.004. Epub 2016 Mar 18 PMID: 27132575. Osterberg EC, Bernie AM, and Ramasamy R (2014) Risks of testosterone replacement therapy in men. Indian Journal of Urology 30(1): 2–7. https://doi.org/10.4103/09701591.124197. Pubchem-1: National Center for Biotechnology Information (2022) PubChem Compound Summary for CID 6013, Testosterone. November 26, 2022. https://pubchem.ncbi.nlm.nih. gov/compound/Testosterone. Pubchem-2: National Center for Biotechnology Information (2022) PubChem Compound Summary for CID 5997, Cholesterol. November 26, 2022. https://pubchem.ncbi.nlm.nih.gov/ compound/Cholesterol. Pubchem-3: National Center for Biotechnology Information (2022) PubChem Compound Summary for CID 5757, Estradiol. November 26, 2022. https://pubchem.ncbi.nlm.nih.gov/ compound/5757. Rao PS (2014) Androgens. Encyclopedia of Toxicology. 3rd edn., vol. 3, pp. 230–233. Elsevier. Starek-Swiechowicz B, Budziszewska B, and Starek A (2021) Endogenous estrogens-breast cancer and chemoprevention. Pharmacological Reports 73(6): 1497–1512. https://doi. org/10.1007/s43440-021-00317-0. Wood RI, et al. (2012) Roid rage in rats? Testosterone effects on aggressive motivation, impulsivity and tyrosine hydroxylase. Physiology and Behavior 110–111: 6–12. https:// pubmed.ncbi.nlm.nih.gov/23266798/.

Anesthetics Samaneh Nakhaeea and Omid Mehrpourb,c, aMedical Toxicology and Drug Abuse Research Center (MTDRC), Birjand University of Medical Sciences, Birjand, Iran; bMichigan Poison & Drug Information Center, Wayne State University School of Medicine, Detroit, MI, United States; c Data Science Institute, Southern Methodist University, Dallas, TX, USA © 2024 Elsevier Inc. All rights reserved. This is an update of F Liu, C Wang, TA Patterson, Anesthetics, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 234–237, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00003-8. This document has been reviewed following US Food and Drug Administration (FDA) policy and approved for publication. However, approval does not signify that the contents necessarily reflect the position or opinions of the FDA. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the FDA.

Anesthetics General anesthetics Inhalation anesthetics Intravenous anesthetics Uses and exposure routes and pathways Toxicokinetics Acute and short-term toxicity of general anesthetics Chronic toxicity (animal/human) Mechanism of toxicity Carcinogenesis, mutagenesis, and impairment of fertility Clinical management Local anesthetic agents Uses and exposure routes and pathways Toxicokinetics Acute and short-term toxicity of local anesthetics Chronic toxicity (animal/human) Carcinogenesis Mutagenesis and impairment of fertility Clinical management Anesthetic agents of plant origin References Further reading

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Abstract Anesthetics are generally categorized according to their functions into two classes: local anesthetics and general anesthetics. Local anesthetics are composed of esters and amides. They are administered to the skin, subcutaneous tissues, and intrathecal and epidural spaces to block pain sensation. Allergic reactions to local anesthetics are rare. General anesthetics are either gases or volatile liquids that evaporate as they are inhaled with oxygen or anesthetic agents that are administered intravenously to produce a state of unconsciousness. However, preclinical data indicate that prolonged exposure to general anesthetics has acute and even long-lasting effects on the developing central nervous system, which has called into question the safety of administering general anesthetics to the pediatric population.

Keywords Anesthesia; Anesthetics; General anesthetics; Local anesthetics

Key points

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Anesthetics can be categorized into two classes based on their functions: general and local anesthetics. General anesthetics are gases or volatile liquids that evaporate as inhaled through a mask and oxygen or are anesthetic agents administered intravenously. Inhalation anesthetics can be toxic to the liver, kidney, or blood cells. Preclinical data indicate that general anesthetics may have acute and sometimes long-lasting effects on the developing CNS. There are controversial findings regarding the potential harmful effects of general anesthetics on human patients.

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It has been suggested avoiding surgical procedures in children under 3 years old, except in emergencies. Systemic toxicity is likely to occur with Local anesthetics which involve the central nervous, cardiovascular, and immune systems. Intravenous lipid emulsion (ILE) can effectively reverse cardiovascular or neurological symptoms of local anesthetic toxicity.

Anesthetics An anesthetic is a chemical that produces a state of anesthesia. The term ‘anesthesia’ originates in Greek, meaning ‘without sensation.’ It is a medical technique generally used to induce a loss of sensation to stimuli in patients who undergo surgery or other medical procedures to avoid pain or distress they may otherwise experience. Anesthetics can be categorized based on their functions into two classes: general and local anesthetics.

General anesthetics General anesthetics induce general anesthesia, a balanced state of unconsciousness accompanied by the absence of pain sensation and paralysis of skeletal muscle over the entire body. General anesthetics are gases or volatile liquids that evaporate as inhaled through a mask and oxygen or are anesthetic agents administered intravenously. Table 1 lists commonly used general anesthetics. The precise mechanism of general anesthesia is not yet fully understood. Most of the currently used general anesthetics may act by enhancing the activity of the inhibitory neurotransmitter g-aminobutyric acid (GABA) in the central nervous system (CNS) or by antagonizing the effect of the excitatory neurotransmitter, N-methyl-D-aspartate (NMDA), on NMDA receptors. Narcotic anesthetics are an important adjunct for general anesthesia. Opioid receptors mediate their analgesic functions in the CNS, spinal cord, and periphery. There are three principal classes of opioid receptors: mu, delta, and kappa. Narcotics may be administered intravenously, intrathecally, or into epidural space. There are four categories of narcotics: natural alkaloids of opium, synthetic derivatives of morphine, synthetic agents resembling the morphine structure, and narcotic antagonists used as antidotes in narcotic overdose cases. Fentanyl (also known as fentanil) is a commonly used synthetic narcotic due to its better lipid solubility, more rapid onset of action, more potent anesthetic effect, absence of histamine release, and independence of renal function for drug clearance. Sufentanil and alfentanil are two examples of newer synthetic narcotics.

Inhalation anesthetics Inhalation anesthetics are compounds that enter the body through the lungs and are carried by the blood to tissues. An ideal inhalation anesthetic should have great potency, low solubility in blood and tissues, resistance to physical and metabolic degradation, and a protective effect and lack of injury to vital tissues. The potency of an inhalation anesthetic is determined by the minimum alveolar concentration (MAC), which is the concentration of an inhaled vapor in the lung at a steady state, at which 50% of subjects do not react to a standard surgical stimulus at 1 atm. A potent volatile anesthetic has a lower MAC value. Some inhalation anesthetics have been reported to cause seizures and agitation and irritate the airways. Sevoflurane has an intermediate solubility in blood and tissues and does not cause respiratory irritation, circulatory stimulation, or hepatotoxicity. Therefore,

Table 1

Commonly used general anesthetics.

Generic name

CAS Registry number

Chemical formula

Dosage forms/routes

Desflurane Enflurane Etomidate Halothane Isoflurane Ketamine hydrochloride Methohexital sodium Midazolam hydrochloride Nitrous oxide Propofol Sevoflurane Thiopental sodium

57041-67-5 13838-16-9 33125-97-2 151-67-7 26675-46-7 1867-66-9 22151-68-4 59467-96-8 10024-97-2 2078-54-8 28523-86-6 71-73-8743

C3H2F6O C3H2ClF5O C14H16N2O2 C2HBrClF3 C3H2ClF5O C13H17Cl2NO C14H17N2NaO3 C18H14Cl2FN3 N2O C12H18O C4H3F7O C11H17N2NaO2S

Volatile liquid/inhalation Volatile liquid/inhalation Injectable/injection Volatile liquid/inhalation Volatile liquid/inhalation Injectable/injection Injectable/injection Injectable/injection; syrup/oral Gas/inhalation Injectable/injection Volatile liquid/inhalation Injectable/injection

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sevoflurane is currently the most popular anesthetic agent in North America for anesthesia induction by inhalation. The depth of anesthesia induced by inhalation anesthetics can be adjusted rapidly by altering the anesthetic-to-oxygen ratio inhaled by the patient.

Intravenous anesthetics Similar to inhalation anesthetics, an ideal intravenous anesthetic agent should have a rapid onset of action; be quickly cleared from the circulation and CNS to facilitate the adjustment of the depth of anesthesia; protect vital tissues; and not affect the circulatory system or have other adverse effects. Among these agents, propofol is commonly used for the intravenous induction of general anesthesia. Propofol has a rapid onset of action because it is lipid-soluble and distributes quickly into the CNS and other tissues. It has greater safety than other intravenous general anesthetics due to its faster clearance. In addition, propofol potentiates the inhibitory effects of GABA receptors in the CNS. Ketamine is labeled a dissociative anesthetic because the anesthetized patient feels consciously detached from the environment before losing consciousness. The dissociative state is indicated by sedation, amnesia, immobility, and marked analgesia. In adult patients, ketamine produces postoperative effects (such as sensory illusions and vivid dreams); therefore, it is not routinely administered to adults. However, it is useful for anesthetizing children, patients in shock, and trauma casualties in war zones where anesthesia equipment may be difficult to obtain. Ketamine acts as a noncompetitive antagonist of NMDA receptors. Intravenous anesthesia is suggested for the induction of anesthesia but not for maintenance. Administration of multiple doses by intravenous injection or a continuous intravenous infusion could result in drug accumulation and delays in recovery from anesthesia.

Uses and exposure routes and pathways In modern medicine, it is very common to use both inhalation and intravenous general anesthetics to achieve balanced anesthesia. Combining both classes of anesthetics maximizes the benefits of each drug and minimizes the dose of both drugs, thus reducing side effects from the anesthesia. Moreover, the patients can better tolerate a combination of anesthetics than an individual anesthetic.

Toxicokinetics Inhalation anesthetics undergo very little metabolism. Inhalation anesthetics are primarily eliminated from the body via exhalation through the lungs, with only a small amount being metabolized because inhalation anesthetics are highly volatile and easily evaporate into the air as breathed in and out. Therefore, they do not undergo significant body metabolism and are eliminated through respiration. Some inhalation anesthetics, such as halothane, can undergo metabolism in the liver, but this is generally a minor elimination pathway. The inhalation anesthetic nitrous oxide is not metabolized in the body. Sevoflurane is an inhalation anesthetic commonly used in clinics, and metabolism studies suggest that approximately 5% of the sevoflurane dose may be metabolized. Cytochrome P450 2E1 is the principal isoform identified for sevoflurane metabolism. Intravenously injected propofol has a high rate of total body clearance; it is rapidly and extensively metabolized in the liver and at extrahepatic sites. Propofol metabolites are inactive glucuronide and sulfate derivatives. Intravenously administered ketamine is metabolized in the liver by the microsomal enzyme system involving hydroxylation and demethylation. The principal metabolite of ketamine is norketamine, which lacks a methyl group compared with ketamine. The concentration of norketamine is about one-third that of ketamine. The cyclohexanone ring of ketamine also undergoes oxidative metabolism to form the second metabolite, dehydronorketamine—the high lipid solubility of ketamine results in a rapid onset of action. Intravenous fentanyl is extensively used in anesthesia and analgesia. Cytochrome P450 CYP3A4 metabolizes fentanyl to norfentanyl, hydroxypropionyl fentanyl, and hydroxypropionyl norfentanyl. None of the metabolites are active. About 75% of administered fentanyl is excreted as metabolites in the urine, and 9% is excreted in the stool.

Acute and short-term toxicity of general anesthetics Inhalation anesthetics can be toxic to the liver, kidney, or blood cells. The inhalation anesthetic with the most incidence of liver injury is halothane, followed by isoflurane, then desflurane. Respiratory depression occurs with all inhalation anesthetics. Malignant hyperthermia is a rare condition caused by an allergic response to a general anesthetic. The signs of malignant hyperthermia include rapid, irregular heartbeat; breathing problems; high fever; and muscle tightness or spasms. These reactions can occur following the administration of general anesthetics, especially halothane. Reduced blood pressure generally can be observed when halothane is administered; however, other inhalation anesthetics can have the same effects, although to a lesser extent.

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Preclinical data since 2002 indicate that general anesthetics can have acute and sometimes long-lasting effects on the developing CNS by increasing neuron apoptosis. Rodent and nonhuman primate models have demonstrated that the neurotoxic effects of anesthetics on developing brains are related to the route of administration and are dose-dependent, exposure duration-dependent, and developmental stage-dependent; the brain growth spurt period is the most vulnerable time. These factors are important because they can help identify exposure thresholds for producing neurotoxicity in the developing nervous system. These findings have called into question the safety of general anesthetics in the pediatric population. However, existing human data are insufficient to support or refute the possibility that neurotoxic effects observed in animals could occur in children. After being exposed to general anesthesia, young children have been observed to exhibit a range of behavioral changes. These changes include the emergence of delirium, which could indicate the presence of toxicity (McCann and Soriano, 2019). Emergence delirium is a type of mental state that young children may experience typically after receiving general anesthesia, and it usually lasts between 10 min and 30 min. It is characterized by a dissociated mental state where the child becomes inconsolable, irritable, and uncooperative and may exhibit behaviors such as thrashing, crying, moaning, or speaking incoherently. While this condition occurs in 5–10% of adult patients, it can affect up to 80% of some pediatric patient populations. The occurrence of delirium after exposure to halothane, isoflurane, sevoflurane, and desflurane can range from 2% to 55%. Delirium is a potential adverse effect of inhalation anesthetics, particularly sevoflurane and desflurane, and is more frequently observed in vulnerable populations, including young children (under 5 years old) and older adults (over 65 years old) (McCann and Soriano, 2019).

Chronic toxicity (animal/human) Traditionally, it has been believed that the effects of anesthesia are completely reversible once the drugs are eliminated from the body. Furthermore, it has been assumed that general anesthetics can provide neuroprotection against ischemia-reperfusion injury to the central nervous system. However, a significant body of animal research suggests that several general anesthetics can cause substantial neuroapoptosis and lead to long-term neurocognitive impairments in developing brains (Chiao and Zuo, 2014; Yu et al., 2017). The results from epidemiological studies and animal models have led to concerns that exposure to anesthetic agents during the early postnatal period could result in long-term cognitive impairments (Jackson et al., 2016). It is possible that several different mechanisms could be responsible for the spatial learning deficits commonly seen in animal studies, although some of these mechanisms may not be directly related to the condition. For example, brain-derived neurotrophic factor (BDNF) has been linked to various mechanistic studies, and the actions of anesthetics on this essential trophic factor are probably significant. In addition, many studies suggest that anesthetics can activate proapoptotic pathways, although it is unclear whether apoptosis is a contributing factor or a result of neurotoxicity caused by anesthetics in pediatric patients (Lee et al., 2015; Jackson et al., 2016). Several retrospective studies have reported a possible link between anesthesia exposure during childhood and subsequent neurocognitive deficits, while others have not found such an association (Chiao and Zuo, 2014; Batista et al., 2015; Fodale et al., 2017; McCann and Soriano, 2019). While most studies have demonstrated long-term effects in children exposed to anesthetics, particularly when exposed multiple times, the findings have not been consistent across all studies. These data discrepancies and interpretations may be due to the presence of confounding variables and a lack of adequate details presented in retrospective studies (Armstrong et al., 2017; Walkden et al., 2019). Although some studies have not found evidence of anesthetic neurotoxicity in routine surgical procedures during early life, these negative findings should be considered in the context of potential complexities, and further investigation is still necessary. Clinicians should be aware of the current research and remain vigilant in monitoring potential neurocognitive deficits in children exposed to anesthetics (McCann and Soriano, 2019). The results of three large retrospective matched cohort studies involving a total of 59,814 children who were exposed to general anesthesia before the age of 4 (including 30,021 who were younger than 2 years old and 9814 who had multiple exposures) showed no significant association between anesthetic exposure and cognitive outcomes in healthy children who were less than 2 years old, even when they were exposed to anesthesia multiple times (Graham, 2017). In a population-based cohort study, researchers retrospectively investigated the possible correlation between exposure to anesthesia during pregnancy, early childhood, or later in life and Autism Spectrum Disorder (ASD). The study included a total of 262 patients diagnosed with ASD, out of which 99 individuals had been exposed to anesthetics prior to their diagnosis. In contrast, among the non-ASD population consisting of 253 children, 110 individuals had been exposed to anesthesia. The study results revealed no statistically significant association between exposure to anesthesia and the development of ASD in either group (P ¼ .2091) (Creagh et al., 2016). From 2001 to 2010, a matched-cohort study was conducted in Taiwan to investigate whether there is a possible connection between exposure to general anesthesia and surgery and autistic disorder. The primary focus of the study was to identify the diagnosis of autistic disorder after the first exposure to general anesthesia and surgery. The study’s findings indicated that there were no significant variations in the incidence of autistic disorder between the exposed group (0.96%) and the unexposed controls (0.89%) (P ¼ .62). Hence, the exposure to general anesthesia and surgery before the age of 2 years and the number of exposures did not seem to correlate with the development of the autistic disorder (Ko et al., 2015). Despite the controversial findings regarding the potential harmful effects of general anesthetics on human patients, the FDA and the International Anesthesia Research Society (IARS) have formed a partnership called SmartTots (Strategies for Mitigating Anesthesia-Related Neurotoxicity in Tots). The group brought together a team of experts to establish guidelines to be followed until more conclusive results from new studies are available. In 2012, they prepared the first agreement, and in 2014, they updated

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the consensus, which suggested avoiding surgical procedures in children under 3 years old, except in emergencies. In addition, they emphasized the importance of conducting new animal studies and randomized clinical trials to obtain more conclusive evidence (Chiao and Zuo, 2014; Batista et al., 2015; Armstrong et al., 2017). Many studies investigating the potential link between anesthetic exposure and neurocognitive deficits contain various confounding factors that may affect the interpretation of results. For example, anesthetics are rarely administered alone, and the studies have instead evaluated the association between surgery and anesthetic exposure and subsequent cognitive/behavioral deficits rather than the specific risks associated with anesthetics themselves. As a result, it can be challenging to distinguish the impact of surgery on neurocognitive development. Additionally, children who require surgery at a young age may differ in several ways from those who do not, and these developmental differences could contribute to neurocognitive deficits attributed to surgery and/or anesthesia. Therefore, a cautious approach is necessary for interpreting the findings of these studies (Belrose and Noppens, 2019; Wu et al., 2019). Elderly patients are another group who are at higher risk of cognitive deficits after surgery and anesthesia. The International Study of Postoperative Cognitive Dysfunction has established the existence of postoperative cognitive dysfunction (POCD) in the elderly. It is estimated that around 50% of elderly patients who undergo surgery may experience some degree of cognitive decline lasting up to 5 years (Armstrong et al., 2017). A case-control study involving 5345 newly diagnosed dementia patients over 50 years old and a control group of 21,380 individuals without dementia found that a history of previous exposure to surgery under general anesthesia may be linked to a higher risk of developing dementia, particularly in individuals who have undergone repeated exposure to general anesthesia. Additionally, subjects who underwent surgery under endotracheal tube intubation general anesthesia and had comorbidities such as stroke, hypertension, diabetes mellitus, and atherosclerosis may also have an increased risk of developing dementia (Chen et al., 2014). A meta-analysis study has suggested that general anesthetics are unlikely to contribute to adult postoperative cognitive decline (POCD) (Guay, 2011). Another meta-analysis of case-control studies, which included 1752 cases and 5261 controls, investigated the possible association between general anesthesia and Alzheimer’s disease (AD). The study found no significant association between general anesthesia and the development of AD (pooled odds ratio ¼ 1.05, P ¼ .43). Subgroup analyses also found no difference in the odds ratio for developing AD after receiving general anesthesia compared to regional anesthesia. Additionally, there was no significant association between cumulative exposure to general anesthesia and AD development (Seitz et al., 2011). While some studies have suggested that anesthetics may not be a significant contributor to cognitive decline, there is still a possibility that they could interact with underlying vulnerabilities in aging brains and contribute to cognitive decline. For instance, volatile anesthetics can bind to and increase the levels of amyloid-b protein, which is known to contribute to the pathology of Alzheimer’s disease (Hussain et al., 2014; Armstrong et al., 2017). Interestingly, the effect of increasing amyloid-ß protein levels is only observed with volatile anesthetics and not with propofol or benzodiazepines (Mandal and Fodale, 2009).

Mechanism of toxicity The mechanisms by which general anesthetics cause acute and prolonged neurotoxic effects are unclear. However, one of the hypotheses for the developing brain regarding the acute neurotoxic effects of anesthetics by antagonizing NMDA receptors suggests that continually blocking NMDA receptors with NMDA receptor antagonists, such as ketamine, may induce a compensatory upregulation of NMDA receptors on the neurons. The upregulation of NMDA receptors allows for accumulating toxic levels of intracellular free calcium. Consequently, neurons are more vulnerable to endogenous glutamate’s excitotoxic effects after withdrawal from the NMDA receptor antagonist. The underlying mechanisms for POCD in the elderly remain unknown. However, in vitro studies demonstrated that some inhalation anesthetics (i.e., isoflurane, isoflurane plus nitrous oxide, sevoflurane, etc.) induced apoptosis and increased amyloid-beta (Ab) formation (Ab is a protein fragment of an amyloid precursor protein; a healthy brain can break down and eliminate it, but in Alzheimer’s disease, the fragments accumulate to form hard, insoluble plaques), providing a potential link between anesthesia administration and Alzheimer’s disease.

Carcinogenesis, mutagenesis, and impairment of fertility In general, inhalation anesthetics are freely transferred to fetal tissues. Animal studies on enflurane did not reveal evidence of carcinogenic or mutagenic effects. Reproduction studies conducted in rats and rabbits at doses up to four times the human dose of enflurane did not reveal evidence of impaired fertility or fetal harm. Isoflurane, enflurane, and sevoflurane do not show teratogenic potential. Chronic occupational exposure to anesthetic gases in operating rooms during pregnancy has raised concern that such exposure may cause congenital abnormalities and spontaneous abortions, but there has been no direct evidence of a correlation between occupational exposure and congenital abnormalities. Furthermore, with good scavenging and low-level exposure to waste, anesthetic gases do not appear to be a risk during occupational exposure.

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Clinical management Although evidence from preclinical studies indicates the neurotoxic effects of general anesthesia on developing animal brains, there is no direct evidence that anesthetics are unsafe for children. Parents of children requiring surgery should consult anesthesiologists or other qualified physicians for advice about an individual child’s situation. During general anesthesia, respiratory and cardiovascular functions should always be monitored. Necessary actions should be taken immediately to resolve any depressant effects.

Local anesthetic agents Local anesthetic agents can cause reversible local anesthesia by inducing the absence of pain sensation without affecting consciousness. All local anesthetics have three components: an aromatic portion, an intermediate chain, and an amine group. The intermediate chain, which connects the aromatic and amine portions, comprises either an ester or an amide linkage. Thus, the local anesthetics are further classified into esters and amides. Anesthetics such as benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine/larocaine, piperocaine, propoxycaine, procaine/novocaine, proparacaine, and tetracaine are esters. Examples of amides include articaine, bupivacaine, cinchocaine/dibucaine, etidocaine, levobupivacaine, lidocaine/lignocaine, mepivacaine, prilocaine, ropivacaine, and trimecaine. Both classes of local anesthetics act mainly by preventing sodium influx through voltage-gated sodium channels in the neuronal cell membrane into the cytoplasm, thus preventing the local membrane from depolarization. Therefore, an action potential cannot be generated and spread; the signal conduction is inhibited, affecting local anesthesia. In general, all nerve fibers are sensitive to local anesthetics. However, nerve fibers with a smaller diameter are more readily blocked than those with a larger diameter. For instance, the pain sensation, transmitted by small and unmyelinated nerves, can be blocked more rapidly than other sensations. In addition, local anesthetics have a higher affinity for binding to activated sodium channels. As a result, an active neuron is more susceptible to the effects of local anesthetics. This phenomenon is known as a state-dependent blockade. The lipid solubility of anesthetics is the most important factor in determining the intrinsic anesthetic potency, which is influenced by the aromatic portion of the molecule. The duration of anesthesia is determined by the extent of local anesthetics binding to proteins, which are immersed in membrane lipids. The greater the binding affinity to nerve proteins, the longer the anesthetic activity will persist. Several studies have evaluated the effectiveness of different additives combined with local anesthetics to expedite the onset of nerve blocks, prolong the duration of the block, or minimize toxicity. For example, sodium bicarbonate has been demonstrated to accelerate the onset of mepivacaine nerve blocks, but it might delay the onset of others. Epinephrine has been proven to lengthen the sensory nerve blockade and postpone systemic absorption of local anesthetics, thereby reducing the likelihood of anesthetic toxicity. Tramadol, buprenorphine, dexamethasone, and clonidine could be useful additives in specific circumstances. However, Midazolam, magnesium, dexmedetomidine, and ketamine are not typically recommended as nerve-block additives due to insufficient supporting evidence, limited effectiveness, and, in the case of ketamine, significant adverse effects. Recent research has suggested that administering additives intravenously or intramuscularly may improve perineural administration while minimizing the possibility of neurotoxicity, contamination, and other risks (Bailard et al., 2014).

Uses and exposure routes and pathways Local anesthetic agents are administered to the areas around the nerves to be blocked (skin, subcutaneous tissues, intrathecal, and epidural spaces). Their activities vary considerably. Topical anesthesia is the administration of local anesthetics to the skin or other body surfaces. Most anesthetics are barely absorbed through intact skin, and the effectiveness of anesthesia is affected. Eutectic mixtures, such as 2.5% lidocaine and 2.5% prilocaine (EMLA), improve the effectiveness of the anesthetic on intact skin by lowering the melting temperature of the mixture compared with that of each anesthetic. Infiltration anesthesia is the injection of local anesthetics into the tissue to be anesthetized. Amide anesthetics with a moderate duration of action are commonly used (i.e., lignocaine, prilocaine, and mepivacaine) to cause infiltration anesthesia for minor surgical procedures. Epidural anesthesia is the administration of local anesthetics to the epidural space between the dura mater and the periosteum lining the vertebral canal. As a result, the conduction is blocked at the intradural spinal roots, and the absence of pain sensation can be achieved. Spinal anesthesia is the application of local anesthetics into the cerebrospinal fluid at the site of the lumbar spine. Intravenous local anesthesia, also known as regional intravenous anesthesia (RIVA), involves the injection of a local anesthetic directly into a vein to numb a specific area of the body. Unlike traditional regional anesthesia, such as a nerve block, RIVA is not limited to a specific nerve distribution but provides anesthesia to an entire limb. The procedure is typically done without a tourniquet and does not require limb exsanguination. In addition, the anesthesia is limited to the area excluded from blood circulation. One restriction that should be considered is that bupivacaine and etidocaine should never be used for local, intravenous anesthesia due to the risk of cardiotoxicity.

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Toxicokinetics All local anesthetics are weakly alkaline. In solution, local anesthetics exist in both nonionized (lipid-soluble) and ionized (watersoluble) forms, with the ratio between the two depending on the pH of the solution. The term pKa of a weak base is defined as the pH at which both forms exist in equal amounts. At physiological pH (7.4), local anesthetics are more ionized than nonionized (as their pKa values are >7.4). However, the proportions vary among the anesthetics. Nonionized anesthetics can pass through the lipid cell membrane more rapidly than ionized ones. Therefore, an anesthetic agent with a higher proportion of nonionized form will reach the target site more quickly and have a faster blocking onset. Ester and amide anesthetics are metabolized through different routes. The metabolism of esters (except cocaine) is hydrolyzed in plasma by the enzyme pseudocholinesterase, which has a short half-life. Cocaine is hydrolyzed in the liver. Ester-type local anesthetics are metabolized in the body and excreted by the kidneys. On the other hand, amide-type local anesthetics are broken down by microsomal enzymes in the liver. This process is relatively slow, which results in longer half-times for amide-type local anesthetics. If they are given repeatedly, they can accumulate in the body.

Acute and short-term toxicity of local anesthetics Allergic reactions to local anesthetics are rare. Esters produce the most anesthetic-induced allergic reactions due to their metabolite, para-aminobenzoic acid (PABA), a well-known allergen. Hypersensitivity to amide local anesthetics is seldom observed. Since there is no cross-allergy between esters and amides, amides can be used as alternatives in patients who show hypersensitivity to esters. Therefore, amides are now more commonly used than esters. Local anesthetics may be toxic if sufficient amounts are absorbed into the systemic circulation or administered improperly. The toxicity can be at local and systemic levels. The local adverse effects of anesthetics may include prolonged anesthesia and paresthesias, which may become irreversible. Local anesthetic systemic toxicity (LAST) is more likely to occur with high lipid solubility agents, such as bupivacaine, etidocaine, and tetracaine (Barletta and Reed, 2019). Several comorbidities can increase the risk of local anesthetic overdose and systemic toxicity. These include cardiac disease, hepatic dysfunction, metabolic syndromes, and pregnancy. Patients who are either very young or very old are also at higher risk for toxicity due to reduced clearance of the anesthetics. In particular, infants younger than 4 months old have low alpha-1-acid glycoprotein plasma concentrations, which can result in lower intrinsic clearance of bupivacaine (Mahajan and Derian). Systemic toxicological effects of local anesthetics involve the central nervous, cardiovascular, and immune systems. The CNS is generally more sensitive to local anesthetics than the cardiovascular and immune systems. Therefore, symptoms and signs of CNS disturbances usually occur earlier, showing excitatory effects in the brain before the depressant effects. Neurotoxicity can result from local anesthetics blocking inhibitory pathways within the brain, which leads to unopposed excitatory activity. This neurotoxicity can result in clinical signs such as twitching and seizures (Barletta and Reed, 2019). Local anesthetic systemic toxicity’s initial signs and symptoms include agitation, dizziness, drowsiness, confusion, auditory changes, tinnitus, dysphoria, metallic taste, perioral numbness, and dysarthria. These signs and symptoms can progress to seizures, respiratory arrest, and/or coma without adequate recognition and treatment (Mahajan and Derian). It has been reported that seizures are the most frequent manifestation of systemic toxicity resulting from local anesthetics (Gitman et al., 2019). When the concentration of local anesthetics in the bloodstream rises, they can induce cardiotoxic effects by blocking sodium channels. This blockade causes a decrease in the rate of rise of the cardiac action potential’s phase 0. As a result, the cardiac system may experience electrocardiogram (ECG) changes, including prolonged PR and QRS intervals (Barletta and Reed, 2019). In the past, studies on local anesthetics suggested that cardiac toxicity usually occurred after signs of CNS toxicity. However, with more potent local anesthetics, cardiac toxicity can happen simultaneously or before CNS toxicity. The first signs of cardiac toxicity are usually hypotension and bradycardia. However, arrhythmias are more commonly seen, and bradyarrhythmias are the most frequent type. Other signs of cardiac toxicity include hypertension, difficulty breathing, pain, abnormalities on the ECG (such as a widened QRS complex and ST-segment changes), tachycardia, asystole, and various types of ventricular arrhythmias (Mahajan and Derian). Cardiac toxicity is more common with bupivacaine, a longer-acting local anesthetic. Bupivacaine blocks inactive sodium channels at 0.2 mg/mL concentration during the cardiac action potential. This blockage occurs in a “fast-in/slow-out fashion,” which means that bupivacaine binds to a large number of sodium channels during the cardiac action potential quickly but takes longer to release from the channels during diastole. Lidocaine at 5–10 mg/mL can also cause significant sodium channel blockade during the cardiac action potential. However, unlike bupivacaine, lidocaine follows the “fast-in/fast-out” principle, which results in quicker recovery and a lower incidence of cardiac toxicity when compared to bupivacaine (Mahajan and Derian). On very rare occasions (160,000 mg kg−1. LD50 Rat dermal: >1320 mg kg−1. According to Wolfe (1989) study (prepared for the USEPA Office of Solid Waste), the sub-chronic (0, 250, 500, and 1000 mg/kg/day for 13 weeks) oral exposures of male and female mice to anthracene have no effects on the respiratory, cardiovascular, gastrointestinal, musculoskeletal, renal, and endocrine systems. No hematological, hepatic, dermal, ocular, body weight, immunological and lymphoreticular, neurological, and reproductive effects were also observed. Due to the absence of any observed organ effects in the sub-chronic study, the no-observed-adverse-effect-level (NOAEL) of anthracene is 1000 mg/kg/day.

Human Anthracene is a photosensitizer, and can further cause acute dermatitis with symptoms of burning, itching, and edema. The effects are more notable when the exposed skin is bare. Furthermore, anthracene is also found to induce lacrimation, photophobia, edema of the eyelids, conjunctival hyperemia, headache, nausea, loss of appetite, slow reactions, and adynamia. The effects caused by acute exposure to anthracene eventually disappear after termination of exposure.

Chronic toxicity (or exposure) Animal Anthracene was found to have no mutagenicity towards Salmonella typhimurium TA100 and TA98 with and without addition of rat liver microsomes (S9) (Bos et al., 1988). Furthermore, it did not exhibit carcinogenicity towards Swiss albino mice. Chronic exposures of low concentrations of anthracene to Drosophila melanogaster larvae were found to significantly increase the formation of nonneoplastic melanotic tumors to the first- and second-generation progeny.

Human Human chronic exposure to anthracene could lead to gastrointestinal tract inflammation, pigment changes in the skin, loss of skin pigment, thinning or patchy thickening of skin, skin warts, skin cancer, and pimples. Repeated exposure to fumes of heated anthracene may cause chronic bronchitis with cough and phlegm. Repeated exposure of male scrotum can cause skin thinning and increased skin pigmentation. A study reported that consumption of anthracene-containing laxatives for a prolonged period of time was correlated to the increased incidence of colon and rectal melanosis (Badiali et al., 1985). The study, however, was found to be inconclusive due to several contributing factors.

Genotoxicity and carcinogenicity Anthracene is generally evaluated as neither a genotoxic nor a carcinogenic substance. The International Agency for Research on Cancer (IARC) assigned anthracene to the Group 3 classification, which means that it is “not classifiable as to its carcinogenicity to humans.”

Toxicogenomics The Comparative Toxicogenomics Database (CTD) of the National Institute of Environmental Health Sciences (NIEHS) lists some genes that interact with anthracene. For example, readers can access the CTD database link here: HYPERLINK “https://ctdbase.org/ detail.go?type¼reference&acc¼21668866” https://ctdbase.org/detail.go?type¼reference&acc¼21668866. To mention a few, the interactions of anthracene with CYP 1A1 and CYP 1B1 genes of Homo sapiens lead to the increased expression of their respective mRNAs (Mujtaba et al., 2011). Anthracene was also found to affect the expression of proteins like the ACP2 protein (Drwal et al., 2017).

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Clinical management No specific treatments have been prescribed. The patient should be moved to fresh air in case of respiratory distress. For additional information on exposure control and management, readers can refer to an updated safety data sheet for Anthracene.

Ecotoxicology Aquatic organisms toxicity The substance is very toxic to aquatic organisms. The substance may cause long-term effects in the aquatic environment. The acute and chronic toxicity of anthracene to fish have been explored in literature (European Chemicals Agency (ECHA), 2008). The 96-h LC50 of anthracene for C. chanos (milkfish) is 0.030 mg/L (Palanikumar et al., 2012). The phototoxicity of anthracene is evident in the UV-enhanced 96-h LC50 of 1.56 mg/L for larval Danio rerio (zebrafish) (Willis and Oris, 2014).

Invertebrate organisms toxicity The acute and chronic toxicity of anthracene to invertebrates have been reported in literature (European Chemicals Agency (ECHA), 2008). Anthracene was found to exhibit lethal toxicity towards Caenorhabditis elegans (nematode) (LC50 ¼ 2561 and 1560 mg/L at 48 h and 72 h, respectively). Toxicity was also reported towards Daphnia magna (water flea) and Artemia salina (brine shrimp) (LC50 ¼ 20 mg/L at 1 h for both). However, anthracene was reported to show no lethal toxicity towards Eisenia fetida (earthworm) (Honda and Suzuki, 2020).

Exposure standards and guidelines The US EPA must be notified if 5000 pounds of anthracene will be released to the environment within a period of 24 h. The Agency for Toxic Substances Disease Registry (US Department of Health, and Human Services, Agency for Toxic Substances Disease Registry (ATSDR), 1995) minimal risk level (MRL) for the intermediate duration (15–364 days) oral exposure to anthracene is 10 mg/kg/ day (hepatic endpoint; uncertainty factor (UF) ¼ 100). The oral reference dose (RfD) according the US EPA Integrated Risk Information System (European Chemicals Agency (ECHA), 2008) is 0.3 mg/kg/day (estimated from the 1000 mg/kg/day NOAEL obtained from the 90-day oral exposure of mice; UF ¼ 3000). The Occupational Safety and Health Administration (OSHA) has defined a permissible exposure limit (PEL) of 0.2 mg/m3 as coal tar pitch volatiles (benzene-soluble fraction) for an 8-h time-weighted average (TWA). National Institute for Occupational Safety and Health (NIOSH) has defined a recommended exposure limit (REL) of 0.1 mg/m3 as coal tar pitch volatiles (cyclohexane-extractable fraction) for a 10-h TWA.

Summary Anthracene is an organic compound under the class of polycyclic aromatic hydrocarbons (PAHs). It does not last long in the atmosphere, but it is persistent in the soil. Several microorganisms have been reported to have the ability to biodegrade it. Although it does not possess genotoxic and carcinogenic properties, it is known to show photo-induced toxicity. Some studies also suggest its ability to induce oxidative stress. Anthracene is known to be toxic to aquatic organisms.

See also: Coke oven emissions; Polycyclic aromatic hydrocarbons (PAHs)

References Badiali D, Marcheggiano A, Pallone F, Paoluzi P, Bausano G, Iannoni C, Materia E, Anzini F, and Corazziari E (1985) Melanosis of the rectum in patients with chronic constipation. Diseases of the Colon and Rectum 28(4): 241–245. Birolli WG, de Santos AD, Alvarenga N, Garcia ACFS, Romão LPC, and Porto ALM (2017) Biodegradation of anthracene and several PAHs by the marine-derived fungus Cladosporium sp. CBMAI 1237. Marine Pollution Bulletin 129(2): 525–533. Bos RP, Theuws JLG, Jongeneelen FJ, and Henderson PT (1988) Mutagenicity of bi-, tri- and tetra-cyclic aromatic hydrocarbons in the “taped-plate assay” and in the conventional Salmonella mutagenicity assay. Mutation Research 204: 203–206. Drwal E, Rak A, Grochowalski A, Milewicz T, and Gregoraszczuk EL (2017) Cell-specific and dose-dependent effects of PAHs on proliferation, cell cycle, and apoptosis protein expression and hormone secretion by placental cell lines. Toxicology Letters 280: 10–19. European Chemicals Agency (ECHA) (2008) Support Document for the Identification of Anthracene as a Substance of Very High Concern. ECHA. Hansch C, Leo A, and Hoekman D (1995) Exploring QSAR—Hydrophobic, Electronic, and Steric Constants. Washington, DC: American Chemical Society 118. Honda M and Suzuki N (2020) Toxicities of polycyclic aromatic hydrocarbons for aquatic animals. International Journal of Environmental Research and Public Health 17: 1363. Ifegwu OC and Anyakora C (2015) Chapter 6: Polycyclic Aromatic Hydrocarbons: Part I. Exposure. Advances in Clinical Chemistry 72: 277–304.

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Kottuparambil S and Park J (2019) Anthracene phytotoxicity in the freshwater flagellate alga Euglena agilis Carter. Scientific Reports 9: 15323. Marzuki I, Asaf R, Paena M, Athirah A, Nisaa K, Ahmad R, and Kamaruddin M (2021) Anthracene and pyrene biodegradation performance of marine sponge symbiont bacteria consortium. Molecules 26(22): 6851. Mujtaba SF, Dwivedi A, Mudiam MKR, Ali D, Yadav N, and Ray RS (2011) Production of ROS by photosensitized anthracene under sunlight and UV-R at ambient environmental intensities. Photochemistry and Photobiology 87(5): 1067–1076. Palanikumar L, Kumaraguru AK, Ramakritinan CM, and Anand M (2012) Biochemical response of anthracene and benzo [a] pyrene in milkfish Chanos chanos. Ecotoxicology and Environmental Safety 75(1): 187–197. Safitri R, Handayani S, Surono W, Astika H, Damayanti R, Kusmaya FD, Rukiah, and Balia RL (2018) Biodegradation of phenol, anthracene and acenaphthene singly and consortium culture of indigenous microorganism isolates from underground coal gasification area. IOP Conference Series: Earth and Environmental Science 306: 012026. US Department of Health & Human Services, Agency for Toxic Substances Disease Registry (ATSDR) (1995) Toxicological Profile for Polycyclic Aromatic Hydrocarbons (Update), NTIS# PB/95/264370. Available at: https://wwwn.cdc.gov/TSP/ToxProfiles/ToxProfiles.aspx?id¼122&tid¼25. (accessed, February 12, 2023) van Hattum B and Montañés JFC (1999) Toxicokinetics and bioconcentration of polycyclic aromatic hydrocarbons in freshwater isopods. Environmental Science & Technology 33(14): 2409–2417. Willis AM and Oris JT (2014) Acute photo-induced toxicity and toxicokinetics of single compounds and mixtures of polycyclic aromatic hydrocarbons in zebrafish. Environmental Toxicology and Chemistry 33(9): 2028–2037. Wolfe G (1989) Subchronic Toxicity in Mice With Anthracene. Final Report. Hazelton Laboratories America, Inc. Prepared for the Office of Solid Waste.

Further reading Anthracene IARC Summary & Evaluation (1983) Vol. 32. Link: https://monographs.iarc.who.int/wp-content/uploads/2018/06/mono32.pdf. Bonnet JL, Guiraud P, and Dusser M (2005) Assessment of anthracene toxicity toward environmental eukaryotic microorganisms: Tetrahymena pyriformis and selected micromycetes. Ecotoxicology and Environmental Safety 60(1): 87–100. Choia J and Oris JT (2003) Assessment of the toxicity of anthracene photo-modification products using the topminnow (Poeciliopsis lucida) hepatoma cell line (PLHC-1). Aquatic Toxicology 65(3): 243–251. Comparative Toxicogenomics Database (2022) National Institute of Environmental Health Sciences. Anthracene. Retrieved from http://ctdbase.org/detail.go?type¼chem&acc¼C034020. Huang X-D, McConkey BJ, Sudhakar BT, and Greenberg BM (1997) Mechanisms of photoinduced toxicity of photomodified anthracene to plants: Inhibition of photosynthesis in the aquatic higher plant Lemna gibba (duckweed). Environmental Toxicology and Chemistry 16(8): 1707–1715. McCloskey JT and Oris JT (1991) Effect of water temperature and dissolved oxygen concentration on the photo-induced toxicity of anthracene to juvenile bluegill sunfish (Lepomis macrochirus). Aquatic Toxicology 21(3–4): 145–156. Occupational Safety and Health Administration (2020) OSHA Occupational Chemical Database (Anthracene). Retrieved from https://www.osha.gov/chemicaldata/835. Purcaro G, Moret S, and Conte LS (2016) Polycylic aromatic hydrocarbons. In: Encyclopedia of Food and Health, pp. 406–418. Elsevier. U.S. Environmental Protection Agency (2008) Integrated Risk Information System (IRIS) for Anthracene. Available at: https://iris.epa.gov/ChemicalLanding/&substance_nmbr¼434. (accessed February 12, 2023) U.S. Environmental Protection Agency (2009) Provisional Peer-Reviewed Toxicity Values for Anthracene. Available at: https://cfpub.epa.gov/ncea/pprtv/documents/Anthracene.pdf. (accessed February 12, 2023).

Relevant websites https://ccme.ca/en/res/polycyclic-aromatic-hydrocarbons-2010-canadian-soil-quality-guidelines-for-the-protection-of-environmental-and-human-health-en.pdf :Canadian Soil Quality Guidelines for the Protection of Environmental and Human Health. https://ccme.ca/en/res/2010-pah-csqg-scd-1445-en.pdf :Carcinogenic and Other Polycyclic Aromatic Hydrocarbons (PAHs) (Environmental and Human Health Effects). https://pubchem.ncbi.nlm.nih.gov/compound/Anthracene :Anthracene Compound Summary. https://comptox.epa.gov/dashboard/chemical/details/DTXSID0023878%20%20 :Anthracene Chemical Details. https://www.osha.gov/chemicaldata/835 :OSHA Occupational Chemical Database (Anthracene). https://wwwn.cdc.gov/TSP/ToxProfiles/ToxProfiles.aspx?id¼122&tid¼25 :Agency for Toxic Substances and Disease Registry. Toxicological Profile for PAHs. https://www.nj.gov/health/eoh/rtkweb/documents/fs/0139.pdf :State of New Jersey. Anthracene, Hazardous Substance Fact Sheet (Revised June 2002). https://rais.ornl.gov/tox/profiles/Anthracene_ragsa.html :Anthracene Toxicity Profile Summary-The Risk Assessment Information System.

Anthrax Ryan E Fabian Campusano and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of K. Shankar, H.M. Mehendale, Anthrax, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 262-263, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00239-6.

Introduction Epidemiology Microbiology Molecular mechanism of toxicity Clinical forms of Anthrax Clinical treatment Potential for use as a biological weapon Conclusion References Further reading

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Abstract Anthrax disease results from introducing Bacillus anthracis spores into host organisms and requires spores to germinate and produce toxins enabling bacterial dissemination. Bacillus anthracis is a gram-positive bacillus that often shows chain-like growth on culture media. Anthrax in humans is associated with agricultural, horticultural, or industrial exposure to infected animals or animal products. Humans are normally diagnosed with three forms of anthrax: cutaneous, gastrointestinal, and inhalational. Although anthrax is a primarily zoonotic disease, but it has long been considered a potential biological weapon due to its ability to cause massive mortality through the intentional release of spores. Anthrax is a Category ‘A’ bioterrorism weapon. All laboratory procedures involving B. anthracis must be performed in Biological Safety Level 2 (BSL 2) microbiology laboratories. Sheep blood agar (5%), McConkey’s agar, and phenyl ethyl agar are used for its isolation and culture. There is one USFDA approved anthrax vaccine available (BioThrax®). However, antitoxins are also available for treatment (CDC, 2020; DHS, n.d.). Epidemiology, microbiology, molecular mechanisms of toxicity, and clinical treatment of anthrax are discussed.

Keywords Biological warfare; Bioterrorism; Botulinum Toxin; Chemical Warfare Agents; Toxins and Other Agents

Key points

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Bacillus anthracis causes the disease anthrax, which is a virulent, contagious, and potentially fatal disease. Anthrax can cause a different diseases, such as, inhalational, cutaneous, and oral/ingestional forms based on the route of exposure of the bacteria’s spores. Anthrax infection follows a complex but organized pathway in its pathogenesis from spore uptake by the specialized immunocompetent cells to ultimate infection. Spores germinate and then transported to local lymph nodes, and at last produce of deadly toxins, leading to systemic spread. Death of the infected host is certain if they remain untreated. The antibiotic Ciprofloxacin is the drug of choice to treat anthrax. There is one vaccine available for anthrax and antitoxins are also available to treat anthrax (Manish et al., 2020).

Abbreviations EF EISD LF PA

Edema factor Exposure-Infection-Symptomatic-illness-Death Lethal factor Protective antigen

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Introduction Bacillus anthracis are gram-positive, spore-forming, rod-shaped bacteria. It causes anthrax disease, which is primarily a zoonotic disease found all over the world. It is generally seen in developing economically challenged countries. It has three different strains, two of which are encapsulated and virulent. The anthrax toxin which causes anthrax disease is made of 3 different proteins: lethal factor, edema factor, and the protective antigen. Clinically anthrax disease can manifest in three ways: cutaneous, inhaled, and gastrointestinal. Regardless of the clinical form, anthrax must be treated promptly for treatment to be successful. Treatment is composed of a cocktail of antimicrobial drugs dependent upon whether meningitis is also present. Anthrax is a category A biological weapon; as such, it is an ideal candidate for biological terrorism (Shankar and Mehendale, 2014).

Epidemiology Anthrax is a zoonotic disease with worldwide distribution. The earliest known description of anthrax is found in the Book of Genesis, wherein the fifth plague is said to have killed Egyptian cattle. Further, there are numerous descriptions of anthrax in animals and humans in Hindu, Greek, and Roman literature. The World Health Organization estimates that the annual global incidence of anthrax infection is between 2000 and 20,000 cases (Simonsen and Chatterjee, 2021). Human Anthrax is most common in enzootic areas in developing countries, among people who work with livestock, eat undercooked meat from infected animals, or work in establishments where wool, goatskins, and pelts are stored and processed. West Africa is the most affected part of the world. Anthrax is also a significant problem in other parts of Africa, Central America, Spain, Greece, Turkey, and the Middle East. In economically advanced countries, where animal anthrax is controlled, the incidence in humans is rare. Further infections have been dramatically reduced by the vaccination of high-risk individuals and improvements in industrial hygiene. Incidence in the United States declined to less than 1 per year until the recent biological terrorist attacks in the fall of 2001. However, most recently, there have been outbreaks of injectional anthrax reported in Europe from 2009 to 2012 resulting in 141 cases across Scotland, England, Germany, France, Wales, and Denmark (Zasada, 2018).

Microbiology Anthrax disease is caused by Bacillus anthracis, a gram-positive, spore-forming, rod-shaped bacterium that primarily infects herbivores such as cattle and deer. B. anthracis is non-motile, catalase-positive, non-hemolytic on blood agar, and frequently occurs in long chains. There are three well-known strains of B. anthracis Ames, Sterne, and Vollum (Goel, 2015). Ames and Vollum, the virulent forms, are surrounded by a capsule. Sterne is toxigenic but avirulent since it lacks a capsule (Goel, 2015). Sporulation occurs in soil and culture media but not in living tissue. Spores are highly resistant to UV light, extremely high temperatures, high pH, drying, high salinity, and routine disinfection methods.

Molecular mechanism of toxicity Anthrax toxin is composed of three proteins: protective antigen (PA; 83 kDa), lethal factor (LF; 90 kDa), and edema factor (92.5; kDa). Individually, none of the three proteins are toxic but interact synergistically with at least one of the others. PA and LF (called LeTx) can cause lethal shock in experimental animals, and a mixture of PA and EF (edema toxin, EdTx) induces edema at the injection site. Since two discrete units of the toxin are required for its action, the term binary toxin has been used for this and other bacterial toxins. Anthrax is unique from other binary toxins in that the binary moieties (EF and LF) interact only after being secreted from the bacteria. The following mechanism is illustrated in Fig. 1. Once the spore germinates inside the host, it produces the tripartite anthrax toxin (Friebe et al., 2016). PA will bind to one of two transmembrane protein receptors, TEM8 or CMG2, via the van Willebrand factor A (Friebe et al., 2016). PA is then cleaved by a furin-like protease to a shorter 63 kDa form (PA63) (Friebe et al., 2016). PA63 will then oligomerize into either heptamers or octamers. The toxin-receptor will cluster, forming lipid raft subdomains on the cellular membrane (Friebe et al., 2016). The bound receptors are then phosphorylated on their tyrosine residues by the proteins Src or Fyn. The LF and EF will bind to the toxin-receptor complex, further oligomerizing it (Friebe et al., 2016). Finally, the complex undergoes clathrin-mediated endocytosis (Friebe et al., 2016). Inside the cellular cytoplasm, EF (a calcium and calmodulin-dependent adenylate cyclase) causes a dramatic increase in intracellular cAMP concentrations, and LF acts proteolytically to cleave specific MAPK kinases.

Clinical forms of Anthrax Anthrax mainly occurs in three forms; cutaneous, inhalation, and gastrointestinal. Exposure to B. anthracis, most likely in an occupational setting, is the cause of cutaneous anthrax. The incubation period varies from 1 to 12 days. In most cases, the disease

Anthrax

Key PA: Protective antigen subunit of anthrax toxin (83kDa) 63 PA : The shorter form of PA subunit (63kDa) LF: Lethal factor subunit of anthrax toxin EF: Edema factor subunit of anthrax toxin vWA: van Willebrand factor A. An extracellular protein bound to the receptor TEM8 and CMG2: Transmembrane receptors Src and Fyn: Intracellular phosphorylating proteins

B. anthracis spore germinates and begins producing anthrax toxin (PA, LF, and EF)

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Receptors are then phosphorylated on tyrosine residues by Src or Fyn

PA binds to vWA domain on TEM8 or CMG2 receptors forming a toxinreceptor complex

LF and EF bind to toxin-receptor oligomer

PA is cleaved by a furin-like protease forming PA63

Toxin-receptor oligomer undergoes clathrin-mediated endocytosis

PA63 oligomerizes into heptamers/octamers

Fig. 1 The Cellular Entry Mechanism of .B. anthracis.

remains localized to the skin lesion. A major diagnostic characteristic is the development of edema around the lesion. Inhalation anthrax is the most lethal form resulting from inhalation of pathogenic endospores. The illness is biphasic after exposure to large numbers of spores. The first phase is characterized by a ‘flu-like’ illness with a nonproductive cough. After several days of apparent improvement, there is a sudden onset of rapidly progressive respiratory failure, acute dyspnea, circulatory collapse, and pleural effusion. The mortality rate is very high despite supportive care and antibiotics, generally within 24 h of the onset of the second stage due to toxemia and suffocation. Gastrointestinal anthrax, although rare, occurs after an incubation period of 1–7 days following ingestions of B. anthracis via contaminated food or drink. Mortality rates are estimated to be between 25% and 60% unless treatment is begun early enough. Severe abdominal pain, fever, nausea, vomiting, and bloody diarrhea manifest during the disease. Death occurs due to toxemia and shock. Cutaneous infection usually occurs via a small cut or abrasion in the skin, allowing spores to invade. There have also been reports of palpebral anthrax, where the infection occurs in the upper eyelid (Goel, 2015). Injectional Anthrax is the latest form of anthrax infection (Zasada, 2018). Injectional Anthrax presents with a severe soft tissue infection like cellulitis or necrotizing fasciitis at the injection site and is often complicated by sepsis and meningitis (Zasada, 2018). This form of anthrax is primarily found in intravenous drug users (Zasada, 2018).

Clinical treatment Prompt clinical diagnosis and treatment with effective antimicrobial drugs are necessary to treat anthrax successfully. Several historical strains produce an inducible b-lactamase and are resistant to penicillin. According to the CDC recommendations, in cases where meningitis is excluded, two agents should be used a bactericidal agent and a protein synthesis inhibitor. If meningitis is suspected or confirmed, two bactericidal agents, a b-Lactam and a fluoroquinolone, and a protein synthesis inhibitor should be used. The preferred bactericidal agents are ciprofloxacin and meropenem. The preferred protein synthesis inhibitor is linezolid. Supportive therapy should be initiated to prevent shock, fluid and electrolyte imbalance, and loss of airway patency. CDC has approved penicillin G procaine and ampicillin for treatment of all age groups if the strain is penicillin-sensitive. Levofloxacin is FDA-approved for treating “inhalational anthrax (postexposure)” in adults aged 18 years or older. Treatment with oral ciprofloxacin or doxycycline for 7–10 days is recommended for localized or uncomplicated cases of naturally acquired cutaneous anthrax, such as that associated with exposure to animals with anthrax or to products such as hides from animals with anthrax. Immunotherapy is not very common, but it can be an adjunct to systemic treatment (Goel, 2015). In the case of injectional anthrax and other severe cutaneous anthrax infections require, surgical debridement of infected tissue.

Potential for use as a biological weapon Anthrax is classified as a category A biological weapon (most dangerous) along with smallpox, plague, clostridium botulinum toxins, filoviruses, etc. B. anthracis has several biological, technical, and virulence characteristics, making it an attractive weapon for bioterrorism. The spores produced by the microbe are highly resistant to temperature, pressure, pH, and ionizing radiations (Goel, 2015). After production and purification, the spores can be easily stored in a dry form with a half-life of approximately 100 years. The most viable weaponized form of anthrax infection is inhalational anthrax because early symptoms present like the

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flu, making diagnosis difficult and delaying treatment (Goel, 2015). The lethal dose of anthrax spores has not been determined due to the difficulty in assessing it but using the Exposure-Infection-Symptomatic-illness-Death (EISD) quantitative model; the ID50 was estimated to be 11,000 aerosolized spores (Goel, 2015).

Conclusion Bacillus anthracis is a hazardous bacteria that causes anthrax disease. Normally, B. anthracis can be found in herbivores like cattle; thus, farmhands are the principal source for it, especially those in developing countries. Tropical warm humid weather also foster survival of anthrax spores. It is a gram-positive bacteria whose spores are highly resistant to many types of extreme environmental conditions. These spores can be stored as a dried powder, weaponizing them as an inhalant. The anthrax toxin is a tripartite toxin whose mechanism is well understood; however, no lethal dose has been established. Anthrax disease is challenging to treat because early symptoms are commonly found in many disorders like the flu. Once diagnosed, the antimicrobial drugs are then selected based on whether or not meningitis is present. Due to its easy storage, potential for weaponization, and difficulty in diagnosing and treating B. anthracis is one of the most dangerous biological terror weapons currently known. There is one anthrax vaccine licensed for use in the United States by the Food and Drug Administration (BioThrax®) (CDC, 2020; DHS, n.d.).

See also: Bio warfare and terrorism: Toxins and other mid-spectrum agents; Botulinum toxin; Chemical warfare; Chemical warfare delivery systems; Immune system

References CDC (2020) Anthrax. Centers for Disease Control and Prevention. Available at: https://www.cdc.gov/anthrax/index.html. DHS (n.d.) http://www.dhs.gov/publication/protecting-responders-health-after-wide-area-aerosol-anthrax-attack Friebe S, van der Goot F, and Bürgi J (2016) The ins and outs of anthrax toxin. Toxins 8(3): 69. Goel AK (2015) Anthrax: A disease of biowarfare and public health importance. World Journal of Clinical Cases 3(1): 20–33. Manish M, et al. (2020) Anthrax prevention through vaccine and post-exposure therapy. Expert Opinion on Biological Therapy 20(12): 1405–1425. https://doi.org/ 10.1080/14712598.2020.1801626. Shankar K and Mehendale HM (2014) Anthrax. In: Encyclopedia of Toxicology, 3rd edn, 262–263. Simonsen KA and Chatterjee K (2021) Anthrax. In: StatPearls, Available at: https://www.ncbi.nlm.nih.gov/books/NBK507773/. Accessed April 25, 2022. Zasada A (2018) Injectional Anthrax in human: A new face of the old disease. Advances in Clinical and Experimental Medicine 27(4): 553–558.

Further reading Artenstein AW and Opal SM (2012) Novel approaches to the treatment of systemic anthrax. Clinical Infectious Diseases 54(8): 1148–1161. Bouzianas DG (2010) Current and future medical approaches to combat the Anthrax threat. Journal of Medicinal Chemistry 53: 4305–4331. Celik E and Gonen T (2021) Cutaneous anthrax on the upper eyelid. Case Reports in Ophthalmology 12(3): 836–840. Collier RJ and Young JA (2003) Anthrax toxin. Annual Review of Cell and Developmental Biology 19: 45–70. Eshraghi B, et al. (2020) Palpebral anthrax, a rare though important condition in villagers: A case report and literature review. International Journal of Infectious Diseases 99: 260–262. Ghosh N, Tomar I, and Goel AK (2012) A field usable qualitative anti-PA enzyme linked immunosorbent assay for serodiagnosis of human anthrax. Microbiology and Immunology. https://doi.org/10.1111/1348-0421.12014. [Epub ahead of print]. Hicks CW, Sweeney DA, Cui X, Li Y, and Eichacker PQ (2012) An overview of anthrax infection including the recently identified form of disease in injection drug users. Intensive Care Medicine 38(7): 1092–1104. Kaur M and Bhatnagar R (2011) Recent progress in the development of anthrax vaccines. Recent Patents on Biotechnology 5(3): 148–159. Oncu S, Oncu S, and Sakarya S (2003) Anthrax—An overview. Medical Science Monitor 9: 276–283. Putzer GJ, Koro-Ljungberg M, Duncan RP, and Dobalian A (2013) Preparedness of rural physicians for bioterrorist events in Florida. Southern Medical Journal 106(1): 21–26. https:// doi.org/10.1097/SMJ.0b013e31827caed2. Steelfisher GK, Blendon RJ, Brulé AS, et al. (2012) Public response to an anthrax attack: A multiethnic perspective. Biosecurity and Bioterrorism 10(4): 401–411. Wang DB, Tian B, Zhang ZP, et al. (2012) Rapid detection of Bacillus anthracis spores using a super-paramagnetic lateral-flow immunological detection system. Biosensors & Bioelectronics (12): 00782–00788. https://doi.org/10.1016/j.bios.2012.10.088. pii: S0956-5663. [Epub ahead of print].

Antibacterial agents Roberto Maldonadoa, Vera Bulakhovaa, Manish Varmab, Numair Mukhtara, Dorina Bircea, and Sidhartha D Raya, aDepartment of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States; bTouro School of Medicine, New York, NY, United States © 2024 Elsevier Inc. All rights reserved.

Introduction Cell wall synthesis inhibitors Background information/mechanism of action Protein synthesis inhibitors Mechanism of action Nucleic acid synthesis inhibitors Mechanism of action Antimetabolites Conclusion References Further reading

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Abstract This article includes a description of the antibacterial agents such as cell wall synthesis inhibitors, nucleic acid synthesis inhibitors, protein synthesis inhibitors, and antimetabolites. All the antibacterial agents used in clinical practice are described. Special attention was placed upon various classes and categories of agents, their mechanism of action, structure, effectiveness, dose, toxicities, spectrum of activity, and approved therapeutic indication. Many antibacterial agents show organ toxicities and many show serious hypersensitivity reactions, occasionally fatal. Clinicians are always on the alert to discover well-know side effects and sometimes unknown side effects. Combination of various antimicrobials are routinely administered now-a-days for better clinical outcomes. Efforts are in place to design, synthesize new antibacterial entities all the time.

Keywords A. baumannii (Acinetobacter baumannii); B. fragilis (Bacteroides fragilis); B. ovatus (Bacteroides ovatus); B. thetaiotaomicron (Bacteroides thetaiotaomicron); B. vulgaris (Bacteroides vulgaris); Bacterial cell wall; C. freundii (Citrobacter freundii); cIAI (Complicated intra-abdominal infections); cUTI (Complicated urinary tract infections); E. cloacae (Enterobacter cloacae); E. coli (Escherichia coli); H. influenzae (Haemophilus influenzae); K. oxytoca (Klebsiella oxytoca); K. pneumoniae (Klebsiella pneumoniae); P. aeruginosa (Pseudomonas aeruginosa); PBP-Penicillin binding proteins; S. aureus (Staphylococcus aureus); S. pneumonia (Streptococcus pneumoniae)

Key points

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Bacterial cell wall synthesis inhibitors provide a powerful tool to prevent/eradicate bacterial infections Recent generations (newer) of antibiotics are mechanism-based drugs which are effective at lower doses and designed to reduce side effects and toxicity In recent years, it has been possible to treat/prevent infections with an aggressive clinical approach when needed (by using combination medications) Progress in this field has generated tons of research-based information regarding possible drug interactions (with food and nutrients, other medications and genetics-based), and has paved the way to develop customized strategies to minimize adverse effects and interactions Despite all these efforts, minimizing evolution of bacterial drug-resistance remains a difficult task, although antibacterial drug therapy regimens were devised in a number of ways to combat or minimize evolution of resistant species Studying ADME characteristics and toxicity profile for each drug and when they are in combinations with another drug, remains ever-challenging

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Introduction Antibiotics are very commonly used anti-infective agents in healthcare. Microbes that are medically significant include diverse groups of bacteria (such as gram positive, gram negative etc.), viruses, chlamydia, mycoplasmas, rickettsia, fungi, and parasites. Antibiotics or antibacterial compounds are either naturally or synthetically produced substances that target bacteria and, thus, are intended to treat and prevent bacterial infections. This review will focus on only antibacterial agents that are approved for treating a wide variety of bacterial infections. Tremendous progress has been made not only in new entity development but also in devising ways to understand their impact on human health. The last two decades have had significant developments of a number of extremely effective new generations of antibacterial agents with improved efficacies and lesser side effects. Significant efforts were placed on designing a combination of antibiotic formulations with enhanced stability in in vivo situations (Brunton, 2022). The rationale behind antibiotics therapy is to destroy the bacterial cell by either preventing cell replication, changing a necessary cellular function, or changing processes within the cell. Antimicrobials are generally grouped into two main categories based on their effects: either bactericidal or bacteriostatic, although many chemotherapy specialists categorize them by narrow spectrum, broad spectrum, and very broad spectrum of activity. Bactericidal antibiotics “kill” bacteria (lyse the cell) whereas bacteriostatic antibiotics “prevent the replication” of bacteria. Some of the bacteriostatic agents in use are as follows: Glycylcyclines: Tigecycline; Tetracyclines: Doxycycline, minocycline; Lincosamides: Clindamycin; Macrolides: Azithromycin, clarithromycin, erythromycin; Oxazolidinones: Linezolid; Sulfonamides: Sulfamethoxazole. Some of the bactericidal agents are: Aminoglycosides: Tobramycin, gentamicin, amikacin; Beta-lactams penicillins, cephalosporins, carbapenems: Amoxicillin, cefazolin, meropenem; Fluoroquinolones: Ciprofloxacin, levofloxacin, moxifloxacin; Glycopeptides: Vancomycin; Cyclic Lipopeptides: Daptomycin; Nitroimidazoles: Metronidazole (Henson et al., 2015; LivTox-NIH-NLM, n.d.; Hutchings et al., 2019; Khardori et al., 2020; Cochrane and Lohans, 2020; Calhoun et al., 2022; FDA-Tigecycline, n.d.). The clinical implications of antibacterial drug’s effectiveness depend on pharmacokinetic (PK) and pharmacodynamic (PD) properties of that agent, which is further complicated by the ability of some bacteriostatic agents when they exhibit bactericidal activity. This type of differential action is not uncommon against particular type or group of bacteria at higher concentrations and vice versa. Although it appears simple but in reality, age, gender, immunocompromised status, stage of illness, pre-existing conditions, metabolic status, pregnancy, drug/nutrient and pharmacogenetics, all play a prominent role in decision making while prescribing a particular type of antibacterial agent to a patient (Werth, 2022a,b,c,d,e,f,g,h,i,j,k). PK and PD parameters are taken into consideration to optimize the effectiveness of antimicrobial compounds by dosing in patients. ADME are the PK parameters that regulate the antibiotic concentration over time, which explain how an antibiotic behaves after entering the body until the parent drug and its metabolites are cleared out from the body (via kidneys, GI tract etc.). In contrast, PD of a drug defines the drug effect within the body when it reaches the infected target tissue. The principles of PD correlates concentration to effect. The two main PD properties of a drug are the maximum effect and the concentration producing 50% of the maximum effect (Werth, 2022a,b,c,d,e,f,g,h,i,j,k; Phillips et al., 2022; Maxwell and Andrade, 2022). Cell wall synthesis inhibitors (vancomycin, which is one of the most important class of antibiotics, and the b-lactams) include different types of penicillins such as natural penicillins (penicillin G and penicillin V(K), antistpahylococcal penicillins (nafcillin, dicloxacillin, oxacillin, cloxacillin, methicillin), aminopenicillins (amoxicillin and ampicillin, amoxicillin/clavulanate, ampicillin/ sulbactam), antipseudomonal penicillins (piperacillin/tazobactam), as well as cephalosporins which are composed of five generations (1st generation: cephazolin, cephalexin; 2nd generation: cefuroxime, cefotetan, cefoxitin; 3rd generation: ceftriaxone, cefotaxime, ceftazidime, ceftazidime/avibactam, ceftolozane/tazobactam; 4th generation: cefepime; 5th generation: ceftaroline; other cephalosporins: cefiderocol); other b-lactams are carbapenems (meropenem, imipenem/cilastatin, doripenem, ertapenem, imipenem/varbobactam, imipenem/cilastatin/relabactam), and monobactam (aztreonam). Other antibacterial medications discussed in this article are not known to be differentiated by generations (Werth, 2022a,b,c,d,e,f,g,h,i,j,k; Wall and Maxwell, 2021; Phillips et al., 2021).

Cell wall synthesis inhibitors Background information/mechanism of action Cell wall biosynthesis inhibitors (CBIs) have traditionally been one of the most efficient classes and broadly used class of antibiotics. The vast difference in composition of cell wall structure and composition of bacteria (procaryotes) and mammals (eucaryotes) provide an unprecedented advantage to design drugs and chemicals that target procaryotic cell wall and spare the eucaryotic cells in vivo. Importance of this class is represented by the b-lactams, and glycopeptide antibiotics. CBIs primarily attack the peptidoglycan layer (composed on N-acetyl muramic acid [NAM], N-acetyl glucosamine [NAG] and 4 or 5 amino acid peptide bonds). The cell wall synthesis inhibitors affect either synthesis or incorporation of precursors or linkages to destabilize or destroy natural configuration of the cell wall ultimately leading to a deformed/dysfunctional cell. Disrupting the cell wall formation or stalling the cell-wall synthesis both work to paralyze bacterial growth and replication. Normally NAM and NAG form the backbone

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of the cell wall by tetra- or pentapeptide linkages. Some of these agents target these polypeptide linkages, which force bacterial cells to form a distorted cell wall which ultimately makes the cell dysfunctional (Hutchings et al., 2019; Khardori et al., 2020; Calhoun et al., 2022). b-lactam antibiotics contain a b-lactam nucleus in their molecular structures. b-lactam is a very large group which contains penicillin derivatives, cephalosporins, carbapenems, and other miscellaneous CBIs. These b-lactam CBIs are bactericidal and act by preventing the synthesis of the peptidoglycan layer. Anti-cell wall properties are facilitated by binding to penicillin-binding proteins (PBPs) located on the plasma membrane of the bacterial cell wall. Porin pores found on the outermost layer of gram-negative organisms which assist in the transport of molecules from outside to inside of the cells. In contrast, gram positive cells have no porin pores which makes it difficult for the antibiotics to penetrate through the cell (Liu and Breukink, 2016; Ciccolini et al., 2016; Gallagher and Macdougall, 2018; Werth, 2022a,b,c,d,e,f,g,h,i,j,k). Glycopeptide antibiotics are composed of glycosylated cyclic or polycyclic non-ribosomal peptides. This class of drugs constrain the synthesis of cell walls in susceptible microbes by obstructing peptidoglycan synthesis. In addition, they bind to the amino acids within the cell wall, inhibiting the appropriate conformation of new units of the peptidoglycan. The overall outcome of this class of CBIs prevents bacteria from synthesizing and surviving in the human body. However, monitoring is required for certain patients with severe impairments. Like all other antimicrobials, these agents can cause organ toxicities and induce severe hypersensitivity reactions (Acharya et al., 2022; Werth, 2022a,b,c,d,e,f,g,h,i,j,k). Glycopeptides have been the choicest antibiotics against drug-resistant gram-positive bacterial infections. This group exhibit unique mechanism of action. They bind to the substrate of cell wall biosynthesis and show substantial half-life in clinical setting. However, resistance to glycopeptides has been reported, which led the development of synthetic and semisynthetic glycopeptide analogues to combat acquired resistance. Recent studies show that glycopeptides are also effective against Gram-negative bacteria, Mycobacteria, and viruses thus expanding their spectrum of activity across the microbial World. The semisynthetic glycopeptides telavancin, dalbavancin, and oritavancin were developed because of the evolution of vancomycin resistance (van Groesen et al., 2022; FDA-oritavancin, n.d.) (Table 1).

Protein synthesis inhibitors Mechanism of action A prominent way that modern medicine protects us from infection specifically bacterial infections is by blocking the protein synthesis capabilities of the pathogens. Microbes that lack cell wall do not respond to cell wall synthesis inhibitors (such as penicillins and cephalosporins). Cell-wall deficient organisms are Mycoplasmas, Rickettsia and Chlamydia. Therefore, these organisms respond to protein synthesis inhibitors. The ribosome is the macromolecular instrument that masterminds the genetic information encoded in the messenger RNA (mRNA) into the polypeptide sequence that comprises the proteins and enzymes of the cell. Bacterial (or procaryotic) 70S ribosomes are comprised of two subunits, a small 30S subunit and a large 50S subunit. Bacterial protein synthesis pathway involves the assembly of 30S and 50S ribosomal subunits to form a complex. Ribosomal complex facilitates the formation of peptide bonds until it reaches a codon in its mRNA; then it instructs to stop synthesis. The drugs classes that target/inhibit these various steps are as follows: tetracyclines, macrolides, lincosamides, oxazolidinones, pleuromutilin, and chloramphenicol. The mechanism of action, spectrum activity, organ toxicity, and structure of protein synthesis inhibitors are described in the table provided below (Ciccolini et al., 2016; Gallagher and Macdougall, 2018; Lenz et al., 2021). The aminoglycoside class of antibiotics consists of many different agents. In the United States, gentamicin, tobramycin, amikacin, plazomicin, streptomycin, neomycin, and paromomycin are approved by the US Food and Drug Administration (FDA) and are available for clinical use. Of these, gentamicin, tobramycin, and amikacin are the most frequently prescribed by intramuscular or intravenous injection for systemic treatment. The most common clinical application (either alone or as part of combination therapy) of the aminoglycosides is for the treatment of serious infections caused by aerobic gram-negative bacilli (Pubchem-Plazomicin, n.d.; Werth, 2022a,b,c,d,e,f,g,h,i,j,k) (Tables 2 and 3).

Nucleic acid synthesis inhibitors Mechanism of action Antibiotics induce bacterial cell death via different types of mechanisms. Like Eukaryotes, procaryotes (bacteria) must also be able to transcribe genetic information which is essential for making proteins that carry out their metabolic pathways and ensuring their survival. Nucleic Acid synthesis inhibitors are antibiotics that halt or prevent bacterial replication by disrupting DNA or RNA pathways. Two classes of bacteria that are categorized as Nucleic Acid synthesis inhibitors are Fluoroquinolones and Rifamycins (Ciccolini et al., 2016; Gallagher and Macdougall, 2018). Fluoroquinolones induce bacterial-cell death via interfering with changes in DNA supercoiling, primarily by binding to topoisomerase II or IV. This leads to the formation of double-stranded DNA breaks and consequently cell death in either a protein

Table 1

demonstrates main characteristics of cell wall synthesis inhibitors. Effectiveness/Structure

CEFIDEROCOL (FETROJAW)

A catechol side chain promotes formation of chelated complexes with ferric iron which allows transport systems to deliver cefiderocol across the outer membrane of gram-negative bacilli.

Potent activity against Gram-negative bacteria producing all four Ambler classes of b-lactamases, including ESBL and carbapenems.

Siderophore Cephalosporin Antibacterial

Binds to PBP which in turn inhibits the final transpeptidation step of peptidoglycan synthesis in bacterial cell walls

Dose/Toxicity

References

Administer 2 g of FETROJA for injection every FDA (Cefiderocol): 8 h by IV infusion over 3 h in patients with https://www.accessdata.fda. gov/drugsatfda_docs/label/ CrCl 60 mL/min. 2019/209445s000lbl.pdf Unlike other b-lactams, cefiderocol contains a chlorocatechol group which allows it to chelate iron. Once bound to ferric iron, cefiderocol can undergo Dosage adjustment is recommended in active transport into bacterial cells through iron channels. Iron channels in patients with renal impairment. PubChem (Cefiderocol): https:// the bacterial outer cell membrane are encoded by
cirA
and
fiu
genes in
pubchem.ncbi.nlm.nih.gov/ Exceeding recommended dose can lead to E. coli
or the
PiuA
gene in
P. aeruginosa
compound/Cefiderocol neurotoxicity. Increased risk in patients #section¼Structures with a history of seizure disorders Syed, Drugs, 2021

PIPERACILLIN (ZOSYNW) 4th generation extended spectrum penicillin

Inhibits the third and last stage of bacterial cell wall synthesis. Cell lysis is then mediated by bacterial cell wall autolytic enzymes such as autolysins (cause bacteria to lyse) and murein hydrolases. Piperacillin exhibits time-dependent killing

S. aureus, A. baumannii, H. influenzae, K. pneumoniae, and P. aeruginosa, and Children with appendicitis and/or peritonitis 9 FDA (Piperacillin): https://www.accessdata.fda. months of age or older, weighing up to E. coli. gov/drugsatfda_docs/label/ 40 kg, and with normal renal function, 2017/050684s88s89s90_ recommended dosage 100 mg piperacillin/ Intra-abdominal infections 050750s37s38s39lbl.pdf 12.5 mg tazobactam per kilogram of body b-lactamase producing isolates of E. coli or members of the B. fragilis group: weight, every 8 h. B. fragilis, B. ovatus, B. thetaiotaomicron, or B. vulgatus 2–9 months of age, recommended dosage, is 80 mg piperacillin/10 mg tazobactam per kg of body weight, every 8 h. Pediatric patients 40 kg and with normal renal function should receive the adult dose. Higher risk of nephrotoxicity or AKI when piperacillin/tazobactam used in combination with vancomycin compared to either agent alone. GI disorders: diarrhea (11.3%), constipation (7.7%), nausea (6.9%), vomiting (3.3%), dyspepsia (3.3%), abdominal pain (1.3%). Renal—increases in SCr and BUN Hepatoxicity: piperacillin/tazobactam a risk factor for renal failure

Antibacterial agents

MOA

528

Drug/Generation/Class

UNASYNW: AMPICILLIN + SULBACTAM 3rd generation broad spectrum penicillin

An irreversible CBI of b-lactamase—Sulbactam can prevent from reducing antibiotic activity. The addition of sulbactam, a b-lactamase inhibitor, to ampicillin extends the spectrum of ampicillin activity against b-lactamase—producing organisms

FDA (Unasyn): Intra-abdominal infections: Adult dosage is 1.5 g (ampicillin https://www.accessdata.fda. Caused by b-lactamase producing strains of E. coli, Klebsiella spp. (including + sulbactam) to 3 g; every 6 h. gov/drugsatfda_docs/label/ K. pneumoniae), Bacteroides spp. (including B. fragilis), and Enterobacter Daily dose in pediatric patients is 300 mg/kg 2012/050608s040lbl.pdf spp. IV infusion every 6 h in equally divided. Gynecological infections: Caused by b-lactamase producing strains of E. coli,
and Bacteroides spp.
(including B. fragilis
).

Liver injury is associated with hypersensitivity NIDDK-LiverTox (NIH/NLM): http://www.ncbi.nlm.nih.gov/ or allergy (eosinophilia, arthralgias and skin books/NBK547872/ rash, and occasionally TEN or SJS) Overdose can lead to GI toxicity (adverse gastritis, stomatitis, black “hairy” tongue, pseudomembranous colitis, and enterocolitis) Neurological adverse reactions due to high CSF levels of beta-lactams

AVYCAZW: (CEFTAZIDIME + AVIBACTAM) 3rd generation cephalosporin

529

Neurotoxicity: confusion with temporospatial disorientation (96%), myoclonus (33%), and seizures (13%)

Antibacterial agents

Exerts a primarily bactericidal effect through In vitro experiments in Gram-negative bacteria such as E. coli, P. aeruginosa, Recommended dosage is 2.5 g (ceftazidime FDA (Avycaz): https://www. accessdata.fda.gov/ A. baumannii, and K. pneumoniae suggest that ceftazidime primarily binds to binding to PBP and the inhibition of cell wall + avibactam) administered every 8 h by IV drugsatfda_docs/label/ PBP3, with weaker binding to PBP1a/1b and PBP2 as well. synthesis. Inhibition of this cross-linking by infusion over 2 h in patients 18 years 2019/206494s005,s006lbl. b-lactam antibiotics greatly compromises with normal renal function. pdf the structural integrity of the bacterial cell Similarly, ceftazidime showed binding to S. aureus PBP 1, 2, and 3 with a much lower affinity for PBP4 wall and leads to aberrant cellular Dose of 62.5 mg/kg to a maximum of 2.5 g PubChem (Avibactam): https:// morphology, cell lysis, and death. (ceftazidime 50 mg/kg and avibactam pubchem.ncbi.nlm.nih.gov/ 12.5 mg/kg to a maximum dose of compound/9835049 Avibactam: Diazabicyclooctanone, nonceftazidime 2 g and avibactam 0.5 g) for b-lactam, b-lactamase inhibitor; When pediatric patients without renal alone has no antibacterial activity at impairment. clinically relevant doses, but in combination aged 2 to 5000 mg/kg

FDA (Ciprofloxacin): https://www.accessdata.fda. gov/drugsatfda_docs/label/ 2016/019537s086lbl.pdf

Nitrofurantoin (No author listed), 2022. https:// pubmed.ncbi.nlm.nih.gov/ 35121574/ >1803 mg/kg FDA (Levofloxacin): https://www.accessdata.fda. gov/drugsatfda_docs/label/ 2008/021721s020_ 020635s57_020634s52_ lbl.pdf

All Gram-negative pathogens and some Gram-positive bacteria Community-acquired pneumonia in hospitalized patients Hepatotoxicity, SJS, TEN. or if atypical pathogens are strongly suspected. (including Staphylococcus aureus, except Streptococcus pneumoniae) and some atypical organisms.

MOXIFLOXACIN (AVELOXW)

Covers all the activities of third generation drugs (Aerobic Gram-Positive and Gram-negative microorganisms) and extra anaerobic activity.

Acute Exacerbation of Chronic Bronchitis (AECB), CNS and gastrointestinal effects 100 mg/kg Community acquired pneumonia (CAP), acute bacterial such as decreased activity, sinusitis and uncomplicated skin and skin structure somnolence, tremor, infections convulsions, vomiting, and diarrhea

FDA (Moxifloxacin): https://www.accessdata.fda. gov/drugsatfda_docs/label/ 2016/021085s063lbl.pdf

DELAFLOXACIN (BAXDELAW)

Broad-spectrum in vitro activity, including activity against Gram-positive organisms, Gram-negative organisms, anaerobes, and atypical respiratory tract pathogens (i.e., Legionella, Chlamydia, and Mycoplasma)

Community-acquired bacterial pneumonia, acute bacterial skin, and skin structure infections

FDA (Delafloxacin): https://www.accessdata.fda. gov/drugsatfda_docs/label/ 2017/208610s000, 208611s000lbl.pdf

Nausea (8%), diarrhea (8%), N/A headache (3%), transaminase elevations (3%), and vomiting (2%)

Antibacterial agents

LEVOFLOXACIN (LEVAQUINW)

541 (Continued )

(Continued) Spectrum of Activity

RIFABUTIN (MYCOBUTINW)

Similar Gram-positive activity to Rifampicin but more potent activity against Mycobacterium spp., Streptococcus spp., and Escherichia coli

Toxicities

LD50 Dose

References

TB in combination; prevent bacterial meningitis

5% GI discomfort (nausea, vomiting)

1570 mg/kg

FDA (Rifamycin): https://www.accessdata.fda. gov/drugsatfda_docs/label/ 2020/050420s084, 050627s027lbl.pdf

4.8 g/kg

FDA (Rifabutin): https://www.accessdata.fda. gov/drugsatfda_docs/label/ 2008/050689s016lbl.pdf

3300 mg/kg

FDA (Rifapentine): https://www.accessdata.fda. gov/drugsatfda_docs/label/ 2010/021024s009lbl.pdf

5% flu-like syndrome T) may have increased progression-free survival time when treating colorectal cancer (Drug Bank: Cetuximab, n.d.; Petrelli et al., 2018; Mayfield et al., 2019; Muraro et al., 2021). Trastuzumab became the first humanized antibody registered for the treatment of solid tumors in 1998. Trastuzumab has a complex mechanism of antitumor action, which is due to the blockade of intracellular signal transduction pathways that are triggered by HER2. As a result of clinical studies, the following effects of trastuzumab have been proven: promotes internalization and degradation of HER2; inhibits proliferation and restores the ability of tumor cells to undergo apoptosis possibly due to blockade of the PI3K/AKT signaling pathway, which also determines the synergy of trastuzumab with chemotherapy; inhibits HER2-regulated angiogenesis; prevents the formation of HER2p95, a truncated active form of the HER2 receptor, which is characterized by the absence of an extracellular domain in the presence of tyrosine kinase activity, thus inhibiting tumor development. Trastuzumab also induces an antitumor immune response by activating antibody-dependent cellular cytotoxicity. The Fc domain is present in the structure of trastuzumab, which is a subclass G1 immunoglobulin. It is recognized by the effector cells of the immune system expressing the Fcg receptor. Due to the binding of natural killer cells to the Fc domain of trastuzumab, tumor cell lysis occurs. Trastuzumab’s overdose experience clinical trials is rather limited. Single dose toxicity has not been determined in humans. However, it can contribute to the development of ventricular dysfunction and congestive heart failure, particularly when used in combination to other cardiotoxic chemotherapies such as anthracycline CTDs. Patients with genotype (heterozygous-G allele) have increased risk of cardiotoxicity (Barok et al., 2014; Bregni et al., 2016; García-Alonso et al., 2020; Drug Bank: Trastuzumab, n.d.).

Restrictions on use Dyspnea at rest due to metastatic lesions or concomitant lung diseases (increased risk of lethal infusion reactions); patients at risk for cardiotoxicity (heart failure, arterial hypotension, coronary artery disease). Safety and efficacy in children have not been determined.

Side effects Digestive tract: nausea, vomiting, dyspeptic symptoms, anorexia, constipation, rarely—liver dysfunction, hepatitis, liver failure, jaundice, pancreatitis.

562

Anticancer therapeutic agents

Cardiovascular system and blood (hematopoiesis, hemostasis): tachycardia, vasodilation, hypotension, heart failure, cardiogenic shock, cardiomyopathy, palpitations; leukopenia, rarely—heart rhythm disturbance, decreased ejection fraction, thrombocytopenia, anemia; decrease in the level of prothrombin. Skin: itching, hyperhidrosis, dry skin, acne, maculopapular rash, nail damage, alopecia.

Signal transduction inhibitors—Tyrosine kinase inhibitors (TKIs) A new class of drugs, aka EGFR tyrosine kinase inhibitors (TKIs) target epidermal growth factor receptors (EGFRs), have shown promising results in clinical trials and on practice (such as erlotinib). Sensitivity to these is determined by the presence of somatic mutations in the EGFR gene in tumor cells, which are small deletions of amino acids 747–750 or point mutations (most often in the form of replacement of leucine for arginine in codon 858 (L858R)). TKI treatments to patients with a confirmed mutation in the EGFR gene is associated with an increase in the frequency of the desired responses, and most importantly contributes to an increase in progression-free life expectancy. In this regard, TKI therapy for non-small cell lung cancer (NSCLC) with a mutation in the EGFR gene has been overwhelmingly successful However, with most recent EGFR family members, such as, osimertinib, response appears to last over 2+ years, although these agents are not free of adverse effects (Gelatti et al., 2019). Extension of the above treatments show first-generation (gefitinib, erlotinib) and second-generation (afatinib, dacomitinib) EGFR-tyrosine kinase inhibitors (TKIs) have become standard-of-care (SoC) first-line treatment for patients with sensitizing EGFR mutation positive advanced NSCLC. However, this protocol is following Phase III trials versus platinum-based doublet chemotherapy. However, majority of patients treated with first-line first- or second-generation EGFR-TKIs somehow develop drug resistance. Osimertinib, a third-generation, central nervous system active EGFR-TKI which potently and selectively inhibits both EGFR-TKI sensitizing (EGFRm) and the most common EGFR T790M resistance mutations, has shown greater effectiveness versus first-generation EGFR-TKIs (gefitinib/erlotinib). Osimertinib has now become the best choice of treatment for patients with advanced NSCLC harboring EGFRm in the first-line setting, and treatment of choice for patients with T790M positive NSCLC following disease progression on first-line EGFR-TKIs. The second-generation EGFR-TKI dacomitinib has also recently been approved for the first-line treatment of EGFRm positive metastatic NSCLC (Gelatti et al., 2019). Zhao et al. published a systematic review and network meta-analysis of this family of drugs and found the following: rash, diarrhea, anorexia, nausea, vomiting, fatigue, stomatitis, anemia, constipation, paronychia, pruritis, dry skin, alopecia, liver enzyme elevation (both ALT & AST), cardiotoxicity, leucopenia, neutropenia, and interstitial lung disease (Zhao et al., 2019; Zhao et al., 2021; Waliany et al., 2021).

Imatinib (Gleevec) The mechanism of action Imatinib (2-phenylaminopyrimidine derivative) is a small molecule kinase inhibitor that revolutionized the treatment of cancer, particularly chronic myeloid leukemia, in 2001. The discovery of imatinib also established a new modality of therapy called “targeted therapy”, since treatment can be personalized specifically to the unique cancer genetics of each patient. Imatinib inhibits the activity of the enzyme tyrosine kinase, which plays a quintessential role in the growth and multiplication of tumor cells. Inhibition of tyrosine kinase leads to demise of these cellular processes (Hochhaus et al., 2016). CYP3A4 is the major enzyme responsible for the metabolism of imatinib. Other cytochrome P450 enzymes, such as CYP1A2, CYP2D6, CYP2C9, and CYP2C19, play a minor role in its metabolism. Imatinib (mesylate) specifically inhibits the BCR-ABL tyrosine kinase, the constitutively active tyrosine kinase created by the Philadelphia chromosome abnormality in CML patients. Although the role of normal BCR is still unclear, ABL activation is overexpressed in various tumors and is heavily implicated in cancer cells growth and survival. Imatinib inhibits the BCR-ABL protein by strongly binding to the ATP pocket in the active site, thus halting downstream phosphorylation of target protein. Imatinib is also known to inhibit of the receptor tyrosine kinases for platelet-derived growth factor (PDGF) and stem cell factor (SCF), c-Kit, and inhibits PDGF- and SCF-mediated cellular events. In clinical studies, Gleevec’s ability to increase the overall survival of patients, as well as the period of progression-free survival, was statistically significant. In addition, with long-term therapy, a decrease in the likelihood of recurrence of the disease has been reported. Although an exemplary compound, its contraindications include imatinib allergy, pregnancy, lactation, and if the patient’s age is $170 billion each year. The vast majority of the current medications are aimed at maintaining HbA1c levels via suppressing hepatic glucose production, activating b-cells to release insulin, increasing tissue sensitivity to insulin, increasing peripheral glucose utilization, and decreasing glucose absorption in the gut. Although most agents are designed to treat hyperglycemia or indirectly lower HbA1c, their long-term effects on the pathophysiology of b-cell function has not been well documented. The major classes of agents that are available include biguanides, sulfonylureas, meglitinides, dipeptidylpeptidase-4 inhibitors (aka DPP-4i), Glucagon like peptide-1 (GLP-1) agonists, Sodium Glucose Cotransporter-2 inhibitors (aka SGLT-2i), alpha-glucosidase inhibitors, thiazolidinediones, and many forms of insulin. All these agents show toxicity and adverse reactions at some point, short-term or long-term. Many agents show drug interactions, and a few are dependent on patient pharmacogenetics. This review is focused on mechanisms of actions of all these classes and their possible adverse effects.

Keywords Albiglutide; Acarbose; Biguanide; Canagliflozin; Cardiomyopathy; Dapagliflozin; dpp-4 inhibitors; Empagliflozin; Exenatide; Glimepiride; Glipizide; glp-1 agonists; Glyburide; HbA1c-glycohemoglobin; Hyperglycemia; Hypoglycemia; Insulin secretagogue; Liraglutide; Liver; Meglitinides; Metformin; Miglitol; Nateglinide; Nephropathy; Neuropathy; Pancreas; Pioglitazone; Repaglinide; Retinopathy; Rosiglitazone; Saxagliptin; sglt-2 inhibitors; Sitagliptin; Sulfonylurea; Type 1 diabetes mellitus (T1DM); Type 2 diabetes mellitus (T2DM); Voglibose; b-cell

Key points

•

⁎

T2DM is an ensemble of metabolic diseases that has reached pandemic proportions. Although T2DM is a preventable disease via appropriate diet control, exercise and life-style modifications, most patients try to manage with medications.

These authors equally contributed in preparation of this manuscript.

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.01134-9

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Anti diabetic agents

Because insulin is quintessential to almost all the tissues in the body, defects in pancreatic insulin production leads to a complex pathology (hyperglycemia, hyperlipidemia, oxidative stress, inflammation and cell death) that requires extensive lifestyle modifications simultaneously with life-long drug therapy. The most viable options are early detection and aggressive management during pre-diabetic stage in high risk T2DM individuals. Prevention strategies may begin with both pharmacologic (specific medications) and non-pharmacologic (phytochemicals and lifestyle modifications) interventions. The above approaches will at least delay or slow the progression of macro- and microvascular complications associated with cardiovascular, renal, ocular and nervous system. The most remarkable progress in DM management in the last three decades has been the development of a variety of medications that has wide-range of targets and suitable for different types of people (children or elderly, obese or non-obese, with or without genetic predisposition). Additional accomplishment has been the different types of formulations and combination therapies which has considerably reduced toxicity, adverse effects, comorbidities and extended life-span in general. The American Diabetes Association criteria for acceptable glycemic control include an HbA1c of less than 7% (53 mmol/mol), premeal glucose levels of 90–130 mg/dL (5–7.2 mmol/L), and peak postprandial glucose of less than 180 mg/dL (10 mmol/L). This review has focused on mechanisms of actions of all the classes of anti-diabetic medications, their side effects and toxicity profiles.

Introduction The disease diabetes is characterized by elevated blood glucose (aka blood sugar). Blood glucose provides energy and comes from the food. Insulin, a pancreatic hormone, facilitates transport of glucose from blood to different cells and tissues throughout the body. Occasionally, the body doesn’t make enough insulin or doesn’t use insulin adequately which leads to glucose accumulation in the blood (high glucose in blood is aka hyperglycemia). Loss of insulin receptors of tissues is also very common. Hyperglycemia is the root cause of all these diabetic complications (Centers for Disease Control and Prevention, 2017). The most common types of diabetes are type 1 (aka T1DM), type 2 (T2DM), and gestational diabetes. Type 1 diabetics do not make enough insulin because body’s immune system (auto antibodies) attacks and destroys insulin-producing b-cells of the pancreas. Due to this, people with T1DM need to take insulin every day to stay alive, and they do not respond to most antidiabetic medications. Insulin promotes synthesis (from circulating nutrients) and storage of glycogen, triglycerides, and protein in its major target tissues: liver, fat, and muscle. The release of insulin from the pancreas is stimulated by increased blood glucose, incretins, vagal nerve stimulation, and other factors. In contrast to T1DM, T2DM individuals either do not make or use insulin adequately. T2DM is the most common type of diabetes, and these individuals respond to all the medications available to various degrees. A third type is called gestational diabetes, and it develops during pregnancy in some women. Although this type of diabetes goes away after the baby is born, it increases the chance of developing type 2 diabetes later in life. The risk factors for type 2 diabetes are: improper diet/nutrition, sedentary lifestyle, genetic predisposition, continued life stress, and lack of physical activity. Over time, hyperglycemia leads to a number of comorbidities. Late clinical manifestations may include a number of pathologic changes that involve small and large blood vessels, cranial and peripheral nerves, the skin, and the eye. These lesions lead to hypertension, end-stage chronic kidney disease, blindness, autonomic and peripheral neuropathy, amputations of the lower extremities, myocardial infarction, and cerebrovascular accidents. In T2DM, end-stage chronic renal disease develops in up to 40% of patients, compared with 3200 mg/kg (oral, mice), >4000 mg/kg (oral, rats), >10,000 mg/kg (oral, rats), and >3000 mg/kg (oral, mice), respectively (Drug Bank: Gliclazide, n.d.). Sulfonylureas are extensively metabolized in the liver by cytochrome CYP2C9 isoform, and hence patients on medications that inhibit or activate this enzyme may be subjected to sulfonylurea toxicity or a lack of efficacy (Gunaratne et al., 2018). Observational studies in T2DM patients have shown that use of Glyburide was associated with a higher risk of developing cancer compared to the other second-generation sulfonylureas (similar observations were noted with patients that didn’t receive Glyburide at all). Although the increase was clinically insignificant, Glyburide’s ability to generate reactive oxygen species (ROS) has been theorized to be the pro-oncogenic trigger (Tuccori et al., 2015). In addition, a study comprising the Drosophila wing spot test has shown that Glimepiride and Glipizide exhibit genotoxic effects, which are dependent on homologous somatic recombination (Gürbüzel et al., 2014) (Table 2).

SGLT-2 Inhibitors SGLT-2 inhibitors (Sodium-glucose co-transporter 2 inhibitors) are used specifically for the treatment of T2DM. Located within the proximal convoluted tubules in the kidneys, SGLT-2 is a low-affinity, high-capacity glucose transporter that contributes to the reabsorption of glucose after it is freely filtered by the renal glomeruli. SGLT-2 is a major renal glucose transporter that is responsible for approximately 90% of renal glucose transport. SGLT-2 inhibitors function through a novel mechanism of reducing renal tubular glucose reabsorption, by lowering blood glucose changing the renal threshold without stimulating the release of insulin. Therapeutic benefits consist of effects on blood pressure and body weight, whereas adverse effects may include increased urination leading to an increase in thirst, increased urinary tract infections, and possible hypotension. This specific class of lipid-lowering agents consists of canagliflozin, dapagliflozin, and empagliflozin, which are all oral medications approved for clinical use but are also available in XR forms. SGLT-2 inhibitors are available as monotherapy or in combination with other diabetes medications such as metformin. For example, when canagliflozin is combined with metformin, the brand name is called Invokamet®, with dapagliflozin it is called Xigduo XR®, and with empagliflozin it is called Synjardy®. SGLT-2 inhibitors inhibit the reabsorption of glucose, which results in lowering of blood glucose levels causing glycosuria coupled with loss of water; this is also called osmotic diuresis. After administration, SGLT-2 inhibitors are metabolized by glucuronidation in the liver and kidneys by uridine diphosphate glucuronosyltransferase-1A9 (UDP-1A9). If metabolized, the metabolites produced from canagliflozin, dapagliflozin, empagliflozin include two O-glucuronides called M7 and M5, 3-O-glucuronide, and 2-O-, 3-O-, and 6-O-glucuronide respectively. SGLT-2 inhibitors are FDA-approved for use with diet and exercise for T2DM patients. Toxicity of SGLT-2 inhibitors consist of fetal risks, especially during 2nd and 3rd trimesters, hypoglycemia, macrovascular outcomes, genital infections, urinary tract infection, increased urination, hypotension, acute kidney injury, dose-related changes in LDL-C, rare diabetic ketoacidosis (may occur with no hyperglycemia), necrotizing fasciitis of the perineum (Fournier’s Gangrene), urosepsis and pyelonephritis, intravascular volume depletion, increased risk of bone fractures and amputations, especially lower-limb (with canagliflozin), CHF hospitalizations (with empagliflozin and canagliflozin in persons with clinical CVD) (Lipscombe et al., 2018). In addition, these agents are contraindicated during lactation, in people with severe hepatic impairment, with renal dysfunction or renal impairment, those in end-stage renal disease and using loop diuretics, in the elderly, those with bladder cancer, those with serious hypersensitivity reactions such as anaphylaxis or angioedema, and any patients on dialysis (Lipscombe et al., 2018; Padda et al., 2022). The most common adverse reactions for SGLT-2 inhibitors with a 5% or greater incidence were female genital mycotic infections, urinary tract infections, increased urination. Empagliflozin causes nasopharyngitis (Table 3).

GLP-1 agonists Glucagon-like peptide-1 (aka GLP-1 agonists, incretin mimetics, GLP-1 analogs, or GLP-1 receptor agonists), are a class of drugs used to manage type 2 T2DM, and in some circumstances, obesity. Exenatide, Lixisenatide, Liraglutide, Albiglutide, Dulaglutide, and Semaglutide are a few examples in this class. Although metformin remains the drug of choice for treating T2DM, the GLP-1 agonist is suitable for patients who are either intolerant to metformin or patients with hemoglobin A1C (HbA1C) (Collins et al., 2023). Studies have shown that GLP-1 agonists can reduce HbA1C levels by 1.0% compared to placebo, although efficacies within the class vary (Morris, 2021; Waldrop et al., 2018). GLP-1 is an incretin hormone produced from proglucagon and secreted by the L-cells of the small intestine in response to food intake or hyperglycemia (Seino et al., 2010). The GLP-1 supports glycemic regulation by acting on the a-cells to inhibit postprandial glucagon release, increases the secretion of insulin from the b-cells of the pancreas, and delays gastric emptying (Collins and Costello, 2023; Seino, 2010). The GLP-1 agonists are also shown to promote pancreatic b-cell proliferation while reducing their apoptotic cell death (Table 4).

Table 2

Mechanism of action and toxicity profile of sulfonylureas.

Name of the drug W

References

Sulfonylureas stimulate pancreatic beta cells to release insulin by binding and inhibiting sulfonylurea receptors (SUR) on pancreatic b-cell membranes, inhibit insulin metabolism in the liver, decrease secretion of glucagon, and increase insulin sensitivity in peripheral tissues. Following figures depict the structure of Tolbutamide, a first-generation sulfonylurea (Upper panel: with the molecular formula C12H18N2O3S), and the structure of Glimepiride, a second-generation sulfonylurea (Lower panel: with the molecular formula C24H34N4O5S). Upper panel: 2-dimensional Structure of Tolbutamide (OrinaseW)

Overdosage of Glimepiride may lead to severe hypoglycemia with coma, seizures, or other neurological impairment. Increased risk of cardiovascular events (especially with Tolbutamide). Liver injury with concurrent fatigue, nausea, abdominal pain, dark urine, itching and jaundice Increased reactive oxygen species and higher instances of cancer (Glyburide) Sulfonylurea-induced hypoglycemic episodes are managed with octreotide (Sandostatin); presence of CYP2C9 3 allele increases risk of T2DM in sulfonylurea treated patients (Ragia et al., 2009).

Costello et al. (2023) PubChem: Tolbutamide (n.d.) PubChem: Glimepiride (n.d.) LiverTox (2018) Tuccori et al. (2015) Ragia et al. (2009)

Lower panel: 2-dimensional Structure of Glimepiride (AmarylW)

Anti diabetic agents

Toxicity Profile

578

Tolbutamide (Orinase ), Chlorpropamide (DiabineseW), Tolazamide (TolinaseW), Glyburide (DiabetaW), Glipizide (GlucotrolW), Glimepiride (AmarylW), Gliclazide (DiamicronW)

Mechanism of Action

Table 3

Mechanism of action and toxicity profile of SGLT-2 inhibitors.

Name of the Drug

Mechanism of Action

Toxicity Profile

References

Canagliflozin (InvokanaW), Dapagliflozin (FarxigaW), Empagliflozin (JardianceW)

SGLT-2 inhibitors inhibit the reabsorption of glucose. This results in the loss of glucose, known as glycosuria, and the loss of water, also known as osmotic diuresis. Figures depict the structure of canagliflozin (upper panel: with a molecular formula C24H25FO5S), whereas lower panel depicts the structure of empagliflozin (with a molecular formula C23H27ClO7) (PubChem; Empagliflozin, n.d.).

Fetal risks (especially during 2nd and 3rd trimesters), hypoglycemia, genital infections, urinary tract infection, increased urination, hypotension, dose-related changes in LDL-C, rare diabetic ketoacidosis (may occur with no hyperglycemia), necrotizing fasciitis of the perineum (Fournier’s Gangrene), urosepsis and pyelonephritis, intravascular volume depletion, increased risk of fractures and amputations with canagliflozin, reduced progression of nephropathy and CHF hospitalizations with empagliflozin and canagliflozin in persons with clinical CVD (Lipscombe et al., 2018). Contraindicated during lactation, with severe hepatic impairment, with renal dysfunction using loop diuretics, in the elderly, dapagliflozin not to be used if bladder cancer, those with serious hypersensitivity reactions such as anaphylaxis or angioedema, and any patients on dialysis (Lipscombe et al., 2018; Padda et al., 2022).

PubChem; Canagliflozin (n.d.) PubChem; Empagliflozin (n.d.) Lipscombe et al. (2018) Padda et al. (2022)

2-dimensional Structure of Canagliflozin (InvokanaW)

Anti diabetic agents 579

2-dimensional Structure of Empagliflozin (JardianceW)

580

Mechanisms and toxicity profiles of GLP-1 agonists.

Name of the drug

Mechanism of Action

Toxicity Profile

References

Exenatide (ByettaW), Lixisenatide (AdlyxinW), Liraglutide (SaxendaW), Albiglutide (EperzanW), Dulaglutide (TrulicityW), Semaglutide (WegovyW)

GLP-1 is a hormone that modulates blood glucose by inhibiting postprandial glucagon release, delaying gastric emptying, and secreting insulin. Figure (upper panel) depicts the structure of Semaglutide (molecular formula C187H291N45O59), whereas lower panel depicts the structure of exenatide (molecular formula C184H282N50O60S). Upper panel: 2-dimensional Structure of Semaglutide (WegovyW)

Gastrointestinal disorders, acute kidney injury, dyspepsia, infection, anaphylaxis (especially with exenatide), acute pancreatitis, and gallstone disease. Contraindicative with a personal and family history of medullary thyroid cancer or MEN2. GLP-1 agonists are also contraindicative in patients with type 1 diabetes. Patients who are pregnant or breastfeeding should not use GLP-1 agonists.

Collins et al. (2023) PubChem: Semaglutide (n.d.) PubChem: Byetta (n.d.) Lipscombe et al. (2018)

Lower panel: 2-dimensional Structure of Exenatide (ByettaW)

Anti diabetic agents

Table 4

Anti diabetic agents

581

DPP-4 Inhibitors Dipeptidyl peptidase 4 (DPP-4) inhibitors, also known as gliptins, are a class of antihyperglycemic drugs used in conjunction with diet and exercise to manage type 2 diabetes mellitus. Sitagliptin, Saxagliptin, Linagliptin, and Alogliptin are a few examples of this drug class. Because the DPP-4 drugs are not recommended as an initial monotherapy, various combination medications are available, including gliptin-metformin, gliptin-pioglitazone, and gliptin-sodium glucose co-transporter-2 inhibitors. Both the glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are considered incretin hormones. GLP-1 as mentioned above is secreted by L-cells of the small intestine whereas, the GIP is secreted by the K-cells of the small intestine. These incretin hormones are released in response to the ingestion of food and stimulate insulin secretion thereby regulating glucose homeostasis. The incretin hormones can stimulate the pancreas to increase the release of insulin and inhibit glucagon secretion. The incretin hormones also increase satiety, decrease appetite, and are responsible for delaying gastric emptying. The DPP-4 is a serine protease that is responsible for the inactivation of GLP-1 and GIP by cleaving the N-terminal dipeptide. These inactivated peptides are then eliminated via the renal system, with the rate of clearance exceeding the glomerular filtration rate, indicating that active transport is involved (Makrilakis, 2019; Kasina et al., 2022). With the exception of saxagliptin, there are no major drug-drug interactions when DPP-4 inhibitors; they are often co-administered with metformin or simvastatin. Saxagliptin undergoes liver metabolism by CYP3A4/5 to its primary metabolite. Saxagliptin accumulation and toxicity is very likely when it is accidentally co-administered with strong CYP3A4/5 inhibitors, such as ketoconazole and clarithromycin (Makrilakis, 2019). Drugs in this class also exhibit antihypertensive, anti-inflammatory, antiapoptotic, and immunomodulatory effects on the heart, kidneys, and blood vessels independent of the incretin pathway. It has been suggested that kidney and liver transplant recipients can benefit from this class if they develop new-onset diabetes after transplantation (NODAT) (Table 5).

Metformin (Biguanides) Biguanides are a group of antihyperglycemic drugs that work in the treatment of T2DM. All biguanides have the same active ingredient (metformin) and are formulated to have varying release mechanisms. Metformin is a first line therapy often used jointly with diet and exercise to lower blood glucose without triggering hypoglycemia. This causes a decrease in insulin resistance and fasting insulin levels, as well as modest weight loss which can be seen in obese patients with T2DM (Drug Bank: Metformin, n.d.). Metformin achieves glycemic control via acting on the liver. It decreases the absorption of glucose in the intestines and increases insulin sensitivity without affecting the secretion of insulin. The mechanism of action involves the inhibition of mitochondrial complex I (of the respiratory chain), which leads to a decrease in mitochondrial ATP. This causes the activation of the AMP-activated protein kinase (AMPK) pathway which is responsible for regulating glucose metabolism. This pathway promotes the inhibition of the fructose-1,6-bisphosphatase, resulting in the blockage of gluconeogenesis, and the phosphorylation of acetyl-CoA carboxylase, resulting in decreased fat synthesis and hepatic lipids, ultimately causing increased insulin sensitivity in the liver (Drug Bank: Metformin, n.d.). In effect, metformin reduces both basal and postprandial blood glucose levels without carrying the risk of hypoglycemia (Corcoran et al., 2022). Metformin’s actions begin within 3 h after ingestion with a half-life of about 20 h. It is not metabolized in the liver or bound to proteins, and thus, is renally excreted as unchanged drug. While it is known to be safe, some side effects include diarrhea, nausea, and vomiting, which occur in up to 30% of patients. Patients may also develop diaphoresis, rhinitis, and vitamin B12 deficiency, although these effects occur less frequently. There is a black box warning for lactic acidosis, which can lead to metabolic acidosis but is rare; usually occurring in hepatically or renally impaired and/or elderly patients. Toxicity is related to metformin overdose, which is associated with lactic acidosis (oral LD50: rat 1 g/kg, mouse 1450 mg/kg, intraperitoneal LD50: rat 500 mg/kg, mouse 420 mg/kg). Furthermore, toxicity is often associated with the potentiation of hypoglycemia only when metformin is used in combination with other antidiabetic drug classes, such as sulfonylureas and insulin (Drug Bank: Metformin, n.d.). Metformin shows drug interaction with carbonic anhydrase inhibitors (increased risk of lactic acidosis), ranolazine, dolutegravir, and cimetidine (which reduce metformin clearance) (Wang and Hoyte, 2019; Akhter and Uppal, 2020; Riomet, 2019) (Table 6).

Meglitinides Meglitinides are a class of short-acting, oral antihyperglycemic agents used for treating adult T2DM patients. As a non-sulfonylurea antidiabetic secretagogue that promote insulin secretion, meglitinide analogs are repaglinide (Prandin®), mitiglinide (Glufast®) (N/A in U.S.), and nateglinide (Starlix®) (Rosa et al., 2014). These drugs that make up the meglitinide class exert their hypoglycemic effect by increasing production of endogenous insulin from the pancreas, thereby decreasing levels of blood glucose in the body (Milner and Akhondi, 2022). This pancreatic beta cell selectivity is characteristic of the meglitinides as there are no biochemical implications pertaining to skeletal or cardiac tissues (PubChem: Repaglinide, n.d.; PubChem: Nateglinide, n.d.). As a representative

582

Mechanisms of actions and toxicity profiles of DPP-4 inhibitors.

Name of the drug

Mechanism of Action

Toxicity Profile

References

Sitagliptin (JanuviaW), Saxagliptin (OnglyzaW), Linagliptin (TradjentaW), Alogliptin (NesinaW)

DPP-4 inhibitors, known as gliptins, prevent the degradation of incretin hormones such as GLP-1 and GIP which increases insulin secretion and delayed glucagon release to maintain glucose homeostasis. Figures below depict the structure of Sitagliptin (upper panel with a molecular formula C16H15F6N5O), and Alogliptin (lower panel with a molecular formula C18H21N5O2.). Upper panel: 2-dimensional Structure of Sitagliptin (JanuviaW)

Upper respiratory tract infection, acute pancreatitis, severe arthralgias. (Especially with saxagliptin, and sitagliptin), urinary tract infection, hypersensitivity reaction including Steven-Johnson syndrome, and heart failure (especially with saxagliptin). The Food and Drug Administration (FDA) issued a warning about a significantly increased risk of heart failure with the use of saxagliptin and alogliptin (Kasina et al., 2022; Packer, 2018). A recent study compared the risk of heart failure between 6330 patients taking saxagliptin alone and 10,162 patients taking saxagliptin with betablockers. The study showed that saxagliptin alone increased the risk of heart failure by 81% versus 18% in patients taking saxagliptin concurrently with beta-blockers (Zannad et al., 2015; Packer, 2018).

Kasina et al. (2022) PubChem: Sitagliptin (n.d.) PubChem: Alogliptin (n.d.) Packer (2018)

Lower panel: 2-dimensional Structure of Alogliptin (NesinaW)

Anti diabetic agents

Table 5

Table 6

Mechanism of action and toxicity profile of metformin.

Name of the Drug

Mechanism of Action

Toxicity

References

Metformin (GlucophageW, Glucophage XRW, FortametW) Glipizide/metformin (MetaglipW) Canagliflozin/metformin (InvokametW) Dapagliflozin/metformin (Xigduo XRW) Empagliflozin/linagliptin/metformin (Trijardy XRW) Metformin/pioglitazone (ActoPlus MetW) Metformin/Sitagliptin (JanumetW) Metformin/Saxagliptin (KombiglyzeW)

Biguanides reduce blood glucose levels by working in the liver to decrease gluconeogenesis, decreasing intestinal absorption, and increasing insulin sensitivity (Corcoran, 2022). Figure below depicts the structure of metformin (with a molecular formula: C4H11N5), 2-dimensional Structure of Metformin (GlucophageW)

Metformin associated lactic acidosis (MALA) most common adverse effect, Gastrointestinal and neurological symptoms, metabolic acidosis, hyperlactatemia (Juneja et al., 2022). Contraindicated in patients with severe renal dysfunction, hypersensitivity to metformin, and metabolic acidosis (Corcoran, 2022). Headache, diaphoresis, rhinitis, and vitamin B12 deficiency (Corcoran et al., 2022). No evidence of association of toxicity with use of metformin during pregnancy; Metformin present in human milk, insufficient data for use in nursing (PubChem: Metformin, n.d.).

Corcoran et al. (2022) PubChem: Metformin (n.d.) PubChem: Janumet (n.d.) Juneja et al. (2022)

Anti diabetic agents 583

584

Anti diabetic agents

member of the meglitinide group of insulin-secreting agents, repaglinide monotherapy has been associated with adverse effects that include hypoglycemia, upper respiratory tract infections, gastrointestinal upset, joint pain, and weight gain (Milner and Akhondi, 2022). Meglitinides can be utilized as monotherapy, when supplemented with regulated diet and exercise, or with other combo medication such as repaglinide plus metformin (PrandiMet®), and with thiazolidinediones such as rosiglitazone plus pioglitazone. Meglitinides are not to be administered in combination with sulfonylureas such as glipizide due to similar mechanisms of action that involve closure of ATP-sensitive potassium channels. Due to the lack of sulfur in its composition, meglitinides are indicated for use in type 2 diabetics that have sulfur allergies. Meglitinides are advantageously utilized in patients with chronic kidney disease and end-stage renal disease as excretion of these drugs is primarily via the liver (Milner et al., 2022). The meglitinide analogs are quickly absorbed and have relatively short half-lives. The active drug peaks in plasma within an hour when taken orally. Absolute bioavailability is 56% for repaglinide and 73% for nateglinide (PubChem: Repaglinide, n.d.; PubChem: Nateglinide, n.d.). In terms of distribution, repaglinide binds primarily to serum albumin 98%. Due to their rapid onset and distinctive duration of effect, the drugs of this class are ideal for therapeutic use in the management of postprandial glucose level spikes and are therefore taken about 30 min before meals (Milner et al., 2022). Metabolism of repaglinide occurs extensively in the liver via oxidation and dealkylation by CYP2C8 and CYP3A4 isoforms, and metabolites are excreted in bile (90% in feces, 8% in urine). This makes the drug suitable for use in patients with renal impairments and in elderly (PubChem: Repaglinide, n.d.). A major dicarboxylic acid derivative metabolite (M2) is formed, and additional oxidation leads to the formation of an aromatic amine derivative (M1) of repaglinide. Phase-II metabolism leads to acyl glucuronide (M7) formation. Metabolites of repaglinide do not exhibit any hypoglycemic effect (DrugBank: Rosiglitazone, n.d.). Nateglinide is metabolized via hydroxylation and glucuronidation by CYP2C9 (70%) and to a lesser extent by CYP3A4 (30%), while the parent compound and its metabolites are excreted primarily in the urine (83%) and in the feces (10%). The main metabolites of nateglinide do not show any glucose-lowering effect, but the parent drug and its minor isoprene metabolite show the most anti-hyperglycemic activity (Backman et al., 2016; PubChem: Nateglinide, n.d.). Repaglinide and nateglinide, phenylalanine derivative of the meglitinides, have been associated with infrequent cases of acute liver injury concurrent with clinical use (PubChem: Repaglinide, n.d.; PubChem: Nateglinide, n.d.). In the case of overdoses, amplified glucose-lowering effects along with prominent hypoglycemic symptoms result. Activated charcoal may be used as an antidote in the case of intentional overdose with significant repaglinide that is caught early, while immediate feeding is effective in relieving symptoms in the case of unintentional, unrestrained ingestion (Milner and Akhondi, 2022). Subcutaneous or intramuscular glucagon injections can also be considered in more severe cases along with octreotide therapy (Fasano and Rowden, 2009). Repaglinide use by pregnant and nursing mothers require precaution because it is a FDA class C category drug (Milner and Akhondi, 2022). Long-term carcinogenicity studies performed in male and female rats revealed no evidence of cancer potential, while there was a more prominent occurrence of thyroid and liver benign adenomas in male rats (FDA.gov: Rosiglitazone, n.d.; Drug Bank: Nateglinide, n.d.). Both in vivo and in vitro studies affirmed that meglitinides are non-genotoxic and that there was no impairment of fertility caused by administration of such agents in rat studies at doses exponentially greater than that of human therapeutic exposure with recommended dose. Due to meglitinides’ mechanism of action depends upon the presence of glucose, the risk of hypoglycemia is not as drastic as compared to sulfonylureas. Meglitinides also are effective in decreasing HbA1c levels and are more influential in diminishing postprandial blood glucose in comparison to other antidiabetic agents including sulfonylureas, metformin, and thiazolidinediones (FDA.gov: Rosiglitazone, n.d.; Drug Bank: Nateglinide, n.d.; DrugBank: Rosiglitazone, n.d.) (Table 7).

Thiazolidinediones (TZDs) TZDs act to decrease insulin resistance. They are ligands of peroxisome proliferator activated receptor gamma (PPARg), members of the steroid and thyroid superfamily of nuclear receptors. These PPAR receptors are found in muscle, fat, and liver. PPARg receptors modulate the expression of the genes involved in lipid and glucose metabolism, insulin signal transduction, and adipocyte and other tissue differentiation. Observed effects of the TZDs include increased glucose transporter expression (GLUT 1 and GLUT 4), decreased free fatty acid levels, decreased hepatic glucose output, increased adiponectin and decreased release of resistin from adipocytes, and increased differentiation of preadipocytes to adipocytes. There are currently two medications in this calss: pioglitazone and rosiglitazone, which can be taken independently or in combination with metformin and sulfonylureas. TZDs function by acting on intracellular metabolic pathways as an agonist for PPARg, a nuclear transcription regulator. This allows regulation of gene expression in insulin sensitive target tissues such as muscle, fat, and liver. The direct actions of TZDs include

Table 7

Mechanism of action and toxicity profile of Meglitinides.

Name of Drug

Mechanism of Action

Toxicity Profile

References

Repaglinide (PrandinW), Mitiglinide (GlufastW) (N/A in U.S.), Nateglinide (StarlixW)

Meglitinides are hormone-secreting agents that work to trigger the release of insulin from the pancreas via action on various beta-cell receptors (Ganesan et al., 2022). Similar to the sulfonylurea class, the hypoglycemic effect of meglitinides involves efflux regulation and inhibition of ATP-sensitive potassium channels. Figures illustrate the chemical structure of repaglinide (upper panel: molecular formula: C27H36N2O4; Mol. wt. 452.6), and lower panel shows the chemical structure of a d-phenylalanine derivative of the meglitinide class, nateglinide (with a molecular formula: C19H27NO3; Mol. wt. 317.4). Upper panel: 2-dimensional structure of Repaglinide (PrandinW)

Severe hypoglycemia, upper respiratory tract infection, sinusitis, gastrointestinal upset, musculoskeletal symptoms. Rare cases of cholestatic or mixed hepatitis associated with repaglinide use. Contraindicated in patients with diabetes mellitus type 1, diabetic ketoacidosis, and in patients with severe liver dysfunction (Milner et al., 2022). Repaglinide: LD50 >1 g/kg (rat)

Ganesan et al. (2022) PubChem: Repaglinide (n.d.) PubChem: Nateglinide (n.d.) Milner et al. (2022) Drugbank.ca (2023) Rosa et al. (2014) Repaglinide. FDA (2019) Nateglinide: FDA (2021) Fasano et al. (2009)

Lower panel: 2-dimensional structure of Nateglinide (StarlixW)

Anti diabetic agents 585

586

Anti diabetic agents

decreasing hepatic and peripheral triglycerides, increasing adiponectin, and promoting lipogenesis (Lebovitz, 2019). Studies show that TZDs decrease HbA1C levels by about 0.5% and may preserve b-cell function better than some medications, such as sulfonylureas since it is a direct insulin sensitizer (Nanjan et al., 2018; Lebovitz, 2019). Along with these functions, TZDs may have anti-inflammatory and anti-cancer effects. TZDs may also be used to treat polycystic ovarian syndrome. Pioglitazone specifically has cardioprotective properties and reduces hepatic fat and may improve liver fibrosis in those with nonalcoholic steatohepatitis. While there is potential, the mechanisms for these additional benefits are still under investigation (Eggleton and Jialal, 2023a).

Bile acid sequestrants (BASs) Three bile acid sequestrants are available in the United States: cholestyramine (Questran), Colesevelam (Welchol) and colestipol (Colestid). Most commonly used BAS to treat T2DM is Colesevelam (Welchol®). Bile acid resins or sequestrants are the oldest and safest lipid lowering agents (primarily remove LDL cholesterol), but they are not so popular because they are not well tolerated by the patients and newer classes of cholesterol lowering drugs are much more potent. BASs are highly positively charged molecules that bind to the negatively charged bile acids in the gut and inhibit cholesterol absorption and reabsorption of bile acids (which is typically 95%). BASs are advantageous because they also show blood glucose lowering ability (exact mechanism remains unknown). BASs prevent bile acid absorption from stomach, so it does not recirculate into blood. This makes the liver pull cholesterol from the blood to synthesize bile acid, which indirectly helps lower blood cholesterol level. Some of the side effects of BASs may include gas and constipation.

Dopamine-2 agonists Dopamine-2 agonists normally reset hypothalamic circadian rhythm. This reset helps reverse insulin resistance and causes a decrease in glucose production in the liver. Bromocriptine (Cycloset®) is the only USFDA-approved dopamine-2 agonist used to treat T2DM.

Conclusion It is well known that T2DM is a preventable disease, therefore lowering the incidence of new T2DM cases could be a key strategy to reduce the global impact of diabetes. Endocrinologists have made major progress in understanding how insulin behaves in our body and learned how to tackle tissue’s unresponsiveness to insulin by different chemical entities. Progress made in antidiabetic drug development in the last 3 decades has provided many hopes for the patients to live a normal and a longer life. Intelligently designed combination therapies have shown tremendous success in patient management in the last two decades. Truncated regimens are not optimal for all patient populations and certainly not affordable in all the parts of the World. Difficult to treat patients, such as those with pre-existing conditions (liver disease, renal failure, pharmacogenetics pre-disposition) might benefit from the newer generations of antidiabetics. Occasionally, even effective treatment strategies fail due to unpredictable factors in a given population. Some drugs often show unpredictable drug–drug interactions, and many agents show desired outcomes but produce toxic side effects on long-term therapy which jeopardize the patient’s ability to complete the full drug regimen. Several other issues that can complicate therapeutic outcomes are a drug’s very short or very long half-life, poor patient compliance, development of insensitivity to the drug during the therapy, immunocompromised status, and low bioavailability. Regardless of all these confounding factors, diabetes care has made unprecedented success in patient management due to these antidiabetic medications (Schalliol et al., 2019; D’Souza et al., 2021; D’Souza et al., 2022) (Table 8).

Table 8

Mechanism of action and toxicity profile of Thiazolidinediones.

Name of Drug

Structure and Mechanism of Action

Toxicity

References

Pioglitazone (ActoplusW, MetW, ActosW, DuetactW, IncresyncW, OseniW, TandemactW) Rosiglitazone (AvandamentW, AvandiaW)

TZDs function as agonists of peroxisome proliferator-activated receptor-gamma (PPARg), a nuclear receptor that regulates gene expression. Following figures depict the structure of pioglitazone (Upper panel: with a molecular formula of C19H20N2O3S), and the lower panel shows the structure of rosiglitazone (with a molecular formula of C18H19N3O3S). Upper panel: Structure of Pioglitazone

TZDs may cause fluid retention, edema, and weight gain. Studies have shown decreased bone density and increased fractures with TZD use. TZDs have teratogenic potential as it can cross the placenta and should not be used by pregnant women. There is also increased correlation with hepatic dysfunction. TZDs, especially Rosiglitazone, should be used in caution for patients with history of congestive heart failure as it is shown to have increased risk for myocardial infarction. Therapy with TZDs may result in ovulation in premenopausal anovulatory women. Most common adverse reactions include upper respiratory tract infection, headache, sinusitis, myalgia, and pharyngitis.

DrugBank: Pioglitazone (n.d.) DrugBank: Rosiglitazone (n.d.) Eggleton and Jialal (2023b) FDA.gov: Pioglitazone (n.d.) FDA.gov: Rosiglitazone (n.d.) Hurren and Dunham (2021) PubChem: Pioglitazone (n.d.) PubChem: Rosiglitazone (n.d.)

The active metabolites of pioglitazone are M-IV and M-III. The enzymes responsible for the metabolism of pioglitazone are primarily CYP2C8, and to a lesser extent, CYP3A4. The oral TDLo observed in mice for pioglitazone is 24 mg/kg administered daily for 4 days, while in rats it is 3 mg/kg administered daily for 6 days. Pioglitazone specifically has shown correlation with increased risk for bladder cancer. TZDs are primarily metabolized in the liver via CYP2C8, with a minor contribution from CYP2C9. Thus, it might interact with CYP2C8 inhibitors and inducers. Studies with rats, mice, and dogs treated with rosiglitazone showed cardiac hypertrophy. The maximum recommended daily dose is 8 mg. Lower Panel: 2-dimensional Structure of Rosiglitazone

Anti diabetic agents 587

588

Anti diabetic agents

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Costello RA, et al. (2023) Sulfonylureas. StatPearls Publishing. PMID: 30020597. D’Souza MS, et al. (2021) Chapter 37—Side effects of insulin and other antihyperglycemic drugs. Side Effects of Drugs Annual. vol. 43, pp. 451–461. Elsevier. https://www. sciencedirect.com/science/article/pii/S037860802100012X. D’Souza MS, et al. (2022) Chapter 28—Side effects of insulin and other antihyperglycemic drugs. Side Effects of Drugs Annual. vol. 44, pp. 397–407. Elsevier. https://www. sciencedirect.com/science/article/pii/S0378608022000022. Derosa G, et al. (2012) a-glucosidase inhibitors and their use in clinical practice. Archives of Medical Science. AMS 8(5): 899–906. PMID: 23185202. Drug Bank: Gliclazide (n.d.) https://go.drugbank.com/drugs/DB01120. Drug Bank: Metformin (n.d.) https://go.drugbank.com/drugs/DB00331. Drug Bank: Nateglinide (n.d.) https://go.drugbank.com/drugs/DB00731. Drugbank.ca (2023) Repaglinide – DrugBank. https://go.drugbank.com/drugs/DB00912. DrugBank: Pioglitazone (n.d.) https://go.drugbank.com/drugs/DB01132. DrugBank: Rosiglitazone (n.d.) https://go.drugbank.com/drugs/DB00412. Eggleton ES and Jialal I (2023a) Stat Pearls. https://pubmed.ncbi.nlm.nih.gov/31869120/. Fasano CJ and Rowden AK (2009) Successful treatment of repaglinide-induced hypoglycemia with octreotide. The American Journal of Emergency Medicine 27(6). 756.e3–e4. https://pubmed.ncbi.nlm.nih.gov/19751648/. Eggleton JS and Jialal I (2023b) Thiazolidinedciones. In: StatPearls. StatPearls Publishing. https://pubmed.ncbi.nlm.nih.gov/31869120/. Fasano CJ, et al. (2009) Successful treatment of repaglinide-induced hypoglycemia with octreotide. The American Journal of Emergency Medicine 27: 6. PMID: 19751648. FDA.gov: Pioglitazone (n.d.) https://www.accessdata.fda.gov/drugsatfda_docs/label/2011/021073s043s044lbl.pdf. FDA.gov: Rosiglitazone (n.d.) https://www.accessdata.fda.gov/drugsatfda_docs/label/2011/021071s039lbl.pdf. Ganesan K, et al. (2022) Oral Hypoglycemic Medications. StatPearls Publishing. PMID: 29494008. Gunaratne K, et al. (2018) Unintentional sulfonylurea toxicity due to a drug-drug interaction: A case report. BMC Research Notes 11: 331. PMID: 29784014. Gürbüzel M, et al. (2014) Genotoxic evaluation of two oral antidiabetic agents in the Drosophila wing spot test. Toxicology and Industrial Health 30(4): 376–383. https://doi.org/ 10.1177/07482337124560. Hemmingsen B, et al. (2016) Insulin secretagogues for prevention or delay of type 2 diabetes mellitus and its associated complications in persons at increased risk for the development of type 2 diabetes mellitus. Cochrane Database of Systematic Reviews 10(10), CD012151. PMID: 27749986. Hurren KM and Dunham MW (2021) Are thiazolidinediones a preferred drug treatment for type 2 diabetes? Expert Opinion on Pharmacotherapy 22(2): 131–133. https://pubmed.ncbi. nlm.nih.gov/33280446/. Juneja, et al. (2022) World Journal of Diabetes 654: 664. PMID: 36159225. Kasina SVSK, et al. (2022) Dipeptidyl Peptidase IV (DPP IV) Inhibitors. StatPearls Publishing. PMID: 31194471. Lebovitz HE (2019) Thiazolidinediones: The forgotten diabetes medications. Current Diabetes Reports 19(12): 151. https://doi.org/10.1007/s11892-019-1270-y. Lipscombe L, et al. (2018) Pharmacologic glycemic management of type 2 diabetes in adults. Canadian Journal of Diabetes 42: 336. PMID: 29650116. LiverTox (2018) Clinical and Research Information on Drug-Induced Liver Injury. National Institute of Diabetes and Digestive and Kidney Diseases; Sulfonylureas, Second Generation. PMID: 31643463. Makrilakis K (2019) The role of DPP-4 inhibitors in the treatment algorithm of type 2 diabetes mellitus: When to select, what to expect. International Journal of Environmental Research and Public Health 16(15): 2720. PMID: 31366085. Masharani U and Kroon L (2020) Chapter 41: Pancreatic hormones and glucose lowering drugs. In: Katzung BG and Vanderah TW (eds.) Basic & Clinical Pharmacology, 15th edn. McGraw Hill. ISBN: 978-1-260-45231-0. Milner Z and Akhondi H (2022) Repaglinide. Stat Pearls. https://www.ncbi.nlm.nih.gov/books/NBK559305/. Milner Z, et al. (2022) Repaglinide. StatPearls Publishing. PMID: 32644731. Morris D (2021) GLP-1 receptor agonists in type 2 diabetes: An underused asset? Updated January 2021. Journal of Diabetes Nursing 25: JDN174. Nateglinide: FDA (2021) https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/021204s025lbl.pdf. Nanjan MJ, et al. (2018) Thiazolidinediones as antidiabetic agents: A critical review. Bioorganic Chemistry 77: 548–567. https://pubmed.ncbi.nlm.nih.gov/29475164/. Packer M (2018) Do DPP-4 inhibitors cause heart failure events by promoting adrenergically mediated cardiotoxicity? Circulation Research 122(7): 928–932. PMID: 29436388. Padda IS, et al. (2022) Sodium-Glucose Transport Protein 2 (SGLT2) Inhibitors. StatPearls Publishing. PMID: 35015430. PubChem; Canagliflozin (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/24812758#section=2D-Structure. PubChem; Empagliflozin (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/11949646#section=2D-Structure. PubChem: Acarbose (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/Acarbose. PubChem: Alogliptin (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/Alogliptin. PubChem: Byetta (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/45588096. PubChem: Glimepiride (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/Glimepiride. PubChem: Janumet (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/Janumet. PubChem: Metformin (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/4091. PubChem: Miglitol (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/441314. PubChem: Nateglinide (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/5311309. PubChem: Pioglitazone (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/Pioglitazone. PubChem: Repaglinide (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/65981. PubChem: Rosiglitazone (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/Rosiglitazone. PubChem: Semaglutide (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/Semaglutide. PubChem: Sitagliptin (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/4369359. PubChem: Tolbutamide (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/Tolbutamide#section=Toxicity. Ragia G, et al. (2009) Pharmacogenomics 10(11): 1781–1787. https://pubmed.ncbi.nlm.nih.gov/19891554/. Repaglinide. FDA (2019). https://www.accessdata.fda.gov/drugsatfda_docs/label/2019/020741s044lbl.pdf. Riomet ER (2019) Package Insert. USFDA. https://www.accessdata.fda.gov/drugsatfda_docs/label/2019/212595s000lbl.pdf (Revised 08/2019).

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Rosa MM, et al. (2014) Commonly used endocrine drugs. Handbook of Clinical Neurology, 120, p. 819. Elsevier. PMID: 24365354. Schalliol L, et al. (2019) Chapter 40—Side effects of insulin and other antihyperglycemic drugs. Side Effects of Drugs Annual. vol. 41, pp. 493–504. Elsevier. https://www. sciencedirect.com/science/article/pii/S0378608019300066. Seino Y, et al. (2010) GIP and GLP-1, the two incretin hormones: Similarities and differences. Journal of Diabetes Investigation 1(1–2): 8–23. PMID: 24843404. Tuccori M, et al. (2015) The use of glyburide compared with other sulfonylureas and the risk of cancer in patients with type 2 diabetes. Diabetes Care 38(11): 2083–2089. PMID: 26341130. Waldrop G, et al. (2018) Incretin-based therapy in type 2 diabetes: An evidence-based systematic review and meta-analysis. The Journal of Diabetic Complications 32: 113–122. PMID: 29074120. Wang GS and Hoyte C (2019) Review of biguanide (Metformin) toxicity. Journal of Intensive Care Medicine 34(11−12): 863–876. https://doi.org/10.1177/0885066618793385. Zannad F, et al. (2015) Heart failure and mortality outcomes in patients with type 2 diabetes taking alogliptin versus placebo in EXAMINE: A multicentre, randomized, double-blind trial. Lancet 385: 2067–2076. PMID: 25765696. Zhang L, et al. (2016) Scientific Reports 6: 32649. https://pubmed.ncbi.nlm.nih.gov/27596383/. Zhang M, et al. (2019) BMJ Open Diabetes Care 7(1), e000717. https://pubmed.ncbi.nlm.nih.gov/31641523/.

Further reading Wishart DS, et al. (2006) Drugbank: A comprehensive resource for in silico drug discovery and exploration. Nucleic Acids Research 34(Database issue): D668–D672. 16381955.

Relevant websites https://www.cdc.gov/diabetes/basics/index.html :Diabetes Basics. https://www.cdc.gov/diabetes/basics/diabetes.html :What is Diabetes.

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Antidotes Christy Turco and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S. Shadnia, L.S. Nelson, Antidotes, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 267–273, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01064-2.

Background Conclusion References Further reading

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Abstract According to the IPCS (International Program on Chemical Safety) definition, an antidote is a therapeutic substance used to counteract the toxic action(s) of a specified xenobiotic. Antidotes are a necessary component of therapeutic processes as they are capable of reversing a toxin’s harmful effect. The purpose of this article is to discuss the different types of antidotes and how they are properly given to reverse poisonings. The mechanism of action of each antidote has been described in this article, besides conventional toxins (i.e., N-acetylcysteine (NAC), calcium, naloxone, oxygen, etc.). Several new antidotes have been added. In 2019, there were 2,148,141 confirmed poisonings in the United States. Of the individuals affected, 46.8% were treated with decontamination methods (i.e., activated charcoal, whole bowel irrigation, etc.), 12.6% were treated therapeutic methods (i.e., N-acetylcysteine, naloxone, oxygen, etc.), and 4.7% were treated with both methods (Chacko and Peter, 2019).

Keywords Activated charcoal; Antidote; Antivenom; Chemical; Heavy metals; Indication; Medicine; N-Acetylcysteine; Naloxone; Opiates; Opioids; Overdose; Poison; Therapeutic drugs; Toxicity; Toxin

Key points

• • •

Toxicity caused by envenomation, microbial toxins, or unintentional ingestion of drugs/chemicals can immediately pose danger to life. Prompt diagnosis, rapid intervention, and use of specific antidote are required to mitigate the toxic effects. Use of toxidromes for diagnosis of the poisoned patient is the best approach. Consulting local and national poison control centers for additional help can be meaningful.

Background Paracelsus, known as the father of toxicology, once stated that “The right dose differentiates a poison from a remedy.” This quote explains how a drug, meant to perform a therapeutic effect, may also contain the ability to become harmful. When the adverse effects become too intense, the help of an antidote is needed. Antidotes exhibit a range of abilities: (i) either prevent absorption of a poison, (ii) bind and neutralize the poison, (iii) antagonize the poisonous effect, (iv) inhibit toxic metabolites from being produced or (v) increase the synthesis of a cytoprotective agent which indirectly reduce toxic effects. Therefore, administration of the proper antidote may be vital to improve the morbidity and mortality of a patient. The decontamination method that is most commonly used is activated charcoal (AC). AC is a very highly adsorbent substance with an extensive surface area. Drugs and other materials may adsorb to the AC via weak intermolecular forces. Non-ionized, organic compounds are able to bind more easily to AC than inorganic compounds. This suggests that, while AC may have a broad range of use, this method may not be optimal for poisoning cases involving metals, hydrocarbons, or caustics. Nevertheless, it is recommended for the patient to ingest about 50 g AC slurry within 1 h of the poison ingestion for optimal adsorption (Juurlink, 2016). Acetaminophen (APAP) is a popular analgesic and antipyretic drug with a potential to cause harmful effects to the liver and kidneys. APAP overdose results in a build-up of the toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI). Usually, the body naturally protects itself from NAPQI via glutathione conjugation. When glutathione is depleted, NAPQI recklessly attacks plasma membrane, macromolecules and micromolecules. N-acetylcysteine (NAC) is the primary antidote used to unstoppable NAPQI from causing liver cell toxicity as it functions as an antioxidant and replenishes the glutathione in the body. NAC should be given intravenously within 8–10 h of APAP ingestion. The dosing of NAC is based on the treatment line on the Rumack-Matthew

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Antidotes for Chemicals—Demonstrates an in-depth explanation of antidotes that are given when toxicity is due to a chemical not otherwise indicated for medicinal purposes. Chemical Indication

Atropine Sulfate

Competitive antagonist at both the central 1. Intravascular injections: 0.05 mg, 1. Adults: IV bolus of 1-2 mg atropine, 1. Organophosphate and peripheral muscarinic 0.3 mg, 0.4 mg, 0.5 mg, 0.8 mg, 3–5 mg IV bolus for severe poisoning. compounds, receptors. and 1 mg 1 mL vials/ampules. Double dose of bolus every 3–5 min carbamates, nerve Premade 5- or 10 mL injections until improvement. Then infusion of agents, or medicinals with 0.1 mg/mL for adults and 10–20% bolus per hour. 2. Pilocarpine 5 mL injections with 0.05 mg/mL 2. Children: Bolus of 0.02 mg/kg up to 3. Clitocybe and Inocybe for children. adult dosage. Double dose of bolus mushrooms 2. AtroPenW Auto-Injector: 0.25 mg every 3–5 min until improvement. 4. Acetylcholinesterase Then IV infusion of 0.025 mg/kg/h. (yellow label), 0.5 mg (blue label), inhibitors 1 mg (dark red label), 2 mg (green 3. Nerve agent dosing a. Conscious adults receive 2 mg of label) atropine IV or IM every 5–10 min 3. DuoDoteW Autoinjector system: until improvement. Unconscious 2.1 mg followed by 600 mg of adults receive higher doses of pralidoxime. 5–15 mg. 4. Oral tablets: 0.3 mg, 0.4 mg, b. Children ages 3–7 years may 0.6 mg receive one DuoDote Autoinjector 5. Eye drops: 1% solution resulting in an atropine dose of 0.08–0.15 mg/kg. Ages 8–14 may receive two DuoDote Autoinjector resulting in an atropine dose of 0.08–0.15 mg/kg/ Children older than 14 may receive three DuoDote Autoinjector resulting in an atropine dose of less than 0.11 mg/kg. Clostridium botulinum Binds to and neutralizes free botulinum 1. Antitoxin heptavalent: 4500 IU of 1. Heptavalent Adult: one vial given IV toxin anti-A, 3300 IU of anti-B, 3000 IU infusion 0.5 mL/min for 30 min, of anti-C, 600 IU of anti-D, 1 mL/min for 30 min, and 2 mL/min 5100 IU of anti-E, 3000 IU of until end of infusion. anti-F, and 600 IU of anti-G 20 mL 2. Heptavalent Children: 0.01 mL/kg/min or 50 mL vials IV infusion for 30 min, and increased 2. BabyBIGW: 100
20 mg 0.01 mL/kg/min every 30 min until rate reaches 0.03 mL/kg/min. immunoglobulin (IgG, trace IgA, Infusion continues until finished. trace IgM) vial. Infants should be given 10% the adult dose 3. Immunoglobulin: IV infusion 25 mg/kg/h for 15 min, then 50 mg/kg/h.

Botulinum antitoxin

Mechanism of Action

Available Dosage Forms

Proper Dosing

Considerations

References

1. Atropine should be given to the endpoint of clearing of pulmonary secretions and halting of bronchoconstriction. 2. Tachycardia and mydriasis are not markers of therapeutic improvement as they may indicate hypoxia, hypovolemia, or sympathetic stimulation 3. Oxygen may be needed in hypoxic patients. 4. Not effective at nicotinic receptors.

Nelson et al. (2019) Lexicomp (2021) UpToDate (2021) Greget et al. (2016)

Nelson et al. (2019) Lexicomp (2021) Yu et al. (2017)

Antidotes

Name of Antidote

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Table 1

Deferoxamine

1. Acute and chronic iron toxicities. 2. Aluminum toxicities

Dimercaprol (British Anti-Lewisite, BAL)

1. Arsenic poisoning 2. Inorganic mercury poisoning 3. Lead poisoning

Edetate Calcium Disodium (Calcium Disodium EDTA, CaNa2 EDTA)

1. Severe lead poisoning.

Parenteral chelator that chelates nontransferrin-bound iron, forms ferrioxamine, and leaves iron bound to transferrin, hemoglobin, and cytochromes untouched. Deferoxamine usually binds to nontransferrin-bound iron after transferrin becomes saturated following an overdose. Deferoxamine also binds to aluminum to form aluminoxamine. This chelation is excreted renally, and is unaffected by kidney disease or hemodialysis. Chelator of certain metals, products are then renally excreted.

1. IV infusion of 15 mg/kg/h for the first 1. Vials with 500 mg or 2 g of 24 h. Start with 5 mg/kg/h and powder used for injection. Add 5 increase to 15 mg/kg/h after 15 min. or 20 mL of sterile water to the 500 mg or 2 g, respectively. This results in a vial with a concentration of 100 mg/mL 2. The solution can be diluted further with 0.9% sodium chloride, dextrose in water, or Ringer lactate for IV administration. 3. Add 2 mL or 8 mL of sterile water to the 500 mg or 2 g vials respectively for IM administration for a concentration of 200 mg/mL Yellow, viscous liquid available in 1. Adults: 3 mL ampules with 100 mg/mL a. Lead: 4 mg/kg deep IM every 4 h dimercaprol, 200 mg/mL benzyl for up to 3 days benzoate, and 700 mg/mL peanut b. Arsenic: 3 mg/kg deep IM every oil. Only given by deep 4 h for 48 h then every 12 h for intramuscular injection. 10 days or until complete recovery. c. Inorganic mercury salts: 5 mg/kg deep IM initially then 2.5 mg/kg every 12–24 h until improvement for up to 10 days. 2. Children: a. Lead: 75 mg/m2 IM every 4 h for 5 days

1. Dimercaprol has a narrow therapeutic window and is made in peanut oil. Therefore, allergies are an issue. 2. Lead poisoning requires dimercaprol to precede the first dose of CaNa2EDTA by 4 h. 3. Urine should be alkalinized to avoid dissociation of the dimercaprol-metal chelate 4. Dimercaprol can only be used for inorganic mercury salts, not organic mercury. 5. Oral succimer is the preferred antidote for arsenic poisoning, mercury poisoning and less severe lead poisoning. However, dimercaprol is given when the gastrointestinal tract is impaired. 1. Replaced by succimer for 1. Adults: 50–75 mg/kg/day as a patients presenting with lead continuous IV infusion for 5 days and concentrations between a 2–4 day rest period to allow for lead 45 and 70 mg/dL. redistribution 2. Dimercaprol should be given 2. Children: 25–50 mg/kg/day as a 4 h before CaNa2EDTA for lead continuous infusion 3. IM can also be given as a 5 mg/mL poisoning with 1 mL of 1% procaine or 1% 3. Disodium EDTA should not be lidocaine solution since IM injection given as it can lead to can be painful. hypocalcemia.

Nelson et al. (2019) Lexicomp (2021) Dawn and Whited (2021)

Nelson et al. (2019) Lexicomp (2021) Bjørklund (2015)

Antidotes

5 mL ampules with 200 mg of Edetate calcium is a chelator of metals. Since most of the lead being CaNa2EDTA per milliliter (1 g per ampule) for IV infusion. chelated is found in bone, edetate calcium disodium is used since it can displace the calcium and form a stable ring with lead. This mechanism is needed to prevent hypocalcemia from occurring.

Nelson et al. (2019) 1. Urine color can be used to Lexicomp (2021) indicate renal excretion of Velasquez and Wray ferrioxamine. If there is (2021) absence of color change then there is very little renal excretion occurring. a. This method cannot be used if urine color was not obtained before deferoxamine administration.

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Table 1

Chemical Indication

Mechanism of Action

Available Dosage Forms

Ethanol

Methanol and ethylene glycol toxicities.

Competitive substrate for alcohol dehydrogenase which will inhibit the metabolism of alcoholic xenobiotics.

1. IV injection: 5–10% solution 2. Oral solution: 20–30%

Fomepizole

Methanol and ethylene glycol toxicities.

Hydroxocobalamin

Cyanide toxicity

Magnesium

1. Repairs xenobiotic-associated hypomagnesemia 2. An adjunctive treatment for cardiovascular toxins 3. Fluoride toxicity 4. Pesticide poisoning 5. Alcohol use disorders.

Proper Dosing

Considerations

Regardless of route, ethanol should be 1. Disadvantages of IV given at a concentration of 100 mg/dL. preparation include problems preparing ethanol solution as a 10% solution is not readily available. This will result in a delay of treatment 2. While ethanol is cheaper and may be easier to give, fomepizole is preferred for alcohol toxicities. 3. Patients with a high tolerance to ethanol (i.e. alcoholics) should be given double the normal dosage to receive a therapeutic effect. Competitive inhibitor of alcohol Vials containing 1.5 mL with a Loading dose of 15 mg/kg IV for 12 h, 1. Vials may solidify at a dehydrogenase. Prevents toxic concentration of 1 g/mL then 10 mg/kg every 12 h for 4 doses. temperature less than 77  F metabolites being made Increase the dose to 15 mg/kg every (25  C). Warming vials will not 12 h if therapy is needed for over 48 h. affect its potency. 2. Fomepizole must be infused for over 30 min to avoid venous irritation and thrombophlebitis. Hydroxocobalamin reacts with cyanide to IV infusion: Cyanokit contains a 1. Adults: Hydroxocobalamin should be 1. Sodium thiosulfate may often form cyanocobalamin (vitamin B12), be given together with 250 mL vial with 5 g of reconstituted with 200 mL of 0.9% hydroxocobalamin through the which is nontoxic. hydroxocobalamin crystalline sodium chloride and given as an IV same IV but the latter is powder. infusion over 15 min. A second considered more important to dosage may be given if needed and administer. may be infused 15 min to 2 h. 2. Red discoloration may occur. 2. Children: 70 mg/kg of hydroxocobalamin up to adult dose may be given. Acts as a calcium channel blocker and a 1. Parenteral: 4% or 8% in 100 mL to 1. IV: at least 4 g/day NMDA antagonist. 1 L volumes 2. IV injection: 50% in water in 2 or 10 mL or 1–2% in 5% dextrose.

References Nelson et al. (2019) Lexicomp (2021) Zakharov et al. (2015a)

Nelson et al. (2019) Lexicomp (2021) Zakharov et al. (2015a)

Nelson et al. (2019) Lexicomp (2021) Zakharov et al. (2015b)

Nelson et al. (2019) Lexicomp (2021) Bryar (2018)

Antidotes

Name of Antidote

Methylene Blue

Methemoglobinemia inducers

Oxidizing agent that is reduced to leukomethylene blue via reacting with NADPH and NADPH methemoglobin reductase. Leukomethylene blue, in turn, reduces methemoglobin to hemoglobin and, therefore, reduces methemoglobin levels.

IV injection available in 1% and 0.05% solution vials.

Oxygen (hyperbaric)

1. Carbon monoxide toxicity 2. Carbon monoxide toxicity with cyanide poisoning 3. Carbon tetrachloride toxicity 4. Methylene chloride toxicity 5. Hydrogen peroxide toxicity 6. Hydrogen sulfide toxicity 7. Respiratory Depression 1. Plutonium toxicity 2. Americium toxicity 3. Curium toxicity

Increases dissolved oxygen levels in plasma. Restores mitochondrial, neutrophil, and immunologic disruptions

Oxygen 100% present in monoplace or multiplace chambers.

Chelator of specific heavy metals and radionuclides.

IV: 200 mg/mL vial with 5 mL

Pentetic Acid or pentetate trisodium

Competitive inhibitor of thyroid uptake of 1. radioiodine. 2.

Pralidoxime Chloride

1. Organophosphate compounds 2. Anticholinesterase toxicity

Removes organophosphate group from carboxylic esterase enzymes

1.

2.

Nelson et al. (2019) Lexicomp (2021) Fife et al. (2016)

Nelson et al. (2019) Lexicomp (2021) Poonam et al. (2018) Nelson et al. (2019) Lexicomp (2021) Leung et al. (2017)

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Radioactive iodine after nuclear disaster

IV Adult: Infusion 1 g given undiluted for Dose is most effective when given 3–4 min or diluted in 100–250 mL in within the first 24 h of dextrose 5%, Ringers lactate, or 0.9% poisoning. sodium chloride for 30 min IV Children: 14 mg/kg 1. Adults older than 40 year have Oral Tablets: 130- and 65 mg 1. Adults 18-40 year: 130 mg tablet a an almost zero risk of getting Oral solution: 65 mg/mL day or 2 mL of oral solution thyroid cancer from 2. Children 3–18 year: 65 mg tablet a radioactive iodine. day or 1 mL of solution 2. Pregnant women and 3. Children 1 month to 3 year: 0.5 mL neonates should receive a solution single dose to prevent harm to 4. Infant from birth to 1 month: 0.25 mL the fetus. solution Vital for nicotinic sites since IV injection: 1 g powder in 20 mL 1. Loading dose of 30 mg/kg for 15–30 min with a maintenance dose atropine is not effective at vials to be reconstituted with of 8–10 mg/kg/h for adults and nicotinic sites. sterile water or 0.9% sodium chloride. 10–20 mg/kg/h for children. Auto-injector: 600 mg

Nelson et al. (2019) Lexicomp (2021) Cooper et al. (2016)

Antidotes

Potassium Iodide

1. Should not be administered 1. Dose given 1 mg/kg IV for 5 min, subcutaneously or followed by a fluid flush to minimize intrathecally injection pain. Dose may be repeated 2. May be diluted in 50 mL 5% in 30–60 min if needed. dextrose in water, should not 2. Neonate dosing is 0.3 to 1 mg/kg. be diluted in sodium chloride May be given intraosseously over as it may affect the solubility 3–5 min into the anterior tibia of a of methylene blue. 6 week old infant. 3. Cannot be given during pregnancy, considered a category X drug. 4. May alter pulse oximeter readings. 5. Ineffective in patients with G6PD deficiency. Hyperbaric oxygen is given in 2.8–3.0 ATA O2 for 45 min.

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Table 1

Chemical Indication

Mechanism of Action

Available Dosage Forms

Proper Dosing

Considerations

References

Prussian Blue

Thallium or cesium toxicities

Capsule: 500 mg

1. Adults: 3 g 3 times a day 2. Children: 1 g 3 times a day

Should be taken with food.

Nelson et al. (2019) Lexicomp (2021) Busquets and Estelrich (2020)

Sodium nitrite and Sodium thiosulfate

Cyanide poisoning

Usually binds to potassium ions, however, binding affinity increases with increasing ionic radius. Prussian blue binds to both thallium and cesium and prevents absorption. 1. Nitrites: oxidizes iron in hemoglobin to produce methemoglobin. 2. Sodium Thiosulfate: binds to cyanide to produce thiocyanate, which is cleared renally.

1. Nitrites: 300 mg in 10 mL injections 2. Thiosulfate: 12.5 g in 50 mL vial for injections 3. NithiodoteW: kit with a nitrite and thiosulfate vial

1. Cyanide antidote kit is no longer available, Nithiodote has replaced the kit. 2. Amyl nitrite is no longer recommended but can be used if nothing else is available.

Nelson et al. (2019) Lexicomp (2021) Zakharov et al. (2015b)

Succimer (Dimercaptosuccinic Acid, DMSA)

1. Lead poisoning 2. Arsenic poisoning 3. Mercury poisoning

Metal chelator that allows metals to be renally excreted.

Capsule: 100 mg

1. Dosing may be repeated based on blood lead concentrations. 2. Preferred metal chelator antidote.

Nelson et al. (2019) Lexicomp (2021) van Eijkeren et al. (2017)

Thiamine (Vitamin B1)

Ethylene glycol poisoning. Binds to ethylene glycol metabolite and assists in clearance

1. Adults: nitrite given IV infusion 2.5–5 mL/min then thiosulfate IV infusion of 50 mL of 25% solution is given. Give both doses again at half initial dose if no improvements are observed. 2. Children: nitrite given IV infusion 0.2 mL/kg (do not exceed10 mL) then thiosulfate is given IV infusion 1 mL/kg (do not exceed 50 mL). Give both doses again at half initial dose if no improvements are observed. 1. Adults: 10 mg/kg 3 times a day for 5 days then 10 mg/kg twice a day for 14 days 2. Children: 350 mg/m2 3 times a day for 5 days, then 350 mg/m2 for 14 days Dose of 100 mg given IV bolus

Antivenom

IV or IM: 50 or 100 mg/mL

Nelson et al. (2019) Lexicomp (2021) Singh et al. (2016) An antitoxin that is used for specific venomous poisonings. The antivenom’s available include snake, spider, and scorpion antivenom. These antidotes contain immunoglobins specific towards Nelson et al. (2019) the animal and venom produced by the animal. Antivenom contains antibodies manufactured inside another animal that was exposed to the toxic venom. The antibodies are then removed from the animal and purified for treatment.

Antidotes

Name of Antidote

Table 2

Antidotes for therapeutic drugs—Demonstrates an in-depth explanation of antidotes that are given when an overdose is due to a therapeutic drug otherwise indicated for medicinal purposes.

Name of Antidote

Mechanism of Action

Available Dosage Forms

N-Acetylcysteine 1. Acetaminophen toxicity 2. Cyclopeptide-containing mushrooms 3. Carbon tetrachloride 4. Chloroform 5. Doxorubicin 6. Amatoxins 7. Valproic acid toxicity

Detoxifies dangerous metabolites via a number of different methods such as, free radical scavenging, increased oxygen distribution, increased ATP production, increased antioxidant, or a change in microvascular tone

Activated Charcoal

Most therapeutic drugs

Adsorbent based on hydrogen, ion-ion, dipole, or van der Waals bonding

Andexanet Alfa (AndexxaW, FDA approval in May 2018)

Intracranial hemorrhage due to factor Xa inhibitor toxicity.

Binds to Xa inhibitors and inhibits activation of thrombin

Benzodiazepine

1. Xenobiotic-induced seizures Agonist of GABA-A 2. Xenobiotic-induced psychomotor agitation 3. Withdrawal from ethanol and other sedative–hypnotics 4. Cocaine toxicity 5. Chloroquine overdose 6. Serotonin toxicity 7. Strychnine poisoning 8. Black widow spider envenomation Regulates action potentials and provides a 1. Calcium channel blocker source of calcium toxicity 2. Beta blocker toxicity 3. Ethylene glycol toxicity 4. Hydrofluoric acid and fluoride agents’ toxicity 5. Phosphate toxicity 6. Citrate toxicity 7. Hypermagnesemia 8. Hyperkalemia

Give treatment for acetaminophen 1. IV injection: 20% 30 mL 1. IV: Loading dose of 150 mg/kg up to 15 g in toxicity if the patient presents with 200 mL of 5% dextrose for 60 min. Then an IV vials to be diluted upon serum levels on or above the plotted infusion maintenance dose of 50 mg/kg up to administration. line on the Rumack-Matthew 5 g in 500 mL 5% dextrose for 4 h. Then a 2. Oral solution: 10% and nomogram or based on patient second IV infusion maintenance dose of 20% 10 mL vials to be usage history. 100 mg/kg up to 5 h in 1 L 5% dextrose for diluted upon 16 h. administration. 3. Oral tablets: 500 mg or 2. Oral solution/tablet: 140 mg/kg as a loading dose then 70 mg/kg every 4 h for 17 doses. 2.5 g tablets that should be dissolved in water before administration. Oral suspensions: Oral suspension: 1 g/kg up to 100 g or 10:1 1. Only give if airway is clear 208 mg/mL activated charcoal:xenobiotic 2. Only give if there are no gastrointestinal issues 3. Not effective in xenobiotics of low molecular weight compounds. Injection: 100 mg vials 1. Xa inhibitor given more than 7 h before Only indicated for rivaroxaban and antidote: apixaban. 2. Loading dose: 400 mg over 15–30 min 3. Infusion dose: 480 mg over 2 h 4. Xa inhibitor given within 7 h of antidote: 5. Loading dose: 800 mg over 15–30 min 6. Infusion dose: 960 mg over 2 h 1. Diazepam IV: 10 mg for 2 min, may redose 1. Diazepam IV injection: after another 2 min 5 mg/mL vials 2. Diazepam for seizures: loading dose of 2. Diazepam rectal gel: 10 mg IV 5 mg/mL 3. Midazolam IM injection: 3. Midazolam IV: 2 mg for 2 min or 5–10 mg IM. May wait 2 min before redosing 1 mg/mL or 5 mg/mL 4. Midazolam for seizures: loading dose of vials 10 mg IM 4. Lorazepam: 2 mg/mL or 5. Lorazepam IV: 2 mg for 1 min. May wait 15 min 4 mg/mL vials for redosing 6. Lorazepam for seizures: 4 mg IV

Calcium Gluconate or Chloride

Therapeutic Drug Indication

1. Calcium chloride IV injection: 10% vials containing 27.3 mg of calcium 2. Calcium gluconate IV injection: 10% vials containing 9.3 mg of calcium 3. Calcium gluconate gel: 2.5% 4. Calcium gluconate eyewash: 1% solution

Proper Dosing

Considerations

References Fisher and Curry (2019) Nelson et al. (2019) Lexicomp (2021)

Juurlink (2016) Nelson et al. (2019) Lexicomp (2021) Nelson et al. (2019) Lexicomp (2021) Connolly et al. (2019)

Nelson et al. (2019) Lexicomp (2021) Haut et al. (2016)

1. IV infusion of calcium given at a rate of Calcium gluconate is only preferred Nelson et al. (2019) 14–36 mg per minute for 10 min. May give when the risk of tissue injury is low. Lexicomp (2021) repeat dosing every 10–20 min for 3–4 doses. St-Onge et al. (2017)

(Continued )

Table 2

(Continued)

Name of Antidote

Therapeutic Drug Indication

Levo-Carnitine

1. Valproic acid toxicity, Promotes production of ATP including hyperammonemia 2. Levocarnitine deficiency

Dantrolene

1. Anesthetic-induced hyperthermia. 2. Neuromuscular blocker toxicity. Digoxin toxicity

Reduces free calcium in plasma which inhibits excitation-contraction

Flumazenil

Benzodiazepine toxicity

Competitive antagonist of benzodiazepine receptors.

Folinic Acid (Leucovorin) and Folic Acid (Vitamin B9)

1. Methotrexate toxicity 2. Dihydrofolate reductase inhibitors 3. Methanol toxicity 4. Arsenic toxicity

Provide active folate needed for metabolic reactions, such as for precursors and methanol elimination. Aids in elimination of arsenic elimination.

Glucagon

1. Beta blocker toxicity 2. Calcium channel blocker toxicity 3. Hypoglycemia 1. Methotrexate toxicity 2. Folate or folate analog toxicity

Digoxin-Specific Antibody Fragments

Glucarpidase

Idarucizumab (PraxbindW, FDA approval in October 2015) High Dose Insulin

Mechanism of Action

Fragments bind to free digoxin and removes the digoxin from its binding sites.

Available Dosage Forms

Proper Dosing

1. IV injection: 1 g/5 mL vials 2. Oral tablets: 250- and 330 mg 3. Oral solution: 100 mg/mL 1. Powder injection: 20 mg 2. Powder suspension: 250 mg

1. IV injection: 50–500 mg/kg/day 2. IV injection for children: 500 mg/kg/day 3. Oral dosing: 50–100 mg/kg/day up to 3 g/day

Nelson et al. (2019) Lexicomp (2021) Brown et al. (2018)

1. IV injection: bolus of 2.5 mg/kg. Redosing may Should not be stored in glass vials be given every 15 min until improvement or 10 mg/kg is given

DigiFab: 40 mg of digoxin-immune ovine immunoglobulin fragments. IV injection: 0.1 mg/mL in 5- or 10 mL vials.

1. Acute digoxin ingestion: 10–20 vials. 2. Chronic digoxin ingestion for adults: 3–6 vials 3. Chronic digoxin ingestion for children: 1–2 vials. 1. IV injection for adults: 0.1 mg/min titration with 1 min between doses, total dose should not exceed 1 mg. 2. IV injection for children: 0.01 mg/kg up to 0.2 mg. 1. IV injection: 10–25 mg/m2 IM or IV every 6 h for 72 h 2. Methanol toxicity IV: 50–70 mg of folic acid every 4 h for 24 h.

Nelson et al. (2019) Lexicomp (2021) Ratto and Joyner (2021) Nelson et al. (2019) Lexicomp (2021) Pincus (2016)

1. Powder for injection: 50-, 100-, 200-, 350 mg vials 2. Oral tablets: 5-, 10-, 15-, 25 mg 3. IV injection: 10 mg/mL Binding to cardiac receptors activates cardiac Injection powder for adenylate cyclase. solution: 1 mg (1 unit) vials Rapidly inactivates methotrexate and folates by cleaving the terminal glutamate residues.

Injection: 1000-unit vials

Life-threatening or uncontrollable bleeding due to dabigatran toxicity

Binds to and inactivates dabigatran

Injection: 2.5 g in 50 mL vials

1. Beta blocker toxicity 2. Calcium channel blocker toxicity

Increases myocardial contractility and improves tissue perfusion

Regular insulin injection: many strengths

Considerations

Each vial of DigiFab binds 0.5 mg of digoxin. 1. Use with caution in patients with benzodiazepine dependence. 2. Role in overdose is uncertain.

References

Nelson et al. (2019) Lexicomp (2021) An and Godwin (2016)

Specific guidelines should be followed Nelson et al. (2019) based on specific methotrexate Lexicomp (2021) levels. Gristan and Moosavi (2021)

1. IV infusion: 50 mg/kg for 3–10 min 2. Repeat doses: either 3–5 mg as needed or infusion of 2–5 mg/h

Concentration of vial should not exceed 1 mg/mL

Nelson et al. (2019) Lexicomp (2021) Overbeek and Shaffer (2020) IV infusion: 50-units/kg for 5 min 1. Dose may be repeated in 24 to Nelson et al. (2019) 48 h if needed. Lexicomp (2021) 2. Does not substitute for leucovorin, Ramsey et al. must be used in conjugation. (2018) 3. Leucovorin should not be given within 2 h of glucarpidase to avoid enzymatic destruction. IV injection: 5 g, may be given as bolus or infusion IV line must be flushed with normal Nelson et al. (2019) saline before drug administration. Lexicomp (2021) Van der Wall et al. (2019)

1. Bolus dose: 1 unit/kg regular insulin with 0.5 g/kg dextrose 2. Maintenance dose: 1 unit/kg/h regular insulin with 0.5 g/kg/h dextrose

Hypoglycemia should be monitored. Dextrose may be given if needed.

Nelson et al. (2019) Lexicomp (2021) Overbeek and Shaffer (2020)

Intravenous Lipid 1. Anesthetic toxicity Emulsion 2. Calcium channel blocker toxicity 3. Cyclic antidepressant toxicity 4. Insecticides 5. Beta blocker toxicity Naloxone 1. Opioid overdose

Mechanism is not completely understood but Parental 20% solution lipid sink model may be most accurate. Lipid sink consists of the emulsion removing lipid-soluble xenobiotics from site of toxicity. Competitive opioid receptor antagonist (mainly the mu receptor)

1. Sulfonylurea-induced hypoglycemia 2. Quinine overdose Anticholinergic syndrome

Somatostatin analog that inhibits insulin secretion

Protamine Sulfate

Unfractionated heparin toxicity

Binds with heparin to form an inactive salt.

Prothrombin Complex Concentrate (PCC)

1. Warfarin toxicity 2. Direct oral anticoagulantinduced coagulopathy

Contains factors II, VII, IX, and X. Provides direct replacement of factors that were previously inhibited.

Pyridoxine (Vitamin B6)

1. Isoniazid (INH) toxicity 2. Gyromitra esculenta mushrooms 3. Hydrazine and methylated hydrazine toxicity 4. Ethylene glycol toxicity 1. Cyclic antidepressants toxicity 2. Salicylate toxicity 3. Methotrexate toxicity 4. Methanol toxicity 5. Ethylene glycol toxicity 6. Type I antidysrhythmic drug toxicity 7. Herbicide toxicity Fluoropyrimidine toxicity

Cofactor for many different enzymatic reactions.

Warfarin overdose or long-acting anticoagulant rodenticide (LAAR)

Promotes activity of factors II, VII, IX, and X

Octreotide Physostigmine

Sodium Bicarbonate

Uridine Triacetate Phytonadione (Vitamin K1)

Reversible inhibitor of acetylcholinesterase

1. Bolus dose: 1.5 mL/kg 2. Maintenance dose: 0.25 mL/kg/min up to 10 mL/kg for 30–60 min

1. Lipid emulsion made up of phospholipids and triglycerides.

Nelson et al. (2019) Lexicomp (2021) Hoegberg et al. (2016)

Jordan and 1. Patients should be properly ventilated before administration of Morrisonponce naloxone. (2021) 2. Dose may be different for Nelson et al. (2019) Lexicomp (2021) opioid-dependent patients Does not replace dextrose as Nelson et al. (2019) SC injection: 1. Adults SC injection: 50 mg every 6 h for 24 h 50–1000 mg/mL 2. Children SC injection: 1.25 mg/kg/day every 6 h treatment therapy for Lexicomp (2021) up to 50 mg hypoglycemia. Long (2020) Injection: 1 mg/mL in 2 mL 1. IV infusion in adults: 1- to 2 mg for 5 min Dose may be repeated after Nelson et al. (2019) ampules 2. IV infusion in children: 0.02 mg/kg (maximum 10–15 min. Lexicomp (2021) is 0.5 mg/dose) for 5 min Wang et al. (2021) Injection: 10 mg/mL in 5- or 1 mg of protamine will neutralize 100 units of Nelson et al. (2019) 25 mL vials heparin Lexicomp (2021) Ranasinghe et al. (2019) Injection: 400-to-600-unit 1. INR ¼ 2–3.9 Dosage is based on INR level and Nelson et al. (2019) vials a. 25 units/kg actual body weight Lexicomp (2021) 2. INR ¼ 4–6 (maximum weight is 100 kg) Bavalia et al. (2020) a. 35 units/kg 3. INR 6 a. 50 units/kg 1. IV injection: 100 mg/mL 1. Adults: 1 g with each gram of INH ingested Benzodiazepine and barbiturates Nelson et al. (2019) in 2 mL vial 2. Children: 70 mg/kg up to 5 g should be given concurrently Lexicomp (2021) 2. Oral tablet: 10–500 mg with pyridoxine to prevent Glatstein et al. tablet seizures. (2018) IV injection: 0.04 mg increased by 0.04 mg up to 1. Injection: 0.4- and 1 mg/mL in 1- and 2 mL 0.12 mg. vials 2. Oral tablet: 50 mg

1. Alkalinizer capable of reversing acidosis 2. Provides sodium replacement in sodium channel blockers

1. Injection: 8.4% solution and 7.5% solution

Competes with fluoropyrimidine metabolite that incorporates itself into RNA

Oral granules: 10 g packet for single dose

1. IV bolus injection: 1–2 mEq/kg for 1–2 min 2. IV infusion injection: 150 mEq in 1 L 5% dextrose and titrate up to achieve serum pH no greater than 7.55

1. Oral granules adults: 10 g orally every 6 h for May give an antiemetic to prevent 20 doses. vomiting. 2. Oral granules children: 6.2 g/m2 up to adult dose. 1. IV and SC: 2 mg/mL and 1. LAAR: 25–50 mg oral tablets 3–4 times a day 10 mg/mL for 1–2 days. 2. Oral Tablets: 5 mg 2. Warfarin: starting dose of 10 mg IV infusion for minimum 20 min. Do not exceed 1 mg/mL

Nelson et al. (2019) Lexicomp (2021) Bruccoleri and Burns (2016)

Nelson et al. (2019) Lexicomp (2021) Ma et al. (2017) Nelson et al. (2019) Lexicomp (2021) Rice et al. (2021)

600

Antidotes

nomogram which is based on the amount of APAP in plasma. Treatment may continue until the poisoned individual’s APAP plasma is below the treatment line (Fisher and Curry, 2019). Opioids are a group of drugs used in severe pain management and are commonly abused. Opioids usually bind and activate opioid receptors, primarily the mu receptor. Activation of these receptors inhibits synaptic neurotransmission in the nervous system. An overdose from an opioid may result in severe respiratory depression and death. Naloxone is the antidote used for an overdose from an opiate. Naloxone is an opioid receptor antagonist and allows for the reversal of the effects brought on by the overdose. This antidote may be dosed using 0.4 mg intravenously for opioid naïve patients, a larger dose should be given if the patient is opioiddependent. Naloxone is a relatively safe substance and may be given to someone suspected of an opioid overdose (Jordan and Morrisonponce, 2021) (Tables 1 and 2).

Conclusion There is overwhelming evidence in toxicology and poisoning management literature suggesting that the correct use of specific antidotes when combined with general supportive care considerably reduce the morbidity and mortality. When teaching and training clinical toxicology or recommending the use of antidotes in poisoned patients, the projected efficacy level of the antidote in question should be discussed. Common antidotes used in poisoning management have been discussed in this chapter.

References An H and Godwin J (2016) Flumazenil in benzodiazepine overdose. CMAJ 188(17–18): E537. Bavalia R, Abdoellakhan R, Brinkman HJM, et al. (2020) Emergencies on direct oral anticoagulants: Management, outcomes, and laboratory effects of prothrombin complex concentrate. Research and Practice in Thrombosis and Haemostasis 4(4): 569–581. Bjørklund G (2015) Clinical use of the metal chelators calcium disodium edetate, DMPS, and DMSA. Saudi Journal of Kidney Diseases and Transplantation 3: 611. https://doi.org/ 10.4103/1319-2442.157416. Brown LM, Cupples N, and Moore TA (2018) Levocarnitine for valproate-induced hyperammonemia in the psychiatric setting: A case series and literature review. Mental Health Clinician 8(3): 148–154. https://doi.org/10.9740/mhc.2018.05.148. Bruccoleri RE and Burns MM (2016) A literature review of the use of sodium bicarbonate for the treatment of QRS widening. Journal of Medical Toxicology 12(1): 121–129. Bryar (2018) Clinical Toxicology 56(8): 725–735. Busquets MA and Estelrich J (2020) Prussian blue nanoparticles: Synthesis, surface modification, and biomedical applications. Drug Discovery Today 25(8): 1431–1443. https://doi. org/10.1016/j.drudis.2020.05.014. Chacko B and Peter JV (2019) Antidotes in poisoning. Indian Journal of Critical Care Medicine 23(supplement 4): S241–S249. https://doi.org/10.5005/jp-journals-10071-23310. Connolly SJ, Crowther M, Eikelboom JW, et al. (2019) Full study report of andexanet alfa for bleeding associated with factor xa inhibitors. The New England Journal of Medicine 380(14): 1326–1335. https://doi.org/10.1056/NEJMoa1814051. Cooper MS, Randall M, Rowell M, Charlton M, Greenway A, and Barnes C (2016) Congenital methemoglobinemia type II—Clinical improvement with short-term methylene blue treatment. Pediatric Blood & Cancer 63(3): 558–560. https://doi.org/10.1002/pbc.25791. Dawn L and Whited L (2021) Dimercaprol. [Updated 2021 Aug 30]. In: StatPearls. Treasure Island, FL: StatPearls Publishing Available from: https://www.ncbi.nlm.nih.gov/books/ NBK549804/. Fife CE, Eckert KA, and Carter MJ (2016) An update on the appropriate role for hyperbaric oxygen: Indications and evidence. Plastic and Reconstructive Surgery 138(3 supplement): 107S–116S. https://doi.org/10.1097/PRS.0000000000002714. Fisher ES and Curry SC (2019) Chapter 10 - Evaluation and treatment of acetaminophen toxicity. Advances in Pharmacology 85: 263–272. https://doi.org/10.1016/bs. apha.2018.12.004. Glatstein M, Carbell G, Scolnik D, Rimon A, Banerji S, and Hoyte C (2018) Pyridoxine for the treatment of isoniazid-induced seizures in intentional ingestions: The experience of a national poison center. The American Journal of Emergency Medicine 36(10): 1775–1778. Greget R, Dadak S, Barbier L, et al. (2016) Modeling and simulation of organophosphate-induced neurotoxicity: Prediction and validation by experimental studies. Neurotoxicology 54: 140–152. https://doi.org/10.1016/j.neuro.2016.04.013. Gristan YD and Moosavi L (2021). Folinic acid. StatPearls. Haut SR, Seinfeld S, and Pellock J (2016) Benzodiazepine use in seizure emergencies: A systematic review. Epilepsy & Behavior 63: 109–117. Hoegberg LC, Bania TC, Lavergne V, et al. (2016) Systematic review of the effect of intravenous lipid emulsion therapy for local anesthetic toxicity. Clinical Toxicology 54(3): 167–193. Jordan MR and Morrisonponce D (2021) Naloxone. In: StatPearls. Treasure Island, FL: StatPearls Publishing. 28722939. Juurlink DN (2016) Activated charcoal for acute overdose: A reappraisal. British Journal of Clinical Pharmacology 81: 482–487. https://doi.org/10.1111/bcp.12793. Kharel H, Pokhrel NB, Ghimire R, and Kharel Z (2020) The efficacy of pralidoxime in the treatment of organophosphate poisoning in humans: A systematic review and meta-analysis of randomized trials. Cureus 12(3): e7174. https://doi.org/10.7759/cureus.7174. Leung AM, Bauer AJ, Benvenga S, et al. (2017) American thyroid association scientific statement on the use of potassium iodide ingestion in a nuclear emergency. Thyroid 27(7): 865–877. Lexicomp (2021) Evidence-Based Drug Treatment Information. Wolterskluwer.com. https://www.wolterskluwer.com/en/solutions/lexicomp (Published 2021). Long N (2020) Sulfonylurea toxicity. Toxicology. Ma WW, Saif MW, El-Rayes BF, et al. (2017) Emergency use of uridine triacetate for the prevention and treatment of life-threatening 5-fluorouracil and capecitabine toxicity. Cancer 123(2): 345–356. Nelson L, Goldfrank L, Howland M, Lewin N, Smith S, and Hoffman R (2019) Goldfrank’s Toxicologic Emergencies, 11th edn New York: McGraw Hill. Overbeek D and Shaffer RW (2020) Management of beta blocker and calcium channel blocker toxicity. In: Evidence-Based Critical Care: A Case Study Approach, p. 57. Springer. Pincus M (2016) Management of digoxin toxicity. Australian Prescriber 39(1): 18–20. https://doi.org/10.18773/austprescr.2016.006. Poonam NT, Devvret KP, and Anju Rani MP (2018) Detoxification of arsenic and chromium through chelators and enzymes: An in-silico approach. International Journal of Applied Engineering Research 13(10): 8249–8271. Ramsey LB, Balis FM, O’Brien MM, et al. (2018) Consensus guideline for use of glucarpidase in patients with high-dose methotrexate induced acute kidney injury and delayed methotrexate clearance. The Oncologist 23(1): 52–61. https://doi.org/10.1634/theoncologist.2017-0243.

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Ranasinghe T, Mays T, Quedado J, and Adcock A (2019) Thrombolysis following heparin reversal with protamine sulfate in acute ischemic stroke: Case series and literature review. Journal of Stroke and Cerebrovascular Diseases 28(10): 104283. Ratto D and Joyner RW (2021) Dantrolene. In: StatPearls. Treasure Island, FL: StatPearls Publishing. 30571019. Rice JH, Akpunonu P, Davis GA, et al. (2021) Intravenous phytonadione administered orally in reducing warfarin-related coagulopathy. Clinical Toxicology 1–3. Singh R, Arain E, Buth A, Kado J, Soubani A, and Imran N (2016) Ethylene glycol poisoning: An unusual cause of altered mental status and the lessons learned from management of the disease in the acute setting. Case Reports in Critical Care 2016: 9157393. St-Onge M, Anseeuw K, Cantrell FL, et al. (2017) Experts consensus recommendations for the management of calcium channel blocker poisoning in adults. Critical Care Medicine 45(3): e306–e315. https://doi.org/10.1097/CCM.0000000000002087. UpToDate (2021) Uptodate.com. https://www.uptodate.com/contents/organophosphate-and-carbamate-poisoning (Published 2021). Van der Wall, Sake J, Lopes RD, Aisenberg J, et al. (2019) Idarucizumab for dabigatran reversal in the management of patients with gastrointestinal bleeding. Circulation 139(6): 748–756. van Eijkeren JC, Olie JDN, Bradberry SM, et al. (2017) Modeling the effect of succimer (DMSA; dimercaptosuccinic acid) chelation therapy in patients poisoned by lead. Clinical Toxicology 55(2): 133–141. Velasquez J and Wray AA (2021) Deferoxamine. In: StatPearls. Treasure Island, FL: StatPearls Publishing. 32491586. Wang GS, Baker K, Ng P, et al. (2021) A randomized trial comparing physostigmine vs lorazepam for treatment of antimuscarinic (anticholinergic) toxidrome. Clinical Toxicology 59(8): 698–704. https://doi.org/10.1080/15563650.2020.1854281. Yu PA, Lin NH, Mahon BE, et al. (2017) Safety and improved clinical outcomes in patients treated with new equine-derived heptavalent botulinum antitoxin. Clinical Infectious Diseases 66(supplement 1): S57–S64. https://doi.org/10.1093/cid/cix816. Zakharov S, Pelclova D, Navratil T, et al. (2015a) Fomepizole versus ethanol in the treatment of acute methanol poisoning: Comparison of clinical effectiveness in a mass poisoning outbreak. Clinical Toxicology 53(8): 797–806. https://doi.org/10.3109/15563650.2015.1059946. Zakharov S, Vaneckova M, Seidl Z, et al. (2015b) Successful use of hydroxocobalamin and sodium thiosulfate in acute cyanide poisoning: A case report with follow-up. Basic & Clinical Pharmacology & Toxicology 117(3): 209–212. https://doi.org/10.1111/bcpt.12387.

Further reading Brvar M, Chan MY, Dawson AH, Ribchester RR, and Eddleston M (2018) Magnesium sulfate and calcium channel blocking drugs as antidotes for acute organophosphorus insecticide poisoning—A systematic review and meta-analysis. Clinical Toxicology 56(8): 725–736. https://doi.org/10.1080/15563650.2018.1446532.

Relevant website https://aapcc.org/ :American Association of Poison Control Centers.

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Antifungal agents Gina Bertellia, Monica Sciturroa, Sidhartha D Rayb, and Mayur S Parmara, aDr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Clearwater, FL, United States; bDepartment of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved.

Background Mechanism of action General Available agents and routes of administration Uses Side effects and adverse reactions Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Toxicogenomics Pharmacokinetics Agents that affect antifungals and drug interactions Clinical management Population at risk Routes of exposure Environmental fate and behavior Ecotoxicology Exposure standards and guidelines Fungicides Resistance Conclusion References

604 605 605 605 606 606 607 608 608 608 608 608 608 609 609 609 609 609 610 610 610 610 610 612 612 613 613 613 613 613

Abstract Fungi are a diverse, heterogeneous group of eukaryotes, serving as friend and foe of humans for centuries. Since ancient times, fungi have been used in food and beverage-making processes and, more recently, have been used for diverse antibiotics production. Fungal infections, or mycoses, are usually classified by the area of the body primarily affected, such as, superficial mycoses, subcutaneous mycoses, opportunistic mycoses, and systemic mycoses. Immunosuppression in general population is not uncommon due to diverse drug and chemical exposures, which has led to increased incidence of invasive fungal diseases. Inadequate antifungal armamentarium, compounded by drug-drug interactions, toxicity, and misc. constraints in administration routes are ongoing challenges for the medical community. The morbidity and mortality caused by invasive fungal infections are increasing across the globe due to increased incidence of community-acquired and nosocomial fungal infections, frequent use of immunomodulatory agents, and the emergence of drug-resistant fungal strains. It has become a top priority for the academia and pharmaceutical industries to discover and develop new antifungal agents to be able to combat drug resistance, and at the same time possess potential broad spectrum of activity with minimum toxicity. Efforts in the past few decades have yielded several classes of antifungal drugs with diverse formulations. Some of the noted antifungal agents are listed here: Anidulafungin, Amorolfine, Amphotericin B, Caspofungin, Ciclopirox, Clotrimazole, Fluconazole, Flucytosine, Griseofulvin, Itraconazole, Ketoconazole, Micafungin, Miconazole, Naftifine, Nystatin, Pimaricin, Posaconazole, Terbinafine, and Terconazole, which are extensively used in medicine.

Keywords Adverse effects; Antifungal agents; Azoles; Drug resistance; Echinocandins; Fungal infections; Pharmacokinetics; Polyenes; Pyrimidines; Therapeutic uses; Toxicity

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00353-5

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Antifungal agents

Key points

• • • • • •

Fungal infections in general population are very common but in immunocompromised population, it is a problem. A large number of antifungal agents with great efficacies are available, and general population can avoid contracting infections by maintaining health and hygiene. Conventional antifungal agents have limitations for use due to the occurrence of drug-resistant strains and toxicity. Morbidity and mortality in immunocompromised patients due to invasive fungal infections is a major concern which is a humongous burden globally to healthcare systems. Available agents to deal with invasive fungal diseases is limited to only three main classes, such as, polyenes, triazoles, and echinocandins; but unfortunately, each suffer from specific drawbacks that are limitations related to spectrum of activity, likelihood of developing resistance, and toxicity. Caution is warranted on the use of certain antifungals in females of childbearing age.

Background Fungal infections, or mycosis, can manifest in several body systems. Most commonly, they affect the skin and mucosal surfaces but have been known to infect the lungs and develop into systemic infections. While evidence of fungal infections has been noted for centuries, particularly in immunocompromised individuals, the development of antifungal agents has significantly lagged behind its antimicrobial counterparts. The need for antifungal agents is increasing as immunomodulatory therapies continue to rise, increasing the risk for fungal infections due to the breakdown of host defense mechanisms. The challenge in discovering and developing antifungal drugs has been targeting the pathogen (fungi, which are eukaryotes) without posing significant toxicity to the host as many fundamental and cellular biological processes are conserved between fungi to humans (Roemer and Krysan, 2014). Another reason being fungi’s slow growth, and their multicellular forms, makes it difficult to develop an experimental strategy to evaluate a potential antifungal agent. Despite these challenges, numerous effective antifungal agents have been developed, and there is ongoing research to reduce host toxicity through different formulations of existing effective drugs. The first antifungal agent, amphotericin B, was marketed in the 1950s for invasive antifungal infections. Unfortunately, it was associated with significant toxicity and side effects (Nett and Andes, 2016). Over the past 50 years, considerable progress has been made to create new classes of antifungals that have an extended spectrum and are relatively safe. Four categories of antifungal agents have since been developed: azoles, echinocandins, polyenes, and pyrimidine analogs. These agents target the fungal cell’s viability and reproduction. Although new formulations have reduced the number of side effects, several toxicities are still associated, most notably nephrotoxicity and hepatotoxicity. Emerging target sites are currently being explored to treat the most invasive fungal infections and overcome the new challenges around the resistance development to existing agents, such as azole and echinocandin (Table 1). Table 1 List of antifungals available for treating a range of fungal infections. Antifungal Agents

CAS #

Anidulafungin Amorolfine Amphotericin B Caspofungin Ciclopirox Clotrimazole Fluconazole Flucytosine Griseofulvin Itraconazole Ketoconazole Micafungin Miconazole Naftifine Nystatin Pimaricin Posaconazole Terbinafine Terconazole

166663-25-8 78613-35-1 1397-89-3 162808-62-0 29342-05-0 23593-75-1 86386-73-4 2022-85-7 2884-22-2 84625-61-6 65277-42-1 235114-32-6 22916-47-8 65472-88-0 1400-61-9 7681-93-8 171228-49-2 91161-71-6 67915-31-5

Antifungal agents

605

Mechanism of action General Difference in the cell membrane sterol’s biosynthesis pathway between fungi (ergosterol biosynthesis) and host have been widely exploited to design effective antifungal agents for fungal treatment. The new agents offer a more targeted and less toxic therapeutic approach. The azole class of antifungals (fluconazole, itraconazole, ketoconazole) inhibits the enzyme lanosterol 14-a-demethylase, which is necessary for the conversion of lanosterol to ergosterol, a major component of fungal cell membranes. The polyenes (amphotericin B, nystatin, pimaricin) interact with ergosterol to form pores within the fungal membrane, allowing H+ and K+ ions to leak out of the cell. Antimetabolite antifungals (flucytosine) are converted by cytosine deaminase into 5-fluorouracil, which competes with uracil and interferes with fungal RNA and protein synthesis. The allylamine (naftifine, terbinafine) and morpholine (amorolfine) antifungals inhibit ergosterol synthesis by inhibiting squalene epoxidase and sterol D14-reductase/D8-reductase enzymes, respectively. Additionally, the echinocandins (caspofungin, micafungin, anidulafungin) inhibit the synthesis of b-glucan in the fungal cell wall via noncompetitive inhibition of the enzyme 1-3 b-glucan synthase. Griseofulvin is not included in these categories whose unique mechanism of action is carried out via binding to polymerized fungal microtubules inhibiting depolymerization and leading to failure in cell replication (Ghannoum and Rice, 1999). Table 2 summarizes the most important mechanism of action of antifungal agents.

Available agents and routes of administration The various agents and routes of administration for each category of antifungals were derived based on their mechanism of action, potency, and side effect profiles. For systemic infections, antifungal agents like azoles, echinocandins, and amphotericin B will likely be given parenterally as this offers the necessary potency to combat these infections. There are also topical preparations of many agents (cream, powder, shampoo, mouthwash, and lozenges) for vaginal, cutaneous, and mucocutaneous infections. Amorolfine and ciclopirox nail lacquers have also been developed for onychomycosis. Table 3 highlight the various route of administration of antifungal agents. Table 2

Antifungal mechanisms of action as per category.

Category

Examples

Mechanism of Action

Azoles: Imidazoles Triazoles Polyenes Allylamine

Clotrimazole, miconazole, ketoconazole Fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole Amphotericin B, nystatin, pimaricin Naftifine, terbinafine

Inhibit conversion of lanosterol to ergosterol by blocking the cytochrome P-450 enzyme.

Morpholine Antimetabolites Echinocandins Miscellaneous

Amorolfine Flucytosine Caspofungin, micafungin, anidulafungin Griseofulvin

Table 3

Promote pore formation in the fungal membrane (which contains ergosterol). Inhibit ergosterol synthesis by inhibiting the fungal enzyme squalene epoxidase. Inhibit ergosterol synthesis. Interferes with DNA and RNA biosynthesis selectively in fungi. Inhibit fungal cell wall synthesis by inhibiting b-glucan synthesis. Inhibit cell replication via interfering with microtubule function and disrupting mitosis.

Antifungal agents routes of administration.

Antifungal Agents

Routes of Administration

Amphotericin B

Parenterally, via direct injection into an infected area, nebulization as prophylaxis for patients with hematologic malignancies and lung transplants Cream, ointment, powder, “swish-and-swallow” oral suspension, vaginal cream, vaginal tablet PO capsule PO capsule or suspension, IV Clotrimazole: shampoo, gel, cream, ointment, solution, vaginal suppository, oral lozenges/troches Ketoconazole: shampoo, cream, and PO capsule Miconazole: oral gel, vaginal suppository, buccal tablets, topical cream, powder, pessaries PO or parenteral

Nystatin Griseofulvin Flucytosine Clotrimazole, ketoconazole, miconazole Fluconazole, itraconazole, terconazole, voriconazole, and Posaconazole Echinocandins: caspofungin, micafungin, anidulafungin Terbinafine Naftifine Amorolfine and ciclopirox

Parenteral PO capsule, cream, spray Cream and gel preparations Topical nail lacquers

606

Antifungal agents

Uses Clinical uses of antifungal agents depend on the site and extent of the infection as well as the virulence of the causative agent. Mycoses can be classified as local (cutaneous, subcutaneous) or systemic (bloodborne). Local, more superficial infections of dermatophytes or Candida usually only require topical formulations of antifungal agents, most commonly nystatin and azoles. Certain local infections, namely tinea capitis, must be treated with PO rather than topical agents to achieve optimal resolution. Treatment and prophylaxis of more severe systemic fungal infections like meningitis and fungemia require PO or intravenous administration of stronger agents like echinocandins or amphotericin B. Additionally, the virulence of an organism may be classified as either primary- arising in a healthy host or opportunistic- arising in hosts with a compromised immune system. Opportunistic infections can be more challenging to treat and require parenteral administration because of the patient’s immunocompromised state. Table 4 highlights the selective therapeutic uses of antifungal agents.

Side effects and adverse reactions While most agents in each antifungal category exhibit similar characteristics, many have their own unique side effect profiles based on differences in metabolism and biochemistry. Side effects often refer to symptoms less harmful than those found in an adverse reaction. Adverse reactions are unexpected negative reactions when the agent is used correctly and tend to be more severe. Adverse reactions are more unpredictable and occur less often than side effects. Hepatotoxicity is one of the most common side effects, most notably in azoles. Gastrointestinal upset (diarrhea, nausea, vomiting) can also frequently occur in those taking echinocandins, itraconazole, terbinafine, and amphotericin B. Other adverse reactions can include hypersensitivity, anaphylaxis, prolonged QTc,

Table 4

Antifungal agents and their associated therapeutic uses.

Antifungal Agents Systemic Amphotericin B Flucytosine

Echinocandins: Caspofungin Micafungin Anidulafungin Triazoles: Fluconazole Itraconazole Voriconazole Posaconazole Isavuconazole

Griseofulvin Terbinafine Local Nystatin Imidazoles: Clotrimazole, ketoconazole, miconazole Terbinafine Naftifine Ciclopirox, amorolfine

Therapeutic Uses Progressive, potentially life-threatening fungal infections: Aspergillosis, cryptococcosis, systemic candidiasis, blastomycosis, coccidioidomycosis, histoplasmosis, zygomycosis. Intrathecally for coccidioidal meningitis. Empirical therapy in the immunocompromised patient. Combination with amphotericin B for cryptococcal meningitis caused by Cryptococcus, and candidiasis resulting in endocarditis, meningitis, peritonitis and cystitis. As monotherapy for the treatment of local, non-life-threatening infections such as genitourinary candida infections. Chromoblastomycosis infections. Invasive candidiasis with activity against fluconazole resistant Candida glabrata and Candida krusei. Disseminated and mucocutaneous candidal infections and invasive aspergillosis (in patients who have failed to respond to amphotericin B, not as primary therapy) Mucocutaneous candidiasis, candidemia, and prophylaxis of candidal infections in bone marrow transplant patients. Esophageal candidiasis. Chronic suppression of cryptococcal meningitis in people living with HIV, candidiasis, onychomycosis, dermatophyte infections (tinea pedis, tinea cruris, tinea corporis), tinea versicolor (widespread), and tinea capitis. Prophylaxis and empirical therapy in the immunocompromised host (allogeneic bone marrow transplant patients). Cryptococcus, sporotrichosis, blastomycosis, coccidioidomycosis, histoplasmosis, candidiasis (oropharyngeal/ esophageal), onychomycosis, dermatophyte infections (tinea pedis, tinea cruris, tinea corporis), tinea versicolor (widespread), and tinea capitis. Invasive aspergillosis (drug of choice), invasive candidiasis, and pseudallescheriasis. Broad spectrum of activity against yeasts and molds. Oropharyngeal candidiasis and prophylactic treatment of invasive aspergillosis and candida infections in immunocompromised patients. Invasive aspergillosis and invasive mucormycosis. Oral treatment of superficial infections—inhibits the growth of dermatophyte infection (tinea capitis) and onychomycosis. Onychomycosis (most effective treatment; better tolerated), dermatophyte infection (tinea capitis), and chromoblastomycosis. Candida infections (mucocutaneous, cutaneous). Dermatophyte infections (tinea pedis, tinea cruris, tinea corporis), tinea versicolor, and vaginal candidiasis. Ketoconazole: systemic Candida spp., Malassezia furfur, and endemic fungal infections such as blastomycosis and coccidioidomycosis. Candida spp., Malassezia furfur, Trichophyton spp., and Microsporum spp. Dermatophyte infections (tinea pedis, tinea cruris, tinea corporis). Trichophyton spp., and Microsporum spp. Dermatophyte infections (tinea pedis, tinea cruris, tinea corporis). Onychomycosis.

Antifungal agents Table 5

607

Side effects and adverse reactions of antifungal agents.

Antifungal Agents

Side Effect

Adverse Reaction

Fluconazole Itraconazole

Headache, alopecia, anorexia, " AST/ALT Nausea, vomiting, skin rash/pruritus, headaches, dizziness, pedal edema, hypertriglyceridemia, hypokalemia, "AST/ALT Hyperbilirubinemia, " AST/ALT Local burning sensation, pruritus

Rare hepatoxicity Hepatotoxicity, congestive heart failure

Posaconazole Ketoconazole Voriconazole Flucytosine

Skin rash, " AST/ALT, and transient visual or auditory hallucinations are frequent after the first dose. Rash, diarrhea, " AST/ALT

Echinocandins

Gastrointestinal upset, flushing (due to histamine release)

Micafungin Amphotericin B Griseofulvin

Diarrhea, nausea, vomiting, fever, headache, arthralgia Nausea, vomiting, rigors, fever, chills, hypoxia, hypotension, hypokalemia, arrhythmias, anemia, IV phlebitis Headaches, cutaneous eruptions, disulfiram-like reaction

Terbinafine

Gastrointestinal upset, dysgeusia

# Ejection fraction, QT prolongation, torsades de pointes Hepatotoxicity, adrenal gland suppression, QT prolongation, torsades de pointes, gynecomastia Bone marrow suppression (Myelosuppression) leads to leukopenia and thrombocytopenia. Infusion-related reaction if rapidly administered (tachycardia, hypotension, thrombophlebitis) Hypersensitivity, anaphylactic reactions, acute hepatitis Nephrotoxicity (amphotericin B deoxycholate formulation) " Warfarin metabolism, hepatoxicity, teratogenic, carcinogenic Hepatotoxicity

and renal injury. Table 5 highlights the side effects and adverse reactions associated with specific antifungal agents (Develoux, 2001; Bormann and Morrison, 2009; Laniado-Laborín and Cabrales-Vargas, 2009; Thompson et al., 2009; Drew, 2021; Lewis and Kauffman, 2021).

Acute and short-term toxicity The United States Department of Occupational Safety and Health Administration (OSHA) defines acute toxicity as any adverse effect following oral or dermal administration of a single dose of a substance, or multiple doses given within 24 h, or an inhalation exposure of 4 h. The most common acute toxicity manifestations are infusion-related reactions. Infusion reactions are mostly associated with Amphotericin B formulations but may occur with any intravenous antifungal administration. Infusion-related reactions include hypotension, urticaria, headache, fever, chills, nausea, vomiting, myalgias, and arthralgias. Short-term or subacute toxicity is defined as adverse effects occurring after multiple or continuous exposure between 24 h and 28 days. The short-term toxicity of antifungal agents occurs through many different mechanisms, including but not limited to inhibition of cytochrome P450 enzymes leading to toxic buildup of metabolites and other medications, direct cytotoxicity, and bone marrow suppression. Tables 6 and 7 highlights some of the reported acute and short-term toxicity associated with antifungal agents in animals (Knasmüller et al., 1997; Shipstone, 2022) and humans (Rosa et al., 1987; Thompson et al., 2009; Grover, 2010; Lewis, 2011; Mounier et al., 2014; Stolmeier et al., 2018; Drew, 2021; U.S. National Library of Medicine, 2018). Table 6

Table 7

Some reported acute and short-term toxicity of antifungals in animals.

Antifungal Agents

Animal

Toxicity

Ketoconazole Itraconazole Voriconazole Voriconazole Amphotericin B Griseofulvin

Dogs

Hepatotoxicosis, inhibition of CYP3A2, inhibition of P-glycoprotein pump

Dogs Rats Rabbits Mice

Hepatotoxicosis, cell necrosis, " ALT, AST Central nervous system toxicity Ocular toxicity Hepatotoxicity

Some of the acute and short-term toxicity of antifungals in humans.

Antifungal Agents

Acute and Short-Term Toxicity

Amphotericin B Triazoles Echinocandins Flucytosine Terbinafine Griseofulvin

Infusion reaction, nephrotoxicity, bone marrow suppression, hepatotoxicity Hepatotoxicity, embryotoxicity, teratogenicity, inhibition of CYP2C9/3A4/2C19, phototoxic reaction (Voriconazole), ocular toxicity (Voriconazole) Hepatotoxicity, infusion reaction1, cardiotoxicity, embryotoxicity, bone marrow suppression Bone marrow suppression, hepatotoxicity, teratogenicity Hepatotoxicity Teratogenicity

608

Antifungal agents

Animal See Table 6.

Human See Table 7.

Chronic toxicity Chronic toxicity occurs when an agent is administered after prolonged or repeated administration. It often involves low exposures over a long period (> 6 months), and effects are often irreversible. Humans and animals develop similar chronic toxicities after the administration of antifungal agents. Ketoconazole, voriconazole, and imidazoles can cause hepatotoxicity, leading to jaundice and increased liver enzymes. Amphotericin can cause nephrotoxicity both in acute and chronic treatment. Tables 8 and 9 highlights some of the reported chronic toxicity associated with antifungal agents in animals (Van Cauteren et al., 1989) and humans (Björnsson, 2016; Cavassin et al., 2021; Drew, 2021).

Animal See Table 8.

Human See Table 9.

Immunotoxicity While the immunotoxic effects of antifungal pesticides have been studied in detail, the immunotoxicity of commercially available antifungal pharmaceuticals has not been well documented in the literature. Flucytosine has been associated with serious and life-threatening bone marrow depression, including leukocytopenia, thrombocytopenia, and pancytopenia. Several studies have shown that serum flucytosine levels above 100 mg/L are toxic and can lead to bone marrow suppression (Drew and Perfect, 2022). Patients who have undergone radiation or myelosuppressive therapy and those with underlying hematologic disorders are at an increased risk of developing bone marrow depression while using flucytosine (Vermes et al., 2000). One study showed amphotericin B had been found to suppress neutrophil functions, including chemotactic responses, chemiluminescence, phagocytosis, and killing during in vitro studies (Marmer et al., 1981). In another study using human peripheral blood mononuclear cells stimulated with C. albicans and Candida krusei, voriconazole and caspofungin were shown to increase the levels of IL-2, IFN-g, and IL-6 (Fidan et al., 2014). Table 8

Table 9

Some of the chronic toxicity of antifungals in animals.

Antifungal Agents

Animal

Toxicity (>6 months)

Voriconazole Itraconazole Ketoconazole

Dogs Rats Cats

Hepatotoxicosis; cell necrosis, " ALT and AST " Serum cholesterol Hepatotoxicity, jaundice

Some of the chronic toxicity of antifungals in humans.

Antifungal Agents

Toxicity (>6 months)

Amphotericin B Imidazoles: Clotrimazole, miconazole, ketoconazole

Nephrotoxicity (amphotericin B deoxycholate formulation) Hepatotoxicity

Antifungal agents

609

Reproductive and developmental toxicity No scientific studies were found that directly assessed the reproductive and developmental toxicities of antifungals in humans. However, multiple studies have found adverse reproductive effects in animals and concluded that caution should be exercised in females of childbearing age when using certain drugs. Embryotoxicity and teratogenicity were most prominently noted when mothers were given extremely high or teratogenic doses of antifungals. Azoles, especially, have been known to cause developmental and reproductive toxicities in rats and humans. Fluconazole, voriconazole, and itraconazole were found to prolong gestation and reduce both implantation and embryo-fetal survival. Case reports of cleft palates and hydronephrosis have also been associated with azoles. High maternal exposure to fluconazole in early pregnancy has also been associated with craniofacial and skeletal abnormalities. One study found that ketoconazole, when administered in high doses to rats on gestational days 6–15 (organogenesis), was also found to cause an increased frequency of skeletal anomalies compared to fluconazole and the control group (Amaral and Junior, 2008). Efinaconazole led to estrous cycle prolongation in female rats but was not teratogenic, and in female rabbits, it did not affect embryo-fetal development even in the presence of maternal toxicity (Glynn et al., 2015). Liposomal nystatin was also found to cause a statistically significant increase in hydrocephaly in rat fetuses when mothers were administered the highest dose of 3.0 mg/kg/day. Little to no male reproductive effects are associated with Nystatin (Larson et al., 2000). Triazoles have been found to increase serum testosterone and reduce both insemination and fertility indices (Goetz and Dix, 2009).

Genotoxicity Limited human studies assessing the genotoxicity of antifungals exist. Metronidazole has been shown to cause chromosomal aberrations in human cells in vitro and germ cell mutations of male rabbit germ cells in vivo. Griseofulvin was found to produce chromosomal aberrations in human leukocyte genes in vitro, as well as gene mutations in mouse lymphoma cells and Chinese hamster cells in vitro. Administration of griseofulvin also produced aneuploidy of syrian hamster embryo cells in vitro (Brambilla et al., 2012). Additionally, one study found that fluconazole administration to mouse bone marrow cells in vivo resulted in chromatid and chromosome breaks, aberrant chromatid exchange, and polyploidy but did not significantly increase the frequency of cancer (Yüzbas¸io glu et al., 2008).

Carcinogenicity With limited human studies, it is difficult to assess the carcinogenicity of antifungals. Griseofulvin and terbinafine have been linked to animal carcinogenicity or gene mutations in mammalian cells. Due to sufficient evidence in experimental animals, griseofulvin has been classified as possibly carcinogenic in humans. More specifically, griseofulvin has been associated with mouse lymphoma, hepatomas, and thyroid tumors, while terbinafine has been associated with liver tumors in rats (Brambilla et al., 2012).

Organ toxicity Hepato- and nephrotoxicity of antifungal agents have been extensively reported in the literature. Amphotericin B, most notably, is associated with nephrotoxicity resulting in increased serum creatinine levels. Amphotericin B deoxycholate has been associated with a reversible, transient decline in glomerular filtration rate in up to 80% of patients. This risk is amplified by diuretic-induced volume depletion or when co-administered with other nephrotoxic agents such as aminoglycosides, cyclosporin, and foscarnet (Drew, 2021). The hepatotoxic effects of azole antifungals is not well understood; however, various studies have demonstrated that mitochondrial toxicity of ketoconazole in human cell models reveals potential mechanisms for this toxicity (Haegler et al., 2017). Voriconazole has been associated with a unique retinoid-like phototoxic reaction presenting with cheilitis, erythema, and blistering. Cessation of voriconazole therapy generally reverses this phototoxic reaction, but recent studies have linked this reaction to developing skin cancer later in life. All patients who receive long-term voriconazole treatment are advised to undergo routine skin cancer screenings (Lewis, 2011). Table 10 highlights some antifungal agents’ organ-specific toxicities (Lewis, 2011; Drew, 2021; Lewis and Kauffman, 2021; Drew and Perfect, 2022).

Toxicogenomics Triazole antifungals myclobutanil, propiconazole, and triadimefon were found to influence gene expression in various biological pathways in rat livers, most notably fatty acid catabolism, steroid metabolism, and xenobiotic metabolism. These biological pathways regulate lipid, sterol, and steroid homeostasis, which could lead to changes in serum testosterone and adverse reproductive outcomes found in previous rat studies (Goetz and Dix, 2009).

610

Antifungal agents Table 10

Organ specific toxicities of antifungal agents.

Organ System

Antifungal Agents

Hepatic Renal CNS Ocular Cutaneous Cardiac Bone Marrow

Azoles, amphotericin B, flucytosine, echinocandins, griseofulvin, voriconazole Amphotericin B, voriconazole Voriconazole Voriconazole Voriconazole Echinocandins, triazoles, amphotericin B, itraconazole Flucytosine, amphotericin B

Pharmacokinetics The routes of administration and elimination are important along with the spectrum of activity for consideration of an antifungal agent. This is crucial when the optimal antifungal therapy for a patient is being determined. Table 11 summarizes the comparative pharmacokinetics of representative antifungal agents (Ashley et al., 2006; Wagner et al., 2006; Bellmann and Smuszkiewicz, 2017).

Agents that affect antifungals and drug interactions Many antifungals have been found to have significant effects on the absorption, effectiveness and disposition of a number of drugs. Interactions affecting the disposition of antifungal agents involve phenytoin, phenobarbital, carbamazepine, rifampin, ritonavir, efavirenz, and other inducers of CYP3A4. Proton Pump inhibitors and H2 receptor blockers can impair absorption of itraconazole and posaconazole, which leads to subtherapeutic levels. While amphotericin B has been known to cause hypokalemia, corticosteroids have an additive effect to this toxicity and, when administered together, contribute to reversible cardiomegaly and congestive heart failure. Table 12 highlights the drug interaction of antifungals with other drugs (Gubbins and Heldenbrand, 2010).

Clinical management Risks of antifungal toxicity can be minimized by dose modification, avoidance of drug interactions, use of pre-hydration and electrolyte supplementation, and laboratory monitoring (Chau et al., 2014). Antifungal therapeutic drug monitoring has become increasingly important for voriconazole, posaconazole, itraconazole, and flucytosine use. These drugs, in particular, have increased inter-patient drug plasma concentrations variability due to inconsistent metabolism, elimination, and possible interaction with other medications. Further studies are needed to develop specific therapeutic drug monitoring assays and refine therapeutic concentration goals (Andes et al., 2009). Table 13 highlights selected antifungals’ therapeutic drug monitoring (Andes et al., 2009; Chau et al., 2014; Drew and Perfect, 2022).

Population at risk As seen in the reproductive and developmental toxicity section, females of childbearing age are at the highest risk for antifungal toxicity. Systemic antifungal agents like amphotericin B have been noted to cause nephrotoxicity and infusion-related toxicity in children. However, if doses are followed correctly and overseen by a medical provider, antifungals should be an effective and safe treatment in children (Watt et al., 2011). Elderly patients are more likely to experience age-related kidney problems and drug-drug interactions and be more sensitive to side effects, which may require antifungal dose adjustments, especially fluconazole (Kauffman, 2001).

Routes of exposure Commonest exposure routes for antifungal drugs are ingestion, injection, and absorption. For commercial fungicides, absorption and inhalation are the main routes of entry into the body. Fungicide formulations penetrate the skin at different rates. Generally, oil-based liquid formulations are readily absorbed, while powders, dust, and granular formulations do not enter easily. Most fungicides currently used in the United States are unlikely to cause severe acute systemic poisoning due to advanced formulations (Reigart and Roberts, 1999). Table 14 highlights antifungal agent exposure routes (Kimakura et al., 2014; Liao and Lam, 2021).

Table 11

Pharmacokinetics of selected antifungal agents.

Pharmacokinetic parameter

Amphotericin B deoxycholate

Fluconazole

Itraconazole

Intravenous standard dose

0.5–1.5 mg/kg/day Loading dose 12 mg/kg once Maintenance dose 6 mg/kg once daily Oral standard dose Depends on Loading dose 200 mg clinical indication b.i.d. Maintenance dose 200 mg once daily—200 mg b.i.d Cmax (mg/mL)

1.7–2.8

9 after 400 mg i.v. 0.3–1.3

Vd (L/kg) Protein binding (%) t 1/2 (h) CL (mL/h/kg)

0.5–2.0 95–99 15–27 10–30

0.7 12 30 15–24

Metabolism

Bile, kidney; no metabolites identified

Elimination

Feces

Mainly unchanged via the kidney, tubular re-absorption Urine

Renal impairment

11 99.8 30 Dose-dependent, highly variable Excessive metabolisms involving CYP3A4 Feces, urine (inactive metabolites) No dose reduction

Isavuconazole

Loading dose 6 mg/kg b.i.d. on day 1 Maintenance dose 4 mg/kg b.i.d.

Loading dose 200 mg t.i.d. on day 1 and day 2 Maintenance dose 200 mg once daily Loading dose Loading dose 200 mg Loading dose 200 mg t.i.d. on (Tinf, 180 min), 70 mg, maintenance dose day 1 and day 2 maintenance 100 mg Maintenance dose dose 50 (70 if (Tinf, 90 min) 200 mg once daily body weight >80 kg) 2.6 7 10

50 mg for prophylaxis, 100 mg for candidaemia, 150 mg for esophageal candidiasis 18 (dose 150 mg)

6.5 98–99 80–120 30–70

0.3 99.9 13–20 12

Loading dose 400 mg b.i.d. on day 1 Maintenance dose 200 mg b.i.d. 4.4 after i.v. administration 4.5 58 6 100

Hepatic metabolism Hepatic involving CYPs (CYPs) metabolism (2C19 >2C9 >3A4) involving UGT, and CYP3A4 Urine Urine

Anidulafungin

Caspofungin

Micafungin

5-flucytosine 25–37.5 mg/kg four times per day

50–100

0.6 99.0 26 15

0.3–2.0 92.4–96.5 8 10

Spontaneous degradation in plasma/feces

Independent from CYP involved/feces cytochrome P450 (CYP)

Minor intestinal

Feces

Feces, urine

Urine

Caution with iv No dose No dose adjustment preparation for ClCr adjustment 0.7 mg/mL (prophylaxis) >1.25 mg/mL (treatment) >3 mg/mL 30–80 mg/mL

>5.5 mg/mL Not Established

Itraconazole Flucytosine

Table 14

>17.1 mg/mL >100 mg/mL

Antifungal routes of exposure.

Route of Exposure

Antifungal Agents

Inhalation Absorption Ingestion Injection

Itraconazole, voriconazole, amphotericin B, commercial fungicides Amphotericin B, pimaricin, micafungin, voriconazole, commercial fungicides Azoles, griseofulvin, commercial fungicides, terbinafine Amphotericin B, azoles, echinocandins, flucytosine

Environmental fate and behavior The use of azoles in medicine and agriculture has led to their accumulation in water, soil, and aquatic organisms. High amounts of azole compounds in domestic sewage and hospital wastewater have been demonstrated in many studies. One major source of azole pollution is the effluent released from wastewater treatment plants. Due to their differing hydrophobicities and biodegradation rates, the removal of azoles at wastewater treatment plants is highly variable. Higher concentrations of azole contamination are seen during the wet season (May) and less during the dry season (November) (Bhagat et al., 2021). Commercial fungicides’ environmental fate and effects have received less attention when compared to insecticides and herbicides.

Ecotoxicology Fungicides like azoles, methyl benzimidazole carbamate, and anilinopyrimidine have long been used to treat fungal infections in plants to improve crop yields. However, fungicide-resistant strains are becoming more prominent due to a short life cycle, abundant sporulation, and long-distance spore dispersal (Brauer et al., 2019). These resistant strains can negatively affect plant health and farming outcomes. LC50 data on commonly occurring azole pesticides, such as, ketoconazole, fadrozole, and itraconazole have not been documented for most fish species (Bhagat et al., 2021). Given the prevalence of azole pesticides in agricultural areas adjacent to water, systematic monitoring of the surrounding fish community is required. Table 15 highlights the measured LC50 concentration for selected azole compounds (Bhagat et al., 2021). Table 15

Measured LC50 concentrations for selected azole compounds.

Test Organism

Life Stage

Azole Compound

LC50 Concentration (mg/L)

Duration (h)

O. latipes G. rarus

Larvae Embryo

O. mykiss Danio rerio

Juvenile Embryo

Fluconazole Triflumizole Epoxiconazole Prochloraz Difenoconazole

>100 7.11 9.67 5.04 2.34

96 72 72 96 96

LC ¼ lethal concentration.

Antifungal agents Table 16

613

Genes involved in antifungal resistance.

Fungal Species

Medication

Gene(s) Involved

Aspergillus fumigatus C. glabrata C. krusei Candida spp. Corynebacterium auris

Azole Echinocandins Fluconazole Azoles Azoles

CYP51A FKS1, FKS2 ERG11p ERG11, ERG3,CDR1, CDR2, SNQ2, ABC1, MDR1, TPO3 ERG11, ERG3, FKS

Exposure standards and guidelines Fungicides Azoles are the main antifungal class used for crops as they are inexpensive, have a broad spectrum of activity, and are effective in plant fungal diseases. They can be used to control rust and mildew affecting fruits, vegetables, and cereal. Most fungicides used today are unlikely to cause severe systemic poisonings due to low inherent toxicity, slow, inefficient absorption, and application methods to which few individuals are exposed. Fungicide poisonings have previously been due to mistaken consumption of seed grain with organic mercury or hexachlorobenzene. These products have since been replaced with safer materials (Reigart and Roberts, 1999). Fungicide accumulated in the environment can be toxic to humans as well as disrupt the ecological balance and lead to the development of pathogenic resistance (Brauer et al., 2019). Exposure to commercial fungicides can be mitigated by reducing the number or the quantity of fungicides applied and increasing the distance from irrigation sources. Once in the water, vegetated ditches, wetlands or vegetated treatment systems are effective measures to reduce concentration (Zubrod et al., 2019).

Resistance Antifungal resistance is a growing concern. Resistance can manifest due to limitations of drug-drug interactions, adverse effects and toxicities that prevent long-term use, acquired resistance following exposure, and the intrinsic mutations of fungal species. Recent trends in acquired antifungal resistance include azole resistance among Candida isolates, azole resistance in Aspergillus fumigatus, and echinocandin resistance in C. glabrata. C. krusei has been shown to possess intrinsic resistance to fluconazole due to reduced susceptibility of ERG11p gene, which encodes the cytochrome P450 enzyme lanosterol demethylase, the target of fluconazole. A recently discovered multidrug-resistant fungus, Corynebacterium auris, has been shown to possess mutations in ERG11, ERG3, and FKS genes making it resistant to most members of the azole family. However, studies have shown retained susceptibility to posaconazole and isavuconazole. With increasing rates of antifungal resistance, new medications with novel mechanisms of action are currently being developed to help clinicians with these emerging challenges. Table 16 provides a summary of the antifungal resistance information below.

Conclusion Antifungal agents are essential for treating a wide range of fungal infections affecting the body locally and systemically. However, there are a number of acute and chronic toxic associations with the use of antifungals in humans and animals. Because of their toxicology and adverse effects, clinicians must be both careful and specific when prescribing antifungals to patients. The agent’s route of exposure, therapeutic range, drug interactions, and organ toxicities are important characteristics to consider. Additionally, a detailed patient history that includes their current medications, allergies, and comorbidities can help to tailor the agent that would be the most beneficial while also providing favorable toxicology.

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Brauer VS, Rezende CP, Pessoni AM, De Paula RG, Rangappa KS, Nayaka SC, Gupta VK, and Almeida F (2019) Antifungal agents in agriculture: Friends and foes of public health. Biomolecules 9(10). Cavassin FB, Baú-Carneiro JL, Vilas-Boas RR, and Queiroz-Telles F (2021) Sixty years of amphotericin B: An overview of the main antifungal agent used to treat invasive fungal infections. Infectious Disease and Therapy 10(1): 115–147. Chau MM, Kong DC, van Hal SJ, Urbancic K, Trubiano JA, Cassumbhoy M, Wilkes J, Cooper CM, Roberts JA, Marriott DJ, and Worth LJ (2014) Consensus guidelines for optimising antifungal drug delivery and monitoring to avoid toxicity and improve outcomes in patients with haematological malignancy, 2014. Internal Medicine Journal 44(12b): 1364–1388. Develoux M (2001) Griseofulvin. Annales de Dermatologie et de Vénéréologie 128(12): 1317–1325. Drew R (2021) Pharmacology of amphotericin B. Drew R and Perfect J (2022) Pharmacology of flucytosine (5-FC). Fidan I, Yesilyurt E, Kalkanci A, Aslan SO, Sahin N, Ogan MC, and Dizbay M (2014) Immunomodulatory effects of voriconazole and caspofungin on human peripheral blood mononuclear cells stimulated by Candida albicans and Candida krusei. The American Journal of the Medical Sciences 348(3): 219–223. Ghannoum MA and Rice LB (1999) Antifungal agents: Mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clinical Microbiology Reviews 12(4): 501–517. Glynn M, Jo W, Minowa K, Sanada H, Nejishima H, Matsuuchi H, Okamura H, Pillai R, and Mutter L (2015) Efinaconazole: Developmental and reproductive toxicity potential of a novel antifungal azole. Reproductive Toxicology 52: 18–25. Goetz AK and Dix DJ (2009) Toxicogenomic effects common to triazole antifungals and conserved between rats and humans. Toxicology and Applied Pharmacology 238(1): 80–89. Grover ND (2010) Echinocandins: A ray of hope in antifungal drug therapy. Indian Journal of Pharmacology 42(1): 9–11. Gubbins PO and Heldenbrand S (2010) Clinically relevant drug interactions of current antifungal agents. Mycoses 53(2): 95–113. Haegler P, Joerin L, Krähenbühl S, and Bouitbir J (2017) Hepatocellular toxicity of imidazole and triazole antimycotic agents. Toxicological Sciences 157(1): 183–195. Kauffman CA (2001) Fungal infections in older adults. Clinical Infectious Diseases 33(4): 550–555. Kimakura M, Usui T, Yokoo S, Nakagawa S, Yamagami S, and Amano S (2014) Toxicity of topical antifungal agents to stratified human cultivated corneal epithelial sheets. Journal of Ocular Pharmacology and Therapeutics 30(10): 810–814. Knasmüller S, Parzefall W, Helma C, Kassie F, Ecker S, and Schulte-Hermann R (1997) Toxic effects of griseofulvin: Disease models, mechanisms, and risk assessment. Critical Reviews in Toxicology 27(5): 495–537. Laniado-Laborín R and Cabrales-Vargas MN (2009) Amphotericin B: Side effects and toxicity. Revista Iberoamericana de Micologí a 26(4): 223–227. Larson JL, Wallace TL, Tyl RW, Marr MC, Myers CB, and Cossum PA (2000) The reproductive and developmental toxicity of the antifungal drug Nyotran (liposomal nystatin) in rats and rabbits. Toxicological Sciences 53(2): 421–429. Lewis RE (2011) Current concepts in antifungal pharmacology. Mayo Clinic Proceedings 86(8): 805–817. Lewis R and Kauffman C (2021) Pharmacology of echinocandins. Liao Q and Lam JKW (2021) Inhaled antifungal agents for the treatment and prophylaxis of pulmonary mycoses. Current Pharmaceutical Design 27(12): 1453–1468. Marmer DJ, Fields BT, France GL, and Steele RW (1981) Ketoconazole, amphotericin B, and amphotericin B methyl ester: Comparative in vitro and in vivo toxicological effects on neutrophil function. Antimicrobial Agents and Chemotherapy 20(5): 660–665. Mounier A, Douma I, El Chehab H, Vie A, Agard E, Russo A, and Dot C (2014) Ocular toxicity of voriconazole: A case report and review of litterature. In: 2014 European Association for Vision and Eye Research Conference, Acta Ophthalmologica. Nett JE and Andes DR (2016) Antifungal agents: Spectrum of activity, pharmacology, and clinical indications. Infectious Disease Clinics of North America 30(1): 51–83. Reigart R and Roberts J (1999) Fungicides. Recognition and Management of Pesticide Poisonings 6th. from https://www.epa.gov/sites/default/files/2015-01/documents/rmpp_ 6thed_final_lowresopt.pdf. Roemer T and Krysan DJ (2014) Antifungal drug development: Challenges, unmet clinical needs, and new approaches. Cold Spring Harbor Perspectives in Medicine 4(5). Rosa FW, Hernandez C, and Carlo WA (1987) Griseofulvin teratology, including two thoracopagus conjoined twins. Lancet 1(8525): 171. Shipstone M (2022) Antifungals for Integumentary Disease in Animals, Merck Manual Veterinary Manual. Stolmeier DA, Stratman HB, McIntee TJ, and Stratman EJ (2018) Utility of laboratory test result monitoring in patients taking oral terbinafine or griseofulvin for dermatophyte infections. JAMA Dermatology 154(12): 1409–1416. Thompson GR, Cadena J, and Patterson TF (2009) Overview of antifungal agents. Clinics in Chest Medicine 30(2): 203–215. v. U.S. National Library of Medicine (2018) Terbinafine. LiverTox: Clinical and Research Information on Drug-Induced Liver Injury. National Institute of Diabetes and Digestive and Kidney Diseases, 2012 U.S. National Library of Medicine. Van Cauteren H, Lampo A, Vandenberghe J, Vanparys P, Coussement W, De Coster R, and Marsboom R (1989) Toxicological profile and safety evaluation of antifungal azole derivatives. Mycoses 32(supplement 1): 60–66. Vermes A, Guchelaar HJ, and Dankert J (2000) Flucytosine: A review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions. The Journal of Antimicrobial Chemotherapy 46(2): 171–179. Wagner C, Graninger W, Presterl E, and Joukhadar C (2006) The echinocandins: Comparison of their pharmacokinetics, pharmacodynamics and clinical applications. Pharmacology 78(4): 161–177. Watt K, Benjamin DK, and Cohen-Wolkowiez M (2011) Pharmacokinetics of antifungal agents in children. Early Human Development 87(supplement 1): S61–S65. Yüzbas¸ioglu D, Unal F, Yilmaz S, Aksoy H, and Celik M (2008) Genotoxicity testing of fluconazole in vivo and in vitro. Mutation Research 649(1-2): 155–160. Zubrod JP, Bundschuh M, Arts G, Brühl CA, Imfeld G, Knäbel A, Payraudeau S, Rasmussen JJ, Rohr J, Scharmüller A, Smalling K, Stehle S, Schulz R, and Schäfer RB (2019) Fungicides: An overlooked pesticide class? Environmental Science & Technology 53(7): 3347–3365.

Relevant websites https://www.merckvetmanual.com/ :Merck Manual. https://medlineplus.gov/ :MedlinePlus, US National Library of Medicine. https://www.uptodate.com :UpToDate.

Antimicrobial agents Ryan E Fabian Campusano, Rodina Abdelhady, David Guirguis, Silvia Abdelmalak, Mariam Shaker, and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved.

Introduction Aminoglycosides Tetracyclines Sulfonamides Fluoroquinolones Macrolides Carbapenems Lincosamides Glycoproteins Anti-mycobacterial agents Isoniazid Rifamycin antibiotics Pyrazinamide Ethambutol Bedaquiline Anti-fungal agents Azoles Polyenes Flucytosine Anti-parasitic agents Benzimidazoles Imidazothiazoles Macrocyclic lactones (Avermectins) Tetrahydropyrimidines Conclusion References Further reading

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Abstract The concept of chemotherapy evolved from Nobel Laureate Paul Ehrlich’s magic bullet concept, over 100 years ago. Next, the discovery of penicillin in 1928 and its subsequent production for mass use formed the basis for antimicrobial therapy which began in 1940s. Subsequently, Streptomycin was discovered in 1944, and since then literally hundreds of antibiotics and antimicrobials have been added or synthesized to combat the microbial World. Another landmark concept (discovery) that was subsequently added following the introduction of these agents was the possibility that their basic structural skeleton could be modified to enhance their efficacy. The biggest challenge facing the 21st century healthcare is the evolution of drug resistant microbes, and in some cases multi-drug resistant microbes. In this article, we have compiled data on the mechanism of toxicity for all the major classes of antimicrobial agents. Most of the antimicrobials are derived from bacteria, fungi or are man-made. The agents are named as antibacterial, antiviral, antifungal, antiprotozoal and anti-chlamydial etc. This is not a comprehensive list of toxicities as each drug class has its own respective chapter in the Encyclopedia of Toxicology. The U.S. Environmental Protection Agency (EPA) regulates antimicrobial products as pesticides, and the USFDA regulates antimicrobial products as drugs/antiseptics. This article has focused on four major classes of microbes (antibacterial, antiviral, antifungal, antiprotozoal) and a list of agents (non-medicinal) that are considered to have non-medicinal antimicrobials.

Keywords Antibiotics; Anti-fungals; Antimicrobials; Antimycobacterial agents; Anti-parasitic agents; Cardiotoxicity; Dermatotoxicity; Gastrointestinal toxicity; Hematological toxicity; Hepatotoxicity; Hypersensitivity; Immunotoxicity; Nephrotoxicity; Neurotoxicity; Pulmonary toxicity

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Key points

• • • • • • • •

These are diverse chemicals that are naturally derived or synthetically produced. Most of the agents are beneficial and work on organismal structures via unique pathways. These agents are used short-term and long-term depending on the type of infection. Both short-term and long-term exposures can cause toxicity in certain populations (age, sex, genetic predisposition and pre-existing condition). Many of these agents are metabolized to more toxic compounds, while many are not. In both cases, agents are cleared from the body. Many agents show target specific toxicity as an individual agent or as a class. Antimicrobials can show drug interactions with other drug or chemical entities; therefore, caution must be taken prior to administration. Many antimicrobials are currently being investigated for their anticancer potential.

Introduction Toxicity and side effects of antibacterial agents on humans have been well documented (Shaeer, 2019; Nau et al., 2020; Phillips et al., 2022; Maxwell and Andrade, 2022; Li et al., 2023).

Aminoglycosides Ototoxicity: Clinically, aminoglycoside drugs enter the inner ear through systemic and topical circulation. In the systemic pathway, the drug passes through the blood-labyrinth barrier (BLB) and enters the inner ear through the stria vascularis. In topical administration, the drug can bypass the BLB into the middle ear and then through the round window into the inner ear. The drug is absorbed either by endocytosis on the apical surface or by transduction channels. Early genetic analysis showed that the susceptibility of aminoglycosides was related to mitochondrial DNA mutations, which inhibited the synthesis of mitochondrial proteins. However, evidence is accumulating to suggest that the overactivation of N-methyl-D-aspartate (NMDA) receptors and the production of free radicals likely contribute to ototoxic mechanisms (Fu et al., 2021). Nephrotoxicity: Due to their nephrotoxic effects, aminoglycosides are concentrated in endosomal and lysosomal vacuoles of nephrons. After glomerular filtration, they are retained in the epithelial lining of the S1 and S2 segments of proximal tubules. Aminoglycosides are attached to cells with lysosomal vacuoles, resulting in a slow rise in serum creatine and a hypoosmolar urinary output occurring within several days of receiving the treatment, potentially resulting in acute kidney injury (McWilliam et al., 2017; Le et al., 2023).

Tetracyclines Hepatotoxicity: High doses of intravenous tetracycline can induce fatty liver disease and may result in severe hepatic dysfunction, acute liver failure, and death. Hepatoxicity is more prevalent among pregnant women taking tetracycline antibiotics, especially during the last trimester or early postpartum period. However, instances of acute fatty liver attributed to intravenous tetracycline have been reported in nonpregnant women, men, and even children. The injury is characterized by the onset of weakness, fever, fatigue, nausea, and abdominal pain after 3 to 10 days of therapy. Laboratory tests show minimal-to-moderate elevations in serum aminotransferase and alkaline phosphatase levels with mild jaundice, hyperammonemia, and coagulopathy (Moseley, 2013; LiverTox, 2019; Wei et al., 2022). Reports of hepatotoxicity were reported in tetracyclines at high intravenous doses, which resulted in severe hepatic dysfunction, acute liver failure, and even death. It is commonly found among pregnant women during the last trimesters or early postpartum periods. Oral dosages of tetracyclines have very rarely caused liver injury compared to other drugs in the family. Being that intravenous tetracycline is no longer given, doxycycline and minocycline have become associated with idiosyncratic liver injury (LiverTox, 2017; Moseley, 2013).

Sulfonamides Hepatotoxicity: Hepatotoxicity from sulfonamides may represent a part of a spectrum of hypersensitivity due to sulfa-derived medications. Sulfonamides have been linked to many cases of DRESS (Drug Rash with Eosinophilia and Systemic Symptoms), Stevens-Johnson syndrome and toxic epidermal necrosis. The severity of injury varies widely. Most instances of sulfonamide-related liver injury are mild-to-moderate in severity and self-limited in the course. Cases with severe cholestasis may be prolonged and lead to vanishing bile duct syndrome. Importantly, sulfonamides can cause acute liver failure, particularly in instances with an abrupt onset and hepatocellular pattern of serum enzyme elevations. Indeed, sulfonamides remain one of the most common causes of drug-induced acute liver failure and account for 5% to 10% of instances (LiverTox, 2017; Chow and Khan, 2022).

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Neurotoxicity: The development of CNS adverse effects may be attributable to the inhibition of g-aminobutyric acid (GABA) and N-methyl D-aspartate (NMDA) receptors. The neurotoxicities reported for fluoroquinolones range from headache to seizure. Headache, restlessness, dizziness, and insomnia are some of the most commonly reported (1% to 6%) CNS adverse effects, whereas seizures and psychosis are rare. Rates of CNS toxicities vary slightly depending on the specific fluoroquinolone, but on average, they occur at a rate of 0.2% to 2%. Underlying CNS disorders are commonly encountered in patients who experience some of the more severe neurotoxicities. Patients with fluoroquinolone-related seizures typically have other risk factors, such as older age, a history of epilepsy, or renal dysfunction. However, one case report demonstrated that seizures might occur without these conditions (Christ, 1990).

Macrolides Hepatotoxicity: Liver injury is typically self-limited cholestatic hepatitis arising within 1 to 3 weeks of starting therapy. Symptoms include fatigue, dark urine, and jaundice, often with right upper quadrant pain and fever. The injury is usually self-limited and benign, but in some instances, there is prolonged cholestasis and persistence of liver test abnormalities beyond 6 months. Liver histology cases generally show vanishing bile duct syndrome or, at least, some degree of bile duct loss. The second form of clinically apparent hepatotoxicity from the macrolide antibiotics is an acute hepatocellular injury that usually arises within days of starting therapy, often with re-exposure. This hepatocellular injury with jaundice can lead to acute liver failure and has appreciable mortality, either death or need for emergent liver transplantation. This hepatocellular pattern occurs most commonly with telithromycin but is also described with azithromycin and less commonly with clarithromycin and erythromycin (LiverTox, 2017; Lenz et al., 2021; Zhang et al., 2020; Zhang et al., 2022).

Carbapenems

Neurotoxicity: Studies have shown that carbapenems induce neurotoxicity by interaction with the g-amino butyric acid receptor A (GABAA) and that this interaction depends primarily on the side chain on the second carbon atom in the carbapenem nucleus (Ragnar, 2000). The more basic this side chain is, the better the binding to the GABAA and the higher the convulsant activity of the compound in animal models. Imipenem and panipenem, which are carbapenems with a relatively high tendency to produce neurotoxicity, have basic C-2 side chains, while meropenem’s side chain is more acidic. Several studies have indicated that the most probable cause of the neurotoxic effects of a carbapenem is the concentration achieved in the brain tissue. b-lactam antibiotics are potentially neurotoxic and may cause seizures if given in high doses relative to renal function and/or body weight. For example, in patients, imipenem–cilastatin has been studied and shown to cause seizures at higher doses (Norrby, 2000).

Lincosamides GI Toxicity: Various antibiotics, including clindamycin, can cause an overgrowth of dangerous bacteria in the large intestine. This may cause either mild diarrhea or a life-threatening condition called colitis (inflammation of the large intestine). Clindamycin is more likely to cause colitis, so it should only be used to treat serious infections that cannot be treated by other antibiotics. There is no antidote for clindamycin toxicity, but the adverse effects will resolve with dose adjustment or discontinuation of the antibiotic. Only supportive care can be given while the patient recovers. The current recommendation is to measure serum electrolytes in patients with vomiting and/or diarrhea and monitor vital signs along with CBC with differential, platelets, LFTs, and renal function in symptomatic patients. Getting an EKG and maintaining continuous cardiac monitoring is also necessary as cardiac arrhythmias may occur. Finally, if colitis is suspected, evaluation for C. difficile toxin will be needed. It is also essential to monitor for severe allergic reactions like DRESS or Steven-Johnson syndrome. In this case, discontinue the antibiotic therapy immediately and treat with: IV fluids, oxygen therapy, diphenhydramine, and corticosteroids. In rare cases, clindamycin toxicity will lead to cardiac arrhythmias and cardiac arrest, requiring advanced cardiovascular life support (Rutt and Wang, 2021; Dilley and Geng, 2022; Murphy et al., 2022).

Glycoproteins Ototoxicity: Bilateral vestibular hypofunction (BVH) is characterized by a bilateral reduction or loss of function of the vestibular organs and/or the vestibular nerves. The symptoms include oscillopsia, imbalance, and frequent falls. Recent studies have shown that glycoproteins, especially vancomycin, can cause this chronic condition. Vancomycin-induced ototoxicity is rare but vestibular damage and cochlear damage associated with tinnitus and sensorineural hearing loss has been reported in humans after administering vancomycin (Uda et al., 2019; LiverTox, 2020). Nephrotoxicity: Vancomycin is commonly associated with nephrotoxicity. Vancomycin toxicity generally manifests as proximal tubular cell injury with or without necrosis and as acute interstitial nephritis. The exact mechanism of nephrotoxic damage caused by this glycopeptide is still unknown. The toxicity is characterized by acute tubular necrosis (ATN) or interstitial damage, yet the majority of biopsy reports have described acute interstitial nephritis (ATIN) rather than ATN (Barceló-Vidal, 2018; Uda et al., 2019; Cao et al., 2022) (Table 1).

Toxicology of major antibacterial agents.

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Table 1

Mechanism of action

Susceptible microbes

Clinical pharmacology

Organ toxicities

Aminoglycosides (Amikacin, Gentamicin, Kanamycin, Neomycin, Plazomicin, Streptomycin, Tobramycin)

Intracellular presence of aminoglycosides generally disturbs peptide elongation at the 30S ribosomal subunit, giving rise to inaccurate mRNA translation and, therefore, biosynthesis of truncated proteins or bearing altered amino acid compositions at particular points. Specifically, binding impairs translational proofreading, leading to a misreading of the RNA message, premature termination, or both, and the translated protein product inaccuracies. Inhibition of bacterial cell wall peptidoglycan synthesis by inhibiting penicillin-sensitive enzymes (transpeptidases, carboxypeptidases) responsible for the final three-dimensional structure of the rigid bacterial cell wall.

Gram (−) Streptomycin used for TB

Not Metabolized Half-life is estimated to be 2.5 h Excretion: Urine (29% to 89% as unchanged drug) small amount (1%) excreted in bile, saliva, sweat, and tears Protein binding: 34%

Nephrotoxicity, Ototoxicity Contact dermatitis by topical Neomycin

Not Metabolized Half-life: 5.8–8.7 h Renal clearance: 0.32 to 0.73 L/h

No Significant Toxicities Reported; allowed in pregnancy; however- it may enter breast milk and alter microbiota of infant.

Not Metabolized Concentrated by the liver in the bile and excreted in the urine and feces at high concentrations in a biologically active form Half life: 6–12 h

Hepatotoxicity

Metabolized into several metabolites: penicilloic acid, 6-aminopenicillanic, both inactive and acid hydroxylated into one or more active metabolites, which are also excreted via urine 560 ml/min renal clearance Nonrenal clearance includes hepatic metabolism and, to a lesser extent, biliary excretion Half-life: 0.4–0.9 h Not Hepatically Metabolized The majority of 5-ASA stays within the colonic lumen and is excreted as 5-ASA and acetyl-5-ASA in the feces Half-life: 5–10 h Primarily metabolized by CYP1A2 Primary metabolites: Oxociprofloxacin and Sulociprofloxacin make up 3–8% Minor metabolites: Desethylene ciprofloxacin and formylciprofloxacin Half-life: 4 h

Hypersensitivity reactions, Gi discomfort- nausea, vomiting and diarrhea, No Significant Toxicities Reported

Cephalosporins (Ceftriaxone, Cefotaxime, Ceftazidime Cefepime Cefazolin Ceftobiparole, Ceftaroline)

Tetracyclines (Tetracycline, Doxycycline, Eravacycline, Minocycline, Omadacycline)

Tetracyclines passively diffuses through porin channels in the bacterial membrane and reversibly binds to the 30S ribosomal subunit, preventing binding of tRNA to the mRNA-ribosome complex, and thus interfering with protein synthesis.

Penicillins Penicillin

Penicillins kill susceptible bacteria by inhibiting the transpeptidase that catalyzes the final step in cell wall biosynthesis, the cross-linking of peptidoglycan.

Tobramycin, Gentamicin, Kanamycin used for Pseudomonas aeruginosa. Ceftobiparole used for MRSA and VRSA Gram (+)/(−) Depending on the generation used: P. mirabilis, E.coli, Klebsiella pneumonia, H. influenza, P. aeruginosa Gram (+)/(−) H. pylori, Treponema, Brucella species Yersinia pestis Bacillus anthracis Plasmodium vivax Mycoplasma, Rickettsia, Chlamydia Gram (+)/(−) Broad spectrum anti-Pseudomonas: Piperacillin, Ampicillin, Enterobacter, Serratia, E.coli, H. influenza etc.

Sulfonamides Sulfasalazine

Sulfonamides inhibit the multiplication of bacteria by acting as competitive inhibitors of p-aminobenzoic acid in the folic acid metabolism cycle.

Gram (+)/(−)

Fluoroquinolones (Ciprofloxacin, Ofloxacin, Delafloxacin, Gemifloxacin, Levofloxacin, Moxifloxacin)

The fluoroquinolones are the only direct inhibitors of DNA synthesis; by binding to the enzyme-DNA complex, they stabilize DNA strand breaks created by DNA gyrase and topoisomerase IV. Ternary complexes of drug, enzyme, and DNA block the progress of the replication fork.

Gram (+)/(−)

Hepatotoxicity

Neurotoxicity

Antimicrobial agents

Class of drug (name of drug)

Macrolides Azithromycin

Macrolides inhibit bacterial protein synthesis. The mechanism of action of macrolides revolves around their ability to bind the bacterial 50S ribosomal subunit causing the cessation of bacterial protein synthesis. Carbapenems inhibit cell wall synthesis by binding to penicillin-binding proteins (PBPs), thus, causing bacterial cell wall defect, bacterial swelling, and killing bacteria.

Gram (+)

Not metabolized Primary Route of Elimination: Biliary excretion as unchanged drug Half-life: 68 h

Hepatotoxicity

Gram (+)/(−)

Neurotoxicity

Lincosamides Clindamycin

Lincosamides prevent bacterial replication in a bacteriostatic mechanism by interfering with the synthesis of proteins. Lincosamides bind close to the peptidyl transferase center on the 23S portion of the 50S subunit of bacterial ribosomes in a mechanism similar to macrolides and streptogramin B.

Gram (+)

Glycoproteins Vancomycin

Inhibits cell wall synthesis by binding to the D-AlaD-Ala terminal of the growing peptide chain during cell wall synthesis, resulting in inhibition of the transpeptidase, which prevents further elongation and cross-linking of the peptidoglycan matrix

Gram (+)

Primarily excreted unchanged but has one metabolite which is microbiologically inactive Primarily Renal Elimination Half Life: 1 h Metabolized by CYP3A4 and, to a lesser extent, CYP3A5 Two inactive metabolites: Clindamycin sulfoxide and N-desmethylclindamycin Elimination: Renal (10%), Biliary (3.6%), with the remainder excreted as inactive metabolites Half-life: 3 h in adults and 2.5 h in children Not metabolized Primarily Renal Elimination Half-life: 4 to 11 h In anephric patients, the average half-life of elimination is 7.5 days

Carbapenems Meropenem

GI toxicity

Nephrotoxicity, Ototoxicity

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Anti-mycobacterial agents Toxicity and side effects of antimycobacterial agents on humans have been well investigated (Onakopoya, 2022a).

Isoniazid Hepatotoxicity: Isoniazid’s (INH) ‘s toxicity is incumbent on the patient’s acetylation status. The onset is insidious and often manifests analogously to viral hepatitis. Usually, this toxicity can be resolved by discontinuing the drug. Treatment with INH is associated with increased AST and ALT and may cause acute liver injury with jaundice. INH is metabolized by N-Acetyltransferase (NAT) and CYP2E1, which produce various metabolites. The hepatoxic metabolites are hydrazine, monomethylhydrazine, and free radicals (LiverTox, 2018). In slow acetylators, NAT cannot clear the toxic metabolites resulting in INH-induced hepatitis (Badrinath and John, 2022). Neurotoxicity: INH has two mechanisms that can cause peripheral neuropathy. INH inhibits pyridoxine species (Vitamin B6) and pyridoxine phosphokinase, the enzyme that activates vitamin B6. Activated pyridoxine is decarboxylated to form gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter. The inhibition of pyridoxine phosphokinase will result in a deficiency of GABA, which may manifest as seizures (Badrinath and John, 2022).

Rifamycin antibiotics Hepatoxicity: The three rifamycin antibiotics used to treat mycobacterial diseases are Rifampin, Rifabutin, and Rifapentine. Out of those three, Rifampin has been best studied. Liver injury from Rifampin is uncommon but well documented, but it can range from moderate to severe. Typically, Rifampin is very well tolerated, even at higher doses, because it is rapidly metabolized in the liver (Beloor, 2022). The mechanism of toxicity is not well understood. Still, it is theorized that the cause of injury is due to idiosyncratic metabolites, which may be directly toxic or induce an immunologic reaction. The mechanisms for Rifapentine and Rifabutin are even less understood than Rifampin, but the mechanisms are theorized to be like Rifampin (LiverTox, 2018).

Pyrazinamide Hepatotoxicity: This drug is used with other anti-mycobacterial drugs to treat tuberculosis. Its use has been associated with a transient and asymptomatic elevation of Alanine transaminase (ALT) and Aspartate transaminase (AST). However, since it is always used in combination, it has not been studied alone. Pyrazinamide-induced hepatotoxicity may manifest similarly to viral-induced acute hepatitis after 4 to 8 weeks of therapy. Clinical manifestations include hypersensitivity reactions, hepatocellular necrosis, and cholestasis. The toxicity mechanism is unknown, but considering the drug is extensively metabolized by the liver and toxic effects increase dose-dependent, it is theorized that the drug and/or its metabolites cause a direct toxic effect (LiverTox, 2020).

Ethambutol Hepatoxicity: This drug is used in combination with other anti-mycobacterial drugs, as more studies need to be done to determine this drug’s hepatoxicity independently. Current data shows transient increases in AST and ALT-associated ethambutol treatment. However, its hepatoxicity mechanism is unknown; it is theorized to be due to hypersensitivities (LiverTox, 2020). Neurotoxicity: One of ethambutol’s most notorious side effects is ocular neuropathy which commonly manifests as a dose and duration-dependent red-green colorblindness (Onakopoya, 2022a). Doses greater than 50 mg/kg. This is reversible with the discontinuation of the drug. Other neuropathies include loss of visual acuity, scotoma, and blurred vision. The mechanism of toxicity is not well understood, but it appears to be due to ethambutol’s ability to chelate copper (Lee and Nguyen, 2021).

Bedaquiline Hepatoxicity: This drug is indicated in multi-drug resistant Tuberculosis infection; as such, it is used in combination with other anti-tuberculosis drugs. Liver function abnormalities only occur in 8% to 12%. The abnormalities can be asymptomatic or manifest as mild to moderate liver injury that resolves without treatment. The clinically significant liver injury occurs very rarely, and it is likely due to a production of a toxic intermediate. Toxicity may also be due to drug-drug interactions, as Bedaquiline is metabolized primarily by CYP3A4 (LiverTox, 2017) (Table 2).

Table 2

Toxicology of anti-mycobacterial agents. Mechanism of action

Susceptible microbes

Mycolic Acid Synthesis Inhibitors (Isoniazid)

Isoniazid is activated by mycobacterial enzyme katG. Activated isoniazid acylates the four positions of the reduced form of Nicotinamide adenine dinucleotide (NADH). The acylated NADH is no longer capable of catalyzing the reduction of unsaturated fatty acids, which are essential for the synthesis of the mycolic acids.

Mycobacteria

Rifampin

Inhibits prokaryotic DNA- dependent RNA synthesis and protein synthesis by blocking RNA-polymerase transcription initiation

Mycobacteria Gram (+)/(−)

Pyrazinamide

Pyrazinamide’s exact mechanism of action is not known. Susceptible strains of M. tuberculosis release pyrazinamidase, which converts PZA to pyrazinoic acid (POA). Conversion of PZA to this active metabolite may be partially responsible for its activity.

Mycobacteria

Ethambutol

The exact mechanism is not known; however, it appears to inhibit RNA synthesis, resulting in impaired cellular metabolism and multiplication.

Mycobacteria

Bedaquilline

Bedaquiline is a diarylquinoline that binds to the C-ring of adenosine 50 -triphosphate (ATP) synthase. Through this binding, it prevents ATP synthase from using the energy from hydrogen and/or sodium electrochemical gradients to produce ATP.

Mycobacteria

DNA-RNA-Protein Synthesis Inhibitor Rifamycin Misc. Anti-Mycobacterials

Organ toxicities Half-life: 1—4 h, depending on the patient’s acetylation rate Primarily Renal Elimination Potent inhibitor of CYP2C19 and an inhibitor of CYP3A4 in vitro. It only weakly inhibits CYP2A6, CYP2C9, and CYP2D6 in vivo Widely distributed Half-life: 45 h Primary metabolites are 31-hydroxy and 25-O-desacetyl rifabutin Renal (53%) and Biliary (5%) Excretion Inducer and substrate of CYP3A4 PZA is widely distributed Half-life: 9—10 h Pyrazinamide is hydrolyzed in the liver to pyrazinoic acid, its major active metabolite Subsequently, pyrazinoic acid is hydroxylated to the main excretory compound Renal Excretion Widely distributed, with high concentrations in the kidneys, lungs, and saliva Partially metabolized in the liver Primarily Renal Elimination Half-life: 3.5 h Volume of distribution is approximately 164 L 99.9% Protein Binding It undergoes oxidative metabolism via the hepatic isoenzyme CYP3A4 to form N-monodesmethyl metabolite (M2) Compared with the parent compound, M2 has 4- to 6-times less antimycobacterial activity and does not contribute significantly to clinical efficacy Primarily Biliary Elimination Half-life: 5.5 months

Hepatoxicity

Hepatoxicity

Hepatoxicity

Optic Neuropathy

Hepatoxicity

Antimicrobial agents

Class of drug (name of drug)

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622

Antimicrobial agents

Anti-fungal agents Toxicity and side effects of antifungals have been well investigated (Nett and Andes, 2016; Tverdek et al., 2016; Van Daele et al., 2019; Nivoix et al., 2020; Johnson, 2021; Burden et al., 2022; Kim et al., 2022; Brantsevich and Brantsevich, 2022; Muhaj et al., 2022; Samuel et al., 2022).

Azoles Hepatotoxicity: Azoles work by various mechanisms to inhibit ergosterol’s biosynthesis. Azoles have been seen to cause liver toxicity when used systemically. The toxicity ranges from mild to severe hepatic reactions, including hepatitis, cholestasis, and fulminant hepatic failure. Hepatic inflammation can occur, especially with voriconazole, but a clear dose relationship is not well established. The incidence of mild transient liver damage associated with azole drugs is 2–12%. The mechanism of liver damage is unknown, but the hepatotoxicity was reversible upon ending therapy. Careful monitoring of liver enzymes is recommended for all patients by receiving azole therapy. Abnormalities in liver enzymes, although reversible and rare after the termination of treatment, are the most common laboratory abnormalities in voriconazole-treated patients. Voriconazole appears to have a higher risk of liver damage than other modern antifungals (Dodds and Perfect, 2022).

Polyenes Nephrotoxicity: Polyene antibiotics are part of the antimicrobial compounds which bind to estrogen and are known for their amphotericin b-induced nephrotoxicity. Polyenes are known to cause azotemia, renal tubular acidosis, impaired renal concentrating ability, and electrolyte abnormalities like hypokalemia and sodium and magnesium wasting. Various abnormalities occur in patients receiving antibiotic treatment (Noor and Preuss, 2022).

Flucytosine Hepatotoxicity: Flucytosine therapy is commonly associated with hepatotoxicity as an adverse reaction with a low incidence rate ranging from 7% and a high 41% in various studies. Flucytosine toxicity can manifest as mild to moderate transaminitis followed by elevated alkaline phosphatase. Extremely rare acute liver injury and hepatic failure can be seen from uncommon elevation in bilirubin. The exact mechanism has not been fully explicated; however, the current evidence is that the toxic effects are not dose-dependent, but they resolve with the discontinuation of therapy (Bennet et al., 2020) (Table 3). Table 3

Toxicology of antifungal agents.

Class of drug

Drug name

Mechanism of action

Susceptible microbes

Clinical pharmacology

Organ toxicities

Azoles (Imidazoles and Triazoles)

Fluconazole

Azoles inhibit 14-demethylase, a CYP-450 enzyme responsible for converting lanosterol to ergosterol, as ergosterol forms a critical part of the fungal cell membrane. Thus, Azoles inhibit the synthesis of ergosterol from increasing cellular permeability.

Candida and Cryptococcus Species

Half-life ranges from 20 to 50 h with an average of 30 h Minimal Hepatic Metabolism The drug is primarily excreted renally as the unchanged drug Inhibitor of CYP2C9, CYP3A4, and CYP2C19 The half-life of 6 h Extensive hepatic metabolism Primarily renally excreted Half-Life: 34 to 42 h Metabolized extensively via the CYP3A4 Excreted both Biliary and renally

Hepatotoxicity

Half-life: 31 h Peak Concentration: 3–5 h post administration Volume of distribution: 500 L Protein Binding: >95% Biliary Elimination

No Significant Toxicities Reported

Voriconazole

Itraconazole

Posaconazole

Aspergillus, Candida, Scedosporium Blastomyces, Candida, Coccidioidomyces, Histoplasma, Paracoccidioides, Sporothrix, and Aspergillus Candida species

Hepatotoxicity

Hepatotoxicity

Antimicrobial agents

Table 3

623

(Continued)

Class of drug

Polyenes

Drug name

Susceptible microbes

Clinical pharmacology

Organ toxicities

Isavuconazole

Aspergillus, Rhizopus, and Mucor species

No Significant Toxicities Reported

Ketoconazole

Blastomyces, Candida, Coccidioidomyces, Histoplasma, Paracoccidioides, Fonsecaea, and Cladophialophora species Aspergillus, Candida, and Cryptococcus species

Half-life: 130 h Extensively metabolized by CYP3A4, CYP3A5, and subsequently uridine diphosphateglucuronosyltransferases (UGT). Biliary and Renal Elimination Widely distributed Biphasic half lives: 2 h and 8h Extensive metabolism by CYP3A4 with minor metabolism by CYP2D6 Primarily biliary elimination Exclusively renal metabolism Biphasic Half-life: 24 h and 15 days Not absorbed or metabolized Eliminated unchanged in feces Half Life: 40–50 h Not hepatically metabolized Anidulafungin undergoes slow chemical degradation at physiologic temperature and pH to a ring-opened peptide that lacks antifungal activity Biliary Excretion Half-life: 9–11 h Metabolized slowly by hydrolysis and N-acetylation Renal (35%) and Biliary (41%) excretion Half life: 14–17 h Micafungin is metabolized to M-1 (catechol form) by arylsulfatase, with further metabolism to M-2 (methoxy form) by catecholO-methyltransferase M-5 is formed by hydroxylation at the side chain (w-1 position) of micafungin catalyzed by cytochrome P450 (CYP) isozymes Primarily Biliary Excretion Prodrug Half life: 2.4 to 4.8 h Flucytosine is deaminated, possibly by gut bacteria or by the fungal targets, to 5-fluorouracil, the active metabolite Primarily renal excretion

Nephrotoxicity

Amphotericin B

Nystatin

Echinocandins

Anti-Fungal Antimetabolite

Anidulafungin

Mechanism of action

Polyenes bind to ergosterol in the fungal cell membrane, which leads to the formation of pores, ion leakage and ultimately fungal cell death Echinocandins inhibit the synthesis of b-glucan in the fungal cell wall via noncompetitive inhibition of the enzyme 1,3-b glucan synthase

Candida Species

Candida Species

Caspofungin

Candida and Aspergillus Species

Micafungin

Candida Species

Flucytosine

Acts directly on fungal organisms by competitive inhibition of purine and pyrimidine uptake and indirectly by intracellular metabolism to 5-fluorouracil.

Candida and Cryptococcus Species

Hepatotoxicity

No Significant Toxicities Reported No Significant Toxicities Reported

No Significant Toxicities Reported

No Significant Toxicities Reported

Hepatotoxicity

624

Antimicrobial agents

Anti-parasitic agents Toxicity and side effects of antiparasitic agents on humans have been well investigated (Onakopoya, 2022b).

Benzimidazoles Dermal toxicity: Thiabendazole can induce dermal toxicity if any residue of the drug is left on the skin for an extended period. Benzimidazoles’ route of administration is oral, and they work by selectively killing parasitic worms. They inhibit fungal growth and kill helminths by inhibiting microtubule assembly, which is thought to involve skin toxicity through this mechanism. The mechanism of action is binding to the beta-subunit of tubulin, which prevents the addition of any other subunits onto the microtubule. The acute toxicity risk associated with this class is low. Studies have resulted in toxic effects in many organs, such as the liver, bone marrow, and gastrointestinal tract. There is currently no antidote for toxicity involving benzimidazoles. Any toxic exposure should be treated symptomatically (Costa, 2013; Zhou, 2016; Fredericks et al., 2017; Chai et al., 2021; Vercruysse and Claerebout, 2022).

Imidazothiazoles Dermal toxicity: Imidazothiazoles can act as an immunomodulator and an immunoenhancer by increasing macrophage and neutrophil chemotaxis and T-cell lymphocyte function, enhancing dendritic cell growth maturation. Studies have shown that the most common adverse effect is severe agranulocytosis, retiform purpura, and seizures. Imidazothiazole toxicity involves cutaneous manifestations containing large hemorrhagic bullae and skin necrosis. The most common area affected is the face’s bilateral side. The exact mechanism of action behind this remains unclear. However, it is thought to be due to vascular straining for fibrin and immune complex-mediated vasculitis with vascular deposits of IgM, IgA, and IgG. There are currently no antidotes for Imidazothiazole toxicity but primarily supportive care, and skin lesions typically resolve when the drug is stopped (Powers et al., 1981; Jenkins et al., 2011; Ching and Smith, 2012; Pugh and Levy, 2016; Mühlig et al., 2019; Vercruysse and Claerebout, 2022).

Macrocyclic lactones (Avermectins) Neurotoxicity: Macrocyclic Lactones are broad-spectrum anti-parasitic agents used orally to treat and prevent human diseases such as lymphatic filariasis to control parasitic infections. The toxic effects seen with the use of Macrocyclic Lactones include severe episodes of confusion, seizures, and hypotension with increasing frequency of use. Studies have shown that individuals with prior ‘leaky’ blood-brain barriers can develop severe neurotoxicity with lethargy. The mechanism of action leading to human neurotoxicity is due to the interaction between Macrocyclic Lactones and inhibitory GABA receptors, which results in the medication being pumped out of the central nervous system by the P-glycoprotein (p-gp) transporter. Macrocyclic Lactones activate chloride channels allowing negative ions entry into the cell, hyperpolarizing the cell membrane and impairing the function of muscle cells, thus killing parasitic worms by inducing toxic paralysis (Yang, 2012; Edwards, 2003; Mealey, 2019).

Tetrahydropyrimidines Minimal toxicity- Tetrahydropyrimidines are a non-absorbable anthelmintic agent that works against intestinal nematodes such as pinworms and roundworms (Zimmerman, 2018). The mechanism of action of Tetrahydropyrimidines is by inhibiting acetylcholinesterase in the nervous system, which disturbs the normal movement of parasites leading to paralysis and are then removed naturally in the stool (Chalasani, 2015). Organ toxicity in humans is not so common with Tetrahydropyrimidines. Symptoms reported were mild nausea and vomiting, diarrhea, abdominal cramps, headache, dizziness, and loss of appetite. Even with an overdose of Tetrahydropyrimidines, minimal toxicity was reported. Overdose symptoms included severe muscle spasms and weakness (Gokbulut and McKeller, 2018; Monteiro et al., 2019) (Tables 4 and 5).

Conclusion With use of any drug, one must consider the risks and benefits associated with the drug, the same applies to antimicrobials. Many antimicrobial agents exert toxic effects due to their mechanisms of action. Other agents have mechanisms of toxicity that are not yet fully understood but must be considered when starting a course of therapy. Many antimicrobial drugs show drug interactions in many forms (synergistic, additive, potentiating etc.), and can occasionally cause severe hypersensitivity reactions, which are sometimes fatal. Intrinsic and idiosyncratic nature of antimicrobials are not uncommon. The potency of antimicrobials against

Table 4

Toxicology of antiparasitic medications.

Class of drug (drug name)

Mechanism of action

Susceptible microbes

Clinical pharmacology

Organ toxicities

Benzimidazoles

Benzimidazoles interrupt parasite energy metabolism by binding to tubulin, disrupting microtubular cell structure, and preventing nutrient uptake and other functions.

Ascariasis (roundworm), enterobiasis (pinworm), hookworm, cutaneous larva migrans, intestinal capillariasis and gnathostomiasis.

Dermal

Imidazothiazoles Levamisole

Imidazothiazoles work as a nicotinic acetylcholine receptor agonist that causes continued stimulation of the parasitic worm muscles, leading to paralysis.

Ascariasis and hookworm infections

Macrocyclic Lactones

Macrocyclic Lactones are anthelminthic agents that bind selectively and strongly to glutamate-gated chloride ion channels that occur in invertebrate nerve and muscle cells. This leads to increased permeability of cell membranes to chloride ions, hyperpolarization of the nerve or muscle cell, and death of the parasite.

Onchocerca volvulus and Strongyloides stercoralis

Rapidly absorbed Some systemic absorption may occur from topical preparations applied to the skin. Half-life is 1.2 h Metabolized almost entirely to the 5-hydroxy form, which appears in the urine as glucuronide or sulfate conjugates Primarily excreted renally Half-life: 4.4–5.6 h Levamisole is rapidly absorbed (2 h) 20–25% protein binding Extensively renally metabolized to form active and inactive metabolites. Half-Life: 18 Hours Primarily hepatic metabolism Ivermectin and/or its metabolites are excreted almost exclusively in the feces

Pyrantel

Enterobius vermicularis

Half Life: 1–2 h Administered orally The poor solubility of the pamoate salt offers the advantage of reduced absorption from the gastrointestinal tract and allows the drug to reach and act against parasites in the large intestine Partly metabolized in the liver Approximately 50% of an oral dose is excreted unchanged in feces; 7% excreted in urine as unchanged drug and metabolites

No Significant Toxicities Reported

Thiabendazole

Avermectins in use are: Ivermectin, Abamectin, Doramectin, Eprinomectin, and Selamectin Tetrahydro pyrimidines

N

No Significant Toxicities Reported

Antimicrobial agents

N H

H

Tetrahydropyrimidines cause the release of acetylcholine and inhibit cholinesterase; act as a depolarizing neuromuscular blocker, paralyzing the helminths.

Dermal

625

626 Table 5

Antimicrobial agents Misc other non-pharmaceutical antimicrobial agents.

Class of compound (agent name)

Mechanism of action/susceptible microbes/uses

Chlorine, chlorine gas and chloride compounds (Sod. Hypochlorite, Ca-hypochlorite, Chloramine-T, Chlorine dioxide, hypochlorous acid); miscellaneous other halogenated products (Fluorine, Bromine)

Most forms of bacteria, virus, fungi, chlamydia, rickettsia, mycoplasmas; effective at ppb to ppm; Cell injury, dissolution of cell wall, cell membrane and other structures; Main uses are chlorination of swimming pools, city water supplies; as sanitizer; treating wounds and hospital equipment Precipitation of cell proteins and many other unknown mechanisms in ointment and salves as skin antiseptics; in surgical-instrument disinfection Denaturation of proteins; interference with metabolism; lysis (dissolving of organism) As skin disinfectants, to form tinctures of antiseptics (used with acetone) Denaturation of proteins; inactivation of cellular metabolites; dissolving of cell wall as skin disinfectants and antiseptics; in sanitizing eating and drinking utensils, food-processing equipment; damage to cell membrane and other constituents Inhibition of cell growth; used as surgical scrubs (used with soaps and detergents); as deodorants Precipitate/coagulate proteins, disrupts cell wall and membrane structure; coagulate used in disinfection of dwellings, ships, storage houses, utensils, clothing; in hospital-instrument sterilization; whole house disinfection and preservation of cadavers/tissues. Precipitation of cell proteins; destruction of cell walls; Extensively used as skin antiseptics

Iodine and iodinated products (Povidone Iodine) Alcohols (e.g., methyl, ethyl and Isopropyl, benzyl alcohol) Cationic, surface-active quaternary ammonium compounds

Bisphenols (2 phenols linked together), chloroxyphenol Aldehydes (e.g., formaldehyde, glutaraldehyde, ortho-phthalaldehyde)

Misc. acids (salicylic and benzoic acids), sulfurous acid, phosphoric acid; in food preservatives (benzoic acid)/ Misc. bases: Sod. Hydroxide, Pot. hydroxide, Misc heavy metals (Mercurin chloride) Misc. gases (Ozone, N2, ethylene oxide) Phenols, Cresol and phenolic compounds; Bisphenol, Hexachlorophene

Misc. chemicals (e.g., acridine, anilides, H2O2, benzalkonium chloride, organomercurials, Biguanides (chlorhexidine); Thymol and Pine oil

Precipitation of cell proteins; Used in cosmetics and deodorants; antiperspirants; skin antiseptics Can kill all types of organisms due to formation of oxyradicals and other nonspecific injury S. epidermidis, P. aeruginosa and many other bacteria, fungi and virus; Bacteriostatic at 0.1 to 1.0%; bactericidal at 1 to 2%; Sporicidal at 5%; precipitates proteins; main uses are damaging cytoplasmic membranes and leakage of cell wall and other structures Inhibition of cell function; combines with essential metabolites; In dentistry as mucous antiseptics, in laboratory media to inhibit growth of unwanted bacteria; chlorhexidine is the active ingredient in preoperative skin preps, surgical hand scrubs, health care personnel handwash products, skin cleansers, acne creams, oral products (mouthwashes), burn ointments, and wound protectants; occasionally used as a preservative.

These agents are used as antiseptics, to sterilize surfaces and differ from chemotherapeutic agents. They can be bacteriostatic or bactericidal depending on the concentration employed. These agents can be either lethal to the microbe or slow down their growth. Sanitizers also fall in this group, although they are used to theoretically reduce the number of microbes to a level that can be considered safe. Sanitizers are extensively used in hospitals, industrial setting to clean the equipment and utensils employed in dairy industry, food-processing plants, eating and drinking establishments, and other places where presence of microbes are not readily apparent. In the antimicrobial World, cleaning, disinfection and sterilization, all connote to different meaning and all these concepts are applied based on circumstances and desired goals. Many agents are added to preserve food or similar products to discourage growth of microbes (high salting, N2 or O3 gassing etc.).

microorganisms depends on a number of factors, some of which are intrinsic qualities of the organism, others of which are the chemical and external physical environment (CDC, 2016). User awareness of these factors can lead to better use of these products and will minimize toxicity and side effects (Alajlan et al., 2022).

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Norrby SR (2000) Neurotoxicity of carbapenem antibiotics: Consequences for their use in bacterial meningitis. Journal of Antimicrobial Chemotherapy 45(1): 5–7. https://pubmed.ncbi. nlm.nih.gov/10629006/. Onakopoya IJ (2022a) Drugs used in the treatment of tuberculosis and leprosy. In: Ray SD (ed.) Side Effects of Drugs Annual, vol. 44, 311–331. https://www.sciencedirect.com/ science/article/abs/pii/S0378608022000174. Onakopoya IJ (2022b) Antihelminthic drugs. In: Ray SD (ed.) Side Effects of Drugs Annual, 44: 333–340. https://www.sciencedirect.com/science/article/abs/pii/ S0378608022000198. Phillips SR, et al. (2022) Side effects of Betalactams and Tetracyclines. In: Ray SD (ed.) Side Effects of Drugs Annual, vol. 44, 261–274. https://www.sciencedirect.com/science/ article/abs/pii/S0378608022000137. Powers LJ, et al. (1981) Effect of structural change on acute toxicity and antiinflammatory activity in a series of imidazothiazoles and thiazolobenzimidazoles. Journal of Medicinal Chemistry 24(5): 604–609. https://doi.org/10.1021/jm00137a022. Pugh JJ and Levy RA (2016) Naegleria fowleri: Diagnosis, pathophysiology of brain inflammation, and antimicrobial treatments. ACS Chemical Neuroscience 7(9): 1178–1179. https:// doi.org/10.1021/acschemneuro.6b00232. Ragnar S (2000) Neurotoxicity of carbapenem antibiotics: consequences for their use in bacterial meningitis. Journal of Antimicrobial Chemotherapy 45(1): 5–7. https://doi.org/ 10.1093/jac/45.1.5. Rutt AL and Wang CE (2021) Reaction to Clindamycin causing Laryngitis and Esophagitis. Ear, Nose, & Throat Journal 100(6): 437–438. https://doi.org/ 10.1177/0145561319875138. Samuel E, et al. (2022) Antifungal drugs. In: Ray SD (ed.) Side Effects of Drugs Annual, vol. 44, 303–309. https://www.sciencedirect.com/science/article/abs/pii/ S0378608022000241. Shaeer KM, et al. (2019) Plazomicin: A next-generation aminoglycoside. Pharmacotherapy 39(1): 77–93. https://doi.org/10.1002/phar.2203. Tverdek FP, Kofteridis D, and Kontoyiannis DP (2016) Antifungal agents and liver toxicity: A complex interaction. Expert Review of Anti-Infective Therapy 14(8): 765–776. https://doi. org/10.1080/14787210.2016.1199272. Uda K, et al. (2019) Ototoxicity and nephrotoxicity with elevated serum concentrations following vancomycin overdose: A retrospective case series. Journal of Pediatric Pharmacology and Therapeutics 24(5): 450–455. https://doi.org/10.5863/1551-6776-24.5.450. Van Daele R, et al. (2019) Antifungal drugs: What brings the future? Medical Mycology 57(Supplement_3): S328–S343. https://doi.org/10.1093/mmy/myz012. Vercruysse J and Claerebout E (2022) Safety of Anthelmintics. Merck Manual. https://www.merckvetmanual.com/pharmacology/anthelmintics/safety-of-anthelmintics. Wei C, et al. (2022) A pharmacovigilance study of the association between tetracyclines and hepatotoxicity based on Food and Drug Administration adverse event reporting system data. International Journal of Clinical Pharmacy 44(3): 709–716. https://doi.org/10.1007/s11096-022-01397-5. Yang CC (2012) Acute human toxicity of macrocyclic lactones. Current Pharmaceutical Biotechnology 13(6): 999–1003. Zhang MQ, et al. (2020) Liver toxicity of macrolide antibiotics in zebrafish. Toxicology 441: 152501. https://doi.org/10.1016/j.tox.2020.152501. Zhang MQ, et al. (2022) A rapid assessment model for liver toxicity of macrolides and an integrative evaluation for azithromycin impurities. Frontiers in Pharmacology 13: 860702. eCollection 2022. https://doi.org/10.3389/fphar.2022.860702. Zhou Y, et al. (2016) Mechanism of action of benzimidazole fungicide on fusarium graminearum: Interfering with polymerization of monomeric tubulin but not polymerized microtubule. Pathophysiology 106(8): 807–813. Zimmerman HJ (2018) Aminoglycosides. Hepatic injury from the treatment of infectious and parasitic diseases. In: Zimmerman HJ (ed.) Hepatotoxicity: The Adverse Effects of Drugs and Other Chemicals on the Liver, 2nd edn, p. 589. Philadelphia: Lippincott. See ref: https://www.ncbi.nlm.nih.gov/books/NBK548232/. LiverTox (2017) Macrolide Antibiotics. LiverTox: Clinical and Research Information on Drug-Induced Liver Injury [Internet] Bethesda (MD): National Institute of Diabetes and Digestive and Kidney Diseases; 2012-[Updated 2017 Aug 10]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK548398/

Further reading Jospe-Kaufman M, et al. (2020) The relationship between the structure and toxicity of aminoglycoside antibiotics. Bioorganic & Medicinal Chemistry Letters 30(13): 127218. https://doi.org/10.1016/j.bmcl.2020.127218. Liver Tox (2017) Clinical and Research Information on Drug-Induced Liver Injury [Internet]. National Center for Biotechnology Information. U.S. National Library of Medicine. June 10, 2017. Pea F (2017) Intracellular pharmacokinetics of antibacterials and their clinical implications. Clinical Pharmacokinetics 57(2): 177–189. https://doi.org/10.1007/s40262-017-0572-y. https://pubmed.ncbi.nlm.nih.gov/28639230/. Verma M, et al. (2014) Moxidectin causes adult worm mortality of human lymphatic filarial parasite Brugia malayi in rodent models. Folia Parasitologica 61(6): 561–570. Werth BJ (2022) Overview of antibacterial drugs. In: Merck Manual (Professional Version), https://www.merckmanuals.com/professional/infectious-diseases/bacteria-andantibacterial-drugs/overview-of-antibacterial-drugs. Westermann GW, et al. (1999) Chronic intoxication by doxycycline use for more than 12 years. Journal of Internal Medicine 246(6): 591–592. https://doi.org/10.1046/j.13652796.1999.00606.x.

Relevant website https://www.accessdata.fda.gov/scripts/cder/daf/ :Drugs@FDA: FDA approved drugs.

Antimicrobial resistance – Impact on humans Arjun Bagaia, Arathi Kulkarnib, and Mayur S Parmara, aDr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Clearwater, FL, United States; bDr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Fort Lauderdale, FL, United States © 2024 Elsevier Inc. All rights reserved.

Introduction Antibacterial agents resistance Cell Wall Inhibitors Glycopeptide b-lactams Others Nucleic acid inhibitors - antifolates Sulfonamides Protein synthesis inhibitors Peptidyl transferases (amphenicols) Lincosamides Protein synthesis inhibitors – other Tetracyclines Macrolides Aminoglycosides Spectinomycin Fluoroquinolones Antifungal agents resistance Azoles Polyenes Flucytosine Echinocandins Antiviral agents resistance Anti-retroviral drugs Non-nucleoside reverse transcriptase inhibitors (NNRTIs) Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) Protease inhibitors Integrase inhibitors Viral entry inhibitors Anti-hepatitis drugs Lamivudine, adefovir, tenofovir NS3 inhibitors NS5A inhibitors NS5B inhibitors Anti-herpes drugs Acyclovir and related derivatives Ganciclovir Foscarnet Anti-influenza drugs Neuraminidase inhibitors Adamantanes Conclusion References

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Abstract Microbes are microscopic organisms that include bacteria, viruses, fungi, protozoa, and helminths. Microbes can have positive or negative effects on human health and well-being. Bacteria normally maintain gut health which is essential for mucosal immunity and health in other organs, although they can cause a myriad of diseases ranging from bloody diarrhea to meningitis. Fungal infections, while rarer relative to bacteria, are common in immunocompromised patients. Fungal infections can be topical or systemic, ranging from rashes to more severe conditions like pneumonia and meningitis. Viruses, which are submicroscopic entities, can cause various diseases, from the common cold to influenza to rabies. These microbes cause significant morbidity and mortality around the globe; for example, the recent COVID-19 pandemic. While antibiotics, antifungals, and antivirals are used to combat these microbes, increasing reliance on these drugs leads to drug resistance. Encyclopedia of Toxicology 4th Edition

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Antimicrobial resistance is one of the more serious public health concerns today and poses a threat to human wellness on a global scale. If left uncontrolled, antimicrobial resistance could be a significant healthcare problem. Additionally, antimicrobial resistance increases treatment complexity and is a growing issue in clinical settings, which presents a significant challenge to the current recommendations for managing antibiotic resistance. Medicinal chemists and pharmaceutical industries need to focus on developing new drugs that will be efficacious while minimizing drug-drug toxicity and adverse drug reactions. Physicians and other health care providers need to stay on alert for indiscriminate overuse of antimicrobials that can give rise to drug resistance; therefore, need to innovate ways of treating infections. A thorough understanding of the mechanism of evolution of antimicrobial resistance is important because it helps healthcare professionals to prevent the spread of antibiotic-resistant bacteria. By understanding the mechanisms of antimicrobial resistance, strategies can be developed to prevent the spread of resistant organisms. New antibiotics can also be synthesized or developed to treat resistant microbes. Additionally, understanding the mechanisms of antimicrobial resistance helps us identify associated risk factors, which can help develop targeted interventions to reduce the risk of antibiotic-resistant infections. This chapter will discuss various antimicrobial classes and the mechanism of drug resistance that impacts human health.

Keywords Antimicrobial resistance; Antibiotics; Antifungal; Antiviral; Mechanism of resistance; Human health

Key points

• • • • • •

Antimicrobial resistance (AMR) is the ability of microorganisms to withstand the effects of antimicrobial drugs, making infections harder to treat and increasing the risk of disease spread, severe illness, and death. The emergence of drug-resistant strains can escalate through inadequate or inappropriate overuse of antimicrobials. AMR is an increasing global health threat that can reduce the effectiveness of antibiotics and antimicrobial drugs. AMR has been linked to increased morbidity, mortality, and healthcare costs. Antifungal resistance is of particular importance as not as many agents can be used. This chapter covers known and newer antibacterial, antifungal, and antiviral resistance mechanisms.

Introduction It is estimated that 700,000 thousand people die due to antimicrobial resistance (AMR) each year, and this number is expected to rise to 10 million people per year by 2050 if AMR is not adequately reduced (O’Neill, 2016). A 2019 study found that lower respiratory infections with antimicrobial resistance accounted for greater than 1.5 million deaths, making lower RTIs the most severe infectious disease associated with antimicrobial resistance (Antimicrobial Resistance, 2022). Antimicrobial resistance is a multifaceted problem stemming from overusing prescription antibiotics, lack of patient compliance, and the abuse of these medications (Ventola, 2015). Misuse of these medications can cause a variety of mutations in antimicrobial agents that diminish their efficacy and ability to fight disease. This chapter highlights antimicrobial resistance, underlying mechanisms, growing concern, and the steps needed to curb antimicrobial resistance. Antibacterial drug exposure yields varied resistance mechanisms because of the wide number of antibacterial drugs in the market today. The first cases of documented antibiotic resistance were in the 1950s with penicillin, which soon carried over to additional antibacterial drugs such as methicillin and vancomycin (Spellberg and Gilbert, 2014). Antimicrobial resistance has also been observed with antifungal and antiviral medications. While antifungal resistance is not seen at the same level as antibacterial resistance, treatment for severe fungal infections is limited; therefore, preservation of antifungal efficacy should be kept in mind (Wiederhold, 2017). Antiviral resistance mechanisms are also seen but can be attributed to the rapid ability of viruses to induce mutations compared to bacterial and fungal agents (Antimicrobial Resistance, 2022). Proper use of antimicrobial agents can help reduce the mortality and morbidity associated with AMR. This combined effort includes providers’ and patients’ appropriate use of antimicrobial guidelines to preserve this drug’s effectiveness. In a 2016 landmark AMR report chaired by O’Neill, the review recommends a three-pronged approach to combat AMR. The report suggests AMR can be reduced by limiting the demand for such antimicrobials by improving hygiene and spreading global awareness, secondly by developing effective antimicrobial drugs for infections that have become resistant to traditionally used agents, and lastly by forming a global coalition to implement guidelines and practices throughout the world (O’Neill, 2016). Overall, AMR is a serious global health threat that undermines the effectiveness of existing drugs and poses challenges for developing new ones. To combat AMR, it is essential to understand the current and emerging mechanisms of resistance that microbes employ to evade the action of antimicrobials. Below we review some of the most common and novel resistance mechanisms. Learning or awareness of these mechanisms can inform and facilitate the design of more potent and durable antimicrobial agents.

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Antibacterial agents resistance Antibacterial resistance in humans is an increasing public health concern. This occurs when bacteria become resistant to antibiotics used to treat bacterial infections. Antibiotic resistance has been described as one of the world’s most pressing public health problems. It limits the effectiveness of antibiotics and increases the risk of infection and death. The emergence of antibiotic-resistant bacteria is a natural phenomenon that can occur when antibiotics are used incorrectly or too often.

Cell Wall Inhibitors Cell wall inhibitors (CWI) act via various mechanisms and can be divided into four broad classes: glycopeptide, intracellular, b-lactams, and other mechanism antibiotics.

Glycopeptide Glycopeptide antibiotics inhibit peptidoglycan synthesis by binding to amino acids within the cell wall and preventing adding new amino acids to the peptidoglycan layer. Some glycopeptide antibiotics include vancomycin, teicoplanin, telavancin, bleomycin, ramoplanin, and decaplanin. Glycopeptide antibiotics were initially developed for multidrug-resistant (MDR) pathogens, but bacterial resistance has also developed for them (Sarkar et al., 2017). Gram-negative organisms are inherently resistant to glycopeptide antibiotics due to their outer cell membrane, which restricts the antibiotic from entering its periplasm (Yushchuk et al., 2020). Common resistance mechanisms include the presence of Van genes in gram-positive organisms. These resistant bacterial genes replace the terminal binding site D-ALA with D-Lac or D-ser, thus decreasing the capacity for glycopeptide antibiotics to bind (Yushchuk et al., 2020) (Tables 1 and 2).

b-lactams

b-lactams are part of the original group of antibiotics used to treat bacterial infection and disease. They can be categorized into penicillins, cephalosporins, carbapenems, and monobactams. All have the same b-lactam ring but differ in the presence (or absence) of another ring fused with the b-lactam. b-lactams work by inhibiting the terminal step of transpeptidation (cross-linking) by binding to Penicillin Binding Proteins (PBPs) that leads to disruption of the cell wall. b-lactams are primarily used against gram-positive bacteria. Resistance has been developed against penicillins by enzymes known as b-Lactamase, which cleave the amide bond of the b-lactam ring. Plasmid-mediated genes are also expressed to produce b-Lactamases and proteins that bind to b-Lactams. PBPs (specific types) can bind to b-Lactams and inhibit their actions. Table 1

Mechanisms of resistance against glycopeptides.

CWI-Glycopeptides Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Staphylococcus aureus

Rare VRSA strains with transposon Tn1546, obtained from the vancomycin-resistant Enterococcus faecalis, can alter cell wall structure and metabolism. The exact resistance mechanism is not well defined yet (Gardete and Tomasz, 2014) Pbp2 upregulation was recognized as three TR-MRSA strains teicoplanin (MICs of 16 or 32 mg/ml) while simultaneously having significant pbp4 downregulation was not noted in these strains. Mutated tcaRAB, vraSR, graSR, and rpoB genes may influence the transcription of the cell wall biosynthesis gene in MRSA (Bakthavatchalam et al., 2019)

Vancomycin

Methicillin-resistant Staphylococcus aureus

Table 2

Teicoplanin

Mechanisms of resistance against bacitracin.

Bacitracin Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Streptococcus suis

Streptococcus suis efflux pump SstFEG is encoded upstream of more common known bacitracin-resistance genes bceAB and bceRS that promotes resistance (Ma et al., 2019) Mutations in alr (rv3423c) which codes for alanine racemase may demonstrate resistance through multiple genes participating in different cellular functions such as lipid metabolism, methyltransferase, the stress response and transport systems in Mycobacterium tuberculosis (Chen et al., 2017) In vitro, its mechanism of resistance demonstrated loss of transport systems required for uptake, leading to decreased cAMP or MurA enzyme mutations (Silver, 2017)

Bacitracin

Mycobacterium tuberculosis

Escherichia coli

Cycloserine Fosfomycin

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Penicillin See Table 3. Cephalosporin See Table 4. Carbapenem See Table 5.

Others Daptomycin: Daptomycin is a cell wall inhibitor that utilizes a lipid tail to insert itself into a bacterial cell membrane. Its lipid tail depolarizes the bacterial cell membrane’s potassium-inactivated surfactant. Daptomycin complexes with calcium in a 1:1 M ratio to form small (14–16 molecules) DAP micelles. This may aid in antimicrobial delivery to the bacterial membrane (Scott et al., 2007). Once in the proximity of the bacterial membrane, DAP undergoes a structural change to insert into the cell membrane (Jung et al., 2004). It is commonly used against gram-positive bacteria (Carpenter and Chambers, 2004). Daptomycin resistance may occur due to changes in bacterial cell walls and cell membranes (Tran et al., 2015) (Table 6). Polymyxin B: Polymyxin B disrupts cell bacterial cell wall membrane. Its overall resistance mechanisms include alterations to reduce the “net negative charge or fluidity of LPS, increase in drug efflux, reduced porin pathway, capsule formation, and hypervesiculation” (Trimble et al., 2016). Polymyxin B is mainly used against gram-negative bacilli (Zavascki et al., 2007) (Table 7).

Nucleic acid inhibitors - antifolates Sulfonamides Sulfonamides are a bacteriostatic type of antibiotic that inhibits bacterial folic acid synthesis. Bacterial resistance to sulfonamides mainly occurs due to mutations in folP gene encoding dihydropteroate synthase (DHPS) involved in nucleotide biosynthesis or through the acquisition of alternative DHPS genes (sul1, sul2, and sul3) (Kim et al., 2019). Sulfonamides can be used against gram-positive and some gram-negative bacteria (Ovung and Bhattacharyya, 2021) (Table 8). Table 3

Mechanisms of resistance against b-lactams.

Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Enterococci species

Enterococci have PBP5 that binds to b-lactams and inhibit their actions (Gagetti et al., 2019). Expression of bla genes, which produces b-lactamases (Gagetti et al., 2019) blaTEM genes in plasmids produce TEM b-lactamases that target b-lactams and b-lactamase Inhibitors. Increases in copy number and transcription levels of the genes increase the activity of b-lactamases (Rodriguez-Villodres et al., 2020)

Piperacillin, Ampicillin

Escherichia coli

Table 4

Ampicillin/Sulbactam, Amoxicillin/ Clavulanic acid, Piperacillin/ Tazobactam

Mechanisms of resistance against cephalosporins.

Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Escherichia coli

Increased production of ampC transcription rate, resulting in increased production of AmpC cephalosporinases (Pfeifer et al., 2010) Mutations in the klu and bla genes result in extended CTX-M cephalosporinase activity (Pfeifer et al., 2010; Lerminiaux and Cameron, 2019)

3rd generation Cephalosporins and cephamycins like Cefoxitin Cefotaxime, Ceftazidime

Kluyvera species and Klebsiella species

Table 5

Mechanisms of resistance against carbapenems.

Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae

The presence of blaKPC genes allows for coding of KPC carbapenemases (Bratu et al., 2005; Villegas et al., 2007; Yigit et al., 2008)

Imipenem, Meropenem

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Mechanisms of resistance against daptomycin.

Daptomycin Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Staphylococcus aureus

Staphylococcus aureus is hypothesized to alter the membrane phospholipid composition, which may decrease the amount of PG available at the membrane or change the fluidity of the membrane, thus interfering with DAP binding and subsequent oligomerization (Miller et al., 2016)

Daptomycin

Table 7

Mechanisms of resistance against polymyxin B.

Polymyxin B Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Salmonella typhimurium

LPS modification: L-Ara4N in lipid A is 4–6 times fold greater than the parent Salmonella typhimurium strain, and the presence of PEtN all contribute to resistance (Olaitan et al., 2014; Vaara et al., 1981; Trent et al., 2001)

Polymyxin B

Table 8

Mechanisms of resistance against sulfonamides.

Name of bacteria that developed resistance

Mechanism of resistance

Bacillus, Pseudomonas and Shigella

Presence of sul genes (Wang et al., 2014)

Protein synthesis inhibitors Peptidyl transferases (amphenicols) Amphenicol inhibits protein synthesis by binding to the 50S subunit and prevents the formation of bacterial protein by inhibiting peptidyl transferase (Pietro et al., 2014). Chloramphenicol, a synthetic amphenicol antibiotic, blocks the binding of transfer RNA to the A site on the 50S ribosomal unit (Oong and Tadi, 2023). Amphenicols can be used against gram-positive, gram-negative, and anaerobic bacteria. Amphenicol resistance is mainly studied in E. coli, where acetyltransferase genes have been shown to confer resistance (Bischoff et al., 2002) (Table 9).

Lincosamides Lincosamides bind to the 50S ribosomal with its mycarose sugar that can inhibit peptidyl transferase, thereby inhibiting bacterial protein synthesis (Tenson et al., 2003). Common lincosamide resistance mechanisms include ribosomal changes through methylation or mutation, efflux pumps, and drug inactivation (Leclercq, 2002). Lincosamides are used against gram-positive cocci, gram-positive bacilli, gram-negative cocci, and some intracellular bacteria (Leclercq, 2002) (Table 10).

Protein synthesis inhibitors – other See Table 11.

Tetracyclines Tetracyclines are broad-spectrum drugs that work by interacting with the 16S rRNA of the 30S ribosomal subunit, specifically the steric hindrance of the drug inhibiting elongation and tRNA docking. In gram-negative bacteria, tetracyclines enter the outer membrane porins OmpF and OmpC as a tetracycline-Mg2+ charged complex (Grossman, 2016). Tetracyclines pass through the Table 9

Mechanisms of resistance against amphenicols.

Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Staphylococcus aureus, Enterococcus, Hemophilus influenzae, Salmonella typhi, and Riemerella anatipestifer Pseudomonas aeruginosa, Vibrio cholerae, and Salmonella typhimurium

catA gene responsible for resistance (Singh et al., 1990) catB gene responsible for resistance (Singh et al., 1990)

Chloramphenicol Chloramphenicol

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Table 10

Mechanisms of resistance against lincosamides.

Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Staphylococcus aureus

Presence of the msr(A) gene coding for the efflux mechanism or via erm gene encoding for enzymes that confer inducible or constitutive resistance (Prabhu et al., 2011)

Clindamycin

Table 11

Mechanisms of resistance against other protein synthesis inhibitors.

Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Staphylococcus aureus

The mupA gene confers high-level resistance (Abdulgader et al., 2020)

Mupirocin

inner membrane by dissociating from magnesium (Grossman, 2016). Resistance against tetracyclines has developed via mutations of the ribosome, ribosomal protection proteins (RPPs), efflux pumps, and tetracycline-inactivating enzymes. These antibiotics are primarily used for gram-positive and gram-negative bacteria and have some activity against intracellular Chlamydia, mycoplasmas, and Rickettsiae (Eliopoulos et al., 2003) (Table 12).

Macrolides Macrolides are antibiotics that target the 50S subunit of the ribosome, blocking peptidyl tRNA translocation. They also target the nascent peptide exit tunnel (NPET), preventing newly formed proteins from exiting (Vázquez-Laslop and Mankin, 2018). Macrolides are useful against gram-negative and gram-positive bacteria (Leclercq, 2002). Resistance has been developed via methylation or mutations of the binding site and efflux transporters similar to lincosamides (Table 13).

Aminoglycosides

Aminoglycosides work synergistically with other antibiotics such as b-lactams. Aminoglycosides are effective against various gram-positive and gram-negative bacteria, especially against the Enterococcus family (Krause et al., 2016). They work by binding to the A-site of the 16S rRNA of the 30S subunit. This results in mistranslation, defective protein synthesis, and bacterial cell death. Some aminoglycosides may also inhibit the initiation or elongation phases of protein translation. Resistance includes drug-modifying enzymes, target site methylation, and efflux pumps (Table 14).

Spectinomycin Spectinomycin binds the 30S bacterial ribosomal unit to inhibit the initiation of protein synthesis and interferes with protein chain elongation. Resistance mechanisms include efflux transporters, alteration of the drug-binding site through chromosomal mutations Table 12

Mechanisms of resistance against tetracyclines.

Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Campylobacter jejuni and Streptococcus species

Ribosomal Protection Proteins (RPPs) such as Tet(O) and Tet(M) catalyze the efflux of tetracyclines from the ribosome via GTPs and prevent the interaction of the tetracyclines with the 16S rRNA (Grossman, 2016) Tetracycline inactivating enzymes like Tet(X) hydroxylates the drug, decreasing its efficacy and leading to resistance (Grossman, 2016)

Tetracycline, Minocycline, Doxycycline All tetracyclines

Bacteroides, Escherichia coli

Table 13

Mechanisms of resistance against macrolides.

Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Staphylococcus species

erm genes code for Erm proteins which dimethylate an adenine residue in the 23S rRNA of the 50S subunit (Vázquez-Laslop and Mankin, 2018; Leclercq, 2002)

Escherichia coli, Mycobacterium avium, Helicobacter pylori

Mutations in the 23S rRNA of the 50S subunit (Leclercq, 2002)

Erythromycin, Clindamycin, Azithromycin Clarithromycin

Antimicrobial resistance – Impact on humans Table 14

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Mechanisms of resistance against aminoglycosides.

Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Enterococcus faecium, Mycobacterium species, Providencia stuartii Staphylococcus aureus, Enterococcus species

Aminoglycoside Modifying Enzymes (AMEs) acetylate the aminoglycosides, inhibiting the drug (Krause et al., 2016)

Gentamicin, Tobramycin, Amikacin

Aminoglycoside Modifying Enzymes (AMEs) phosphorylate the aminoglycosides, reducing the interactions between the drug and the rRNA (Krause et al., 2016)

Amikacin, Gentamicin B, Kanamycin, Neomycin

in the ribosomal 16S rRNA genes, and modification of the drug by aminoglycoside modifying enzymes (Bakthavatchalam et al., 2019). Spectinomycin exhibits broad-spectrum activity against gram-positive and gram-negative bacteria (Dharuman et al., 2021) (Table 15).

Fluoroquinolones Fluoroquinolones are antibiotics that target bacterial DNA gyrase (GyrA2GyrB2 heterodimer) and DNA topoisomerase IV (ParC2ParE2 heterodimer) (Hooper and Jacoby, 2016). Binding occurs at the protein-DNA interface, preventing DNA double-strand breaks and DNA resealing during DNA replication. They also stabilize the catalytic intermediate covalent complex of enzyme and DNA, resulting in the failure of movement of the replication fork. Resistance involves mutations in DNA gyrase and DNA topoisomerase, increased production of efflux pumps, and intrinsic resistance genes (qnr genes) (Hooper and Jacoby, 2016). Fluoroquinolones have been effective against aerobic gram-positive and gram-negative bacteria (Duggirala et al., 2007) (Table 16).

Antifungal agents resistance Antifungal resistance is a serious challenge for the medical community. This occurs when a fungus develops resistance to an antifungal drug, making it ineffective in treating infections. This resistance can occur naturally or can be acquired through mutations or the transfer of genetic material from another organism. As antifungal resistance increases, it becomes increasingly difficult to treat fungal infections, leading to longer recovery times and a higher risk of complications. For example, Candida albicans is one of the most common fungi in humans and can cause various serious infections, including skin, mouth, and vaginal infections. When C. albicans becomes resistant to antifungal medications, it can cause severe complications and even death.

Azoles Azoles mechanism of action is through inhibition of lanosterol through C14a demethylase in fungi (Kanafani and Perfect, 2008). Resistance to azoles stems from dysfunctions in permeability, uptake systems, presence of multidrug resistance gene products of the P-glycoprotein type. Alteration in cytochrome P450-dependent 14 alpha-demethylase can also induce resistance (Marichal and Vanden Bossche, 1995). Azoles have been shown to have a wide spectrum of action (Table 17). Table 15

Mechanisms of resistance against spectinomycin.

Name of bacteria that developed resistance

Mechanism of resistance

Escherichia coli

Resistant ribosomes, and all map in a locus (spc) counterclockwise to and 70% cotransducible with the classical str locus (Kim et al., 2019)

Table 16

Mechanisms of resistance against fluoroquinolones.

Name of bacteria that developed resistance

Mechanism of resistance

Antibiotic

Escherichia coli

Mutations in the DNA gyrase or DNA topoisomerase enzyme, specifically GyrA and ParC domains (Hooper and Jacoby, 2016) Expression of norA, norB, and norC increases production of their respective efflux pumps (Hooper and Jacoby, 2016)

Norfloxacin, Enoxacin

Staphylococcus aureus

Norfloxacin, Ciprofloxacin, Sparfloxacin, Moxifloxacin

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Polyenes Polyenes disrupt the fungal cell membrane by forming porin channels (Kanafani and Perfect, 2008). Polyenes are classified as having a wide spectrum of action, while Amphotericin B has the broadest spectrum of action, being fungicidal (Carolus et al., 2020). Filamentous fungi may be more likely than yeasts to have more resistance against polyenes (Kanafani and Perfect, 2008). Aspergillus, specifically Aspergillus terreus, is typically resistant to amphotericin B, and resistance to other aspergillus species is growing. Clinicians use a MIC ⩾1.0 mg/ml to indicate resistance to amphotericin B. Mechanisms are still being explored, but defects in ERG3 gene, which is implicated in ergosterol biosynthesis, may lead to resistance mechanisms observed in polyenes (Kanafani and Perfect, 2008) (Table 18).

Flucytosine Flucytosine’s mechanism of action is through inhibiting DNA and RNA synthesis. It is primarily used to treat severe systematic Candida and Cryptococcus infections (Padda and Parmar, 2023). Acquired mutations include mutations in cytosine deaminase or uracil phosphoribosyl transferase (Kanafani and Perfect, 2008). Some yeast strains develop resistance to flucytosine because of impaired cellular uptake through a mutation in cytosine permease (Kanafani and Perfect, 2008) (Table 19).

Echinocandins Echinocandins inhibit the synthesis of b(1,3)-D-glucan found in the fungal cell wall (Kanafani and Perfect, 2008). Echinocandins are especially effective against Candida and Aspergillus species (Kanafani and Perfect, 2008). Mechanisms of echinocandin resistance may occur due to amino acid changes in the Fks subunit of glucan synthase, but resistance mechanisms are still being studied (Perlin, 2015) (Table 20). Antifungal resistance is a significant concern as not many antifungal medications are used to combat systematic fungal illnesses. While the current antifungals have shown great efficacy, resistance to these agents may allow invasive fungal infections to cause more mortality and morbidity. Especially since serious invasive fungal infections typically have only a few agents for treatment. A global effort to combat antifungal resistance will require coordination from clinicians, agricultural environments, policymakers, and researchers. These systematic efforts can let fungal agents have continued and effective use. Table 17

Mechanisms of resistance against azoles.

Name of fungal strain that developed resistance

Mechanism of resistance

Antifungal

Candida glabrata

Increased expression of the drug: H+ antiporters CgAqr1, CgTpo1_1, CgTpo3, and CgQdr2 (Costa et al., 2016) Econazole efflux in select mutant strains (Feng et al., 2018)

Clotrimazole

Mycobacterium tuberculosis and Mycobacterium bovis Candida Candida

Table 18

Elevated Cap1 and MDR1 mRNA levels may confer resistance (Feng et al., 2018) Mutations in Cap1 and Mrr1 may confer resistance (Feng et al., 2018)

Econazole Fluconazole Itraconazole, Voriconazole

Mechanisms of resistance against polyenes.

Name of fungal strain that developed resistance

Mechanism of resistance

Antifungal

Leishmania promastigotes

Mechanisms include a change in the sterol content in the plasma membrane (TMA)-DPH environment, sensitivity to lower K+ levels, and the presence of SCMT gene (Ellis, 2002)

Amphotericin B

Table 19

Mechanisms of resistance against flucytosine.

Name of fungal strain that developed resistance

Mechanism of resistance

Antifungal

Candida albicans

Substitution of cytidylate for thymidylate at nucleotide position 301 leads to cysteine replacing arginine at amino acid position 101 in UPRT in FUR1 gene, disrupting the enzyme’s quaternary structure (Hope et al., 2004)

Flucytosine

Antimicrobial resistance – Impact on humans Table 20

637

Mechanisms of resistance against echinocandins.

Name of fungal strain that developed resistance

Mechanism of resistance

Antifungal

Candida species

Point mutations in Fks1 gene of the b(1,3)-D-glucan synthase complex may be associated with resistance to echinocandins (Balashov et al., 2006)

Caspofungin

Antiviral agents resistance Antiviral resistance is becoming increasingly common as viruses evolve and develop mechanisms to evade the effects of antiviral medications. As a result, treating viral infections is becoming more complicated and challenging as physicians are forced to choose from limited therapeutic options. Antiviral resistance may have significant effects on public health, leading to longer-lasting and more severe infections that can affect large populations.

Anti-retroviral drugs Non-nucleoside reverse transcriptase inhibitors (NNRTIs) Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) are used in the management of the human immunodeficiency virus (HIV) by forming a hydrophobic pocket in HIV viral replication which slows HIV viral DNA synthesis. These agents are unique in that they do not need to be phosphorylated. The mechanism of resistance for NNRTIs is amino acid substitution mutations that prevent NNRTIs from binding to the NNRTI-binding pockets (NBP), preventing proper alignment for subsequent phosphodiester bond formation (Iyidogan and Anderson, 2014) (Table 21).

Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs)

Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs) lack a 3-hydoroxy group and prevent the 30 to 50 phosphodiester bond forming in HIV viral DNA replication. There are two main mechanisms of resistance for NRTIs. Those are mutations in the NRTIs that decrease the affinity of the NRTI for the vDNA molecule via amino acid substitution mutations and nucleotide excision of the region containing the NRTI (Iyidogan and Anderson, 2014) (Table 22).

Protease inhibitors Protease inhibitors are used in managing HIV by blocking viral maturation by inhibiting the enzyme protease. The mechanism of resistance for protease inhibitors is amino acid substitution mutations that lead to the reduced binding affinity of the protease inhibitor. Specifically, the catalytic activity is reduced due to changes in electrostatic and hydrophobic interactions. Other resistance mechanisms include increased protease activity via non-active site pocket residues, mutations altering the Gag-Pol frameshift leading to increased expression of pol products, and Gag mutations at non-cleavage sites leading to increased access to viral protease (Iyidogan and Anderson, 2014) (Table 23).

Table 21

Most commonly observed NNRTIs-resistant mutations.

Virus

Resistant Mutations/Polymorphisms

Antiviral

HIV HIV HIV

CYP2B6 genetic polymorphism (Maseng et al., 2021); K103N, Y181C, G190A (Iyidogan and Anderson, 2014) CYP2B6 genetic polymorphism (Maseng et al., 2021); K103N, Y181C, G190A (Iyidogan and Anderson, 2014) Y188L (Martin et al., 2020)

Nevirapine Efavirenz Doravirine

Table 22

Most commonly observed NRTIs-resistant mutations.

Virus

Resistant Mutations/Polymorphisms

Antiviral

HIV HIV HIV

K65R (Iyidogan and Anderson, 2014) V75M (Davarpanah et al., 2018) L180M/T184L/M204V + A200V (Liang and Zheng, 2020)

Tenofovir, Lamivudine, Emtricitabine, Abacavir, Didanosine Stavudine Tenofovir

638

Antimicrobial resistance – Impact on humans

Table 23

Most commonly observed protease inhibitors-resistant mutations.

Virus

Resistant Mutations/Polymorphisms

Antiviral

HIV

V32I (Iyidogan and Anderson, 2014)

HIV

D30N/N88D, leading to evolution of the p1-p6 cleavage site (Kolli et al., 2014); D30N (Iyidogan and Anderson, 2014) R8, I50, I84, D25, A28 interactions (Shabanpour et al., 2022)

Atazanavir, Darunavir, Fosamprenavir, Lopinavir/Ritonavir, Tipranavir, Indinavir Nelfinavir

HIV

Darunavir

Integrase inhibitors Integrase inhibitors are used in the management of HIV by preventing viral mRNA synthesis. These inhibitors block the viral enzyme integrase that is used for viral synthesis. The mechanism of resistance for protease inhibitors is amino acid substitution mutations that lead to decreased sensitization of the integrase inhibitors (Iyidogan and Anderson, 2014) (Table 24).

Viral entry inhibitors Viral entry inhibitors are used in the treatment of HIV. These agents block the HIV virus from infecting the cell. Maraviroc, a type of viral entry inhibitor, binds CCR5 and blocks viral attachment. Whereas enfuvirtide binds GP41 and blocks viral penetration. The mechanism of resistance against Maraviroc involves switching tropisms from CCR5 to CXCR4 co-receptors. The mechanism of resistance against enfuvirtide involves amino acid substitutions preventing enfuvirtide from binding to HR1 region of gp41, thus allowing for viral fusion (Table 25).

Anti-hepatitis drugs Lamivudine, adefovir, tenofovir Lamivudine, adefovir, and tenofovir are used in the treatment of hepatitis B. These agents inhibit viral replication. Lamivudine competes with cytosine triphosphate for incorporation into the viral DNA strand to inhibit viral DNA synthesis. Adefovir and tenofovir inhibit hepatis B polymerase. Amino acid substitution mutations inhibit their ability to inhibit reverse transcriptase, thus allowing for HBV replication by allowing the conversion of ssDNA to ssRNA to dsDNA (Bang and Kim, 2014) (Table 26).

NS3 inhibitors NS3A/4B protease inhibitors are used in the treatment of hepatitis C. These agents block hepatitis C translation and replication by interfering with pathogen recognition signaling pathways. The resistance mechanism for NS3 protease inhibitors is amino acid substitution mutations that prevent inhibition of NS3 cleavage into NS5B, thus allowing for viral RNA replication (Kim et al., 2016) (Table 27).

NS5A inhibitors NS5A inhibitors are used in the treatment of hepatitis C. These agents block hepatitis C translation and replication by binding to NS5A and preventing RNA from binding. The mechanism of resistance for NS5A inhibitors is amino acid substitution mutations that allow for NS5A to participate in viral RNA replication and viral assembly (Kim et al., 2016) (Table 28). Table 24

Most commonly observed integrase inhibitors-resistant mutations.

Virus

Resistant Mutations/Polymorphisms

Antiviral

HIV HIV HIV

Y143R/H/C, Q148R/H/K (Iyidogan and Anderson, 2014) R263K (Charpentier and Descamps, 2018); Q148R/H/K (Iyidogan and Anderson, 2014) T66I/R263K, S153A/R263K (Oliveira et al., 2018); Q148R/H/K (Iyidogan and Anderson, 2014)

Raltegravir Dolutegravir Elvitegravir

Table 25

Most commonly observe viral entry inhibitors-resistant mutations.

Virus

Resistant Mutations/Polymorphisms

Antiviral

HIV

G36D/S, I37V, V38A/M/E, Q39R, Q40H, N42T, N43D (Iyidogan and Anderson, 2014)

Enfuvirtide

Antimicrobial resistance – Impact on humans Table 26

639

Most commonly observe resistance mutations against lamivudine, adefovir, and tenofovir.

Virus

Resistant Mutations/Polymorphisms

Antiviral

Hepatitis B Hepatitis B Hepatitis B

M204I/V (Bang and Kim, 2014); F160M/M184V (Yasutake et al., 2020) N236T (Bang and Kim, 2014); A181V/N236T (Zhao et al., 2018) A194T (Bang and Kim, 2014)

Lamivudine Adefovir Tenofovir

Table 27

Most commonly observed NS3-resistant mutations.

Virus

Resistant Mutations/Polymorphisms

Antiviral

Hepatitis C Hepatitis C Hepatitis C

D168E/V, Q80R, R155K (Kim et al., 2016) D168A/V/Y, Y56H (Kim et al., 2016) N84T, mutation in the enterovirus D68 2A protease (Musharrafieh et al., 2019); R155K (Kim et al., 2016)

Simeprevir Paritaprevir Telaprevir

NS5B inhibitors NS5B Inhibitors are used in the treatment of hepatitis C. These agents have an RNA-dependent RNA polymerase activity that blocks hepatitis C viral replication and is the gold standard treatment for hepatitis C. The mechanism of resistance for NS5B RNA-dependent RNA polymerase is amino acid substitution mutations that allow for NS5B to participate in the synthesis of (+) and (−) strands of viral RNA (Kim et al., 2016) (Table 29).

Anti-herpes drugs Acyclovir and related derivatives Acyclovir and its derivatives are primarily used in the treatment of herpes simplex virus (HSV) and the varicella-zoster virus (VZV). These agents are viral thymidine kinases that add 3 phosphate groups in viral synthesis and inhibit viral DNA polymerase from preventing viral replication. There are several mechanisms of resistance against acyclovir and its derivatives related to viral thymidine kinase. These mechanisms affect the synthesis or activity of the thymidine kinase, thus allowing for viral DNA synthesis (Jiang et al., 2016) (Table 30).

Ganciclovir Ganciclovir is primarily used in treating cytomegalovirus (CMV) by inhibiting viral DNA polymerases. Its resistance mechanism includes amino acid substitution mutations of viral thymidine kinase that prevent phosphorylation of several viral proteins and phosphorylation of ganciclovir, allowing for viral DNA synthesis. Mutations also affect DNA polymerase, resulting in viral DNA synthesis (Hakki and Chou, 2011) (Table 31). Table 28

Most commonly observed NS5A-resistant mutations.

Virus

Resistant Mutations/Polymorphisms

Antiviral

Hepatitis C Hepatitis C

M28T (Kim et al., 2016) Q30E/R, Y93C/H/N (Kim et al., 2016)

Daclatasvir, Ombitasvir Ledipasvir

Table 29

Table 30

Most commonly observed NS5B-resistant mutations.

Virus

Resistant Mutations/Polymorphisms

Antiviral

Hepatitis C Hepatitis C Hepatitis C

M414T, S556G (Kim et al., 2016) A421V, P495L/S (Kim et al., 2016) S282T (Itakura et al., 2020)

Dasabuvir Beclabuvir Sofosbuvir/Ledipasvir

Most commonly observed resistant mutations against acyclovir and related derivatives.

Virus

Mechanism of resistance/Resistant Mutations/Polymorphisms

Antiviral

HSV

Decreased synthesis of viral thymidine kinase (Jiang et al., 2016)

Acyclovir and related derivatives

640

Antimicrobial resistance – Impact on humans

Foscarnet Foscarnet is used in the treatment of CMV and HSV. This agent is a pyrophosphate analog that inhibits viral DNA polymerase. Amino acid substitution mutations of viral DNA polymerase, thus not being inhibited by the pyrophosphate analog foscarnet and allowing viral DNA replication via DNA polymerase (Crumpacker, 1992) (Table 32).

Anti-influenza drugs Neuraminidase inhibitors Neuraminidase inhibitors are used in the treatment of influenza virus. These agents cleave sialic acid residues to prevent the viral release of progeny. An amino acid substitution mutation changes the active site of influenza A/B N1 neuraminidase preventing the binding of neuraminidase inhibitors to N1 neuraminidase and thus allowing for sialic acid cleavage and subsequent release of viral progeny (Lampejo, 2020) (Table 33).

Adamantanes Adamantanes are used in the treatment of the influenza virus. These agents block the M2 protein proton channel and inhibit viral replication. The resistance mechanism involves an amino acid substitution in the M2 protein, thus allowing for continued viral uncoating and subsequent release of viral RNA into the host cell (Lampejo, 2020) (Table 34).

Conclusion Antimicrobial resistance is a global dilemma that could affect human generations to come and is an inevitable part of microbial evolution. From beta-lactams to fluoroquinolones, bacteria develop ways to become resistant or less susceptible to these antimicrobial agents. Common resistance mechanisms include intrinsic genetic opsonins, genetic mutations, mutations in efflux pumps, inactivating drug modifications, and mutations in cellular components (e.g., ribosomes). Standardized global practices can help prevent further resistance from occurring. From reducing overprescribing to formulating new innovative antibiotics—tackling AMR can be approached in multiple facets (Gil-Gil et al., 2019). Medicinal chemists and scientists can work on modifying chemical Table 31

Most commonly observed resistant mutations against ganciclovir.

Virus

Resistant Mutations/Polymorphisms

Antiviral

CMV

Mutations in the UL54 (DNA Polymerase) exonuclease domain region (Chou, 2021)

Ganciclovir

Table 32

Most commonly observed resistant mutations against foscarnet.

Virus

Resistant Mutations/Polymorphisms

Antiviral

CMV, HSV

Mutations in UL30 and UL54 genes lead to changes in DNA polymerase (Zarrouk et al., 2021)

Foscarnet

Table 33

Most commonly observed neuraminidase inhibitors-resistant mutations.

Virus

Resistant Mutations/Polymorphisms

Antiviral

Influenza A Influenza A

H275Y (Lampejo, 2020) R292K (Lampejo, 2020)

Oseltamivir, Peramivir Oseltamivir, Zanamivir

Table 34

Most commonly observed adamantanes-resistant mutations.

Virus

Resistant Mutations/Polymorphisms

Antiviral

Influenza A

S31N (Lampejo, 2020)

Amantadine

Note: Amino acid substitution mutations are represented by the substituted amino acid (first letter), the location of the mutation (the number), and the new amino acid (the second letter). For example, A56E represents a substitution of alanine to glutamic acid at location 56 of the polypeptide sequence: Alanine ¼ A, Arginine ¼ R, Asparagine ¼ N, Aspartic Acid ¼ D, Cysteine ¼ C, Glutamic Acid ¼ E, Glutamine ¼ Q, Glycine ¼ G, Histidine ¼ H, Isoleucine ¼ I, Leucine ¼ L, Lysine ¼ K, Methionine ¼ M, Phenylalanine ¼ F, Proline ¼ P, Serine ¼ S, Threonine ¼ T, Tryptophan ¼ W, Tyrosine ¼ Y, Valine ¼ V.

Antimicrobial resistance – Impact on humans

641

moieties that essentially “bypass” the resistance but can perform similar mechanisms of action. It is important to decrease the use of agents for these purposes to decrease further resistance. Hopefully, with these recommendations and persistent changes, the surge of antibiotic resistance can decline.

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(2003) The mechanism of action of macrolides, lincosamides and streptogramin B reveals the nascent peptide exit path in the ribosome. Journal of Molecular Biology 330(5): 1005–1014. Tran TT, Munita JM, and Arias CA (2015) Mechanisms of drug resistance: daptomycin resistance. Annals of the New York Academy of Sciences 1354: 32–53. Trent MS, Ribeiro AA, Lin S, Cotter RJ, and Raetz CR (2001) An inner membrane enzyme in Salmonella and Escherichia coli that transfers 4-amino-4-deoxy-L-arabinose to lipid A: Induction on polymyxin-resistant mutants and role of a novel lipid-linked donor. The Journal of Biological Chemistry 276: 43122–43131. Trimble MJ, Mlynárcik P, Kolárˇ M, and Hancock RE (2016) Polymyxin: Alternative mechanisms of action and resistance. Cold Spring Harbor Perspectives in Medicine 6. Vaara M, Vaara T, Jensen M, Helander I, Nurminen M, Rietschel ET, and Mäkelä PH (1981) Characterization of the lipopolysaccharide from the polymyxin-resistant pmrA mutants of Salmonella typhimurium. FEBS Letters 129: 145–149. Vázquez-Laslop N and Mankin AS (2018) How macrolide antibiotics work. Trends in Biochemical Sciences 43: 668–684. Ventola CL (2015) The antibiotic resistance crisis: Part 1: Causes and threats. P & T: A Peer-Reviewed Journal for Formulary Management 40: 277–283. Villegas MV, et al. (2007) First identification of Pseudomonas aeruginosa isolates producing a KPC-type carbapenem-hydrolyzing beta-lactamase. Antimicrobial Agents and Chemotherapy 51(4): 1553–1555. Wang N, Yang X, Jiao S, Zhang J, Ye B, and Gao S (2014) Sulfonamide-resistant bacteria and their resistance genes in soils fertilized with manures from Jiangsu Province, Southeastern China. PLoS One 9: e112626. Wiederhold NP (2017) Antifungal resistance: current trends and future strategies to combat. Infect Drug Resist 10: 249–259. Yasutake Y, Hattori SI, Tamura N, Matsuda K, Kohgo S, Maeda K, and Mitsuya H (2020) Structural features in common of HBV and HIV-1 resistance against chirally-distinct nucleoside analogues entecavir and lamivudine. Scientific Reports 10: 3021. Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle JW, Steward CD, Alberti S, Bush K, and Tenover FC (2008) Novel carbapenem-hydrolyzing b-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 52(2): 809. https://doi.org/10.1128/AAC.01445-07. Yushchuk O, Binda E, and Marinelli F (2020) Glycopeptide antibiotic resistance genes: Distribution and function in the producer actinomycetes. Frontiers in Microbiology 11: 1173. Zarrouk K, Zhu X, Pham VD, Goyette N, Piret J, Shi R, and Boivin G (2021) Impact of amino acid substitutions in region II and helix K of herpes simplex virus 1 and human cytomegalovirus DNA polymerases on resistance to foscarnet. Antimicrobial Agents and Chemotherapy 65: e0039021. Zavascki AP, Goldani LZ, Li J, and Nation RL (2007) Polymyxin B for the treatment of multidrug-resistant pathogens: A critical review. Journal of Antimicrobial Chemotherapy 60(6): 1206–1215. https://doi.org/10.1093/jac/dkm357. Zhao L, Li X, Cheng Y, Chen R, Shao J, Zhou Y, Li Q, Liao H, Zhao Y, Liu L, Su H, Liu Y, Liu Y, and Xu D (2018) Hepatitis B virus rtA181T/sW172non-stop mutation may increase resistance fold to adefovir- and entecavir-resistant mutants compared to rtA181T/sW172 mutation. Antiviral Research 154: 26–34.

Antimicrobial resistance and the environment Matías Giméneza,b, Fernanda Azpirozc, Josefina Veraa, and Silvia B Batistaa, aLaboratorio Microbiología Molecular, Departamento de Bioquímica y Genómica Microbiana, Instituto de Investigaciones Biológicas Clemente Estable, Ministerio de Educación y Cultura, Montevideo, Uruguay; bLaboratorio de Genómica Microbiana, Institut Pasteur Montevideo, Uruguay; cSección de Fisiología & Genética Bacterianas, Facultad de Ciencias, UdelaR, Montevideo, Uruguay © 2024 Elsevier Inc. All rights reserved.

Introduction Antimicrobials, antibiotics, definitions and mechanisms of action Antibiotic resistance and associated vulnerabilities Mechanisms of antibiotic resistance, evolution and transmission Involvement of the environment in the appearance of “novel” ARGs in human and animal bacterial pathogens Strategies for the study of AMR in different scenarios Procedures for the analysis and management of antibiotic resistance in the environment Identification of critical sites exposed to anthropogenic contamination Epidemiological surveillance Risk assessments Management of contaminated environments Future perspectives References

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Abstract Antibiotic resistance is identified as one of the main causes of death worldwide. It is estimated that in 2019, 1.27 million deaths were directly associated with this problem and the number is expected to increase. To face this health emergency, the challenge was included within the global strategy called “One Health”, aimed at optimizing and ensuring human, animal and environmental health. However, antibiotic resistance in the environment has been little explored. Recently, novel strategies of study are being incorporated, intending to understand the role of the environment in the appearance, transmission and maintenance of antibiotic resistance in bacteria.

Keywords Antimicrobials; Antibiotics; Environment; Evolution; Horizontal transfer; Mobile genetic elements; “One Health”; Antibiotic resistance genes; Surveillance; Transmission events

Key points

• • • • •

Within the broad set of compounds classified as antimicrobials, bacterial resistance to antibiotics has emerged as a growing problem. The use of antibiotics promotes the selection of resistant microorganisms either by mutations in the involved genes or by capture of specific resistant genes contained in mobile genetic elements, acquired by horizontal transfer. Antibiotic resistance affects the health of humans, animals and the environment. This problem is being addressed through a multidimensional and global strategy, called “One Health”. The role of the environment in the emergence and spread of antibiotic resistance has been relatively little explored. Lately, new strategies have been incorporated to analyze this topic. The strategy to address this issue comprises: identifying the sites with the highest risk of contamination, having surveillance systems as well as protocols to assess the integrated risk and finally, designing technological solutions to control antibiotic resistance spread.

Introduction Antimicrobials, antibiotics, definitions and mechanisms of action Antimicrobial compounds cover a wide group of substances of natural, semi-synthetic and synthetic origin, which can kill or inhibit the growth of different microorganisms, with greater or lesser specificity. They comprise antiviral, antibacterial, antifungal and

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antiparasitic compounds that are used to combat viruses, bacteria, fungi and parasites, respectively. Since 21st century, an increasing number of infections caused by microorganisms no longer susceptible to these compounds raised antimicrobial resistance (AMR) as one of the main public health problems. This concern is especially important in the case of antibacterial compounds, as they threat the effective prevention and treatment of bacterial infections. Thus, this chapter focuses on the resistance to these type of antimicrobial compounds and its relationship with the environment. Antibiotics are antimicrobial compounds originally synthesized by microorganisms, which exert a deleterious effect on bacteria. The discovery of the first antibiotic, the penicillin, occurred by a random event in the laboratory of Dr. Alexander Fleming in 1928. Upon returning from vacation, and examining some petri dishes with bacterial isolates grown on solid media, Fleming’s group observed the presence of growth inhibition halos around a Penicillium notatum contamination. Then, thanks to the ability of those researchers to assess the implications of that event, a new era of advances in human health and animal production began (Leisner, 2020). In the following years, diverse lines of research were developed aiming at the production of various natural and semi-synthetic antibiotics as well as synthetic antibacterial compounds, which could be obtained at industrial level. Thus, these three types of compounds are globally referred as antibiotics. Antibiotics are usually classified considering the group of target bacteria. Thus, they have been catalogued into narrow-spectrum antibiotics (affecting Gram-negative or Gram-positive bacteria), broad-spectrum (which affect a wide range of Gram-negative and Gram-positive bacteria) and extended-spectrum (applied for those antibiotics which undergo chemical modifications that increase the spectrum of target bacteria). These compounds exert their function through a variety of strategies, including inhibition of cell wall synthesis (such as penicillin), inhibition of protein synthesis (aminoglycosides), inhibition of DNA synthesis (fluoroquinolones), RNA synthesis inhibition (rifampicin), mycolic acid synthesis inhibition (isoniazid), and folic acid synthesis inhibition (trimethoprim) (Munita and Arias, 1982). In natural environments, there is still an open debate whether these secondary metabolites function for competing and/or communicating with other organisms in the community. A group of peptides with antimicrobial activity, synthesized by prokaryotic and eukaryotic organisms, has recently been proposed as one of the most promising alternatives to classical antibiotics. These antimicrobial peptides can affect the growth of target organisms through different mechanisms, such as altering membrane permeability or other intracellular functions, and acting on bacterial biofilm formation (Rima et al., 2021).

Antibiotic resistance and associated vulnerabilities The synthesis of antibiotics is naturally associated with the expression of resistance by the microorganisms potentially affected. Antimicrobial resistance can be intrinsic when the microorganism is normally non-susceptible to a given antibiotic, whereas acquired AMR occurs when it appears as a consequence of genome evolution. These defence mechanisms have become a problem of global importance with the widespread use of antibiotics in human health, animal production, and environmental waste management. The industrial production of these compounds and their intensive use in humans for the treatment of bacterial infections as well as in animals for therapy, prophylaxis and growth promotion, cause a qualitative and quantitative change in the situation. Humans and animals using antibiotics have become bioreactors that promote the selection of microorganisms resistant to antibiotics, due to the incorporation of mutations in some of their own genes or by capture of specific resistant genes harbored in mobile genetic elements (MGEs), which are usually acquired by horizontal transfer. In recent years, the emergence of pathogenic bacteria resistant to various antibiotics to which they originally exhibited sensitivity, revealed that the problem of antibiotic resistance must be considered as a priority. Thus, the issue of AMR has been incorporated into the discussions of international organizations, within the framework of the “One Health” concept (https://www.who.int/news-room/questions-and-answers/item/ one-health). The “One Health” approach defines the need to comprehensively consider human, animal and environmental health, in order to define strategies aimed at detecting, preventing, and mitigating threats to global health (United Nations Environment Programme, 2022) (Fig. 1). A recent article indicates that AMR is the main cause of human deaths in the planet. According to a study published in The Lancet covering data from 204 countries and territories, about 4
95 million people died from bacterial infections associated with AMR, and 1.27 million of deaths were caused directly from AMR in 2019. The main organisms responsible for 80% of these 1.27 million deaths belonged to six pathogens, including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa (Murray et al., 2022). This group of microorganisms has been named ESKAPE and the World Health Organization (WHO) has declared it as top priority for research in the context of AMR global crisis. The problem of AMR emergence correlates with the socio-economic profile of the different countries, beyond resolutions that international organizations recommend adopting. In a similar way to what happens with global climate change, the measures to be adopted require political decisions that imply allocating enough funds to face the problem and eventually modify production models of each country, as well as improve the systems of public health and urbanization. These measures should be aimed at developing surveillance protocols and improving drug production to assure the access to all who require these compounds, including second- and third-line antibiotics. Another area to analyze is livestock production managements that prevent the use of antibiotics to promote growth, as well as reducing overcrowding conditions for breeding, which makes it necessary to supply antimicrobials for prophylaxis. Finally, policies should also be promoted to ensure access to drinking water, sanitation systems and wastewater treatment that effectively reduce the load of pathogenic microorganisms and antibiotic resistance genes (ARGs) that can be discharged in the environment.

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Fig. 1 Emergence and dissemination of antimicrobial resistance genes. Sites of waste disposal appear as a hotspot for the emergence of new AMR bacteria.

It is important to highlight the contribution of the recent COVID-19 pandemic accelerating the spread of AMR. Several aspects could explain this issue. One of them is the notable increase in the use of hand sanitizers, disinfectants and environmental cleaners that introduce instability in the microbial genome through DNA damage. As a consequence, mutagenic mechanisms are induced in bacteria with the concomitant appearance of mutant clones, some of them eventually resistant to antibiotics (Lobie et al., 2021). Another relevant aspect is the high number of antibiotic treatments prescribed to patients with COVID-19. Specifically, review studies have shown that antibiotics were administered in nearly 70% of COVID-19 hospital admissions and between 80% and 100% of COVID-19 intensive care unit admissions. In this sense, Langford et al. (Langford et al., 2022) recently published a systematic review and meta-analysis of data from 148 studies and more than 360.000 patients where a low prevalence of bacterial co-infection and a moderate prevalence of secondary infection in hospitalized patients with COVID-19 were identified. Undoubtedly, the overuse and/or misuse of antibiotics to treat patients with COVID-19 worsened the emergence and dissemination of AMR in pathogenic bacteria. In addition, other aspects such as scientific efforts concentrated to fight against this viral infection and the alteration or even halt of preventive and control programs for public health priority diseases, also contributed to the already existing AMR global crisis (Lobie et al., 2021; Langford et al., 2022).

Mechanisms of antibiotic resistance, evolution and transmission Bacteria have evolved efficient mechanisms of resistance, including increased efflux, decreased uptake, enzymatic inactivation, target modification, biofilm formation, and even global modifications of metabolic network. These mechanisms exhibit a greater or lesser specificity for their target drug, involve one or more genes, and can be expressed simultaneously in the same microorganism (Larsson and Flach, 2022) (Table 1). As previously mentioned, there would be two major steps responsible for the “evolution of AMR”. The first involves point mutations in specific genes, with originally diverse functions, selected in bacteria exposed to strong selective pressures, such as those that can be experienced in human or animal body under treatment with antibiotics. These events would involve pathogenic or commensal bacteria, and always imply “permanent” changes of non-reparable consequences. A remarkable characteristic of the environment, such as soil and water, is the presence of a great diversity of genes with the natural potential to be modified by point mutations (Larsson and Flach, 2022). By exposing the host to a selective pressure imposed by the presence of an antibiotic, the mutations that confer some level of resistance to the bacteria could be selected. In addition, there are some studies that identified the ability of some bacteria to increase resistance against some antibiotics by successive passages in culture media without the antimicrobial compound. This evolution of resistance would be associated with the selection of phenotypes with better fitness for adaptation to the environment, which generate pleiotropic effects that eventually modify the

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Table 1

Summary of known antibiotic resistance mechanisms described, including genes and molecular processes involved.

Resistance Mechanism

Gene

Molecular process involved

Antibiotic class

References

Antibiotic modification

bla aph, ant, aac

Hydrolysis Phosphorylation, nucleotidyl transfer, acetylation Phosphorylation, methylation

Beta-lactams Aminoglycosides

Naas et al. (2017) Wachino et al. (2020)

Macrolides

Leclercq (2002); Schroeder and Stephens (2016) Markley and Wencewicz (2018)

mph, erm, mtg

Target site modification

Target site protection Antibiotic efflux

tet (X), tet (47–56) lnu fos cat rmt, arm mcr gyr, par cfr sul qnr tet (M,O,S,T,W) ABC-F (family)

Oxidation

Tetracyclines

Nucleotidyl transfer Glutathione transfer Acetylation Methylation Phosphoethanolamine transfer Type II topoisomerase Methylation Alternative DHPS Topoisomerase binding protein Ribosome binding protein Ribosome binding protein

mexAB-OprM emrE acr tet (A,B,C) mefA Msr (A,B) cmlA

Hydrophobe efflux Small Multidrug Efflux H+ antiporter H+ antiporter H+ antiporter Drug exporter 1 H+ antiporter

Lincosamides Fosfomycin Chloramphenicol Aminoglycosides Colistin Quinolones, Fluoroquinolones Phenicol, Lincosamide, Streptogramin Sulfonamides Quinolones, Fluoroquinolones Tetracyclines Macrolides, Lincosamides, Phenicols, Streptogramins Multi-resistance Macrolides, Sulfonamides, Tetracyclines Multi Resistance Tetracyclines Macrolides Macrolides Phenicol, Oxazolidinone

Leclercq (2002) Galindo-Méndez et al. (2022) Lin et al. (1996) Gutierrez et al. (2012) Liu et al. (2016) Correia et al. (2017) Long et al. (2006) Sköld (2000) Correia et al. (2017) Markley and Wencewicz (2018) Sharkey et al. (2016) Van Bambeke et al. (2000) Van Bambeke et al. (2000) Van Bambeke et al. (2000) Van Bambeke et al. (2000) Van Bambeke et al. (2000) Van Bambeke et al. (2000) Van Bambeke et al. (2000)

antibiotic resistance profile. There is also evidence that resistance mechanisms are easier to be selected when embedded in a low-diversity community (Klümper et al., 2019). Thus, the appearance and evolution of antibiotic resistance mechanisms may not necessarily be associated with the selective pressure exerted by the presence of that compound in the environment (Knöppel et al., 2017). In spite of that, it is generally accepted that the probability of these events occurring in the environment is relatively low, although it should not be clearly ruled out. The second mechanism requires that those genetic determinants, that potentially contribute to AMR, are associated with MGEs, allowing horizontal gene transfer (HGT) between bacteria, even phylogenetically distant ones. For this, AMR genes usually associate with insertion sequences, transposons, genomic islands (Baquero et al., 2021) or are captured by integrons as gene cassettes. Then, when these genetic platforms are incorporated into MGEs such as plasmids, especially conjugative or movilizable, the antibiotic resistance transfer could be possible. This process is favored in certain environments where there is an abundance of microorganisms containing MGEs, such as fecal bacteria. In case the mobile element with the resistance gene enters in a pathogenic bacteria capable of maintaining it in successive generations, the cycle would be complete, i.e., an AMR clone originates. It should be noted that a single MGE can contain multiple ARGs, therefore the presence of a selective pressure associated with a single gene located in this MGE would lead to the transfer of the remaining genes contained in the element. Thus, these genetic clusters would be able to disseminate ARGs not necessarily associated with a specific environmental condition. In addition, it is common to find the same type of ARGs associated to different MGEs, increasing their possibilities of transmission to different bacteria. Finally, the potential contribution of extracellular DNA in the transfer of antibiotic resistance, especially in some niches such as aquatic systems, should be also considered. This extracellular DNA could be incorporated into bacterial cells by natural transformation. Depending on the environment, the importance of this ARG dissemination mechanism should be taken into account when evaluating mitigation measures (Zarei-Baygi and Smith, 2021). The entry of the AMR pathogenic bacteria into the host, named as “transmission event,” can involve various environmental sources, such as humans, animals, water, soil, food, etc. (Larsson and Flach, 2022). These events are very common and relatively easy to control once the source of contamination is identified. In this sense, several protocols have been designed for risk assessment of bacteria expressing resistance genes.

Involvement of the environment in the appearance of “novel” ARGs in human and animal bacterial pathogens In hypothesis, environmental microbiomes could be involved in the emergence of “novel” resistance genes, in their subsequent association with mobilizable elements, and finally in their dissemination to potential human and animal bacterial pathogens by

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HGT. Recently, modeling studies based on bioinformatics data, analyzed the involvement of the environment in each of these processes. According to these reports, the environment would only play a minor role in the emergence of “novel ARGs,” which seem to be restricted to some niches. Initially, human and animal intestine was proposed as likely the environment for the appearance of ARGs in pathogens (Bengtsson-Palme et al., 2021; Berglund et al., 2023). More recently, new evidence is pointing to wastewaters as the sites of emergence and mobilization of known ARGs associated to MGEs (Berglund et al., 2023). Concerning the origin of ARGs, some authors postulate that this kind of genes have been in the environment for a long time and that the use of new antibiotics will inevitably confront this gene pool, leading eventually to its flow towards pathogenic microorganisms (D’Costa et al., 2011; Hu et al., 2017). In this sense, the fact that some ARGs present in pathogenic bacteria are orthologous to resistance and detoxification genes of original antibiotic-producing microorganisms, suggests that these ARGs could be incorporated from these antibiotic producers by HGT (Benveniste and Davies, 1973; Marshall et al., 1998). More recently, there is a growing understanding that many ARGs might have arisen in nature because they are involved in other as yet unknown functions unrelated to antibiotics. These functions could give some advantage to bacteria, thus contributing to the maintenance and dissemination of resistance genes (Aminov and Mackie, 2007; Bhullar et al., 2012). A possible example could be a carbapenem-hydrolyzing oxacillinase, OXA-54, encoded by a chromosomal gene of the aquatic bacteria, Shewanella oneidensis, which shares 92% identity with the plasmid-encoded OXA-48 from a clinical K. pneumoniae isolate (Poirel et al., 2004a, 2004b). By in silico analysis, presence of OXA-like encoding genes in the genome of the majority of Shewanella species was detected, most of them exhibiting high similarity to OXA-48 (Tacão et al., 2018). These results strongly suggest that these environmental bacteria, susceptible to carbapenems, may be the source of carbapenem-hydrolyzing oxacillinases. In relation to ARG dissemination, the three HGT mechanisms (transformation, transduction and conjugation) have been analyzed in different types of ecosystems, including soil, aquatic environments, and biofilms. These studies have been developed using molecular tools, including sequencing, PCR and some techniques that allow monitor the HGT events such as the use of gfp gene and cytometry (Li et al., 2018). In some cases, biotic and abiotic factors (e.g. stress, SOS response, the presence of sub-inhibitory concentrations of antibiotics, quorum sensing system) that affect the process of lateral gene transfer were identified (Aminov, 2011). Recently, a systematic review about acquisition, diversity and spread of AMR between humans, animals and the environment concludes that there is no substantial evidence-based knowledge on the routes and directions of AMR spread among these three compartments. The analysis, based on 89 publications that investigate isolates present simultaneously in the three compartments, revealed a very limited AMR spread between environments; i.e., only in contexts of very close contact such as people living in close proximity to animals or occupationally exposed people. Hence, metagenomics analyses reported low similarity between environmental and human or animal ARG clusters, indicating certain specificity of these genes to particular environments and thus uncommon horizontal transfer events of ARGs among different environments. Nonetheless, it is clear that natural environments may be reservoirs of precursor ARGs that can eventually emerge in human pathogens (Meier et al., 2022). In this case, broad host range plasmids play a crucial role in HGT events between phylogenetically distant bacteria. Therefore, it is essential to obtain quantitative data from different environments using a global approach, especially with an epidemiological design. This strategy will make it possible to monitor the emergence and possible spread of antibiotic resistance from and to the environment.

Strategies for the study of AMR in different scenarios Clinical microbiology has various applications, including diagnosis, identification of treatments to be carried out and monitoring of infectious outbreaks. Classically in this field, the study of AMR in pathogens that cause human and animal diseases involves the isolation of bacteria in different culture media. Analyses are mainly based on morphological characterization of clinical isolates and on the phenotypic study of additional characteristics, including the antibiotic susceptibility profile (Vargas et al., 2005). Molecular analysis including PCR of the AMR genes harbored by the isolates is also used. This approach, relatively standardized, has been complemented by sequencing the genomes of these organisms (World Health Organization (WHO), 2020). Through this molecular technique, it is possible to identify the location and geographic spread of AMR clones, compare the organization of resistance genes between different clones and even, in some cases, presume the historical routes of how these genetic determinants were acquired and arranged. In addition, genome information makes it possible to identify and monitor the emergence of an infectious outbreak, as well as to elucidate mechanisms and targets for the design of new drugs, vaccines, and detection tests (Rossen et al., 2018). This historical line of work has defined the profile of ARGs annotated and deposited in specific databases: CARD, https://card.mcmaster.ca/, NDARO, https://www.ncbi.nlm.nih.gov/pathogens/antimicrobial-resistance/, and ARDB, https://ardb. cbcb.umd.edu/. It should be noted that these databases mainly contain clinical ARGs, which consequently determines a bias when looking for resistance genes in the environment. More recently, metagenomic has been incorporated as a clinical tool (Chiu and Miller, 2019). Hence, its use made it possible to identify neuroleptospirosis in a patient and define the appropriate antibiotic treatment against the infection (Wilson et al., 2014). In other cases, for example, a directed procedure named as “capture probe enrichment” has been designed to characterize antimicrobial resistance gene variants, such as those involved in the resistance to ciprofloxacin (Stefan et al., 2016). Regarding the study of environmental bacteria that express ARGs, the approach poses new challenges. On one hand, the work focuses on monitoring the environment to estimate the presence and abundance of ARGs from anthropogenic sources (industrial installations, sewage water, aquaculture facilities, farms for intensive animal husbandry, etc.) and their potential health risk. In this

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case, several strategies could be used, including culture-dependent methods and molecular techniques involving the quantification of resistance genes by qPCR and high-throughput sequencing (Karkman et al., 2018). As a result, some genes have been proposed as indicators of anthropogenic contamination, such as taxonomic genes for specific groups of bacteria (Gammaproteobacteria, Firmicutes), some ARGs, and the intI1 gene encoding the integrase of the class 1 integron (Berendonk et al., 2015). It should be noted, however, that the environmental quantification by qPCR of some indicator genes can lead to problems associated with specificity. Precisely, some primer pairs used to detect indicator genes were designed considering available databases that, as previously mentioned, mainly contain sequences of clinical origin and a low representation of environmental ones. Thus, amplified fragments could comprise not only clinical but also environmental genes, generating an overestimation of the anthropogenic contamination (Antelo et al., 2015; Gillings et al., 2015; Stedtfeld et al., 2017). Lowering of costs of sequencing techniques has allowed the incorporation of metagenomic as a monitoring strategy (Fresia et al., 2019). Some projects have been developed on a global scale aimed at monitoring wastewater using these methods (Hendriksen et al., 2019; Karkman et al., 2020). One of the advantages of using metagenomics for pathogen surveillance is the accuracy of the data obtained. Genome-centric metagenomic analysis enables the identification of the pathogenic clones that circulate between different environments and even the detection of AMR genetic determinants spread into and from natural environments (Salazar et al., 2022). On the other hand, the search for “novel” antimicrobial resistance genes in the environment also involves a challenge, in this case associated with establishing search criteria. First of all, it should be considered that the largest proportion of bacteria in the environment cannot be cultivated in the laboratory. Therefore, the isolation of bacteria and the evaluation of their antibiotic resistance profile present great limitations. Metagenomics bypasses this requirement and allows the identification of genes that are not necessarily similar to those present in the clinic. However, the search for “novel” resistance genes in the environment using this approach requires the design of bioinformatic protocols followed by mandatory verification of the associated phenotype. Other strategy, named as functional metagenomics, incorporates the use of a bacterial host to clone fragments of total DNA from the environment in order to identify new potential resistance genes through the phenotype they confer (dos Santos et al., 2017). With this information, it has been possible to detect ARGs in total DNA (from eukaryotes, bacteria and even phages) purified from environmental samples (Hu et al., 2017; Brown-Jaque et al., 2018; Azziz et al., 2019).

Procedures for the analysis and management of antibiotic resistance in the environment Within the “One Health” strategy, the following steps have been proposed aimed at controlling the spread of AMR in the environment. In the first place, the sites of greatest exposure to the flow of antibiotic resistant bacteria (ARB), ARGs and even antibiotics, must be identified. Then, it is necessary to have adequate surveillance systems, followed by the design of procedures that allow estimating associated risks. Finally, the approach should be completed with a battery of technological solutions aimed at controlling AMR spread (Berendonk et al., 2015).

Identification of critical sites exposed to anthropogenic contamination Between critical sites, wastewater systems are one of the most important sources, which are usually associated with hospitals, cities, animal husbandry and industries of different kinds. Another relevant source of contamination, allowed in many countries, is the use of antibiotics as growth promoters or as preventives during intensive animal production characterized by extremely crowded conditions. In addition, the manure and slurry containing these drugs could be used as fertilizers. These compounds, usually with minimal modifications, could be discharged into the environment generating a selective pressure suitable for the appearance of antibiotic resistance. Particularly, wastewater treatment plants (WWTP) have been identified as hotspots for plasmid DNA transfer events. Quantification of transfer events have estimated between 3 and 50 conjugation events per 100,000 recipient cells (Li et al., 2018). These environments allow ecological connections between bacteria with different evolutionary backgrounds, including human pathogens, which evidence a great diversity of potential recipient bacteria for conjugative plasmids. Moreover, there is evidence that most of the plasmids in sewage samples are not described yet and could act as a bridge between clinical and environmental ARG pools, called resistomes (Kirstahler et al., 2021). Regarding the other relevant source of contamination, although not updated, a global description of the consumption of antibiotics, including data of their use in animal production, is available from the efforts of the One Health Trust (The Center for Disease Dynamics, Economics & Policy) (https://resistancemap.onehealthtrust.org/Animals.php). According to these data, the amount of antibiotics used in animal production is far higher than that consumed for human health (Polianciuc et al., 2020).

Epidemiological surveillance According to Diallo et al. resistance surveillance systems are defined as “a structured and systematic procedure to measure the prevalence or incidence of antibiotic resistance through continuous or periodical surveillance performed with a defined methodology and with specified indicators” (Diallo et al., 2020). The data obtained from these studies allow to visualize the AMR situation in a site and eventually to design treatment protocols. Classically, surveillance studies of antibiotic resistance in bacteria have focused especially on humans and to a lesser extent on animals and few on the environment. According to the information retrieved from publications in PubMed up to the year 2019, this recent study concluded the need to incorporate epidemiological surveillance

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of animals and environment, standardize protocols and combine molecular techniques together with culture-dependent strategies (Diallo et al., 2020). It also determines the need for this type of monitoring to be coordinated globally and to assure the availability of the information. The concentration of antibiotics should also be determined at some critical sites, such as industrial effluents in drug manufacturing plants, effluents from animal husbandry establishments, hospitals, etc. However, this does not seem so critical, judging from a study that showed that the increase in ARGs concentration in wastewater was correlated with the abundance of ARB contained in fecal material rather than with the presence of antibiotics released in the environment. Thus, in this report authors suggest to determine, at least in some sites, the abundance of the crAssphage virus as a marker of human fecal contamination (Karkman et al., 2019). During the last years, there have been global efforts on epidemiological surveillance. The most comprehensive one is GLASS, which was launched in 2015 and is coordinated by WHO. This effort looks for the standardization of surveillance systems at a global scale, along with capacity building and data sharing (https://www.who.int/initiatives/glass). In this sense, it is important to recognize that “One Health” issues should be approached at a global scale. Countries or areas with the worst sanitary conditions and antibiotic overuse are hotspots for emergence of newly resistance clones or genes that could have an impact at a global scale.

Risk assessments The “Integrated Risk Assessments” defined by the European Medicines Agency (https://www.emea.europa.eu), related with the evolution and emergence of antibiotic resistance in the environment, consider two main elements: the development in the environment of ARB in the presence of sub-inhibitory concentrations of antibiotics in microbial communities, and the transfer of ARGs from anthropogenic sources (such as treated wastewater, manure or others) to commensal or pathogenic bacteria that cause human or animal infections (Berendonk et al., 2015). The threat/concern of environmental contamination by antibiotics should not be considered linearly associated with their concentration. On the contrary, low concentration of an antibiotic, even combination of some of them, could promote the selection of resistant bacteria. Moreover, the presence of other abiotic factors, such as heavy metals, could also affect the selection process. Therefore, based on these considerations, validated models to quantitatively estimate the probability of ARGs dispersion from anthropogenic sources have been postulated.

Management of contaminated environments The removal of antibiotics, ARB and ARGs from the environment has been analyzed in several studies. Most of these articles focus on wastewater treatment through different strategies. The constructed wetlands (CWs) have been evaluated using different loading speeds and substrates. In some cases, positive results were obtained when analyzing biological and chemical parameters (Chen et al., 2016). One of the major disadvantages of CWs and associated algae treatment systems are related to the limited removal capacity, considering the high contamination levels in some sites. As a chemical strategy, the use of nano-materials as catalysts for redox reactions (advanced oxidation process, AOP) has been proposed, which are mainly aimed at the degradation of antibiotics. Other chemical and physical treatments include the single or combined use of chlorination techniques, application of UV light, ozonation, biochar adsorption techniques, photocatalysis and Fenton reactions, that imply the exposure of wastewater to microwaves and alkaline media. The efficiency of these systems has been assessed in different contexts. In any case, in addition to the efficiency in real systems, biosafety of the eventual sub-products generated during the treatment should be investigated. In this sense, it should be taken into account that this type of management must be designed considering two main issues: the costs of installation and monitoring of the systems, and the possibility of a global application, including low- and middle-income countries. Regarding manure management, which is usually used as soil fertilizer, aerobic and anaerobic digestion have been proposed as a strategy to reduce the load of ARGs and antibiotics in them. Silage, lime addition, composting, have also been tested. Anyway, studies on different types of manure, management and effects on the soil should be developed (Tyrrell et al., 2019).

Future perspectives In summary, antibiotic resistance in the environment has become an extensive area of basic and applied research. The challenge covers different aspects of the problem, including methodological strategies, identification of parameters to be monitored to detect HGT, and the emergence of “novel” ARGs from the environmental gene pool. At the same time, the risks associated with antibiotic resistance at a global level require a rapid transmission of clear knowledge to societies. They would somehow be in charge of demanding decision-making from their respective governments and public health institutions.

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Antimony Shayne C Gad, Gad Consulting Services, Raleigh, NC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.C. Gad, Antimony, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 274–276, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00815-0.

Chemical profile Background Uses/occurrence Exposure Toxicokinetics (ADME) Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines PubChem URL Reference Further reading

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Abstract Antimony (Sb; CASRN 7440-36-0) is a silvery semimetal element with chemical properties similar to lead, arsenic, and bismuth. In nature, it is found associated with sulfur as stibnite. Antimony is used in white metal, a group of alloys having relatively low melting points. White metal usually contains tin, lead, or antimony as the chief component. The introduction of antimony into the human environment is overwhelmingly the result of human activity; approximately half of the antimony used in the United States is recovered from lead-based battery scrap. Most information regarding antimony toxicity has been obtained from industrial exposures. Occupational exposures usually occur through inhalation of dusts containing antimony compounds. Antimony is absorbed slowly through the oral route, and many antimony compounds are gastrointestinal irritants. The toxicity of Sb is a function of the water solubility and the oxidation state (+3 or −3) of the Sb species in question. The American Conference of Governmental Industrial Hygienists classifies antimony as a suspected human carcinogen.

Keywords Antiparasitic; Batteries; Emetic; Flameproofing; Stibnite; White metal

Chemical profile

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Valence States: 0, −3, +3, +5. Chemical/Pharmaceutical/Other Class: Metals. Name: Antimony Synonyms: Stibium.

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Antimony

CAS Number: 7440-36-0. Molecular Formula: Sb Chemical Structure:

Background Antimony (Sb) has been known since antiquity, and its early use as a cosmetic continues today; often mixed with lead or other heavy metals as an eye-liner-type cosmetic known as kohl. Believed to possess powers to shield the eye from the sun and disease, it served purposes in both cosmetics and mysticism. Antimony has been found in many artifacts in the Middle East and seems to have been used in the creation of small personal ornamentation or vessels.

Uses/occurrence Antimony is used in white metals, which are a group of alloys having relatively low melting points. White metal usually contains tin, lead, or antimony as the chief component (e.g., the alloys Britannia and Babbitt). Antimony is used as a hardening alloy for lead, especially in storage batteries and cables, bearing metal, type metal, solder, collapsible tubes and foil, sheet and pipe, semiconductor technology, and pyrotechnics. It is also used in thermoelectric piles and for blackening iron or coatings. Antimony-containing compounds are used in materials for refrigerators, air conditioners, aerosol sprays, paints, and flameproofing agents. Approximately half of the antimony used in the United States is recovered from lead-based battery scrap. Antimony is also used medicinally, e.g., antimony potassium tartrate (APT) as an emetic and the more soluble pentavalent antimony compounds such as sodium stibogluconate and stibosamine as antiparasitic agents to different leishmaniasis vectors. The pentavalent forms are more physiologically tolerated than the trivalent forms.

Exposure The emission of antimony into the human environment is overwhelmingly the result of human activity, with the emission of antimony trioxide being the most significant source. Antimony trioxide is emitted as a result of coal burning, or with fly ash when antimony-containing ores are smelted. In addition, medicines containing antimony are administered orally. Antimony is present in food and drinking water, mostly in the low mg kg−1 wet weight range or less, including vegetables grown on Sb-contaminated soils. Daily oral uptake of Sb ranges from 10 to 70 mg day−1 and appears to be significantly higher than exposure by inhalation. Absorption is poor via the gastrointestinal (GI) route, though onset of symptoms is rapid if sufficient exposure is reached and can result in death. Antimony poisoning symptoms are quite similar to those of arsenic, including vomiting, diarrhea, colic, and metallic taste, but it is far less toxic. Due to the poor absorption of antimony by the GI tract, exposure to antimony is primarily of concern via the inhalation route, particularly in workplaces in which antimony is used. Air concentrations of up to 10 mg m−3 have been recorded in workplaces where antimony is used, particularly smelting works and abrasives production. Worldwide, airborne antimony concentrations are approximately 0.001 mg/m−3.

Toxicokinetics (ADME) Normally, antimony is absorbed slowly when ingested or administered orally. Absorption of all valent states of antimony in the GI tract is low with 5–20% absorption seen in animal studies. Four people with involuntary intoxication of APT showed an absorption rate of 5%. Antimony is more readily absorbed through the respiratory tract. Antimony can concentrate in lung tissue, the thyroid gland, the adrenal glands, the kidneys, and the liver. The trivalent compounds of antimony concentrate in the red blood cells and liver, and the pentavalent compounds concentrate in the blood plasma. Both forms are excreted in feces and urine, but generally, more trivalent compounds are excreted in urine and more pentavalent compounds in feces. Presumably by reacting with the sulfhydryl groups, antimony can inhibit oxidative and phosphorylating enzymes like monoamine oxidase, succinoxidase, pyruvate brain oxidase, and phosphofructokinase. Inhibition of these enzymes can alter activities such as glucose metabolism and nerve transmission. Ten percent of the trivalent form is excreted by the kidney in 24 h; 50–60% of the pentavalent form is found in the urine within 24 h.

Mechanism of toxicity The toxicity of Sb is a function of the water solubility and the oxidation state of the Sb species under consideration. Antimony(III) is generally more toxic than antimony(V) and inorganic forms are thought to be more toxic than organic forms. Stibane gas (SbH3)

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when inhaled is the most toxic antimony compound. Antimony toxicity often parallels that of arsenic, although antimony salts are less readily absorbed than arsenic. It is presumed that antimony, like arsenic, complexes with sulfhydryl groups of essential enzymes and other proteins. By analogy, antimony can uncouple oxidative phosphorylation, which would inhibit the production of energy necessary for cellular functions. Antimony’s trivalent compounds are more toxic than its pentavalent compounds.

In vitro toxicity data Has anti-trypanosomal activity and is cytotoxic against human leukemia cell lines.

Acute and short-term toxicity Animal Minimal intraperitoneal lethal dose for rats injected with antimony metal was shown to be 10 mg 100 g−1 weight. Animals showed dyspnea, weight loss, hair loss, and myocardial insufficiency. Post mortems showed cardiac lesions with liver and spleen damage. Death was attributed to myocardial edema. The LD50 for APT in rabbit and rats is about 115 mg kg−1 and about 600 mg kg−1 in mice. A study where 400 mg kg−1 APT was given to mice and rats for 14 days showed stomach and liver lesions (Tylenda et al., 2015).

Human Accidental poisonings can result in acute toxicity, which produces vomiting and diarrhea similar to arsenic poisoning. Most information regarding antimony toxicity has been obtained from industrial exposures. Occupational exposures usually occur through inhalation of dusts containing antimony compounds. Six workers exposed to antimony trisulfide (used as a pigment and in match production) at concentrations greater than 3.0 mg m−3 in a factory that produces grinding wheels experienced heart complications and died, and the rest of the population working in the environment showed limited cardiovascular changes. Inhalation of antimony hydride (stibine gas) can lead to hemolytic anemia, renal failure, and hematuria. Stibine gas is produced when antimony alloys are treated with acids. Minimal oral lethal doses of APT in children are thought to be 300 mg and adults 1200 mg.

Chronic toxicity Animal Rats exposed to a dose level of 4.2 mg m−3 airborne antimony trioxide dust for 1 year were reported to develop lung tumors; at a dose level of 1.6 mg m−3, lung tumors were not found. Guinea pigs exposed to airborne antimony trioxide developed interstitial pneumonia. Oral feeding of antimony to rats does not induce an excess of tumors or teratogenesis (Tylenda et al., 2015).

Human Inhalation of antimony compounds produces different effects at different concentrations. Chronic inhalation of low concentrations causes rhinitis and irritation of the trachea. At high concentrations, acute pulmonary edema occurs, and bronchitis may occur (the bronchitis may lead to emphysema). Inhaled antimony concentrates in lung tissue; as a result, pneumoconiosis with obstructive lung disease has been recorded. The available data on antimony as a human carcinogen are inconclusive. In addition, a temporary skin rash, called ‘antimony spots,’ can occur in persons chronically exposed to antimony in the workplace. Reoccurring oral exposure to therapeutic doses of antimony(III) was associated with optic nerve destruction, uveitis, and retinal bleeding. The symptoms of exposure included headache, coughing, anorexia, troubled sleep, and vertigo.

Immunotoxicity Transient immunologic reactions, including tachyphylaxis and anaphylactoid reactions, may occur. Increased polymorphonuclear neutrophils and cytokines have been measured in persons exposed to zinc oxide fumes. Very little information is available on the immunotoxicity of antimony.

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Reproductive and developmental toxicity A few studies suggest that antimony can cause reproductive toxicity. A study in rats given antimony trioxide at 209 mg m−3 for 63 days inhibited the ability of two-thirds of the rats to conceive. Rabbits given metallic antimony of 5–55 mg every other day for 30, 60, or 90 days showed an increase in abortions. Women working at an antimony metallurgic plant where they were exposed to antimony trioxide, antimony pentasulfide, and metallic antimony showed higher rates of spontaneous abortions and menstruation difficulties.

Genotoxicity The compounds SbCl3 and SbCl5 were reported to be genotoxic in the rec-assay with Bacillus subtilis. Sb(III)acetate enhanced the simian-adenovirus-7-mediated transformation of SHE-cells, and enhanced rates of chromosomal breaks in human leukocytes after treatment with APT. However, another study showed that SbCl3 did not induce DNA/protein-crosslinks in V79-cells and peripheral human lymphocytes. In a study done on workers exposed to antimony trioxide, increased oxidative damage to DNA was seen in the workers compared to controls, though there was not an increase in sister chromatid exchange or micronuclei. Overall, due to the lack of human studies, antimony genotoxicity in humans is inconclusive.

Carcinogenicity The International Agency for Research on Cancer categorizes antimony as a possible carcinogen (group 2B). While current data on carcinogenicity of antimony in humans have been inconclusive, rat studies done with antimony trioxide and antimony trisulfide have shown lung tumor formation. An increase in the incidence of lung cancer has been reported in workers exposed to APT dust, but not other cancers.

Organ toxicity Thyroid, liver, heart, lung, spleen, stomach.

Interactions Forms a complex with hydroquinone resulting in increased in vitro cytotoxicity.

Clinical management The oil-soluble BAL (British anti-Lewisite; 2,3-dimercaptopropanol) administered intramuscularly appears to be the antidote of choice for antimony poisoning. The antidotal action of BAL depends on its ability to prevent or break the union between antimony and vital enzymes.

Environmental fate and behavior Antimony is found naturally in the Earth’s crust and can be released into the environment as windblown dust or sea spray or from volcanic eruptions or forest fires. However, the emission of antimony into the environment is overwhelmingly the result of human activity, with the emission of antimony trioxide, tetroxide, and pentoxide forms being the most significant. Antimony trioxide is emitted as a result of coal burning, or with fly ash when antimony-containing ores are smelted. Humans are exposed to low amounts of antimony from the air, drinking water, and food contaminated with soil. Antimony concentration in the atmosphere is thought to be 1.4–55 ng m−3. The more water soluble forms of antimony are very mobile in aqueous media while the less soluble forms of antimony are found attached to particles of soil, clay, and sediment in rivers and lakes. The concentration of antimony in the Pacific Ocean was found to be 0.2 mg L−1 and in the Rhine river at 0.1 mg L−1. The trivalent state of antimony is the form most often released by anthropogenic activities. In terms of soil concentrations, it was reported by a US Geological Survey to be less than 1–8 ppm in soil, with an average of 0.48 ppm. Studies have estimated an exposure of less than 5 mg day−1 on average from food and water and appears to be significantly higher than exposure by inhalation. Antimony does not bioaccumulate in food sources. The food contamination from soil is thought to be in the low mg kg−1 wet weight range. Drinking water contamination of antimony is thought to be from the metal plumbing and fittings, in which case it is thought to be the less toxic antimony(V) form. The US

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Environmental Protection Agency standard for antimony in the drinking water is 6 mg L−1. Occupational exposures to antimony as well as therapeutic administration are more likely to be higher and thus may produce toxic effects.

Ecotoxicology Soil toxicity studies show EC20 values for the following invertebrates: Enchytraeus crypticus (enchytraeid), 194 mg kg−1 dry weight (dw); Folsomia candida (springtail), 81 mg kg−1 dw; and Eisenia fetida (earthworm), 30 mg kg−1 dw. These values are well above the background concentrations for antimony in US soils.

Exposure standards and guidelines The American Conference of Governmental Industrial Hygienists (ACGIH) and the Occupational Safety and Health Administration (OSHA) in the United States have the following airborne exposure limits: OSHA Standard: Permissible exposure limit—8 h time-weighted average (TWA) ¼ 0.5 mg m−3. ACGIH threshold limit value: 8 h TWA ¼ 0.5 mg m−3 (antimony and compounds, as Sb). ACGIH classifies antimony as a suspected human carcinogen. In the United States, antimony is listed as a Clean Air Act hazardous air pollutant generally known or suspected to cause serious health problems. Antimony and its compounds are listed as Clean Water Act toxic pollutants, subject to effluent limitations. The Federal Drinking Water Standards is 6 mg L−1.

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/5354495

See also: Antimony trioxide; Metals

Reference Tylenda CA, Sullivan DW Jr., and Fowler BA (2015) Antimony. In: Nordberg G, Fowler B, and Nordberg M (eds.) Handbook on the Toxicology of Metals. 4th edn, vol. II, pp. 565–579. Waltham, MA: Elsevier, Inc.

Further reading Fowler BA, et al. (2013) Antimony, 6th edn Patty’s Toxicology, 6th edn, vol. 1, 491–497. Tylenda CA and Fowler BA (2007) Antimony. In: Nordberg GF, Fowler BA, Nordberg M, and Friberg LT (eds.) Handbook on the Toxicology of Metals, 3rd edn, pp. 353–366. San Diego: Academic Press. Sundar S and Chakravarty J (2010) Antimony toxicity. International Journal of Environmental Research and Public Health 7(12): 4267–4277. Winship KA (1987) Toxicity of antimony and its compounds. Adverse Drug Reactions and Acute Poisoning Reviews 2: 67–90.

Relevant websites https://wwwn.cdc.gov/TSP/ToxProfiles/ToxProfiles.aspx?id¼332&tid¼58 :Agency for Toxic Substances and Disease Registry. Toxicological Profile for Antimony. https://www.echemportal.org/echemportal/substance-search :eChem Portal Homepage, Search for: Antimony. https://echa.europa.eu/registration-dossier/-/registered-dossier/16124 :Registration Dossier-ECHA (europa.eu).

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Antimony trioxide Shayne C Gad, Gad Consulting Services, Raleigh, NC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.C. Gad, Antimony Trioxide, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 277–279, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00816-2.

Chemical profile Background Uses/occurrence Exposure Toxicokinetics (ADME) Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines PubChem URL References

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Abstract Antimony trioxide (ATO) contains the semimetallic element antimony and is the predominant form of antimony released into the environment from human activities. Primarily, it is emitted as a result of coal burning or with fly ash when antimony-containing ores are smelted. Commercially, antimony trioxide is used as a flame retardant, in pigments, and as a catalyst in plastic production. Human exposure can occur from inhalation, dermal, and oral routes, with occupational exposure the most likely route. Though poorly absorbed after oral exposure, eye and respiratory irritation occur from dust and air. The International Agency for Research on Cancer classified antimony as a group 2B carcinogen meaning it is potentially carcinogenic to humans. Carcinogenicity data for ATO in humans at this point is inconclusive, while some animal studies did show carcinogenic potential.

Keywords Antox; BAL (British anti-Lewisite); Flame retardant; Fly ash; Pigment; Thermogrand B

Chemical profile

• • • • • •

Chemical/Pharmaceutical/Other Class: Metal oxide Name: Antimony trioxide Synonyms: Antimony white, Antimony oxide, Antox, Thermogrand B, ATO, Diantimony trioxide, Antimony (3+) oxide CAS Numbers: 1327-33-9, 1309-64-4 Molecular Formula: Sb2O3 Chemical Structure: varies

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Background Antimony trioxide (ATO) is a white, odorless, crystalline powder. This amphoteric oxide is not readily soluble in water, but will dissolve in sodium hydroxide solution or in mineral acids. In nature, it is found as the orthorhombic mineral valentinite and the octahedral mineral senarmontite. ATO is an industrial chemical produced worldwide with a 2005 production estimate of 120,000 tons per year. Several processes are used to produce ATO, with the most common being the smelting of stibnite ore at very high temperatures to produce a crude antimony trioxide, which is then further purified by sublimation to separate out other components like arsenic trioxide, a common contaminant in antimony ores (ATSDR, 2019; Fowler et al., 2012).

Uses/occurrence Commercially, the most common use for antimony trioxide is to provide flame-retardant properties to textiles, paper, rubber, adhesives, and plastics. Added to ceramics and glass, ATO adds opacity, hardness, and resistance to acids. It is also used as a turbidifier in white enamel, pigments, and munitions and as a catalyst in the production of polyethylene terephthalate (PET plastic) (Fowler et al., 2012).

Exposure The most significant source of human exposure occurs occupationally to workers exposed to dust and fumes such as in ATO smelting plants, or in plants making or using antimony trioxide in other ways. Occupational exposure to ATO occurs mainly through inhalation, though mucociliary action may cause gastrointestinal effects. Lung, eye, and dermal irritations have been reported. Occupational exposure can be monitored by urine testing.

Toxicokinetics (ADME) Animals fed ATO showed antimony in the thyroid, lungs, adrenal glands, blood, heart, hair, spleen, kidneys, and liver. Clearance from these tissues over time showed tissue-specific differences, with slower clearance from blood, lungs, liver, and spleen. In another study, ATO persisted the longest in the thyroid. The low solubility of antimony trioxide causes antimony trioxide particles to persist longer in lung tissue when inhaled than other soluble forms of antimony. That ATO can be absorbed through the lungs into the body is based on evidence of finding elevated antimony concentrations in the urine and blood of antimony smelting workers. Women working in antimony smelting plants have shown elevated antimony in placenta, cord blood, urine, and breast milk. Evidence also shows a relatively long half-life of antimony trioxide in the lungs of exposed workers. ATO has been detected in the lungs of these workers long after exposure has been terminated. Animal studies have shown that antimony retention in the lungs depends on the size of the particle (with larger particles being cleared more rapidly) as well as the solubility of the antimony compound. While persistence of antimony in lung tissue has been demonstrated in humans, antimony trioxide is thought to be cleared from other affected organs such as liver and kidney more rapidly. Trivalent antimony readily leaves the plasma but remains in the circulation bound to erythrocytes and is excreted in the bile after conjugation with glutathione (Lylenda et al., 2015).

Mechanism of toxicity Antimony (Sb) toxicity often parallels that of arsenic, although antimony salts are less readily absorbed than arsenic. It is presumed that antimony, like arsenic, complexes with sulfhydryl groups of essential enzymes and other proteins. By analogy, antimony can uncouple oxidative phosphorylation, which would inhibit the production of energy necessary for cellular functions. Mechanisms of toxicity of the ATO form of antimony are not as readily apparent (Lylenda and Fowler, 2007).

In vitro toxicity data Clastogenic and cytogenic.

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Acute and short-term toxicity Animal Antimony trioxide has an oral LD50 in rats of >20,000 mg kg−1. The relatively low toxicity of this compound is due to its extremely low solubility in water. Mouse intraperitoneal LD50 ¼ 172 mg kg−1. Studies where cats and dogs were exposed orally to ATO at 100 mg kg−1 showed no toxic effects; however, animal studies done on acute exposure to ATO by inhalation did show toxicity. For instance, studies on guinea pigs exposed to 45 mg kg−1 ATO by inhalation for 33–106 h exhibited interstitial pneumonitis and fatty degeneration of the liver. Inconsistent hematological effects of ATO inhalation on animals were also reported. Antimony trioxide is a mild eye irritant in rabbits.

Human In humans, the primary sites affected by antimony trioxide acute toxicity are pulmonary and gastrointestinal. Other sites affected are heart, liver, kidney, and mucous membrane. Antimony spots have been reported, which is a rash consisting of pustules and papules near sweat and sebaceous glands on skin. Symptoms related to human exposure to antimony trioxide in the workplace are mainly irritation of mucous membranes in eyes, mouth, nose, throat, and upper respiratory tract. It has been reported that smelter workers exposed to antimony trioxide frequently experienced symptoms of rhinitis, pharyngitis, laryngitis, gastroenteritis, pneumonitis, and bronchitis. Other common symptoms listed were weight loss, nausea, diarrhea, vomiting, and abdominal cramps. A particular incident was reported where numerous people became ill after drinking lemonade that had been left in a white enamel tub overnight. The illness was thought to have been caused by the antimony trioxide that had leached into the acidic lemonade from the tub. The tub contained 2.88% of antimony trioxide while the resulting lemonade had 0.013% antimony. Acute symptoms included nausea, vomiting, burning stomach pain, and colic. Most recovered within 3 h.

Chronic toxicity Animal Given the prevalence of human occupational exposure to ATO, numerous animal studies have been done to assess chronic ATO inhalation exposure. Animals chronically exposed to antimony trioxide showed myocardial damage and fatty degeneration of the liver as well as incidences of anemia, decreased white cell count, and polymorphonuclear leukocyte reduction. Chronic exposure leads to reproductive and developmental effects.

Human Antimony pneumoconiosis (or antimoniosis) is a frequent outcome in workers chronically exposed to antimony trioxide through inhalation. This often symptomless condition is characterized by diffuse punctate spots of less than 1 mm seen on lung X-rays. A study done on workers in antimony smelting plants who were exposed to ATO dust at antimony concentrations of 0.08–138 mg m−3 for 1–15 years showed no significant problems with pulmonary function. Pneumoconiosis was present in 3/13 of the workers, with five additional workers with suspected pneumoconiosis. Other studies done on people with chronic occupational exposure to ATO showed that the number of spots shown on X-rays correlated with the amount of time the person was exposed and the concentration of ATO retained in the lungs. They found these lung changes could occur only after a few years of exposure to ATO. In another study, 51 workers who worked at a smelting plant for 9–31 years and were exposed to dust particles containing up to 88% antimony trioxide and 8% antimony pentoxide showed pneumoconiotic changes after 10 years of employment there. The most common symptom among workers was chronic coughing and frequent symptoms included conjunctivitis, upper airway inflammation, chronic bronchitis, chronic emphysema, and pleural adhesions. No malignant lesions were found in this study.

Immunotoxicity No human data publicly available, studies have been performed.

Reproductive and developmental toxicity It was reported that women working in an antimony smelting plant had higher incidences of spontaneous abortions, higher rates of late stage abortions, higher rates of premature births, and more gynecologic problems than a control group of women. Fumes that the women were exposed to were thought to contain mostly antimony trioxide with antimony pentasulfide and metallic dust. Other

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reproductive effects included lower birth weights of the offspring and at 3 months and 1 year. Increases in antimony content of blood, urine, breast milk, and placenta relative to control, unexposed group were noted.

Genotoxicity The in vivo genotoxicity of antimony trioxide was studied using single- and repeat-dose mouse bone marrow micronucleus tests, and the rat liver unscheduled DNA synthesis assay. All three studies were negative. In contrast, chromosomal damage by antimony trioxide was reported in mouse bone marrow cells after repeat dosing but not after single dosing. Positive results were observed with antimony trioxide in the in vitro cytogenetic assay with human lymphocytes and the sister chromatid exchange assay with V79-cells, but not in the L5178Y mutation assay. Thus ATO may be considered clastogenic with in vitro studies (Leonard and Gerber, 1996; Pohanish, 2002).

Carcinogenicity Antimony trioxide has been classified by the American Conference of Governmental Industrial Hygienists (ACGIH) as group A2, a suspected human carcinogen and by the International Agency for Research on Cancer into group B2 (potential carcinogen). In a rat study, doses of antimony trioxide as high as 4.5 mg m−3 delivered by inhalation for 12 months did not produce cancer. However, other studies did show increased lung tumors in rats exposed to 4.2 mg m−3 antimony trioxide 6 h day−1, 5 days week−1 for 1 year. The inconsistencies in the animal studies as well as a lack strong evidence in humans are the reasons behind these classifications. Studies done by Health Canada suggest that ATO could be a ‘threshold carcinogen.’ It may not cause lung tumors due to DNA effects, but due to impaired lung clearance, particle overload, and subsequent inflammatory response leading to fibrosis and tumor formation (Leonard and Gerber, 1996; Lylenda and Fowler, 2007).

Organ toxicity Pulmonary and cardiovascular.

Clinical management The oil-soluble BAL (British anti-Lewisite; 2,3-dimercaptopropanol) administered intramuscularly appears to be the antidote of choice for antimony poisoning. The antidotal action of BAL depends on its ability to prevent or break the union between antimony and vital enzymes (Dart, 2004).

Environmental fate and behavior Antimony trioxide is a form of antimony that is released into the atmosphere primarily from human activities in high-temperature industrial processes such as coal burning or with fly ash when antimony-containing ores are smelted. Antimony trioxide is the most significant form of antimony present in the atmosphere. The combustion of products coated with ATO for its flame-retardant properties and fossil fuel combustion will also release ATO into the atmosphere. Antimony compounds suspend to air particles and, with a half-life of 30–40 days in the air, are capable of traveling long distances through the atmosphere. Current estimates of atmospheric antimony are around 0.001 mg m–3. ATO is a form of antimony that is relatively insoluble in water. The presence of ATO in bodies of water is due to its absorption onto clays, dirt, and other sediment, especially in areas where sediments accumulate, such where a contaminated river flows into a reservoir, lake, or river bend. Soil persistence of ATO is expected to be higher near production facilities or waste disposal sites (Lylenda and Fowler, 2007).

Ecotoxicology ATO does exhibit some potential to persist in the environment, especially in soils and aquatic sediments. The levels of ATO are not thought to be high enough to be considered hazardous to aquatic or terrestrial organisms. The potential for bioaccumulation is low. LD50 for Lepomis macrochirus (bluegill sunfish) is >530 mg l−1 96 h–1 and the LD50 for Pimephales promelas (fathead minnow) is >833 mg l−1 96 h−1 (ATSDR, 2019).

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Exposure standards and guidelines US Occupational Safety and Health Administration standard for antimony: 0.5 mg m3 8h/day exposure limit for 40 h/workweek (Pohanish, 2002). ACGIH threshold limit value: 8 h time-weighted average: 0.5 mg m3 (antimony and compounds, as Sb). The NIOSH recommended REL TWA is set at 0.5 mg m3. In the United States, antimony is listed as a Clean Air Act hazardous air pollutant generally known or suspected to cause serious health problems. Antimony and its compounds are listed as Clean Water Act toxic pollutants, subject to effluent limitations. The Federal Drinking Water Standard is 6 mg l−1.

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/14794#section¼Toxicity.

See also: Antimony; Metals

References ATSDR (2019) Toxicity profile for antimony and related compounds. Dart RC (2004) Medical Toxicology, 3rd edn Philadelphia: Lippincott, Williams, & Wilkins. Fowler BA, Madden EF, and Chou S (2012) Arsenic, antimony, and bismuth. In: Patty’s Toxicol, 6th edn, pp. 471–510. New York: Wiley. Leonard A and Gerber GB (1996) Mutagenicity, carcinogenicity and teratogenicity of antimony compounds. Mutation Research 366(1): 1–8. Lylenda CA and Fowler BA (2007) Antimony. In: Nordberg GF, Fowler BA, Nordberg M, and Friberg LT (eds.) Handbook on The Toxicology of Metals, 3rd edn, pp. 353–366. San Diego: Academic Press. Lylenda CA, Sullivan DW, and Fowler BA (2015) Antimony. In: Fowler GF, Fowler BA, and Nordberg M (eds.) Handbook on the Toxicology of Metals, 4th edn, pp. 565–579. San Diego: Academic Press. Pohanish RP (2002) Sittig’s Handbook of Toxic and Hazardous Chemicals and Carcinogens. Norwich, NY: Noyes Publishing, imprint of William Andrew Publishing.

Relevant websites https://www.echemportal.org/echemportal/substance-search :eChem Portal Substance Search page, search for ‘antimony trioxide’ or CASRN. www.EPA.gov :Environmental Protection Agency homepage, search for: ‘antimony trioxide.’

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Antiprotozoal medicines Preeti Patela, Amritaparna Senguptab, Ashish Patelc, and Sidhartha D Rayd, aStatPearls, Monroe, NJ, United States; bXcovery Holding Inc, Edison, NJ, United States; cB.J.Medical College, Gandhinagar, Gujarat, India; dDepartment of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Published by Elsevier Inc.

Introduction Antimalarial drugs Aminoquinolines Amodiaquine Chloroquine Hydroxychloroquine (HCLQ) 8-Aminoquinolines Primaquine Tafenoquine Amino alcohols Halofantrine Lumefantrine Biguanides Proguanil Cinchona alkaloid Quinine Diaminopyrimidines Pyrimethamine Mannich base Pyronaridine Naphthoquinone Atovaquone Quinoline-methanol Mefloquine Sesquiterpenes Artesunate Artemether Sulfonamides Tetracyclines Miscellaneous other antiprotozoal drugs Benzimidazole derivatives Albendazole Mebendazole Nitroimidazole derivatives Benznidazole Metronidazole Tinidazole Nitrofuran derivatives Nifurtimox Aminoglycoside antibiotics Paromomycin Quinoline derivatives Praziquantel Tetrahydropyrimidine derivatives Pyrantel Miscellaneous agents Nitazoxanide Background Toxicokinetics Adverse drug reactions (ADRs) Overdose and management

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Eflornithine Background Toxicokinetics Adverse drug reactions Overdose and management Pentamidine Background Toxicokinetics Adverse drug reactions (ADRs) Overdose and management Triclabendazole Background Toxicokinetics Adverse drug reactions Overdose and management Conclusion References

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Abstract Parasitic infections affect more than 2 billion people globally each year. These infections are common in underdeveloped, tropical and subtropical countries and cause substantial morbidity and mortality. Single-celled organisms called protozoa are primarily responsible for parasitic infections, which can live and multiply in the host body. These infections routinely cause significant morbidity and mortality if left untreated, especially in developing countries. Since 1900, many medicines have been developed to treat and prevent these infections worldwide. In order to combat these infections, efforts are in place by health organizations, such as, WHO and CDC, and significant resources are allocated for antiparasitic medication discovery. Healthcare professionals must be aware of antiprotozoal drug’s potential toxic effects prior to treating these debilitating infections. A risk-benefit analysis must be considered prior to mass exposure. This chapter focuses on toxicokinetic, adverse effects, organ toxicology, and overdose of a host of antiprotozoal medications listed below: 4-Aminoquinolines (amodiaquine, Chloroquine, Hydroxychloroquine), 8-Aminoquinolins (primaquine, tafenoquine), Amino alcohols (halofantrine, lumefantrine), biguanides (proguanil), Cinchona alkaloid (quinine), Diaminopyrimidines (pyrimethamine), mannich base (pyronaridine), Naphthoquinone (atovaquone), Quinoline-methanol (mefloquine), sesquiterpenes (artesunate, artemether), Sulfonamides, tetracyclines, Miscellaneous other anti-protozoal drugs: benzimidazole derivatives (albendazole, mebendazole), Nitroimidazole derivatives (benznidazole, metronidazole, tinidazole), Nitrofuran derivatives (nifurtimox), Aminoglycoside antibiotics (paromomycin), Misc quinoline derivatives (praziquantel), Tetrahydropyrimidine derivatives (pyrantel), Miscellaneous others (nitazoxanide, eflornithine, pentamidine, triclabendazole).

Keywords 4-Aminoquinolines; 8-Aminoquinolins; Amino alcohols; Aminoglycoside antibiotics; Benzimidazole derivatives; Biguanides; Cinchona alkaloid; Diaminopyrimidines; Helminths; Mannich base; Naphthoquinone; Nitrofuran derivatives; Nitroimidazole derivatives; Parasites; Protozoans; Quinoline derivatives; Quinoline-methanol; Sesquiterpenes; Sulfonamides; Tetracyclines; Tetrahydropyrimidine derivatives; Worms

Key points

• • • • • •

Parasitic infections, protozoal and non-protozoal combined, cause millions of mortalities every year and present some of the most difficult challenges to the medical community at-large. Current therapeutic protocols often require prolonged treatment, which frequently leads to drug-induced toxicity and side effects resulting in injury to the host. Acquired drug resistance during the therapy becomes additional concerns. In order to combat such situations, combination therapies are being used in treating several infections. Many current treatment protocols show reduced toxicity, and many mono- or combination therapies exhibit activity against multiple cellular target sites within parasites, including inhibition of enzyme function, cell membrane perturbation, and alterations to metabolic pathways, minimizing resistance generation. Antiparasitic agents available are highly effective at targeting a range of key parasites, including the causative agents of malaria, trypanosomiasis, leishmaniasis, amoebiasis, toxoplasmosis and other orphan diseases. For example, there are a large number of medications available to treat various forms of malaria. Despite numerous agents available to treat parasitic infections, diagnosis and treatment remains ever-challenging.

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Introduction Parasites have coevolved with humans and other organisms. Several of them colonize the human body and establish a symbiotic relationship, whereas many parasites cause severe and lethal infections. Parasites utilize a range of sophisticated mechanisms to defeat the physical, biochemical and immunologic fences that humans or other biological organisms have to protect themselves. For example, parasites (ameba) enter the CNS via the olfactory nerve, nematodes and cestodes, enter the host via the bloodstream and thus require prior skin or mucosal injury. Accidental bite of a vector or secretion of proteolytic enzymes can also facilitate entry. Occasionally, parasites are transported in a phagocytic cell to remote areas of the body. In order to combat its own destruction, parasites often use molecular mimicry and/or secrete agents that modulate the host immune system. Yet, another stunning mechanism reported in the literature is that Leishmania and Plasmodium species inhibit host cell apoptosis in order to assure their propagation within the host. Regardless, growth and survival of protozoans depend on environmental, hygienic standards of the surrounding and health conditions of the host. Normally they demonstrate a high propensity to infect immunocompromised individuals. Range of infection can be asymptomatic to fatal. They are categorized based on their main mode of locomotion: (i) Sarcodina includes amoebas such as Entamoeba (dysenteric liver abscess), Dientamoeba (colitis), Naegleria, and Acanthamoeba (central nervous system and corneal ulcers); (ii) Mastigophora: show flagellar movement, and include Giardia lamblia (diarrhea), Trypanosoma cruzii (sleeping sickness and Chagas disease), Leishmania donovanii (visceral, cutaneous and mucocutaneous leishmaniasis), and Trichomonas vaginalis (trichomoniasis, a sexually transmitted infection); (iii) Apicomplexa and Sporozoa: These organisms move via their apical region: include Babesia (babesiosis), Plasmodium (malaria), Toxoplasma (Toxoplasmosis), Isospora, Sarcocystis, and Cryptosporidium, which cause diarrhea; (iv) Ciliophora: use their cilia for movement, such as, Balanidium (dysentery). Treatment options and the consequences of treatments are discussed in the text. Helminths are parasitic worms. They transmit via accidental ingestion, skin penetration, a vector bite, or consumption of the host as food. Transmission is highly dependent on climate, hygiene, and exposure to vectors. Helminths classify as: (i) Trematodes (diseases caused by trematodes include schistosomiasis, fascioliasis, clonorchiasis, and paragonimiasis); (ii) Cestodes cause cysticercosis, echinococcosis (hydatid disease), diphyllobothriasis, and hymenolepiasis; (iii) Nematodes, aka roundworms cause intestinal infections, such as, enterobiasis, ascariasis, ancylostomiasis, strongyloidiasis, and trichinosis. Infections that directly attack the tissues include loiasis, onchocerciasis (river blindness), lymphatic filariasis (elephantiasis), and toxocariasis. Toxicity and side effects of medications that are used to treat various infections caused by these parasites are described in several recent reviews (Black and Ray, 2020, 2021; Onakopoya, 2020, 2021, 2022) (Tables 1 and 2). Parasite infections affect more than 2 billion people globally. These infections are common in underdeveloped tropical and subtropical countries and cause substantial morbidity and mortality. Single-celled organisms called protozoa are preliminarily responsible for parasitic infections, which can live and multiply in the host body. These diseases can be divided into groups based on where they cause infection in the human body (Kappagoda et al., 2011).

•

Systemic (blood or tissue) infections like malaria, sleeping sickness, chagas disease, babesiosis, toxoplasmosis, and leishmaniasis are transmitted by bites of infected insects such as mosquitoes, sandflies, or ticks. • Malaria caused by P. falciparum, P. vivax, P. ovale, P. malaria, P. knowlesi. • Sleeping sickness (African Trypanosomiasis) caused by Trypanosoma brucei. • Chagas disease (American Trypanosomiasis) caused by Trypanosoma cruzi. • Babesiosis caused by Babesia microti. • Toxoplasmosis caused by Toxoplasma gondii. • Leishmaniasis caused by L. donovani, L. infantum/chagasi, L. braziliensis, L. Mexicana, L. panamensis, L. guyanensis, L. major, L. tropica, or L. aethiopica. • Protozoans that infect the genital area are normally sexually transmitted (cause STDs): • Trichomoniasis caused by Trichomonas vaginalis. • Intestinal protozoa infections are mainly caused by the fecal-oral route (ingestion of contaminated food or water) or personto-person contact with an infected person. • Amoebiasis/amoebic dysentery caused by Entameoba histolytica. • Cryptosporidiosis caused by Cryptosporidium. • Giardiasis caused by Giardia lamblia This chapter focuses on names and classes of specific antiparasitic drugs and chemicals, their toxicokinetics, adverse reactions, toxicology, and overdose management strategies (Tables 1 and 2). Readers are requested to visit: www.cdc.gov/parasites/ for better understanding on parasitic diseases, their life cycle, human/animal susceptibilities, and treatments available in various parts of the world.

Antimalarial drugs Malaria is a disease caused by multiple species of Plasmodium, such as, P. falciparum, P. vivax, P. ovale, P. malaria, and P. knowlesi, and transmitted to people through the bites of infected female Anopheles mosquitoes. It is a life-threatening disease, and approximately 241 million malaria cases were reported in 2020 worldwide (WHO: Malaria, 2021). Out of this 627, 000 patients died from the disease and its complications. The WHO African Region has approximately 95% of malaria cases and 96% of malaria deaths. In 2020, 80% of all malaria deaths in the WHO African Region were in children under five. It is prevented in endemic areas through vector control and

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Table 1

Antimalarial medicines.

Classification

Mechanism of action

Name of drug

Therapeutic use/dosage

References

4-Aminoquinolines

4-Aminoquinolines act by increasing the concentration of the drug in the digestive vacuole of the intraerythrocytic parasite. The most recent hypothesis is the inhibition of the haem polymerase of the parasite, leading to the accumulation of soluble haem toxic for the parasite It is a rapidly acting erythrocytic schizontocide, but slower than chloroquine or quinine, effective against chloroquine-sensitive as well as resistant Plasmodia. The mechanism of action is still not known. However, some studies suggest that mefloquine targets the 80S ribosome of the Plasmodium falciparum, inhibiting protein synthesis and causing subsequent schizonticidal effects It is an erythrocytic schizontocide, more toxic and less effective than chloroquine

Chloroquine

300 mg base (500 mg) orally, once/week; Prophylaxis in areas with chloroquine-sensitive malaria 25–30 mg/Kg over 3 days Orally

Pussard and Verdier (1994), Goel and Gerriets (2022)

Mefloquine

684 mg base (¼750 mg salt) orally as an initial dose, followed by 456 mg base (¼500 mg salt) orally given 6–12 h after initial dose Total dose ¼ 1250 mg salt Prophylaxis in areas with mefloquine-sensitive malaria

White (1994), Burke (1993), Ahmed et al. (2021)

Mepacrine (Atabrine, Quinacrine) Quinine

100, 300 mg tab; 600 mg/week for prophylaxis; 3 g over 6 days for clinical cure 542 mg base (650 mg salt) orally three times daily (infections acquired outside Southeast Asia) to 7 days (infections acquired in Southeast Asia)

Gibb et al. (1985)

Chloroguanide (Proguanil)

250 mg atovaquone and 100 mg proguanil hydrochloride: 1 tablet orally, daily for an adult

Grieshaber et al. (2005)

Pyrimethamine

It is not suitable to treat malaria alone. 25 mg/ week is given for 10 weeks as a prophylactic dose after leaving the malarious area Duration of action is short, and it is non-cumulative. Therefore, it is occasionally used in treatment in combination with Sulfonamide 30 mg base (52.6 mg salt) orally, daily

Madireddy et al. (2022), Kemnic and Coleman (2022)

Quinoline-menthol

Acridine Cinchona Alkaloid

Biguanides

Diaminopyrimidines

8-Aminoquinoline

Sulfonamides and Sulfone

Cinchona Alkaloids (Quinine) interfere with Plasmodium falciparum’s ability to dissolve and metabolize hemoglobin. As with other quinoline antimalarial drugs, the precise mechanism of action of quinine has not been fully resolved, although in vitro studies indicate it inhibits nucleic acid and protein synthesis and inhibits glycolysis in Plasmodium falciparum Biguanides are prophylactic antimalarial drugs that stop the malaria parasite, Plasmodium falciparum and Plasmodium vivax from reproducing once into the red blood cells. It does this by inhibiting the enzyme dihydrofolate reductase, which is involved in reproducing the parasite It inhibits the dihydrofolate reductase of Plasmodium and, hence, blocks the biosynthesis of pyrimidines and purines, which are required for DNA synthesis and cell multiplication. In addition, it leads to failure of nuclear division at the time of schizont formation in erythrocytes and the liver

Primaquine’s mechanism of action is unclear. It may be acting by generating reactive oxygen species or by interfering with the electron transport in the parasite. Also, primaquine can bind to and alter the properties of protozoal DNA They compete with para-aminobenzoic acid (PABA) for incorporation into folic acid. The action of sulfonamides exploits the difference between mammal cells and other kinds of cells in their folic acid metabolism. All cells require folic acid for growth. Folic acid (as a vitamin) diffuses or is transported into human cells. However, folic acid cannot cross bacterial (and certain protozoan) cell walls by diffusion or active transport. For this reason, bacteria must synthesize folic acid from p-aminobenzoic acid Sulfonamides/dapsones are not particularly effective antimalarial drugs in their own right; they form supra-additive, synergistic combination die to sequential block

Amodiaquine

Trimethoprim

Primaquine

Sulfadoxine

Suilfadoxine 500 mg + Pyremethamine 25 mg tab

Sulfamethopyrazine

Sulfamethopyrazine 500 mg + Pyrimethamine 25 mg tab Dapsone 100 mg + Pyrimethamine 25 mg tab

Dapsone

Paintaud et al. (1994)

Kemnic and Coleman (2022)

Alving et al. (1955)

McIntosh (2000), Pearson and Hewlett (1987) Lee et al. (2001), Williams et al. (2000)

Antiprotozoal medicines Table 1

669

(Continued)

Classification

Mechanism of action

Name of drug

Therapeutic use/dosage

References

Tetracyclines

Tetracycline diffuses passively via porin channels in the bacterial membrane. It reversibly binds to the 30S ribosomal subunit, preventing the binding of tRNA to the mRNA-ribosome complex and thus interfering with protein synthesis

Tetracycline

Shutter and Akhondi (2022)

Artesunate gets metabolized to the active DHA. When the endoperoxide bridge of DHA reacts with heme, it generates free radicals, which inhibit the synthesis of protein and nucleic acid of the Plasmodium parasites during all erythrocytic stages. Reactions with these free radicals can also lead to the alkylation of parasitic proteins such as EXP1, a glutathione S-transferase, and calcium adenosine triphosphatase The mechanism of action of halofantrine might be similar to that of quinine, chloroquine, and mefloquine, which form toxic complexes with ferriprotoporphyrin IX that damage the membrane of the parasite

Artemisinin (Artesunate, Artemether)

Oral: 250 mg 4 times daily  7 days IV: dosage same as for oral 100 mg given once daily. A person should take the first dose one or 2 days before travel to an area where malaria may occur and continue taking medicine every day throughout travel and for 4 weeks after the person leaves the endemic malaria area Based on weight, a 3-day treatment schedule with 6 oral doses is recommended for adult and pediatric patients. The patient is administered the initial dose, followed by the second dose 8 h later, then one dose twice daily for the following 2 days 2 tab of 250 mg each (8 mg/kg) X Three doses are given at 8 h intervals

Newer Drugs

Doxycycline

Halofantrine

Cunha et al. (1982), Patel and Parmar (2022)

Adebayo et al. (2020), Esu et al. (2019)

Overington et al. (2006), Imming et al. (2006), Blauer (1988), Egan et al. (1999), Bouchaud et al. (2009)

preventative chemotherapy, which includes: mass drug administration (MDA), intermittent preventive treatment of infants (IPTi), intermittent preventive treatment of pregnant women (IPTp) chemoprophylaxis, seasonal malaria chemoprevention (SMC), and vaccines. Infants, children 10% of patients taking pentamidine, and manifest as renal insufficiency, increased serum creatinine, sterile abscess, necrosis, pain, and induration at the injection site.

Overdose and management A 17-month-old infant inadvertently received 1600 mg of intravenous pentamidine isethionate, which followed renal and hepatic impairment, hypotension, and cardiopulmonary arrest. Treatment included cardiopulmonary resuscitation, epinephrine, atropine, and intubation. In addition, a 4-h course of activated charcoal hemoperfusion. Activated charcoal detoxification accompanied reduced the pentamidine serum concentration and stabilized patient’s condition. The patient recovered from these AEs but later died due to an unknown cause (Watts et al., 1997).

688

Antiprotozoal medicines

Triclabendazole Background It is approved for fascioliasis in adult and pediatric patients 6 years and older. It is available as a 250 mg scored tablet, and the recommended total dosage is 10–30 mg/kg. It should be taken with food for better absorption and decrease gastrointestinal adverse effects.

Cl Cl

Cl

N

O

N H

S

Toxicokinetics Food may increase the rate and extent of absorption when administered orally. It is primarily metabolized via CYP1A2 enzymes into active sulfoxide metabolite and reaches peak plasma concentration in 3 h. It has an elimination half-life of approximately 8 h. In humans, plasma protein binding is about 98%. There is no excretion data available for humans.

Adverse drug reactions Common adverse reactions associated with triclabendazole are abdominal pain, nausea, decreased appetite, hyperhidrosis, urticaria, and headache. In addition, a transient increase in total bilirubin and liver enzymes is reported in patients taking triclabendazole.

Overdose and management Nausea was reported following ingestion of approximately 54 mg/kg of triclabendazole (approximately 2.7 times the recommended dose). The patient recovered following osmotic diuresis. Another case report of the patient experienced toxic retinopathy related to overdosage of praziquantel and triclabendazole (Ma and Peng, 2019) In clinical studies performed on dogs, QTc prolongation and anemia were reported at doses 1.1 times the MRHD based on BSA comparison. For patients with a history of QT interval prolongation or when other concomitant QTc-prolonging drugs are used, it is recommended to monitor ECG.

Conclusion Despite having made much progress over the past few decades, a proven magic bullet for any severe parasitic infection has not become available. The goal of finding a single factor or unified theory of pathogenesis for any particular disease does not appear to be likely within foreseeable future. A promising option would be to block a single important parasitic factor or developing a host of target oriented neutralizing and stabilizing protocols may be sufficient to tip the pathogenic balance in a patient and slow or halt the disease progression. In this arena, next-generation sequencing, omics technology, and integrated imaging approaches hold great potential to aid in the identification of pathogenic factors involved in parasitic diseases, where timely diagnosis appears to be the key. If major gaps are filled in these areas, many viable treatment options can be made available.

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Antiviral agents Kenny Lee, Roberto Maldonado, Saqib Khan, and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved.

Introduction Classification of antiviral agents Conclusion References Further reading

691 692 712 712 716

Abstract This article introduces a description of antiviral agents. Major groups are tricyclic amines, guanosine analogs, interferons, pyrophosphate derivatives, nucleoside analogues, nucleotide analogues, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors. The categories discussed include mechanism of action, effectiveness/indication, therapeutic range and toxic effects. Also referenced in this article are the disinfectant and sanitizing agents available to limit viral spread. Special attention is placed on the effectiveness of chemical agents in disrupting a virus’s cellular makeup and the potential toxicities that occur when using these chemical disinfectants.

Keywords Antiviral; Cardiovascular toxicity; Hepatotoxicity; Nephrotoxicity; Neurotoxicity; Teratogenicity; Tricyclic Amines; Guanosine Analogs; Interferons; Pyrophosphate Derivative; Nucleoside Analogues; Nucleotide Analogue; Nucleoside Reverse Transcriptase Inhibitors; Non-Nucleoside Reverse Transcriptase Inhibitors; Protease Inhibitors; Antiviral Chemical Disinfectant

Key points

● Antiviral agents include tricyclic amines, guanosine analogs, interferons, pyrophosphate derivatives, nucleoside analogues, nucleotide analogues, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors. ● Majority of antiviral agents were developed to target HIV. ● Nephrotoxicity is the primary adverse reaction of many antiviral medications. ● Chemical disinfectants are available to limit the spread of viruses. ● Agents for hepatitis treatment include NS3/4A protease Inhibitor, NS5A replication complex inhibitors and NS5B polymerase inhibitors.

Introduction Antiviral diseases are increasing in the new era, and so are the development of vaccines to control viral infections effectively. There are approximately 50 drugs that the FDA has approved since 2014, and more than half are targets for HIV. Antiviral agents’ main objectives are to minimize harm to the host system and eradicate deadly viral infections. Still, the replications of viruses in the host system represent a more complicated therapeutic challenge than bacteria and fungi. Antiviral drugs penetrate and disrupt the intracellular replication of the virus but, at the same time, may harm normal host physiology. Due to these concerns, antiviral agents have a narrower therapeutic index than antibacterial drugs. Moreover, organ toxicities are the primary adverse reaction of antiviral medicines in humans and animals. Practitioners have been plagued with the issue of drug resistance for decades. As a countermeasure, combination therapy is used to offset or minimize such situations in the treatment of viral infections. Viral transmission can occur via close human-to-human contact or from contaminated surfaces. Consequently, careful disinfection or sanitization is essential to limit the viral spread. Many disinfectants/sanitizing agents/biocidal agents are available that can inactivate viruses. Still, their effectiveness depends on many factors, such as the concentration of the agent, reaction time, temperature, and organic load. Therefore, knowledge of the target virus and careful control of the treatment conditions are vital to effective disinfection. Many disinfectants/sanitizing agents inactivate viruses by chemically modifying their surface groups. They are fast-acting and highly successful toward most viruses but are restricted by their higher toxicity and toxic effects. On the other hand, disinfectants like alcohols and surfactants that rely on dissolving the lipid envelopes tend only to show effectiveness toward a narrower range of viruses and may require longer exposure durations but are often more bio-friendly.

Encyclopedia of Toxicology 4th Edition

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692 Antiviral agents

Classification of antiviral agents

Class/Drug

MOA

Tricyclic Amines Amantadine (GocovritW)

Uncertain antiviral activity; primarily interferes with viral replication.

Indication/structure

Dosage form/toxicology

References

Oral capsules and tablets Oral syrup Currently no minimum toxicity dose is listed. Know the patient’s history, mental illness, and seizures particularly before proceeding with therapy. Toxicities: acute psychosis, Neurotoxicity, cardiovascular toxicity, coma, or death. No antidote is available. Side effects resolve with discontinuation of therapy.

FDA3: Amantadine (2017)

Inhibitory effect early in the viral replicative cycle, inhibiting the uncoating of the virus.

Oral tablets, Syrup

FDA 26: Rimantadine (2006); Pubchecm (28) (n.d.)

Specified by the virion M2 gene. Important role in susceptibility of influenza A virus.

Toxicities: Neurotoxicity, seizures, and Gastroenteritis.

Hayden et al. (1981)

HCV Genotype PEGASYS Oral tablet

Graci and Cameron (2006) FDA 24: Ribavirin (2011)

Disrupts transmembrane domain of viral M2 protein; prevents infectious viral nucleic acid entry into the host cell. Inhibits assembly of influenza A virus isolates (H1N1, H2N2, and H3N2) during replication. Influenza B is structurally different M2 protein, which makes amantadine ineffective Rimantadine (FlumadineW)

(Pubchem: Amantadine, 2022) Indications: Parkinson disease: Drug-induced extrapyramidal reaction Influenza A: No longer recommended due to high resistance levels.

Hayden et al. (1981) Pubchem (4) (2022)

Blocks ion channels formed by M2 protein that spans the viral membrane. (Pubchem: Rimantadine, 2022) Indications: Treatment of illness caused by strains of influenza A virus in adults and children. No activity against influenza B. Guanosine Analogs Ribavirin Several mechanisms of actions lead to (CopegusW) inhibition of viral RNA and protein synthesis.

COPEGUS Dose Oral tablet divided in 2 doses

Both indirect mechanisms (inosine monophosphate dehydrogenase inhibition, immunomodulatory effects) and direct mechanisms (interference with RNA capping, polymerase inhibition, lethal mutagenesis).

Pubchem (26) (2022)

Should never be given as monotherapy.

Antiviral agents

Toxicities: Primarily Hemolytic anemia, myocardial infarction (BBW), Teratogenic effects, Pancytopenia, Pancreatitis, and Hepatic decompression.

(Pubchem: Ribavirin, 2022)

693

Indications: 5 years of age and older with chronic HCV infection with compensated liver disease and have not been previously treated with interferon alpha. (Continued)

Indication/structure

Peginterferon a-2a binds to the human type 1 interferon receptor. Dimerization activates multiple intracellular signal transduction pathway mediated by the JAK/STAT pathway. Combination of PEG-IFN a-2a and ribavirin more effective at inhibiting HCV RNA replication than either agent alone.

Inhibits HBV replication; Induces gene transcription; interferes with oncogene expression, and changes cell surface antigen expression; Cytotoxic activity of macrophages increase.

References

Subcutaneous

FDA 24: Pegasys (2011)

Toxicities: Fatal or life threatening neuropsychiatric, autoimmune, ischemic, and infectious disorders (BBW).

Pubchem (19) (2022)

Bone marrow suppression results in severe cytopenia. Myositis, hepatitis, thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura, psoriasis, rheumatoid arthritis, interstitial nephritis, thyroiditis, and systemic lupus erythematosus have also been reported. (Pubchem Interferon alpha 2a, 2022)

Interferon a-2b (PEG-Intron™)

Dosage form/toxicology

Indications: For treatment of chronic hepatitis C, hairy cell leukemia, AIDS-related Kaposi’s sarcoma, and chronic myelogenous leukemia. Also, for the treatment of oral warts arising from HIV infection. Indications: For the treatment of chronic hepatitis C in patients with compensated liver disease who have not been previously treated with interferon alpha and are at least 18 years of age.

[https://www.accessdata.fda.gov/ drugsatfda_docs/label/2002/ pegihof101602lb.htm]

Considerations in HIV patients: Neutropenia and thrombocytopenia occur with a greater incidence May result in serious infections or bleeding. (FDA: Pegasys, 2011)

IM, SC Toxicities: CNS depression: Psychiatric symptoms, suicidal or homicidal ideation or aggressive behavior. Dyspnea, pulmonary infiltrates, pneumonia, bronchiolitis obliterans, interstitial pneumonitis, pulmonary hypertension, and sarcoidosis resulting in respiratory failure or patient deaths. Autoimmune disorders: thyroiditis, thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura. Ischemic and hemorrhagic cerebrovascular events reported. Pancreatitis and ulcerative or hemorrhagic/ ischemic colitis, severe decreases in neutrophil or platelet counts reported

FDA: Intron A (2018) [https://www.accessdata.fda.gov/ drugsatfda_docs/label/ 2018/103132Orig1s5199lbl.pdf] Intron A, Interferon (interferon a-2b) (2018) Pubchem (20) (2022)

Antiviral agents

Interferons Interferon a-2a (PegasysW)

MOA

694

Class/Drug

Interferon alfacon-1 (InfergenW)

Binds to type I interferon receptors (IFNAR1 and IFNAR 2c) activate two Jak (Janus kinase) tyrosine kinases (Jak1 and Tyk2).

Combination: SC given Orally w/ribavirin.

(Kegg Drug; Interferon alfacon-1, n.d.)

Toxicities: Hypotension, arrhythmia, tachycardia, cardiomyopathy, angina pectoris, and myocardial infarction. Dyspnea, pulmonary infiltrates, pneumonia, bronchiolitis obliterans, interstitial pneumonitis, pulmonary hypertension and sarcoidosis.

Indications: Chronic hepatitis C in patients 18 years of age or older with compensated liver disease.

Chronic hepatitis C patients and hepatic decompensation when treated with interferon alphas.

Phosphorylated INFAR receptors bind to Stat1 and Stat2 which dimerize and activate multiple (
100) immunomodulatory and antiviral proteins.

FDA 17: Alfacon-1 Melian and Plosker (2001) Kegg Drug; Interferon alfacon-1 (n.d.) [https://www.genome.jp/dbget-bin/ www_bget?D02744+D02745 +D02747+D02748+D03304+D03305]

Bone marrow suppression and Colitis has been reported.

Interferon alpha-n3 (Alferon NW)

Interferes with oncogene expression, gene transcription, alters cellular differentiation and cell surface antigen expression. Increases phagocytic activity of macrophages, may inhibit cell growth.

Indications: For the intralesional treatment of refractory or recurring external condylomata acuminata [Alferon N (interferon alfa n3), n.d.].

Ophthalmologic Disorders Macular edema SC

Alferon, 2009

Toxicities: Hypersensitivities, papular rash on neck, photosensitivity,

1[https://www.wellrx.com/ALFERON% 20N/monographs/]

depression, nervousness, insomnia

2[https://go.drugbank.com/drugs/ DB00018]

pharyngitis, dysuria, hypotension, and vasovagal reaction has been reported.

Pyrophosphate Derivative Foscarnet Inhibits replication of herpes viruses including sodium cytomegalovirus (CMV) and herpes simplex (Foscavir W) virus types 1 and 2 (HSV1 and HSV-2).

Indications: Indicated for the treatment of CMV retinitis in patients with acquired immunodeficiency syndrome (AIDS). Combination therapy indicated for patients who have relapsed after monotherapy with either drug.

FDA 15: Foscavir (2012)

Toxicities: Renal impairment is a major toxicity. Frequent monitoring of serum creatinine with dose adjustments for changes in renal function and adequate Hydration is imperative.

Pubchem (16) (2022)

Associated with changes in serum electrolytes: hypocalcemia, hypophosphatemia, hyperphosphatemia, hypomagnesemia, and hypokalemia.

695

Does not require activation by thymidine kinase or other kinases and therefore is active in vitro against HSV TK deficient mutants and CMV UL97 mutants.

(Pubchem (16), 2022)

IV

Antiviral agents

Selective inhibition at the pyrophosphate binding site on virus-specific DNA polymerases at concentrations that do not affect cellular DNA polymerases.

3[https://www.sciencedirect.com/topics/ medicine-and-dentistry/alphan3interferon]

Anemia reported in 33% of patients. Granulocytopenia reported in 17% of patients. (Continued)

Class/Drug

MOA

Indication/structure

References

Oral capsules, tablets, suspension, IV Toxicities: Potential risk of thrombotic thrombocytopenic purpura [TTP]/hemolytic uremic syndrome

FDA 36: Acyclovir Pubchem (2) (2022)

Antiviral agents

Neurotoxicity. Renal failure, in some cases resulting in death, has been observed with acyclovir therapy. (FDA: Zovirax, 2019)

(Pubchem: Acyclovir, 2022)

Valacyclovir (ValtrexW)

Rapidly converted to acyclovir which has demonstrated antiviral activity against HSV types 1 (HSV-1) and 2 (HSV-2) and VZV both in cell culture and in vivo.

Indications: Herpes Zoster Infections (shingles) and Genital Herpes Chickenpox: (FDA Zovirax, 2019)

Inhibitory activity of acyclovir is highly selective due to its affinity to thymidine kinase (TK) encoded by HSV and VZV. (FDA: Valtrex, 2019)

(Pubchem: Valacyclovir, 2022) Indications: Cold Sores (Herpes Labialis) Genital Herpes Treatment in immunocompetent patients (initial or recurrent episode) Suppression in immunocompetent or HIV-infected patients Reduction of transmission Herpes Zoster Pediatric Patients Cold Sores (Herpes Labialis) Chickenpox (FDA: Valtrex, 2019)

Oral suspension

FDA 34: Valacyclovir

Most effective when administered within 48 h of onset of signs and symptoms (FDA VALTREX, 2008) Toxicities: Thrombotic Thrombocytopenic Purpura/ Hemolytic Uremic Syndrome (TTP/HUS) TTP/ HUS, in some cases resulting in death, has occurred in patients with advanced HIV disease and also in allogeneic bone marrow transplant and renal transplant recipients.

Pubchem (34) (2022)

Acute Renal Failure, CNS, hallucinations, confusion, delirium, seizures, and encephalopathy. (FDA: Valtrex, 2019)

696

Nucleoside Analogues Highly selective to the enzyme thymidine Acyclovir kinase (TK) encoded by HSV and VZV. (ZoviraxW) Stops replication of herpes viral DNA in 3 ways: (1) competitive inhibition of viral DNA polymerase, (2) incorporation into and termination of the growing viral DNA chain, and (3) inactivation of the viral DNA polymerase. (FDA: Zovirax, 2019)

Dosage form/toxicology

Ganciclovir (CytoveneW)

Oral, IV Toxicities: Hematologic Toxicity: Granulocytopenia, anemia, thrombocytopenia, and pancytopenia have been reported.

Inhibits replication of human CMV. Virustatic activity due to inhibition of the viral DNA polymerase, pUL54, by ganciclovir triphosphate. (FDA: Ganciclovir, 2017)

FDA 16: Ganciclovir (2017) Pubchem (17) (2022)

Impairment of Fertility: inhibition of spermatogenesis in males and suppression of fertility in females. Fetal Toxicity, mutagenesis and carcinogenesis

(Pubchem: Ganciclovir, n.d.)

Valganciclovir (ValcyteW)

Prodrug, converted to ganciclovir in intestine and liver

Indications: Indicated for the treatment of CMV retinitis in immunocompromised adult patients, including patients with acquired immunodeficiency syndrome (AIDS). Prevention of CMV disease in adult transplant recipients at risk for CMV disease. (FDA: Ganciclovir, 2017)

Inhibits viral DNA polymerases resulting in chain termination. Inhibits replication of human CMV in cell culture and in vivo (FDA: Valcyte, 2018)

Overdose causes irreversible pancytopenia, acute renal failure requiring hemodialysis, neutropenia, thrombocytopenia, hepatitis, and seizures (FDA: Ganciclovir, 2017)

Oral tablet

FDA 33: Valganciclovir

Hematologic toxicity: Severe leukopenia, neutropenia, anemia, thrombocytopenia, pancytopenia, and bone marrow failure including aplastic. Impaired fertility: Inhibition of spermatogenesis in males and suppression of fertility in females.

Pubchem: Valacyclovir, 2022

Fetal toxicity Mutagenesis and carcinogenesis (FDA: Valcyte, 2018)

Antiviral agents

(Pubchem Valcyte, 2022) Indicated: CMV retinitis in patients with acquired immunodeficiency syndrome (AIDS). Prevention of CMV disease in kidney, heart, or kidney-pancreas transplant patients at high risk (FDA: Valcyte, 2018)

697

(Continued)

698

MOA

Famciclovir (FamvirW)

Prodrug of penciclovir, selectively inhibits viral DNA replication in herpes simplex virus (HSV) type 1 (HSV-1), HSV-2, and varicella zoster virus (VZV) (FDA: Famciclovir, 2011)

Indication/structure

Dosage form/toxicology

References

Oral tablet

FDA 13: Famciclovir (2011) Pubchem: Famciclovir, 2022

Toxicities: N/A Acute renal failure may occur in patients with underlying renal disease who receive higher than recommended doses. FDA 13: Famciclovir (2011) (Pubchem: Famciclovir, 2022)

Penciclovir (DenavirW)

Inhibitory activity against various Herpesviridae, including herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus, and Epstein-Barr virus. (FDA: Penciclovir, 2018)

Indications: Herpes labialis (cold sores) Genital herpes Herpes zoster (shingles) HIV-Infected Adult Patients Recurrent episodes of orolabial or genital herpes (FDA: Famciclovir, 2011) Cream 0.1% Toxicities: N/A Used for herpes labialis on lips and face. Avoid application in or near the eyes since it may cause irritation. (FDA: Penciclovir, 2018)

(Pubchem: Penciclovir, 2022) Indications: Recurrent herpes labialis (cold sores) in adults and children 12 years of age and older. (FDA: Penciclovir, 2018)

FDA 23: Penciclovir (2013) Pubchem (25) (2022)

Antiviral agents

Class/Drug

Trifluridine (ViropticW)

Inhibition of viral replication. Incorporated into viral DNA during replication resulting in formation of defective proteins and an increased mutation rate. Reversibly inhibits thymidylate synthetase, an enzyme required for DNA synthesis. (Viroptic (trifluridin))

Ophthalmic solution:1%

Viroptic (trifluridin) (n.d.)

Toxicities: Use >21 days should be avoided because of potential ocular toxicity (Viroptic (trifluridin)).

https://reference.medscape.com/drug/ viroptic-trifluridine-ophthalmic343587#5.

Intravenous infusion

PubChem (6) (2022)

Toxicities: Dose-dependent nephrotoxicity. Proteinuria is an early indicator of nephrotoxicity.

FDA 5: Cidofovir (2018)

Pubchem (34) (2022)

(Pubchem: Trifluridine, 2022) Indications: Indicated: Keratoconjunctivitis and recurrent epithelial keratitis due to herpes simplex virus, types 1 and 2 (Viroptic (trifluridin)). Nucleotide Analogue Cidofovir Selective inhibition of viral DNA synthesis (VistideW) through suppression of cytomegalovirus replication. (FDA 5: Cidofovir, 2018)

Hematological Toxicity—neutropenia may occur. Ocular Hypotony Metabolic acidosis association with liver dysfunction & pancreatitis have been reported (Pubchem: Cidofovir, 2022)

(FDA 5: Cidofovir, 2018)

(Continued)

Antiviral agents

Indications: Indicated for the therapy of CMV retinitis in patients with AIDS. (FDA 5: Cidofovir, 2018)

699

MOA

Indication/structure

Capsule, tablet, syrup, & injectable solution

Pubchem (37) (2022)

Tοxicities Hematologic toxicity or bone marrow suppression

FDA 35: Zidovudine (2005)

Symptomatic myopathy

ZDV-TP is also a weak inhibitor of the cellular DNA polymerases a and g.

Lactic acidosis and severe hepatomegaly with steatosis

(FDA: Zidovudine/AZT, 2005)

(Pubchem: Zidovudine/AZT) Indications: It is indicated for HIV-1 infection in combination with other antiretroviral agents. It is also indicated for the prevention of maternal-fetal HIV-1 transmission. (FDA: Zidovudine/AZT, 2005) Didanosine/DDI (VidexW)

References

A synthetic nucleoside analogue, which inside of a cell, it is converted to the active metabolite, dideoxyadenosine 50 triphosphate. Inhibits the activity of HIV-1 RT by two ways. First, it competes with the natural substrate, deoxyadenosine 50 -triphosphate with 30 -hydroxyl group. And second, its incorporation into viral DNA causing termination of viral DNA chain elongation

HIV-1/HCV coinfected patient receiving ribavirin and zidovudine, the exacerbation of anemia has been reported Hepatic decompensation has occurred in co-infected patients with HIV-1/HCV receiving combination of antiretroviral and interferon alfa Immune reconstitution syndrome (FDA: Zidovudine/AZT, 2005) Capsule, powder for oral solution, tablet for oral suspension Toxicities: Pancreatitis fatal and nonfatal pancreatitis Lactic acidosis and severe hepatomegaly with steatosis Retinal changes and optic neuritis Hepatic impairment and toxicity (FDA: Didanosine/DDI, 2005)

(Pubchem: Didanosine/DDI, 2022) Indications: It is indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents

Pubchem (9) (2022) FDA 8: Didanosine (2005)

Antiviral agents

Nucleoside Reverse Transcriptase Inhibitors A synthetic nucleoside analogue which is Zidovudine/AZT phosphorylated to its active 50 -triphosphate (RetrovirW) metabolite (ZDV-TP) inside of a cell, where it inhibits RT via DNA chain termination after nucleotide analogue is integrated.

Dosage form/toxicology

700

Class/Drug

Emtricitabine (EmtrivaW)

Capsule, & suspension

It is a synthetic nucleoside analog of cytidine which is phosphorylated to form a metabolite, emtricitabine 50 -triphosphate.

Pubchem (11) (2022) FDA 11: Emtricitabine (2011)

Toxicities: Lactic acidosis/severe hepatomegaly with steatosis

It inhibits the HIV-1 RT activity by competing with cytidine and by being incorporated into nascent viral DNA leading to chain termination.

Patients co-infected with HIV and HBV: must be tested for chronic HBV before the start of treatment. Hepatic function should be closely monitored. (FDA: Emtricitabine, 2003)

The metabolite is a weak inhibitor of mammalian DNA polymerase a, b, e, and mitochondrial DNA polymerase g. (FDA: Emtricitabine, 2003) (Pubchem: Emtricitabine, 2022)

Stavudine (ZeritW)

A nucleoside analogue of thymidine which is phosphorylated to the active metabolite stavudine triphosphate. It inhibits the HIV-1 RT activity by competing with the natural substrate thymidine triphosphate and by incorporation into viral DNA leading to DNA chain termination.

Indications: It is indicated for the treatment of HIV-1 Infection in combination with other antiretroviral agents. (FDA: Emtricitabine, 2003)

Capsule, & powder for oral solution Toxicities: Lactic acidosis/severe hepatomegaly with steatosis

Pubchem (31) (2022) FDA 30: Stavudine (2006)

Hepatic impairment and toxicity Neurologic symptoms (Motor weakness)

It also inhibits cellular DNA polymerases b and g and the synthesis of mitochondrial DNA is reduced. (FDA: Stavudine, 2006)

Peripheral neuropathy Fatal and nonfatal pancreatitis (FDA: Stavudine, 2006) (Pubchem: Stavudine, 2022)

(Continued)

Antiviral agents

Indications: It is indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents (FDA: Stavudine, 2006)

701

702

MOA

Tenofovir disoproxil fumarate (VireadW)

It is an acyclic nucleoside phosphonate diester analog of adenosine monophosphate. Tenofovir diphosphate inhibits the activity of HIV-1 RT and HBV RT by competing with the natural substrate deoxyadenosine 50 -triphosphate and by DNA chain termination after being incorporated into DNA.

Indication/structure

Dosage form/toxicology

References

Tablet, & powder for oral

Pubchem (32) (2022)

Toxicities Lactic acidosis/severe hepatomegaly with steatosis

FDA 31: Tenofovir disoproxil fumarate (2001)

Severe acute exacerbation of hepatitis New onset or worsening renal impairment

Tenofovir diphosphate is a weak inhibitor of mammalian DNA polymerases a, b, and mitochondrial DNA polymerase g

Decreases in bone mineral density Immune reconstitution syndrome (Pubchem: Tenofovir, 2022)

Lamivudine (EpivirW)

It is nucleoside analogue reverse transcriptase inhibitor. (FDA: Lamivudine, 2017)

Indications: It is indicated in combination with other antiretroviral agents for the therapy of HIV-1 infection and chronic HBV (FDA: Tenofovir, 2001)

Tablet & oral solution Toxicities Hepatic decompensation has occurred Pancreatitis Immune reconstitution syndrome Redistribution/accumulation of body fat Lower virologic suppression rates (FDA: Lamivudine, 2017)

(Pubchem: Lamivudine, 2022) Indications: It is indicated in combination with other antiretroviral agents for the treatment of HIV-1 infection. (FDA: Lamivudine, 2017)

Pubchem (21) (2022) FDA 19: Epivir (2005)

Antiviral agents

Class/Drug

Entecavir (BaracludeW)

It is a guanosine nucleoside analogue with activity against HBV RT. After it is phosphorylated to the active triphosphate form, it competes with the natural substrate deoxyguanosine triphosphate. Leading to the inhibition of all three activities of the HBV RT, namely, base priming, reverse transcription of the negative strand from the pregenomic messenger RNA, and synthesis of the positive strand of HBV DNA. Entecavir triphosphate is a weak inhibitor of cellular DNA polymerases a, b, and d and mitochondrial DNA polymerase g.

Abacavir (ZiagenW)

It is a carbocyclic synthetic nucleoside analogue which is converted to the active metabolite, carbovir triphosphate (CBV-TP). CBV-TP inhibits the activity of HIV-1 RT both by competing with the natural substrate dGTP and by its incorporation into viral DNA leading to the termination of DNA chain elongation.

Tablet & oral solution

Pubchem (12) (2022)

Toxicites: After discontinuation, severe acute exacerbations of HBV infection have occurred.

FDA 11: Emtricitabine (2011)

If lactic acidosis and severe hepatomegaly with steatosis are suspected, the therapy should be suspended. (FDA: Entecavir, 2011)

(Pubchem: Entecavir, 2022) Indications: It is a nucleoside analogue indicated for the therapy of chronic HBV infection in adults

Oral solution & tablet Toxicities: Hypersensitivity reaction

Pubchem (1) (2022) FDA 1: Abacavir (2002)

Lactic acidosis Severe hepatomegaly, steatosis have been reported Immune reconstitution syndrome (FDA Abacavir, 2002)

CBV-TP is a weak inhibitor of cellular DNA polymerases a, b, & g

(Pubchem: Abacavir, 2022)

Adefovir (HepseraW)

Tablet

Pubchem (3) (2022)

Toxicities Severe acute exacerbations of hepatitis Nephrotoxicity

FDA 2: Adefovir (2003)

Antiviral agents

It is an acyclic nucleotide analog of adenosine monophosphate. It is phosphorylated to the active metabolite which inhibits HBV DNA polymerase (reverse transcriptase) by competing with the natural substrate deoxyadenosine triphosphate and by causing DNA chain termination after its incorporation into viral DNA.

Indications: It is indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents

Lactic acidosis and severe hepatomegaly with steatosis Do not administer Adefovir concurrently with other tenofovir containing products.

Adefovir diphosphate is a weak inhibitor of human DNA polymerases a and g

Untreated HIV may result in HIV resistance

703

(Pubchem: Adefovir, 2022) Indications: It is indicated for the treatment of chronic HBV (>12-year-old) (Continued)

704 Antiviral agents

Class/Drug

MOA

Indication/structure

Non-Nucleoside Reverse Transcriptase Inhibitors It is a non-nucleoside reverse transcriptase Nevirapine inhibitor (NNRTI) of human (ViramuneW) immunodeficiency virus type 1 (HIV-1), which binds to reverse transcriptase (RT) blocking the activity of RNA-dependent and DNA-dependent DNA polymerases. However, Nevirapine does not inhibit the HIV-2 RT and eukaryotic DNA polymerases (such as human DNA polymerases a, ß, g, or d)

Efavirenz (SustivaW)

It is a non-nucleoside reverse transcriptase inhibitor of HIV-1.

Dosage form/toxicology

References

Oral suspension, tablet IR & tablet ER Toxicities: Hepatotoxicity

Pubchem (24) (2022) FDA 22: Nevirapine (2004)

Rash: Fatal and non-fatal skin reactions has been reported

(Pubchem: Nevirapine, 2022) Indications: Indicated in combination with other antiretrovirals for the treatment of HIV-1 infection

It noncompetitively inhibits reverse transcriptase (RT) of HIV-1.

Capsule & tablet Toxicities: Hepatotoxicity Rash Serious psychiatric symptoms

However, it does not inhibit the HIV-2 RT and human cellular DNA polymerases a, b, g, and d

Nervous system symptoms (Pubchem: Efavirenz, 2022) Indications: Indicated in combination with other antiretroviral agents for the treatment of HIV-1 infection

Contraindicated in pregnant woman. Convulsions Elevation of total cholesterol and triglyceride

Pubchem (10) (2022) FDA 9: Efavirenz (2011)

Rilpivirine (EdurantW)

It is a diarylpyrimidine NNRTI of HIV-1. It inhibits the replication of HIV-1 by inhibiting the HIV-1 RT non-competitively.

Tablet Toxicities: Depressive disorders have been reported

It does not inhibit the human cellular DNA polymerases a, b and g

Redistribution/accumulation of body fat

Pubchem (27) (2022) FDA 26: Rilpivirine (2013)

Immune reconstitution syndrome (FDA: Rilpivirine, 2013)

(Pubchem: Rilpivirine, 2022)

Etravirine (IntelenceW)

It is an NNRTI of HIV-1 which binds directly to RT & blocks the RNA-dependent and DNA-dependent DNA polymerase activities by causing a disruption of the enzyme’s catalytic site.

Indications: It is indicated for the treatment of HIV-1 infection in combination with other antiretroviral agents for naïve adult patients

Tablet Toxicities: Severe, potentially life-threatening, and fatal skin reactions have been reported.

Pubchem (13) (2022) FDA 12: Etravirine (2008)

Hypersensitivity reactions have also been reported and were characterized by rash.

It does not inhibit the human DNA polymerases a, b, and g

Immune Reconstitution Syndrome (FDA: Etravirine, 2008)

Antiviral agents

(Pubchem: Etravirine, 2022) Indications: It is indicated for the treatment of HIV-1 infection in antiretroviral treatment of experienced patients ages 6 years and older, who have evidence of viral replication and HIV-1 strains resistant to a NNRTI and other antiretroviral agents

705

(Continued)

706

MOA

Delavirdine (RescriptorW)

It is a NNRTI of HIV-1. It binds directly to RT and blocks RNA-dependent and DNA-dependent DNA polymerase activities.

Indication/structure

HIV-2 RT and human cellular DNA polymerases a, b, and g are not inhibited by delavirdine

Dosage form/toxicology

References

Tablets

Pubchem (8) (2022)

Toxicites: Immune reconstitution syndrome

FDA 7: Delaverdine (2007)

Redistribution/accumulation of body fat

(Pubchem: Delavirdine, 2022)

Severe skin rash (FDA: Delavirdine, 2007)

Indications: It is indicated for the treatment of HIV-1 infection. It must be given in combination with at least 2 other active antiretroviral agents Protease Inhibitors Indinavir Binds to the protease active site and inhibits (CrixivanW) the proteolytic cleavage, resulting in the formation of immature non-infectious viral particles

Capsule (Lexicomp Indinavir, 2022)

LivTox, 2017

Toxicities: Hepatotoxicity with resulting hyperbilirubinemia

FDA 18: Indinavir (2002)

Nephrolithiasis/urolithiasis (FDA: Indinavir, 2010)

(Pubchem: Indinavir, 2022) Indications: Treatment of HIV infection in combination with other antiretroviral agents; no longer recommended for use in the treatment of HIV in the US (FDA Indinavir, 2017)

Lexicomp (4) (2022) Pubchem (18) (2022)

Antiviral agents

Class/Drug

Saquinavir (InviraseW)

A peptide-like substrate analogue that binds to the protease active site and inhibits the activity of HIV protease. It prevents cleavage of the viral polyproteins resulting in the formation of immature non-infectious virus particles (FDA: Saquinavir, 2010)

Capsule and Tablet

FDA: Saquinavir, 2010

Toxicities: Altered cardiac conduction: Saquinavir/ritonavir QT interval prolongation.

Lexicomp (8) (2022) Pubchem (30) (2022)

Immune reconstitution syndrome Photosensitivity reaction FDA 29: Saquinavir (2010)

(Pubchem: Saquinavir, 2022)

Ritonavir (NorvirW)

A peptidomimetic inhibitor of both the HIV-1 and HIV-2 proteases. It renders the enzyme incapable of processing the Gag-pol polyproteins resulting in production of non-infectious immature HIV particles

Indications: Treatment of HIV-1 infection in adults in combination with ritonavir and other antiretroviral agents

Tablet, solution, and packet (Lexicomp Ritonavir, 2022)

CDC (2) (2022) FDA 28: Ritonavir (2011)

Toxicities: Retinal toxicity (Tu et al., 2016)

Lexicomp (7) (2022)

Hepatotoxicity

Pubchem (29) (2022)

Pancreatitis

(Tu et al., 2016)

Hemophilia PR interval prolongation (FDA: Ritonavir, 2011) (Pubchem: Ritonavir, 2022)

Antiviral agents

Indications: Use in combination with other antiretroviral agents for treatment of HIV-infection. Use in combination with nirmatrelvir for COVID-19 treatment (CDC, 2022) (Continued)

707

Nelfinavir (ViraceptW)

An inhibitor of the HIV-1 protease. It prevents cleavage of the gag and gag-pol polyprotein resulting in the production of immature non-infectious virus

Indication/structure

Dosage form/toxicology

References

Tablet and Powder

FDA 21: Nelfinavir (2005)

 Powder formulation available in Canada (Lexicomp Nelfinavir, 2022)

Lexicomp (6) (2022) Pubchem (23) (2022)

Toxicities: Immune Reconstitution Syndrome Diabetes mellitus: new onset or exacerbation (FDA: Nelfinavir, 2005)

(Pubchem: Nelfinavir, 2022)

Atazanavir (ReyatazW)

An azapeptide HIV-1 protease inhibitor. It selectively inhibits polyproteins in HIV-1 infected cells preventing formation of mature virions

Indications: Treatment of HIV infection with other antiretroviral agents

Capsule and Packet (Lexicomp Atazanavir, 2022)

FDA 4: Atazanavir (2008)

Toxicities: Cardiac conduction abnormalities

Lexicomp (1) (2022)

Severe skin reactions Hepatotoxicity hyperbilirubinemia Granulomatous interstitial nephritis in CKD patients Nephrolithiasis and Cholelithiasis Diabetes mellitus: new onset or exacerbation (Pubchem: Atazanavir, 2022) Indications: Treatment of HIV-1 infection in combination with other antiretroviral agents; coadministration of Reyataz/ritonavir is recommended for patients with prior virologic failure (FDA: Atazanavir, 2008)

Immune reconstitution syndrome with combination antiretroviral therapy

Pubchem (5) (2022)

Antiviral agents

MOA

708

Class/Drug

Darunavir (PrezistaW)

Tablet and Suspension (Lexicomp Darunavir, 2022)

Inhibitor of the HIV-1 protease. It selectively inhibits the Gag-Pol polyproteins preventing the formation of mature virus particles

Toxicities: Hepatotoxicity

Exhibit activity against HIV-1 and laboratory strains of HIV-2 in acutely infected T-cell lines

FDA 6: Darunavir (2008) Lexicomp (2) (2022) Pubchem (7) (2022)

Severe skin rash including erythema multiforme and SJS. Diabetes mellitus: new onset or exacerbation (FDA: Darunavir, 2008)

(Pubchem: Darunavir, 2022)

Fosamprenavir (LexivaW)

A prodrug of amprenavir; converted by cellular phosphatases. It is an inhibitor of HIV-1 protease. It binds to the active site and prevents Gag and Gag-Pol polyproteins processing resulting in the formation of immature non-infectious viral particles

Indications: Treatment of HIV infection with strains resistant to more than one protease inhibitor

Tablet and Suspension (Pubchem: Fosamprenavir, 2022) Toxicities: Carcinogenicity: hepatocellular adenomas and hepatocellular carcinomas in male mice at highest dose (Clinical Guidelines, 2022) Immune reconstitution syndrome Lipid Elevations

Clinicalinfo.hiv.gov: Fosamprenavir, 2018 https://clinicalinfo.hiv.gov/en/ guidelines/perinatal/fosamprenavirlexiva-fpv FDA 14: Fosamprenavir (2006) Lexicomp (3) (2022) Pubchem (15) (2022)

Nephrolithiasis

Antiviral agents

(Pubchem: Fosamprenavir, 2022) Indications: Treatment of HIV infection in adults. Not recommended for protease inhibitor-experienced patients with once-daily administration

709

(Continued)

Lopinavir/ Ritonavir (KaletraW)

Inhibits HIV protease, prevents cleavage of Gag-Pol polyprotein, resulting in non-infectious viral particles (FDA: Lopinavir/Ritonavir, 2000)

Indication/structure

Dosage form/toxicology

References

Tablet and Solution (Pubchem (15) (2022))

FDA 20: Lopinavir/Ritonavir (2000)

Toxicities: Hepatotoxicity

Lexicomp (5) (2022) Pubchem (22) (2022)

Immune reconstitution syndrome Myocardial infarction with cumulative use of lopinavir/ritonavir Pancreatitis in patients with increased triglycerides. (FDA: Lopinavir/Ritonavir, 2000)

(Pubchem: Lopinavir/Ritonavir, 2022)

Tipranavir (AptivusW)

A non-peptidic HIV-1 protease inhibitor. It inhibits the processing of Gag and Gag-Poly proteins in HIV-1 infected cells, preventing formation of mature virions

Indications: Treatment of HIV infection in combination with other antiretroviral agents

Capsule and Solution (Pubchem: Tipranavir, 2022) Toxicities: US Boxed Warning: Hepatotoxicity US Boxed Warning: Fatal and nonfatal intracranial hemorrhage Diabetes mellitus: new onset or exacerbation

(Pubchem: Tipranavir, 2022) Indications: Treatment for HIV-1 infected adult patients with strains resistant to multiple protease inhibitors when co-administered with 200 mg of ritonavir

Urticarial rash, maculopapular rash and possible photosensitivity Hyperlipidemia Immune reconstitution syndrome

FDA 32: Tipranavir (2005) Lexicomp (9) (2022) Pubchem (33) (2022)

Antiviral agents

MOA

710

Class/Drug

Chemical Disinfectants (CDC guidelines) Chemical Activity Ethyl Alcohol (60–80%)

Isopropyl Alcohol Chlorine Dioxide

Formaldehyde Glutaraldehyde Hydrogen Peroxide

It is a potent virucidal agent activity against lipophilic viruses, namely, herpes, vaccinia, and influenza virus. It also has activity against hydrophilic viruses, such as adenovirus, enterovirus, rhinovirus, and rotaviruses. It inactivates HBV, the herpes virus, HIV, rotavirus, echovirus, and astrovirus. However, it lacks activity against HAV or poliovirus It is the extensively active against lipid viruses, HBV, and the herpes virus. However, it does not have any activity against the non-lipid enteroviruses Products containing chlorine dioxide claim that it is a fungicidal, sporicidal, tuberculocidal, and virucidal. Overall, they work. (600–800 ppm bactericidal; 50–200 ppm for sanitizing); Regular household bleach is good when diluted 1:100. Contact time is important: surfaces When exposed to superoxidized water for approx. 5-min, presence of bacteria or viruses were not detected on artificially contaminated endoscopes. After 7 min, there was no HBV-DNA detected on an endoscope experimentally contaminated with HBV-positive mixed sera Formaldehyde is a bactericide, tuberculocidal, fungicide, virucide and sporicide. After 10 min, 2% of formalin inactivates all virus, however, it takes 8% of formalin to inactivates poliovirus Glutaraldehyde (>2% aqueous solutions pH 7.5–8.5) with sodium bicarbonate is very effective in killing bacteria in 2 min while killing M. tuberculosis, fungi, and viruses within 10 min Hydrogen Peroxide has germicidal, bactericidal, virucidal, sporicidal, and fungicidal properties. Within 1 min, 0.5% accelerated hydrogen peroxide, showed bactericidal and virucidal activity while within 5 min, it showed mycobactericidal and fungicidal activity. many studies have demonstrated that it takes approx. 6–8 min for 3% hydrogen peroxide to show activity against three serotypes of rhinovirus. As the concentration decreases to 1.5% and 0.75%, it takes 18–20 and 50–60 min, respectively, to show against rhinovirus.

Iodophors Phenolics

Quaternary Ammonium Compounds

A study has shown that after 10-min of exposure to hydrophilic viruses, such as coxsackie B4, echovirus 11, and poliovirus 1–12% ortho-phenylphenol, it did not show any activity against them. However, when these three viruses were exposed to 5% phenol, it showed a very potent activity against them Quaternary Ammonium Compounds are commonly used disinfectants in hospitals, day care centers, restaurants, and homes. Quaternary Ammonium Compounds are shown to be fungicidal, bactericidal, and virucidal against lipophilic (enveloped) viruses; However, they are not sporicidal, tuberculocidal or virucidal against hydrophilic (nonenveloped) viruses UV kills bacteria and viruses very easily, however, it is weak against bacterial spores Use of microwave can be fungicidal, bactericidal, sporicidal and virucidal, although it depends on the length of exposure

Antiviral agents

Ultraviolet Radiation (UV) Microwave

Other studies have shown that more than 99.9% poliovirus and HAV are inactivated in 30 min of exposure to Hydrogen Peroxide In in vitro studies, iodophors were shown to be bactericidal, mycobactericidal, and virucidal. However, some fungi and bacterial spores require prolonged contact times for Iodophors to be effective Phenolics are shown to be bactericidal, fungicidal, virucidal, and tuberculocidal.

711

712

Antiviral agents

Few more antiviral agents are available on the market for the treatment of hepatitis C virus. They include NS3/4A protease Inhibitor, NS5A replication complex inhibitors and NS5B polymerase inhibitors. Grazoprevir and paritaprevir are NS3/4A protease inhibitors that are used with other agents. Ledipasvir, a NS5A inhibitor, is used in combination with sofosbuvir, a NS5B polymerase inhibitor, for the treatment of Hepatitis C virus genotype 1, 4, 5, or 6. There is a diverse spectrum of disinfectants, sanitizing agents, biocidal agents, and antiviral drugs that are widely used today. While each category shares common side effects and toxicities, specific agents within each category can have different toxic clinical presentations. Understanding the usage, the mechanism of action, and its toxicities will help with selecting the best agents for the prevention and treatment of viral infections. The effort can also assist in selecting the appropriate drug for repurposing in the presence of novel virus infections. Describe the different forms of interferons and their medical indications.

Conclusion Virologists have made significant progress in understanding how viruses behave in our bodies, but viral diseases continue to surrender millions of lives each year. Progress made in antiviral development in the last 30 years together with the agents on hand has considerably reduced the duration of therapy and in situations has reduced the duration of illnesses. However, there is a minimum length of treatment. In order to counter this challenge, combination therapy has evolved and has shown tremendous success in the last two decades. Truncated regimens are not optimal for all patient populations and certainly not affordable in all parts of the world. Difficult to treat patients, such as those with pre-existing conditions (liver disease, renal failure, pharmacogenetics), might benefit from the newer generations of antivirals. Occasionally, even effective treatment strategies fail due to unpredictable factors in a given population, such as, the opioid epidemic. Despite the unprecedented success of antivirals, new targets and compounds can still provide prospects to improve patient care. Some antivirals often show unpredictable drug–drug interactions. Many agents show desired outcomes but produce toxic side effects on long-term treatment, which jeopardize the patient’s ability to complete the full drug regimen. Several other issues that can complicate therapeutic outcomes are drug’s very short or very long half-life, poor patient compliance, development of drug resistance during the therapy, immunocompromised status, low bioavailability, inaccessible anatomical locations like the CNS, lymphatic system, and synovial fluid, and long latency of the virus. Development of broad-spectrum antivirals has been challenging due to these confounding factors (Othumpanagat and Noti, 2021; Kim, 2021; Chakravarty and Vora, 2021; Samuel et al., 2022; Ray et al., 2022; Zhang et al., 2022).

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Antiviral agents

713

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Antiviral agents

Pubchem 32: Tenofovir (2022). https://pubchem.ncbi.nlm.nih.gov/compound/Tenofovir#section¼2D-Structure. Pubchem 33: Tipranavir (2022). https://pubchem.ncbi.nlm.nih.gov/compound/54682461#section¼2D-Structure. Pubchem 34: Trifluridine (2022). https://pubchem.ncbi.nlm.nih.gov/compound/Trifluridine#section¼2D-Structure. Pubchem 3: Adefovir (2022). https://pubchem.ncbi.nlm.nih.gov/compound/Adefovir#section¼2D-Structure. Pubchem 4: Amantadine (2022). https://pubchem.ncbi.nlm.nih.gov/compound/Amantadine. Pubchem 5: Atazanavir (2022). https://pubchem.ncbi.nlm.nih.gov/compound/148192#section¼2D-Structure. Pubchem 35: Valacyclovir (2022). https://pubchem.ncbi.nlm.nih.gov/compound/Valacyclovir#section¼2D-Structure. Pubchem 37: Zidovudine/AZT (2022). https://pubchem.ncbi.nlm.nih.gov/compound/35370#section¼2D-Structure. Pubchem: Ganciclovir n.d. https://pubchem.ncbi.nlm.nih.gov/compound/135398740 Pubchem10: Efavirenz (2022). https://pubchem.ncbi.nlm.nih.gov/compound/Efavirenz#section¼2D-Structure. Ray SD, et al. (2022) Focus on pharmacogenomics, phytonutrient–drug interactions and COVID-19 vaccines: Perspectives on ADRs, ADEs, and SEDs. Side Effects of Drugs Annual. 44: pp. xxv–lii. Elsevier. https://www.sciencedirect.com/science/article/pii/S0378608022000484. Samuel E, et al. (2022) Chapter 21—Antiviral drugs. In: Ray SD (ed.) Side Effects of Drugs Annual, vol. 44, 291–301. https://www.sciencedirect.com/science/article/pii/ S037860802200023X. Tu Y, et al. (2016) Retinal toxicity with Ritonavir. International Journal of Ophthalmology 9(4): 640–642. Published 2016 Apr 18 10.18240/ijo.2016.04.29. Viroptic (trifluridine ophthalmic); MedScape. https://reference.medscape.com/drug/viroptic-trifluridine-ophthalmic-343587#5. Zhang B, et al. (2022) Side effects, toxicity and ADRs of monoclonal antibodies in multiple organ systems. Side Effects of Drugs Annual. 44: pp. 447–460. Elsevier. https://www. sciencedirect.com/science/article/pii/S0378608022000253.

Further reading Alferon N (2023) (IFN alfa n3) 2. https://go.drugbank.com/drugs/DB00018. Alferon N (2003) (IFN alfa n3). https://www.wellrx.com/ALFERON%20N/monographs/. CDC 1: Chemical Disinfectants (2016). https://www.cdc.gov/infectioncontrol/guidelines/disinfection/disinfection-methods/index.html. FDA 17: Infergen (2010). https://www.accessdata.fda.gov/drugsatfda_docs/label/2010/103663s5069Lbl_2.pdf. FDA 27: Rimantadine (2010). https://www.accessdata.fda.gov/drugsatfda_docs/label/2010/019649s015lbl.pdf. FDA 33: Valcyte (2010). https://www.accessdata.fda.gov/drugsatfda_docs/label/2010/021304s008,022257s003lbl.pdf. FDA 34: Valtrex (2005). https://www.accessdata.fda.gov/drugsatfda_docs/label/2008/020487s014lbl.pdf. FDA 36: Zovirax (2008). https://www.accessdata.fda.gov/drugsatfda_docs/label/2005/018828s030,020089s019,019909s020lbl.pdf. Pubchem 36: Valganciclovir. (2022). https://pubchem.ncbi.nlm.nih.gov/compound/Valganciclovir#section¼2D-Structure.

Anxiolytics Parna Haghparasta, Thao Nguyena, and Sidhartha D Rayb, aDepartment of Pharmacy Practice, West Coast University, School of Pharmacy, Los Angeles, CA, United States; bDepartment of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of D. Fuentes, S.D. Ray, C.P. Holstege, Anxiolytics, Editor (s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 280–286, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00241-4.

Chemical profile Background Uses/occurrence Toxicokinetics (ADME) Mechanism of toxicity Acute and chronic toxicity Animal Human Reproductive and developmental toxicity Lactation Genotoxicity Carcinogenicity Interactions Clinical management Environmental fate and behavior Exposure standards and guidelines Geriatrics Pediatrics/neonates Conclusion References Further reading

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Abstract Benzodiazepines (BZDs), also known as anxiolytics, are prescribed to manage anxiety. BZDs are currently among the most frequently prescribed drug class worldwide. Since this is a medication, it exhibits multiple routes of exposure to humans. Some of the most popular BZDs are: Diazepam (Valium®), Flurazepam (Dalmane®), Chlordiazepoxide (Librium®), Clonazepam (Klonopin®), Alprazolam (Xanax®), Midazolam (Versed®), Triazolam (Halcion®), Lorazepam (Ativan®), Oxazepam (Serax®), Temazepam (Restoril®), Quazepam (Doral®), Clorazepate (Tranxene®) and Estazolam (ProSom ®). BZDs bind to the GABAA receptor complex, resulting in sedation, muscle relaxation, anxiolysis, and anticonvulsant effects. BZDs are metabolized predominantly in the liver by oxidation and/or conjugation. Elderly patients taking BZDs are at higher risk for falls and fractures, as well as toxic drug accumulation from poor hepatic function. The carcinogenicity of BZDs is still unclear, but a study has demonstrated possible association between developing specific cancers following long-term BZD exposure. Approximately 1 in 100 women took benzodiazepine or atypical antipsychotic medicines during pregnancy, and researchers found a small increased risk for some birth defects following use of these medicines during pregnancy. Flumazenil is the best antidote for BZD poisonings. This chapter reviews the different BZDs, their pharmacokinetics, toxicity, and mutagenicity.

Keywords Anti-anxiety; Anxiolytics; Benzodiazepines; Carcinogenicity; Dependence; Drug abuse; Flumazenil; Insomnia; Tolerance; Toxicity; Withdrawal

Key points

• •

Benzodiazepines are effective treatment for situational anxiety, chronic anxiety disorders, insomnia, alcohol withdrawal syndromes, and catatonia. BZDs are also useful adjuncts in the treatment of anxious depression and mania, and for medically ill patients. Tolerance develops over time to sedation and possibly psychomotor impairment, but not to the anxiolytic effect of benzodiazepines.

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00817-4

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Benzodiazepine misuse is not uncommon. Members of this class are usually not lethal in overdose except when ingested with other substances, such as, barbiturates, tricyclic antidepressants, alcohol and opioids. Benzodiazepines is a unique class of psychotropic medication, the mechanisms of action of which are clearly worked out, therefore, their use in clinical practice is accomplished with considerable precision.

Chemical profile

Diazepam: ValiumW; 7-Chloro-1,3 dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one Chlordiazepoxide: LibriumW; 3H-1,4-Benzodiazepin-2-amine, 7-chloro-N-methyl-5-phenyl, 4-oxide; 7-Chlor-2-methylamino-5-phenyl-3H-1,4-benzodiazepin-4-oxide Flurazepam: DalmaneW; 7-Chloro-1-[2-(diethylamino)ethyl]-5-(2-fluorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one Clonazepam: KlonopinW; 5-(2-Chlorophenyl)-7-nitro-1,3-dihydro-2H-1,4-benzodiazepin-2-one Midazolam: VersedW; 8-Chloro-6-(o-fluorophenyl)-1-methyl-4H-imidazo [1,5-a] [1,4] benzodiazepine Alprazolam: XanaxW; 8-Chloro-1-methyl-6-phenyl-4H-s-triazolo [4,3-a] [1,4]benzodiazepine Clobazam: OnfiW; SympazanW; 7-chloro-1-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-2,4-dione Triazolam: HalcionW; 8-chloro-6-(o-chlorophenyl)-1-methyl-4H-s-triazolo[4,3-a][1,4] benzodiazepine Lorazepam: AtivanW; Lorazepam IntensolW; 7-chloro-5-(2-chlorophenyl)-3-hydroxy-1,3-dihydro-1,4-benzodiazepin-2-one Oxazepam: SeraxW; 7-chloro-3-hydroxy-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one Temazepam: RestorilW; 7-chloro-1,3-dihydro-3-hydroxy-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one Quazepam: DoralW; QuaziumW; 7-chloro-5-(2-fluorophenyl)-1-(2,2,2-trifluoroethyl)-3H-1,4-benzodiazepine-2-thione Clorazepate: TranxeneW; 7-chloro-2-oxo-5-phenyl-1,3-dihydro-1,4-benzodiazepine-3-carboxylic acid Estazolam: ProSom W; 8-chloro-6-phenyl-4H-[1,2,4]triazolo[4,3-a][1,4]benzodiazepine

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Name: Benzodiazepines

Name

CAS Number

Diazepam Chlordiazepoxide Flurazepam Clonazepam Midazolam Alprazolam Clobazam Triazolam Lorazepam Oxazepam Temazepam Quazepam Clorazepate Estazolam

439-14-5 58-25-3 17617-23-1 1622-61-3 59467-70-8 28981-97-7 22316-47-8 28911-01-5 846-49-1 604-75-1 846-50-4 36735-22-5 23887-31-2 29975-16-4

•

Synonyms:

Anxiolytics

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Anxiolytics

•

719

CAS Number:

Diazepam: C16–H13–Cl–N2–O Chlordiazepoxide: C16–H14–Cl–N3–O Flurazepam: C21–H23–Cl–F–N3–O Clonazepam: C15–H10–Cl–N3–O3 Midazolam: C18–H13–Cl–F–N3 Alprazolam: C17–H13–Cl–N4 Clobazam: C16–H13–Cl–N2–O2 Triazolam: C17–H12–Cl2–N4 Lorazepam: C15–H10–Cl2–N2–O2 Oxazepam: C15–H11–Cl–N2–O2 Temazepam: C16–H13–Cl–N2–O2 Quazepam: C17–H11–Cl–F4–N2–S Clorazepate: C16–H11–Cl–N2–O3 Estazolam: C16H11ClN4

(Molecular weight 284.75) (Molecular weight 299.76) (Molecular weight 387.88) (Molecular weight 315.71) (Molecular weight 325.77) (Molecular weight 308.77) (Molecular weight 300.74) (Molecular weight 343.20) (Molecular weight 321.20) (Molecular weight 286.71) (Molecular weight 300.74) (Molecular weight 300.74) (Molecular weight 314.72) (Molecular weight 294.74)

Background Benzodiazepines (BZD) are currently among the most frequently prescribed drug class worldwide. In the United States, more than 1 in 20 people fill a prescription for it every year and it is the third most misused prescription drug class. According to 2018 American Association of Poison Control Centres’ TESS (Toxic Emergency Surveillance System), sedative-hypnotics ranked No. 1 in the top-25 fatality list. COVID19 did influence BZD use. All benzodiazepines are classified as scheduled IV. There has been a significant increase in deaths and emergency room visits that is concurrent with the growing number of prescriptions. The misuse potential of BZDs has grown overtime due to co-ingestion of other substances like opioids—leading to higher death rates from overdose. BZD misuse in the U.S. is mostly seen in young adults (ages 18–25 years old) (Votaw et al., 2019). Benzodiazepines (BZD) are a very large family of central nervous system (CNS) medications. The brand names in this category, Diazepam (Valium®), Flurazepam (Dalmane®), Chlordiazepoxide (Librium®), Clonazepam (Klonopin®), Alprazolam (Xanax®), Midazolam (Versed®), Triazolam (Halcion®), Lorazepam (Ativan®), Oxazepam (Serax®), Temazepam (Restoril®), Quazepam (Doral®), Clorazepate (Tranxene®) and Estazolam (ProSom ®) have been around for a long time while Clobazam (Onfi®) is the most recently approved BZD. All BZDs are classified as schedule IV-controlled substances in the United States, as they have the capability of causing dependence, tolerance, and abuse. Chronic exposure-associated toxic effects are secondary to the presence of the drug and metabolites and include depressed mental status, ataxia, vertigo, dizziness, fatigue, impaired motor coordination, confusion, disorientation, and anterograde amnesia. Paradoxical effects of psychomotor excitation, delirium, and aggressiveness may also occur. These chronic effects are more common in the elderly, children, and patients with renal or hepatic disease (Gaudreault et al., 1991).

Uses/occurrence Benzodiazepines are extensively used medications and can be used for management of anxiety (short term), seizure, status epilepticus, alcohol withdrawal, muscle spasms (short term), insomnia (short term), sedation, chemotherapy-induced nausea and vomiting (as prophylaxis). BZDs are preferred over barbiturates since these are less likely to produce tolerance and physical dependence and have better safety profile. Although all benzodiazepines possess anxiolytic effects, not all of them are approved by the USFDA for this indication. BZDs, specifically approved by FDA for anxiety include diazepam, alprazolam, lorazepam, chlordiazepoxide, clonazepam, oxazepam and clorazepate. When dosed appropriately, all BZDs, have similar anxiolytic and sedative effects (Melton and Kirkwood, 2017). A comprehensive list of BZDs, including those that are not specifically approved for anxiety are listed below.

Benzodiazepine

Time to peak concentration

Elimination half life

Duration of action

Formulations

Diazepam Chlordiazepoxide Clonazepam Clobazam Quazepam

Oral: 1–1.5 h 0.5–2 h 1–4 h 0.5–4 h 2h

Up to 48 h 24–48 h 30–40 h 36–42 h 39 h

Long-Acting Long-Acting Long-Acting Long-Acting Long-Acting

Oral, Intramuscular, Intravenous, Nasal (Valtoco), Rectal Gel Oral Oral Oral Oral (Continued)

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(Continued) Benzodiazepine Clorazepate Alprazolam Triazolam Lorazepam Oxazepam Temazepam Flurazepam Midazolam Estazolam

Time to peak concentration 1–2.5 h Immediate release: 1–2 h Extended release: 5–11 h Within 2 h Oral: 2 h 3h 1.5 h 30–60 min Nasal: 0.5 h IV: 3–5 min IM (adults): 0.5 h 2h

Elimination half life

Duration of action

Formulations

20–160 h 11.2 h

Long-acting Intermediate-Acting

1.5–5.5 h 12 h 8.2 h 8.8 h 2.3 h Nasal: 2.1–6.2 h IM, IV, Oral: 3–4.2 h

Intermediate-Acting Intermediate-Acting Intermediate-Acting Intermediate-Acting Short-Acting Short-Acting

Oral Oral, Nasal (Staccato—currently awaiting Phase 3 clinical trials) Oral Oral, Intramuscular, Intravenous Oral Oral Oral Nasal, Intramuscular, Intravenous, Oral (pediatrics only)

10–24 h

Intermediate

Oral

Diazepam (Valium®), which is a BZD derivative, is the frontrunner in this family because it has a variety of indications—anxiety disorders, alcohol withdrawal syndrome, acute repetitive seizures, skeletal muscle spasms, and as a sedative prior to surgical procedures, it is very well known for its psycholeptic and anxiolytic actions. It is a crystalline solid, very slightly soluble in water, soluble in alcohol, and freely soluble in chloroform. Diazepam is excreted in the breast milk in significant amounts and in sweat in nanogram quantities. Valium is contraindicated in patients with a known hypersensitivity to diazepam, pediatric patients, and patients with myasthenia gravis, severe respiratory insufficiency, severe hepatic insufficiency, pregnancy, and sleep apnea syndrome. Diazepam is not classifiable as to its carcinogenicity in humans (Group 3). The most encountered adverse events are ataxia, euphoria (3%, rectal gel), incoordination (3%, rectal gel), somnolence, rash (3%, rectal gel), and diarrhea (4%, rectal gel) (Micromedex Solutions, 2020a; USFDA, 2008). Chlordiazepoxide (Librium®) used as an anxiolytic as well as in alcohol withdrawal syndromes. As with other BZDs, chlordiazepoxide therapy is not associated with serum aminotransferase or alkaline phosphatase elevations. Clinically apparent liver injury from this BZD has been reported but is very rare. The typical anxiolytic activity of the BZDs is mediated by their ability to enhance gamma-aminobutyric acid (GABA)-mediated inhibition of synaptic transmission through binding to the GABAA receptor. Use of chlordiazepoxide in the United States began in 1960s, and it enjoyed popularity for many years. The most common side effects of chlordiazepoxide are dose related and include drowsiness, lethargy, ataxia, dysarthria, and dizziness. Tolerance develops to most of these side effects and to the anxiolytic effects (Micromedex Solutions, 2020b; USFDA, 2016b). Flurazepam (Dalmane®), is available in multiple generic forms (former brand name Dalmane®). Use of flurazepam in the United States began in 1970 for the short-term management of insomnia. Like chlordiazepoxide, flurazepam was an extensively prescribed medication for sleep but no longer has the same popularity. It is an orally administered BZD for treating insomnia. Flurazepam exposure has not been reported to be associated with serum aminotransferase or alkaline phosphatase elevations. Clinically apparent liver injury has been reported very rarely. The sedating and soporific activity (depressing physiological or psychological activity or response) of this compound follows the BZD-footprints as described previously. Safety and efficacy of flurazepam in children younger than 15 years of age have not been established. The residual effects of a single dose are more prominent after diazepam, and lorazepam, than after flurazepam and triazolam. Upon repeated administration, the effects of flurazepam may persist for over 10 h (Micromedex Solutions, 2020c; USFDA, 2007a). Clonazepam (Klonopin®) was approved as an antiepileptic agent in the United States in 1997; more than 20 million prescriptions are filled annually. Clonazepam is currently approved for management of panic disorders, absence seizures and myoclonic seizures in children as well as Lennox-Gastaut syndrome. It is effective in status epilepticus, but diazepam and lorazepam are preferable because of their longer half-lives. Side effects are dose related and include drowsiness, lethargy, ataxia, dysarthria, and dizziness. Tolerance develops to these side effects, but tolerance may also develop to the antiseizure effects. Clonazepam as with other BZDs is rarely associated with serum alanine transaminase (ALT) elevations, and clinically apparent liver injury from clonazepam is extremely rare formation. Overdose may produce many adverse effects, such as, somnolence (
37% of patients), confusion, ataxia, diminished reflexes, or coma (Micromedex Solutions, 2020d; USFDA, 2013). Midazolam (Versed®) acts at the level of the limbic, thalamic, and hypothalamic regions of the CNS through potentiation of GABA (inhibitory neurotransmitter), primarily by decreasing neural cell activity in all regions of CNS. Anxiety is decreased by inhibiting cortical and limbic arousal. Midazolam promotes relaxation through inhibition of spinal motor reflex pathway and depresses muscle and motor nerve function directly. It also acts as an anticonvulsant and augments presynaptic inhibitions of neurons, limiting the spread of electrical activity. It has greater affinity (2) for benzodiazepine receptors than does diazepam and has more potent amnesic effects. It is short acting and roughly three to four times more potent than diazepam. Therefore, it is primarily used for procedural sedation/induction of amnesia and status epilecticus. Decreased respiratory rate (23%) and apnea (15%) are the two prime adverse effects (Micromedex Solutions, 2020e; USFDA, 2017).

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Alprazolam (Xanax®) is an orally available BZD used predominantly to treat anxiety and panic disorders. Alprazolam came into use in the United States in 1981, and more than 40 million prescriptions are filled every year. Extended-release forms are also available. A nasal spray formulation of alprazolam has also been developed under the brand name (Staccato®). It is designed as a single-dose inhalation to stop a seizure that lasts for more than 5 min. Staccato is currently pending to be in Phase 3 of clinical trials. The most common side effects of alprazolam are dose related and include drowsiness, lethargy, ataxia, dysarthria, and dizziness. Tolerance develops to these side effects, but tolerance may also develop to the anxiolytic effects. Alprazolam like other BZDs is rarely associated with serum ALT elevations, and clinically discernible liver injury. Liver injury is usually mild to moderate in severity and self-limited. Among all BZDs, alprazolam is the most frequently prescribed medication in several continents. It has also been hypothesized that alprazolam is relatively more toxic than other BZDs. The paradoxical effects associated with alprazolam are aggression, rage, twitches, tremor, mania and agitation, and hyperactivity. Miscellaneous other effects include slurred speech, suicidal ideation, disinhibition, change in libido, skin rash, anterograde amnesia, concentration problems, and urinary retention (Micromedex Solutions, 2020f; USFDA, 2011). Clobazam (Onfi®) is indicated for the management of seizures in patients 2 years or older with Lennox-Gastaut syndrome (LGS). It was synthesized in 1966 with the intention of having less side effects than other BZDs while achieving greater efficacy. In the 1970s it was approved for treating anxiety in Australia and France and later marketed for seizures in 1984. In 2011, it was approved in the U.S. in the form of oral tablet and suspension, and oral film as Sympazan® to be used as adjunctive therapy for epilepsy associated LGS. Adverse effects include somnolence, lethargy, constipation, drooling, and pyrexia. Clobazam has exhibited fewer adverse reactions compared to other BZDs, while being equally efficacious in treating anxiety. That is due to a minor difference in its structure—with nitrogen atoms at the 1st and 5th positions of the BZD ring while typical BZD have them at the 1st and 4th positions (Micromedex Solutions, 2020g; Gauthier and Mattson, 2015; USFDA, 2016c). Triazolam (Halcion®), a short acting BZD. It was introduced in 1986, and used as a hypnotic to treat insomnia. Some countries withdrew it from the market in 1991 due to concerns of the frequent occurrence of adverse psychiatric effects. Triazolam is indicated for oral short-term insomnia for no more than 7–10 days. If insomnia does not resolve after 7–10 days, patients must be re-evaluated for underlying psychiatric illnesses. Side effects such as amnesia, dizziness, and drowsiness are dose-related due to triazolam’s high potency (Micromedex Solutions, 2020h; USFDA, 2016d). Lorazepam (Ativan®) was introduced in 1984 to treat anxiety disorders. It is available as both oral tablet and concentrated solution, as well as intramuscular and intravenous injection to be used prior to undergoing an anesthetic procedure. It is also approved for treating status epilepticus. Adverse effects include weakness, dizziness, and sedation being the most frequent (Micromedex Solutions, 2020i; USFDA, 2007b). Oxazepam (Serax®) is used for management of alcohol withdrawal syndrome and anxiety. It was introduced in 1968 and is available in capsule and tablet forms. The most common side effect of oxazepam is transient mild drowsiness. Psychiatric patients may also experience mild paradoxical reactions during the first 2 weeks of therapy that may be secondary to anxiety relief (Micromedex Solutions, 2020j; USFDA, 2001). Temazepam (Restoril®) was introduced in 1991 and is an oral BZD capsule that is indicated for short-term treatment of anxiety. Similar to triazolam, temazepam should only be prescribed for periods of 7–10 days. Adverse effects of temazepam are typically mild and include drowsiness, lethargy, and blurred vision (Micromedex Solutions, 2020k; USFDA, 2016a). Quazepam (Doral®) was approved in the US in 1985 for treatment of insomnia. The most common adverse reactions include drowsiness, headache, fatigue, dizziness, dry mouth. It has been shown to be effective for both acute and chronic insomnia. Close monitoring and low initial doses for elderly patients on quazepam is still recommended. It should not be used for more than 10 days for insomnia (Micromedex Solutions, 2020l; USFDA, 2016e). Clorazepate (Tranxene®) was approved in the US in 1972 under the brand name Tranxene® for the treatment of anxiety, seizures and alcohol withdrawal syndrome. It may also be used as a muscle relaxant and anticonvulsant for epilepsy. Common adverse effects include drowsiness, dizziness, and stomach upset. It is contraindicated in patients with acute narrow angle glaucoma (Micromedex Solutions, 2020m; USFDA, 2010). Estazolam (ProSom®) was approved for short term management of insomnia in patients that have difficulty falling asleep and staying asleep. This BZD is classified as sedative/hypnotic and not does not have a specific indication for anxiety (Micromedex Solutions, 2021).

Toxicokinetics (ADME) Representative examples, diazepam and lorazepam, both are well absorbed from the gut. Some of these agents are also absorbed quickly via intramuscular routes. Plasma concentration of the BZDs and their metabolites exhibits considerable inter-patient variation. Onset and duration of action vary depending on the BZD and the route of administration. BZDs are widely distributed in body tissues and cross the blood-brain-barrier (BBB). A variety of other BZDs, such as, chlordiazepoxide, clorazepate, flurazepam, quazepam, clonazepam, midazolam and triazolam, undergo variety of biotransformations and form metabolites (Melton and Kirkwood, 2017).

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Anxiolytics

BZDs are primarily metabolized via two processes: hepatic oxidation by CYP450 enzymes and/or glucuronide conjugation (with the exception of clonazepam which undergoes nitro-reduction) and are excreted renally (Melton and Kirkwood, 2017). Most BZDs that undergo oxidation can lead to accumulation of active metabolites or the parent drug, whereas BZDs that undergo conjugation that yield inactive metabolites have a better safety profile. BZDs with no active metabolites that are considered safer in elderly and those with liver disease are referred to as the “LOT” BZDs. The “LOT” benzodiazepines are lorazepam, oxazepam and temazepam. Diazepam acts within 30 min and has a long duration of action of up to 48 h. Other BZDs on the market—oxazepam and temazepam—are metabolites of diazepam. Liver CYP450 enzymes metabolize diazepam and very little unchanged drug is eliminated in the urine. Hepatic n-demethylation results in the formation of the active metabolite desmethyldiazepam (or nordiazepam). This metabolite is hydroxylated to form oxazepam, which is conjugated to oxazepam-glucuronide (minor metabolite is temazepam). The main active substances found in blood are diazepam and desmethyldiazepam. Urinary excretion of diazepam is primarily in the form of sulfate and glucuronide conjugates, and accounts for most of the ingested dose. Diazepam given intramuscularly reaches peak concentrations within approximately 2 h. Diazepam is bound to proteins at 95–98%. Diazepam is metabolized in the liver via CYP2C19 and CYP3A4, undergoing extensive oxidation and demethylation, as well as 3-hydroxylation and glucuronidation. Metabolites of diazepam include 3-hydroxydiazepam (temazepam) and 3-hydroxy-N-diazepam (oxazepam) (Micromedex Solutions, 2020a). Chlordiazepoxide is metabolized extensively in the liver and has a very long half-life. The liver injury from this BZD is probably due to a rarely produced intermediate metabolite, desmethylchlordiazepoxide. Due to its half-life of 48 h, and both of its metabolites being as active as the parent compound, it is not a commonly prescribed BZD anymore and particularly in those with liver impairment, chlordiazepoxide may take longer to metabolize. It has now been replaced by other BZDs with more favorable ADME characteristics, half-life, and tolerance (Micromedex Solutions, 2020b). Flurazepam binds to an allosteric site on GABA-A receptors. Binding potentiates the action of GABA on GABA-A receptors by opening the chloride channel within the receptor, causing chloride influx and hyperpolarization. Flurazepam (Dalmane®), is metabolized extensively in the liver to its active metabolite, which is then excreted in the urine. About 70% of flurazepam is converted to metabolites during its first pass through the intestine and liver. There are at least six nonconjugated metabolites of the drug, of which five are more potent in executing various activities than is flurazepam. The active metabolites in flurazepam are desalkylflurazepam and N-1-hydroxyethylflurazepam. Both hydroxyethyl flurazepam (the major metabolite) and N-desalkyl flurazepam are active metabolites found in the blood. The N-desalkyl metabolite is slowly excreted in the urine as the conjugated form. Not only are they an order of magnitude more potent than is flurazepam in animal tests, but also, they reach higher concentration in plasma than does flurazepam (Micromedex Solutions, 2020c). Clonazepam (Klonopin®), on the other hand, is rapidly and completely absorbed after oral administration; bioavailability is
90%. Plasma concentrations of clonazepam peak within 1–4 h after oral administration (approx. 85% remain bound to plasma proteins). It is completely metabolized, with less than 2% unchanged, and excreted in the urine. Biotransformation occurs mainly by reduction of the 7-nitro group to the 4-amino derivative. This derivative can be acetylated, hydroxylated, and glucuronidated. CYP3A is thought to be involved in oxidation and reduction of Klonopin. The elimination half-life of clonazepam is typically 30–40 h. Clonazepam kinetics are dose independent throughout the dosing range. There is no evidence that it induces its own metabolism or that of other drugs in humans. To avoid excess accumulation of metabolites, caution should be exercised in the administration of this drug to patients with impaired renal function (Micromedex Solutions, 2020d). Midazolam (Versed®) is primarily metabolized in the liver and gut by human CYP3A4 to its pharmacologic active metabolite, a-hydroxymidazolam, followed by glucuronidation of the a-hydroxyl metabolite that is present in unconjugated and conjugated forms in human plasma. The a-hydroxymidazolam glucuronide is then excreted in urine. No significant amount of parent drug or metabolites is extractable from urine before beta-glucuronidase and sulfatase deconjugation, indicating that the urinary metabolites are excreted mainly as conjugates. Midazolam is also metabolized to two other minor metabolites: 4-hydroxy metabolite (about 3% of the dose) and 1,4-dihydroxy metabolite (about 1% of the dose) are excreted in small amounts in the urine as conjugates (Micromedex Solutions, 2020e). Alprazolam (Xanax®) is metabolized in rat and human liver by P4503A1 and P4503A4, respectively, to 4-hydroxy alprazolam (4-OHALP, pharmacologically less active) and alpha-hydroxy alprazolam (alpha-OHALP, pharmacologically more active), and relative amounts of alpha-OHALP formed in the brain have been found to be higher than in liver. The half-life of alprazolam is approximately 12 h, and it is primarily excreted through kidneys (80%) with 7% excreted through feces (Micromedex Solutions, 2020f). Clobazam (Onfi®) is metabolized in the liver to N-desmethylclobazam and is primarily excreted in the urine. Clobazam is demethylated by both CYP3A4 and CYP2C19 to be activated to N-desmethylclobazam and is further metabolized to inactive 40 -hydroxy-N-clobazam by CYP2C19. Mutations in CYP2C19 may lead to more adverse effects in those who carry the mutated allele due to increased concentrations of N-desmethylclobazam—a mutation that would primarily affect those in Asian populations. Those with the  1/ 2 genotype (intermediate metabolizers) have shown to have 2 times more the concentration of N-desmethylclobazam while concentrations in those with the  2/ 2 genotype (poor metabolizers) are 3–5 times higher (Pharmacogenetics considerations). Clobazam’s protein binding is approximately 80–90% and N-desmethylclobazam’s is approximately 70%. Approx. 2% of clobazam is excreted in urine, 1% in feces and remaining unchanged (Micromedex Solutions, 2020g). Triazolam (Halcion®) undergoes glucuronidation that leads to inactive metabolites that are primarily excreted in urine. Hydroxylation by CYP4503A is the initial step in triazolam metabolism. Medications that strongly inhibit CYP3A oxidative metabolism are contraindicated (drug interactions likely). Triazolam is contraindicated in pregnant patients, and it undergoes glucuronidation that

Anxiolytics

723

leads to inactive metabolites that are primarily excreted in urine. Hydroxylation by CYP4503A is the initial step in triazolam metabolism. Medications that strongly inhibit CYP3A oxidative metabolism are contraindicated (Micromedex Solutions, 2020h). Lorazepam (Ativan®) undergoes hepatic rapid conjugation into its major metabolite, lorazepam-glucuronide as 3-O-phenolic glucuronide and is excreted in urine. Accumulation in plasma is rare—even up to 6 months of administration—making it one of the safer BZDs for long-term use. Unlike most other BZDs, its metabolism does not involve oxidation by CYP450 enzymes and would not be affected by hepatic disease. Lorazepam taken orally reaches peak concentrations in approximately 2 h and has a 90% bioavailability. It possesses a protein binding capacity of 85–91% (Micromedex Solutions, 2020i). The metabolism of oxazepam involves glucuronidation in the liver. Its major metabolite, oxazepam-glucuronide, is inactive and is excreted in the urine. Oxazepam’s kinetics makes it preferable for BZD pharmacotherapy for elderly patients. There was no difference in its metabolism in the elderly compared to younger patients. Its elimination half-life is approximately 8.6 h, and the metabolites are found in urine after renal excretion (Micromedex Solutions, 2020j). Temazepam (Restoril®) is metabolized via conjugation and has only 8% first pass metabolism. Its major and minor metabolites are the O-conjugate of temazepam and the O-conjugate of N-desmethyl derivative. Both metabolites are inactive and do not accumulate in the plasma. The half-life for both metabolites is approximate 2 h. Temazepam is renally excreted, and men have been shown to have greater clearance of temazepam compared to women (Micromedex Solutions, 2020k). Quazepam (trx. insomnia) is extensively metabolized in the liver and slowly eliminated due to the long elimination half-life of the parent compound (25–41 h). It forms two active metabolites, 2-oxoquazepam (28–43 h) and N-desalkyl-2-oxoquazepam (72–84 h). It is excreted renally (31%) and in feces (23%). In elderly patients, the elimination half-life of the metabolite N-desalkyl2-oxoquazepam is twice as long in than it is in younger adults (Micromedex Solutions, 2020l). Clorazepate (trx. Anxiety, alcohol withdrawl syndrome) is metabolized by the liver (active metabolite, nordiazepam, and inactive metabolite, 3-hydroxynordiazepam). Nordiazepam has an approximate half-life of 2 days and is longer in elderly patients (
120 h). The protein binding of clorazepate’s active metabolite is 97–98%. When nordiazepam is hydroxylated to 3-hydroxynordiazepam, it is excreted in the urine (Micromedex Solutions, 2020m). Estazolam has a linear kinetics with 93% protein bound in the plasma. Estazolam is extensively metabolized to two metabolites, 4-hydroxy estazolam (major) and 1-oxo-estazolam. Estazolam is eliminated via hepatic metabolism and is largely eliminated in the urine. The main pharmacotherapeutics of the drug comes from the parent drug (Micromedex Solutions, 2021).

Mechanism of toxicity The advantage of using BZDs is that they have a larger therapeutic index compared to barbiturates. BZDs require greater dose increments than barbiturates to reach CNS depression effects such as anesthesia and coma (Suddock and Cain, 2020). The exact sites and mode of action of the BZDs have not been elucidated. Pharmacologically, these agents act on the BZD binding site on the chloride channels of GABA, an inhibitory neurotransmitter receptor in the CNS. Action on this receptor increases the frequency of the chloride channel opening, which hyperpolarizes the cells and prevents nerve firing and stimulation. CNS depression results from this effect via depression of the reticular activating system and spinal cord reflexes. Allosteric interaction of central BZD receptors with GABAA receptors and subsequent opening of chloride channels are involved in eliciting the CNS effects of the drugs. These drugs appear to act at the limbic (behavioral and emotional responses), thalamic (body’s information relay station), and hypothalamic (helps control the pituitary gland and regulates many body functions) levels of the CNS. In usual therapeutic doses, BZDs appear to have very little effect on the autonomic nervous system, respiration, or cardiovascular system (Bergmann et al., 2013).

Acute and chronic toxicity Animal The toxic effects of diazepam, chlordiazepoxide, and nitrazepam on the spermatozoa of mice disclosed different types of abnormalities involving both shape and size of the sperm head. The incidences of abnormal sperm heads were significantly high after diazepam treatment. All three drugs produced maximum effects at week 6 (Pubchem). LD50 values for some of the well-known BZDs reported in the literature (Pubchem):

Diazepam LD50 rat oral 352–710 mg kg−1 LD50 mouse oral 48–278 mg kg−1 LD50 mouse i.p. 47–220 mg kg−1 LD50 rat i.p. 46500 mg kg−1 LD50 rabbit i.v. 9 mg kg−1 Chlordiazepoxide LD50 rat oral 548 mg kg−1 LD50 rat i.p. 143 mg kg−1 LD50 rat i.v. 165 mg kg−1 LD50 mouse oral 260 mg kg−1 LD50 mouse i.p. 207 mg kg−1 LD50 mouse i.v. 95 mg kg−1

(Continued )

724

Anxiolytics (Continued) Flurazepam LD50 mouse i.p. 290 mg kg−1 LD50 mouse oral 870 mg kg−1 LD50 mouse i.v. 84 mg kg−1 Clonazepam LD50 mouse i.p. 13300 mg kg−1 LD50 rat i.p. 14200 mg kg−1 LD50 mouse oral 2 g kg−1 LD50 rat oral >15 g kg−1 Midazolam LD50 mouse i.m. 50 mg kg−1 LD50 mouse i.v. 50 mg kg−1 LD50 rat oral 215 mg kg−1 LD50 rat i.v. 75 mg kg−1 LD50 rat i.m. 50 mg kg−1 Alprazolam LD50 mouse oral 1020 mg kg−1 LD50 rat oral >2000 mg kg−1 LD50 mouse i.p. 540 mg kg−1 LD50 rat i.p. 610 mg kg−1 i.p., intraperitoneally; i.v., intravenous; i.m., intramuscular; s.c., subcutaneous.

A number of repeated-dose studies have been carried out. In general, toxic effects have not been remarkable. In a 3-month study in rats and a 6-month study in dogs, some increase in liver size was seen, together with an increase in blood cholesterol; in the dogs an elevation of plasma alanine aminotransferase activity was observed. There was no increase in tumor frequency after feeding diazepam to rats and mice for 104 and 80 weeks, respectively.

Human IV administration of even therapeutic doses of BZDs may produce apnea and hypotension in certain recipients. BZDs primarily target the CNS, causing respiratory depression and consciousness. The BZDs have a low order of toxicity unless ingested with other CNS depressants. Deep coma is rare. The BZDs have been known to cause dose-dependent adverse CNS effects. BZD overdosage may result in sedation, somnolence, impaired coordination, slurred speech, confusion, coma, and diminished reflexes. Diplopia, dysarthria, ataxia, and intellectual impairment are not uncommon. Hypotension, seizures, respiratory depression, and apnea may also occur. Although cardiac arrest has been reported, death from overdose of BZDs in the absence of concurrent ingestion of alcohol and other CNS depressants is rare. Outcomes are usually favorable when used alone. Deep coma and other manifestations of severe CNS depression are rare (Gaudreault et al., 1991). Anxiolytic and possibly paradoxical CNS stimulatory effects of BZDs are postulated to result from release of previously suppressed responses (disinhibition). After usual doses of BZD for several days, the drugs cause a moderate decrease in rapid eye movement (REM) sleep. REM rebound does not occur when the drugs are withdrawn. Stages 3 and 4 sleep is markedly reduced by usual doses of the drugs; the clinical importance of these sleep stage alterations has not been established. The onset of impairment of consciousness is relatively rapid in BZD poisoning. Onset is more rapid following larger doses and with agents of shorter duration of action. The most common and initial symptom is somnolence. This may progress to coma Grade I or Grade II following very large ingestions. Severe anaphylactic reactions following intravenous administration of diazepam have been reported. Intravenous formulations of lorazepam and diazepam may contain propylene glycol, which may contribute to these types of reactions. When large doses of lorazepam have been infused chronically, there are multiple reports of the development of a syndrome consisting of a hyperosmolar state with metabolic acidosis and cardiovascular compromise. This syndrome has been attributed to propylene glycol, the diluent in lorazepam. Effects are merely extensions of pharmacological effects. Nausea, vomiting, dizziness, drowsiness, miosis, and gastric distention may be seen (Haybarger et al., 2016). There is a broad spectrum of signs and symptoms associated with acute BZD toxicity. Lethargy, ataxia, nystagmus, diplopia, amnesia, slurred speech, confusion, hypotonia, hypotension, hypothermia, coma, respiratory depression, and even death is not uncommon. Rarely, paradoxical excitation may occur at lower doses. Toxic doses for each agent have not been clearly established. CNS depression and respiratory depression may occur with toxic doses, especially when co-ingested with other sedative and hypnotic medications, or substances like ethanol. The elderly and very young children are more susceptible to the CNS depressant action. Intravenous administration of even therapeutic doses of BZDs may produce apnea and hypotension. In severe cases, hypotension, coma, and hypothermia may be seen. However, the autonomic nervous system is not compromised by the co-ingestion of those agents and adverse extrapyramidal side effects are not produced. Because the brainstem—which controls the respiratory center has low-density BZD binding sites, without co-ingestion of other substances, BZDs rarely demonstrate incidence of respiratory compromise (Suddock and Cain, 2020).

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Reproductive and developmental toxicity Generally, BZDs are not recommended in pregnancy due to risk of fetal toxicity and congenital malformations. Anxiety, the most frequent condition for which benzodiazepines are prescribed, occurs commonly, and BDZ use has considerably increased during pregnancy. Use of benzodiazepines during pregnancy is associated with preterm delivery and low birth weight. They have metabolites that cross the placenta, distribute into the amniotic fluid in animals and humans and are highly bound to plasma proteins. For example, the concentration of diazepam in fetal circulation has been reported to be equal to or greater than the maternal plasma concentration and midazolam has been detected in maternal venous serum, umbilical venous serum, umbilical arterial serum, and amniotic fluid in humans. BZDs may cause fetal toxicity when administered to pregnant women, but potential benefits from use of the drug may be acceptable in certain conditions despite the possible risks to the fetus. However, flurazepam, triazolam and temazepam are contraindicated and patients who are pregnant or planning to become pregnant should not take those drugs. In the case of clobazam and oxazepam, credible, well-controlled studies in pregnant women are not available while animal studies have shown fetal abnormalities and toxicity with clobazam (Gauthier and Mattson, 2015; Ram and Gandotra, 2015; NCBI, 2012; USFDA, 2008; 2016c). BZDs should be avoided during the first trimester and at delivery. Malformation and CNS dysfunction have been described in infants born to mothers using BZDs during pregnancy. BZDs can induce withdrawal symptoms as neonatal side effects include “floppy infant syndrome.” Symptoms of “floppy infant syndrome” in new-borns may consist of increased sensitivity to cold, low muscle development, and neurological depression that is observed at the time of delivery. Withdrawal symptoms (bradycardia, seizures, irritability, etc.) have been observed from 3 weeks to few months after birth (Ram and Gandotra, 2015). Both animal data and human epidemiological studies suggest that BZDs are teratogens. Although its teratogenic effects have been observed with BZD use, overall exposure to BZDs during pregnancy was rare—according to the National Birth Defects Prevention Study published in 2019. The study used logistic regression to analyze data from 1997 to 2011 and investigate the relationship between birth defects and using BZDs. It was found that BZD exposure was uncommon and was more common in educated non-Hispanic white mothers in the control group who smoked and were also on antidepressants (Tinker et al., 2019).

Lactation Generally, BZDs have shown low incidence of adverse effects in lactation—especially with short-term use. With long-term use, signs and symptoms should be monitored in infants. Therefore, in lactation, only low-dose, short-acting BZDs such as alprazolam and lorazepam should be preferred for treatment. There is no wait time needed to resume breastfeeding after administration of short-acting BZDs while in longer-acting BZDs like diazepam, mothers should wait 6–8 h to resume (Ram and Gandotra, 2015; Drugs and Lactation Database (LactMed), 2019, 2020). Liquid chromatography/tandem mass spectrometry was used to develop a quantification method for human breast milk and was applied to measure alprazolam concentrations. Generally, a relative infant dose of 10 mm) compared to long chrysotile fibers of all widths (ECHA, 2021). Although there are studies that demonstrated the association of lung cancer mortality with the exposure to thin long fibers, there are also assays where it is not observed this correlation across fiber groups and sizes (ECHA, 2021).

Other cancers The evidence of pharynx, esophagus, stomach and colorectum cancers is not clear enough, so it is needed more studies to clarify its correlation with asbestos exposure. IOM indicated that there was not a higher risk of pharynx cancer associated with more extreme exposures. The IOM observed that the case-control studies were inconsistent, and there was not enough evidence for a dose-response relationship. In addition, IOM estimates a relative risk of esophagus cancer from any asbestos exposure. Current studies show a non-significant increase in mortality from esophagus cancer in men after asbestos exposure (ECHA, 2021).

Asbestos

827

Organ toxicity (pulmonary, neurotox, hepatotox, kidney, endocrine disruption, Skin, etc.) Asbestos inhalation can lead to pulmonary fibrosis or also called asbestosis. This disease often begins at the subpleural level at the respiratory bronchioles with a latency of 20–30 years, which is often mild but can progress to diffuse pulmonary fibrosis. Diagnosis of asbestosis is supported by the presence of asbestos bodies in the alveoli or interstitium. In addition, one assay fixed an unofficial lower limit for the recognition of asbestosis in >10 fibers/year (Omland et al., 2018). There is also consistent evidence of the fibrogenic effect of all types of asbestos (Hadrup et al., 2019). Pleural plaques are the most common asbestos-related pleural lesions. In people exposed to asbestos daily, it is more probable to suffer pleural abnormalities. However, there is needed more studies to confirm the health significance. Asbestos exposure is correlated with retroperitoneal fibrosis, a rare condition which need more studies to be understood. In addition, there is evidence of cardio- and cerebrovascular diseases after asbestos exposure, affecting the right ventricular function of the heart which results in a cardiac insufficiency (ECHA, 2021).

Interactions There are several evidences about the multiplying effect of tobacco smokes and asbestos exposure. The biological mechanism of lung carcinogenesis consist of additional mutations, an increase in genetic changes and a release of growth factors, producing cell proliferation and carcinoma (Klebe et al., 2019). In 14 case-control studies between 1985 and 2010 in Europe and Canada, there were 17,705 lung cancer cases and 21,813 controls with detailed information on smoking habits (never smoker, former smoker, current smoker) as well as cumulative asbestos exposure data. It was demonstrated an additive effect between smoking and asbestos exposure for all major lung cancer subtypes among men and for squamous-cell lung carcinoma among women. In addition, it shows a high risk of lung cancer and its subtypes at relatively low levels of cumulative exposure (>0.5 ff/mL-years), which persisted at least up to 40 years after last exposure.

Toxicogenomics There is a direct interaction between asbestos fibers and chromosomes. It produces a disruption of chromosomal structure which is inherit by daughter cells, double strand breaks in DNA along with intrachromosomal deletion and DNA mutations. There is also evidence of lagging chromosomes or the trisomy of chromosome 11, latest found in in six out of eight Syrian hamster embryo cell lines derived immediately after asbestos exposure (Gupta et al., 2022). In Table 2 are reported some examples of gene interaction compiled in CTD (Comparative Toxicogenomics Database). Table 2

Top interacting genes involved in the exposure to asbestos (CTD).

Gene

Inter-actions

Examples of interaction

Species

References

TNF

104

99

IL6

72

CXCL8

71

MAPK1

56

MAPK3

53

TGFB1

46

SPP1

44

JUN

37

EGFR

34

Homo sapiens Mus musculus Homo sapiens Homo sapiens Homo sapiens Rattus norvegicus Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens Mus musculus Mus musculus Rattus norvegicus Homo sapiens Homo sapiens Homo sapiens Mus musculus Mus musculus Homo sapiens Homo sapiens Rattus norvegicus

Huang et al. (2012) Fukagawa et al. (2008) Pietarinen-Runtti et al. (1996)

IL1B

Increases expression of TNF mRNA Increases expression of TNF protein Increases activity of SOD2 protein Increases susceptibility to Vit K3, Increases expression of IL1B mRNA Increases expression of IL1B protein Increases secretion of IL1B protein Increases expression of NOS2 mRNA Increases expression of IL6 protein Increases expression of IL6 mRNA Increases expression of CXCL8 mRNA Increases expression of CXCL8 protein Decreases expression of MAPK1 mRNA Increases activity of MAPK1 protein Increases activity of MAPK3 protein Increases phosphorylation of MAPK1 protein Decreases expression of TGFB1 mRNA Increases expression of TGFB1 mRNA Increases activity of SPP1 protein Increases expression of SPP1 mRNA Increases expression of JUN mRNA Decreases phosphorylation of JUN protein Decreases phosphorylation of EGFR protein increases expression of EGFR mRNA

Zhang et al. (1993) Gavett et al. (2016) Zhang et al. (1993) Choe et al. (1998) Leivo-Korpela et al. (2012) Huang et al. (2012) Huang et al. (2012) Comar et al. (2014) Huang et al. (2012) Chernova et al. (2017) Chernova et al. (2017) Dai and Churg (2001) Kim et al. (2012) Pass et al. (2005) Rehrauer et al. (2018) Shukla et al. (2001) Baldys et al. (2007) Baldys and Aust (2005) Cyphert et al. (2012)

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Asbestos

Clinical management Epidemiologically, asbestos fibers could cause asbestosis (pulmonary fibrosis), pleural abnormalities such as effusion and plaques, and malignancies such as mesothelioma and bronchogenic carcinoma. To diagnose disease provoked by asbestos two main methods are performed, chest radiographs and pulmonary function tests. Other procedures sometimes used are: bronchoalveolar lavage, blood analyses, lung biopsy or colon cancer screening (ATSDR, 2018). Asbestosis, the main pulmonary disease caused by asbestos, has some treatments to reduce the symptoms, such as the administration of an oxygen therapy or a pulmonary rehabilitation. Antibiotics, chemotherapy, radiotherapy and surgical interventions are used to treat oncogenic effects (NHS Choices, 2019; PubChem, 2022).

Environmental fate and behavior Environmental asbestos contamination can affect the genetic diversity of natural populations, which would lead to differential mortality of genotypes. In long term, this will decrease genetic diversity resulting in a decline in population size (Ben-Shlomo and Shanas, 2011). Chrysotile is the only form still produced in US because of its properties of strength, thermostability and low-cost properties. The industrial production using asbestos has caused an airborne to the environment and its deposition in soil or water. The concentration in US cities is approximately 2–4 ng/m3 in areas with a far asbestos source. Vehicular traffic or mining operations can resuspend asbestos fibers but, phytoremediation is a current strategy to avoid the dispersion of asbestos fibers with a stabilization of topsoil (Gonneau et al., 2017). There is not volatilization or degradation in soil or water surface. Inhalation of ambient air and ingestion of contaminated water or food cause the asbestos exposure to general population. It is also present in some talc powders and vermiculite in low levels, so it is considered a low exposure risk (PubChem, 2022).

Ecotoxicology In a plant health study, asbestos itself does not affect germination and root growth (Gonneau et al., 2017). There is also no evidence in asbestos bioaccumulation in aquatic organisms (PubChem, 2022). Juvenile green sunfish (Lepomis cyanellus) and larval coho salmon (Oncorhynchus kisutch) were exposed to levels of chrysotile asbestos found in the Great Lakes basin (Belanger et al., 1986). Different mortality between control and treated groups were not observed, but behavioral stress effects appeared in both species.

Exposure standards and guidelines Asbestos is considered a potential carcinogen and NIOSH recommends that exposures should be reduced to the lowest concentration. National Archives and Records Administration’s Electronic Code of Federal Regulations (ECFR) propose an exposure standard of 104; 218 >176 in positive mode ([M + H]+), (EURL Data Pool, 2023) (Table 1).

Background Atrazine is used as a herbicide to protect many crops, including corn, sorghum, tea, sugarcane, and various fruit crops. In fact, atrazine is one of the most widely used herbicides in the world. However, is a restricted-use pesticide, only available for registered professionals. The European Commission excluded atrazine from a re-registration process in 2003. Their residues and metabolites had the potential to persist in fields and surface waters for many years (Singh et al., 2018). It is a carcinogen that can disrupt the endocrine system of frogs at concentrations detected in the environment (Hayes et al., 2006). The World Health Organization (WHO) has indicated a 2 mg/L guideline. In the European Union, the Groundwater Directive sets a precautionary quality standard of 0.1 mg/L for all pesticides (European Union, 2006).

Uses Atrazine (1912-24-9) is a commonly used herbicide to control pre- and post-emergence grasses and broadleaf weeds. For decades, atrazine has been one of the most heavily used agricultural herbicides in the United States and is used extensively worldwide to control pre- and postemergence grasses and broadleaf weeds. The annual usage of atrazine in US agriculture has been ranked number two among conventional pesticides (behind glyphosate) based on active ingredients used (70–80 pounds annually) over approximately the last decades (Grube et al., 2011). Atrazine is primarily used on various field crops such as corn (approximately 75% of the field corn acreage grown in the United States), sorghum, sugarcane, wheat, and nuts (Singh et al., 2018). It is also used in industrial applications.

Environmental fate and behavior Atrazine is highly persistent in the environment (half-life: 41–231 days) due to its resistance to abiotic hydrolysis (stable at pH 5, 7, and 9) and to direct aqueous photolysis (stable under sunlight at pH 7) (Singh et al., 2018; Chang et al., 2022). The main proposed degradation pathways are dealkylation, dechlorination, and deamination. Dealkylation and dechlorination are seen as the dominant mediating pathways in biological and chemical processes, respectively (Rowe et al., 2007). Moreover, the compound has a limited volatilization potential and bioaccumulation. In addition, is moderately susceptible to aerobic biodegradation, which is the main route of dissipation of atrazine. A colder climate makes atrazine even more persistent in the environment (Kookana et al., 2010). Atrazine does not get adsorbed to soil particles strongly and therefore has a relatively high potential to contaminate ground and surface waters despite its moderate solubility in water (Chang et al., 2022).

Exposure and exposure monitoring The ocular and dermal routes are the primary exposure pathways. Ingestion and inhalation are other possible routes of exposure.

Atrazine

847

Toxicokinetics Atrazine has the potential to be absorbed through the gastrointestinal tract, through the intact skin, and by inhalation. The percentage absorbed through a dermal application is increased with time and decreased with dose. However, the majority (65–95%) of atrazine applied on the skin was recovered in the water used for washing or was found associated with the skin at the site of exposure. Once absorbed, it follows first-order distribution kinetics and undergoes N-dealkylation and dechlorination of the triazine ring. The highest level of atrazine is noted in the red blood cell followed by the lungs, liver, spleen, and kidneys. The half-life of atrazine in the tissues is 2 g/kg. The dermal LD50 and inhalation LC50 (1 h) values in rats are >3 g/kg and 700 mg m3, respectively. The oral LD50 values in mice and rabbits are 1.8–4.0 g/kg and 750 mg/kg, respectively. Atrazine was negative in primary skin irritation and dermal sensitization tests. Rats exposed to high dosages of atrazine showed changes in arousal and motor function, dyspnea, hypothermia, and spasms. With lethal oral dosages, death occurred rapidly (within 12–24 h). A 90-day subchronic oral study in rats and a 21-day dermal study in rabbits provided no observed adverse effect levels (NOAELs) of 3.3 and 100 mg/kg day, respectively.

Human There have been relatively few recorded cases of human poisonings among occupationally exposed workers in the United States. One death was reported following extensive dermal exposure. Dermal exposure to atrazine can cause skin rash, erythema, blisters, and edema. Ocular irritation, chest pains, a feeling of tightness in the chest, nausea, and dizziness have also been reported after ocular, oral, or inhalation exposures.

Chronic toxicity (or exposure) Animal About 40% of rats died, with signs of respiratory distress and paralysis of the limbs following oral administration of 20 mg/kg day atrazine for 6 months. Structural and chemical changes were noticed in various organs including the heart, liver, and ovaries. Dogs treated with 3.65 mg/kg day atrazine in the diet for 52 weeks showed various treatment-related cardiac changes including EKG alterations, moderate to severe atrial dilation, and enlarged hearts. Histopathology revealed cardiac myolysis and focal atrophy. The NOAEL for atrazine in dogs of both sexes was established at about 5 mg/kg day.

Human The carcinogenic effect of high doses of atrazine noted in female Sprague-Dawley rats is a strain-, sex-, and tissue-specific response that appears to have low biological relevance in humans due to the differences in the endocrine control of reproductive senescence (Simpkins et al., 2011). While considerable debate still exists on the carcinogenic potential of atrazine, the potential impact of chronic exposure on human health appears more likely on reproduction and development via effects on endocrine signaling and not carcinogenicity (Jowa and Howd, 2011).

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Atrazine

Immunotoxicity Atrazine has been reported to cause changes in immunological parameters in adult female B6C3F1 mice. Acute atrazine (100–300 mg/kg, ip) exposure decreased the percentage of CD4+ and CD8+ T cells in the thymus and the nucleated cells in the spleen. Splenic NK cell activity and the IgG1/IgG2a responses to KLH were all decreased by atrazine in a dose-dependent manner (Pruett et al., 2009). Both acute and chronic (28 days) exposure to atrazine caused changes in white blood cell count and lymphocyte phenotypes in the blood and spleen (Pruett et al., 2009). NK cell activity was decreased to a greater extent with chronic exposure. Prenatal/lactational exposure has been shown to cause changes in immune function in adult offspring in a gender- and age-specific manner (Stoker et al., 1999).

Reproductive toxicity Prenatal developmental toxicity study in female Sprague-Dawley rats exposed to atrazine during gestation day 6 through day 15 demonstrated maternal and developmental NOAELs of 25 mg/kg day. Using both Long-Evans, and Sprague-Dawley female rats, atrazine was found to disrupt the hypothalamic control of pituitary-ovarian function as indicated by alteration in LH and prolactin serum levels. Females treated with atrazine (75, 150, and 300 mg/kg day for 21 days by gavage) showed irregular cycles and repetitive pseudopregnancies (Cooper et al., 2000). Maternal exposure to atrazine during lactation may result in prostatitis in adult male offspring due to atrazine’s suppressive effect on suckling-induced prolactin release (Stoker et al., 1999).

Genotoxicity Based on evidence derived from a large array of assays such as bacterial reverse mutation test, mammalian bone marrow chromosome aberration test, dominant lethal assay, and UDS assay, atrazine was concluded to lack mutagenic potential.

Carcinogenicity When CD-1 mice of both sexes were treated with atrazine in the diet at dose levels of 10–3000 ppm daily for 91 weeks, no treatment-related increase in tumor incidence was noted when compared with controls. Neither male nor female Fischer 344 rats nor male Sprague–Dawley rats were given atrazine at a maximum tolerated dose in the diet for 24 months exhibited any increase in the incidence of tumors of any type. However, mammary tumors were observed in female Sprague-Dawley rats after 24 months of dietary administration of high levels of atrazine. The mechanism of tumorigenesis in female Sprague-Dawley rats appears to be mediated via suppression of the LH ‘surge,’ leading to persistent elevation of estrogen and prolactin. The differences in response to the carcinogenic effect of high levels of atrazine observed in mice versus rats and male (and of primary importance, humans) versus female Sprague-Dawley rats appear due to differences in endocrine control mechanisms affecting reproductive senescence and the development of the mammary tumors during aging (Simpkins et al., 2011).

Clinical management Treatment is symptomatic.

Ecotoxicology Atrazine, with acute oral LD50 values of 4900 mg/kg, is practically nontoxic to birds. The compound is slightly toxic to aquatic animals. Rainbow trout and midge, the most sensitive freshwater species tested, have 96 and 48 h LC50 values of 5.3 and 0.72 mg/L, respectively. The most sensitive marine animals tested were the spot fish (Leiostomus xanthurus) with a 96 h LC50 value of 8.5 mg/L and the copepod (Acartia tonsa) with a 96 h LC50 value of 88 mg/L. Atrazine has low acute toxicity potential in bees (oral LD50 >100 mg per bee) (Pesticide Properties Database (PPDB), 2023; Turner, 2015). The reported toxicity to several aquatic species is shown in Table 2.

Exposure standards and guidelines The United States Environmental Protection Agency (USEPA) and the Joint Meeting on Pesticides define an acute reference dose (RfD) of 0.1 mg/kg day, and chronic RfD is 0.018 mg/kg day. The ACGIH TLV–TWA for atrazine is 2 mg/m3 (inhalable particulate

Atrazine Table 2

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Ecological toxicity (Turner, 2015; Pesticide Properties Database (PPDB), 2023).

Mammals (Rat)—Acute oral LD50 (mg/kg) Birds Acute LD50 (mg/kg) Bobwhite quails Mallard ducks Coturnix japonica Earthworms (Eisenia fetida)—LC50 (mg/kg) Honeybees (Apis spp.) mg/bee (worst case from 24, 48, 72 h) Fish Acute 96-h LC50 (mg/L) Rainbow trout Oncorhynchus mykiss Aquatic invertebrates (Daphnia magna) Acute at 48-h EC50 (mg/L) Chronic 21 day NOEC (mg/L)

1869–3090 940 >2000 4237 78 mg/kg soil >100 (contact and oral) 11 4.5 85 0.25

matter). Moreover, the USEPA defines the maximum contaminant level for atrazine in drinking water as 3 mg/L (Code of Federal Regulation, 40 CFR 141.61, 2013), while the European Union establish the 0.1 mg/L level (European Union, 2020).

References Breckenridge CB, Werner C, Stevens JT, and Sumner DD (2008) Hazard assessment for selected symmetrical and asymmetrical triazine herbicides. In: Le Baron HM, McFarland JE, and Burnside OC (eds.) The Triazine Herbicides, pp. 387–398. The Netherlands: Elsevier. Chang J, Fang W, Chen L, Zhang P, Zhang G, Zhang H, Liang J, Wang Q, and Ma W (2022) Toxicological effects, environmental behaviors and remediation technologies of herbicide atrazine in soil and sediment: A comprehensive review. Chemosphere 307: 136006. Code of Federal Regulation, 40 CFR 141.61 (2013) Maximum Contaminant Levels for Organic Contaminants. https://www.govinfo.gov/content/pkg/CFR-2013-title40-vol24/pdf/CFR2013-title40-vol24-sec141-61.pdf (accessed April 2023). Cooper RL, Stoker TE, Tyrey L, Goldman JM, and McElroy WK (2000) Atrazine disrupts the hypothalamic control of pituitary-ovarian function. Toxicological Sciences 53(2): 297–307. EURL Data Pool (2023) https://www.eurl-pesticides-datapool.eu/Member/Compound (accessed March 2023). European Union (2006) Directive 2006/118/EC of the European Parliament and of the Council of 12 December 2006 on the Protection of Groundwater against Pollution and Deterioration, OJ L 372, 27.12. pp. 19–31, 32006L0118 European Union (2020) Directive 2020/2184 of the European Parliament and of the Council of 16 December 2020 on the Quality of Water Intended for Human Consumption (recast), L435/1. Grube A, Donaldson D, Kiely T, and Wu L (2011) Pesticides Industry Sales and Usage: 2006 and 2007 Market Estimates. U.S. Environmental Protection Agency. Hayes TB, Stuart AA, Mendoza M, Collins A, Noriega N, Vonk A, Johnston G, Liu R, and Kpodzo D (2006) Characterization of atrazine-induced gonadal malformations in African clawed frogs (Xenopus laevis) and comparisons with effects of an androgen antagonist (Cyproterone Acetate) and exogenous estrogen (17b-Estradiol): Support for the demasculinization/ feminization hypothesis. Environmental Health Perspectives 114(supplement 1): 134–141. Jowa L and Howd R (2011) Should atrazine and related chlorotriazines be considered carcinogenic for human health risk assessment? Journal of Environmental Science and Health. Part C 29: 91–144. Kookana R, Holz G, Barnes C, Bubb K, Fremlin R, and Boardman B (2010) Impact of climatic and soil conditions on environmental fate of atrazine used under plantation forestry in Australia. Journal of Environmental Management 91(12): 2649–2656. Pesticide Properties Database (PPDB) (2023) Agriculture and Environment Research Unit (AERU), University of Hertfordshire, Atrazine. http://sitem.herts.ac.uk/aeru/ppdb/en/Reports/ 43.htm (accessed February 2023). Pruett SB, Fan R, Zheng Q, and Schwab C (2009) Patterns of immunotoxicity associated with chronic as compared with acute exposure to chemical or physical stressors and their relevance with regard to the role of stress and with regard to immunotoxicity testing. Toxicological Sciences 109(2): 265–275. Rowe AM, Brundage KM, and Barnett JB (2007) Developmental immunotoxicity of atrazine in rodents. Basic and Clinical Pharmacology and Toxicology 102: 139–145. Simpkins JW, Swenberg JA, Weiss N, Brusick D, Eldridge JC, Stevens JT, Handa RJ, Hovey RC, Plant TM, Pastoor TP, and Breckenridge CB (2011) Atrazine and breast cancer: A framework assessment of the toxicological and epidemiological evidence. Toxicological Sciences 123: 441–459. Singh S, Kumar V, Chauhan A, Datta S, Wani AB, Singh N, and Singh J (2018) Toxicity, degradation and analysis of the herbicide atrazine. Environmental Chemistry Letters 16(1): 211–237. Stoker TE, Robinette CL, and Cooper RL (1999) Maternal exposure to atrazine during lactation suppresses suckling-induced prolactin release and results in prostatitis in the adult offspring. Toxicological Sciences 52(1): 68–79. Tchounwou PB, Wilson BA, Ishaque AB, and Schneider J (2001) Atrazine potentiation of arsenic trioxide-induced cytotoxicity and gene expression in human liver carcinoma cells (HepG2). Molecular and Cellular Biochemistry 222(1–2): 49–59. Turner JA (2015) The Pesticide Manual: A World Compendium, 17th edn. Bath, UK: British Crop Protection Council & Royal Society of Chemistry.

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Atropine Amanda Lofton Scotta and Timothy J Wiegandb, aAllston, MA, United States; bUniversity of Rochester Medical Center and Strong Memorial Hospital, Rochester, NY, USA © 2024 Elsevier Inc. All rights reserved. This is an update of A.L. Scott, Atropine, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 339–341, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00695-3.

Chemical profile Background Uses Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Exposure standards and guidelines References

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Abstract Atropine is the racemic mixture of L- and D-hyoscyamine and possesses 50% of the antimuscarinic potency of L-hyoscyamine. Atropine is used in the management of sinus bradycardia with hemodynamic instability and in the treatment of peptic ulcer disease, irritable bowel syndrome, urinary incontinence, and organophosphate and carbamate poisoning. The mainstay of exposure or overdose treatment is supportive care. Physostigmine, a cholinesterase inhibitor, can be given to patients to reverse signs and symptoms of the anticholinergic toxidrome.

Keywords Anticholinergic; Antimuscarinic; Belladonna alkaloid; Datura; Mydriatic-cycloplegic; Nerve gas antidote; Urinary antispasmodic

Glossary Bradycardia Resting heart rate less than 60 beats per minute (general). Cycloplegia Paralysis of the ciliary muscle of the eye, resulting in a loss of accommodation. Mydriasis Dilation of the pupil (general). Racemic Relating to a chemical compound that contains equal quantities of dextrorotatory and levorotatory forms and therefore does not rotate the plane of incident polarized light. Stenosis Constriction or narrowing of a duct or passage; a stricture. Toxidrome Set of clinical signs that suggest a specific class of poisoning.

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Key points

•

Atropine is the racemic mixture of L- and D-hyoscyamine it has antimuscarinic effects and is used in the management of sinus bradycardia with hemodynamic instability and in the treatment of peptic ulcer disease, irritable bowel syndrome, urinary incontinence, and organophosphate and carbamate poisoning.

Abbreviations IP LD50

Intraperitoneal Median lethal dose, lethal dose 50%

Chemical profile

• • • • • •

Name: Atropine CAS RN: 51-55-8 Synonyms: AtroPen; Atropine sulfate; Benzeneacetic acid, alpha-(hydroxymethyl)-8-methyl-8-azabicyclo(3,2,1)oct-3-yl ester, endo-(+-)- ; dl-Hyoscyamine; Tropine tropate Chemical/Pharmaceutical/Other Class: Antimuscarinic agent; Anticholinergic agent Chemical formula: C17-H23-N-O3 Chemical structure:

Background Atropine is the racemic mixture of L- and D-hyoscyamine and possesses 50% of the antimuscarinic potency of L-hyoscyamine. Atropine is derived from components of the Belladonna plant and is also present in other plants from the Solanaceae family. Women in ancient times often dripped the plant’s juices into their eyes, causing mydriasis and thereby enhancing their beauty. In Italian, Belladonna translates to ‘beautiful lady.’ In the United States, the atropine autoinjector has been in use since 1973 for the treatment of exposures to chemical warfare nerve agents and insecticides (Mercey et al., 2012).

Uses Atropine is used in the management of sinus bradycardia with hemodynamic instability and in the treatment of peptic ulcer disease, irritable bowel syndrome, urinary incontinence, and organophosphate and carbamate poisoning (Bucaretchi et al., 2012). It is also present in ophthalmic preparations to induce mydriasis and cycloplegia (Chia et al., 2012). Atropine is often administered preoperatively to decrease secretions.

Environmental fate and behavior Free atropine is only slightly soluble in cold water. It melts at 115  C but decomposes upon boiling. Environmental monitoring of atropine is not routinely performed by regulatory bodies. Hazardous short term degradation products are not likely to occur. Accidental environmental exposure may occur through intentional or unintentional ingestion of toxic plants of the Solanaceae family, such as the Deadly nightshade (Gaire and Subedi, 2013).

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Exposure and exposure monitoring Ingestion is the most frequent route of exposure. Exposure can also occur following instillation of eye solutions and via subcutaneous, intramuscular, intravenous, and inhalation routes. Accidental overdosage and adverse effects may occur when atropine is administered for the treatment of organophosphate or carbamate insecticide poisoning (Robenshtok et al., 2002). Atropine exposure can also occur from ingestion of plants containing atropine such as Datura stramonium (Gaire and Subedi, 2013). Monitoring of atropine levels in blood or bodily fluids is not employed in standard exposure management. Environmental monitoring of atropine is not common practice.

Toxicokinetics In therapeutic doses, atropine is well absorbed. In toxic doses, absorption may be prolonged secondary to decreased gastric motility. Atropine is 18% bound to plasma protein and its volume of distribution ranges from 2 to 4 l kg−1. Atropine is metabolized in the liver to tropic acid, tropine, esters of tropic acid, and glucuronide conjugates. Elimination follows first-order kinetics. Approximately 30–60% is excreted unchanged in the urine. Drug clearance is dependent on glomerular filtration. The elimination half-life is 2–3 h in adults but may be longer in children.

Mechanism of toxicity Atropine competitively antagonizes acetylcholine at the neuroreceptor site. Atropine prevents acetylcholine from exhibiting its usual action but does not decrease acetylcholine production. Cardiac muscle, smooth muscle, and the central nervous system are most affected by the antagonism of acetylcholine.

Acute and short-term toxicity Animal Animals are at risk for anticholinergic poisoning from atropine. Toxicity is similar to that in humans. Gastrointestinal decontamination and supportive care should be employed. There is interspecies variability and variability based on route of exposure to atropine. The rat LD50 oral is 500 mg kg−1; the LD50 IP is 280 mg kg−1, and the LD50 IV is 73 mg kg−1.

Human Overdosage of atropine results in signs and symptoms consistent with the anticholinergic toxidrome. Signs and symptoms have been reported following the ingestion of as few as four to five drops of 4% ocular atropine solution (Hoefnagel, 1961). Patients exhibit warm, flushed, and dry skin as a result of peripheral vasodilatation. Mydriasis occurs due to antagonism of acetylcholine in the muscles of the iris. Urinary retention, thirst, delirium, hallucinations, and decreased bowel sounds may occur. Tachycardia with ensuing hypertension can appear secondary to vagal blockade. The anticholinergic toxidrome may be delayed and can occur in cycles. Severe intoxications may progress to seizures, coma, and arrhythmias. Ingestion of plant material containing atropine causes anticholinergic toxicity similar to pharmaceutical atropine.

Chronic toxicity Animal A juvenile pygmy sperm whale (Kogia breviceps) was treated with several doses of atropine to relieve symptoms of pyloric stenosis. The animal developed signs and symptoms of anticholinergic toxicity including hyperexcitability, ascending weakness, vomiting, and aspiration of seawater. Symptoms resolved after administration of physostigmine.

Human Chronic ingestion of greater than therapeutic amounts of atropine may produce symptoms of the anticholinergic toxidrome.

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Atropine

Immunotoxicity Atropine is not known to cause specific immunotoxic effects. As with exposure to any agent, humans may be at risk for hypersensitivity reactions to atropine.

Reproductive and developmental toxicity Atropine carries FDA pregnancy category C rating. Atropine may be used during pregnancy as a preoperative, preanesthetic agent to reduce salivation and bronchial secretions. Atropine rapidly crosses the human placenta. In one study of 44 healthy pregnant women, a maximum umbilical to maternal vein ratio of 1.27 was observed 6 min after administration of 0.01 mg kg−1 intravenously. The corresponding umbilical and maternal vein atropine levels were 22 and 17 nmol l−1 respectively. Intramuscular injection produced lower concentrations. Another study administered labeled atropine intravenously prior to delivery to quantify placental transfer and fetal distribution of the drug. The concentrations in the umbilical vein 1 and 5 min after injection were 12% and 93%, respectively, of the corresponding maternal value. Concentrations in the umbilical artery were approximately 50% of those in the umbilical vein during the same period. Studies have shown that administration of atropine to a pregnant woman during the last trimester can mask the effects of vagal stimulation on the fetal heart, producing tachycardia within 5 to 30 min after injection. Limited data have shown that atropine can suppress fetal breathing, although fetal hypoxia has not been observed. Atropine could reduce lower esophageal sphincter pressure enough to predispose the newborn to aspiration. Uterine contractility does not appear to be significantly affected by atropine, perhaps due to a decrease in the sensitivity of muscarinic receptors on myometrial tissue during pregnancy. Multiple prospective cohort studies have monitored tens of thousands of mother-child pairs in which the mother was exposed to atropine during pregnancy. Overall, these data do not support an association between the use of atropine and congenital defects. Anticholinergic agents can inhibit lactation in animals, via inhibition of growth hormone and oxytocin secretion. These agents can also reduce serum prolactin in non-nursing women, but decreased prolactin levels in an established nursing mother should not affect her ability to breastfeed. In theory, long-term use of atropine may reduce milk production or milk letdown, but a single systemic or ophthalmic dose should not interfere with breastfeeding.

Genotoxicity Studies of the mutagenicity of atropine in Escherichia coli have demonstrated no effects on cell DNA. Atropine is not expected to cause chromosome abnormalities at clinically relevant doses.

Carcinogenicity No large-scale or long-term studies have been performed examining the carcinogenicity of atropine. In one isolated rat study, 25 mg kg−1 administered twice daily for 5 days to virgin female rats demonstrated no carcinogenic effect.

Clinical management Basic and advanced life support measures should be utilized as necessary for atropine exposure. Gastric decontamination procedures should be employed based on the patient’s history and current symptomatology. Activated charcoal can be given to adsorb atropine if patients present shortly after an oral ingestion of products containing atropine. The mainstay of treatment is supportive care. Physostigmine, a cholinesterase inhibitor, can be given to patients to reverse signs and symptoms of the anticholinergic toxidrome. However, the administration of physostigmine may be contraindicated in the patient who has also been exposed to a tricyclic antidepressant, or another agent known to cause QRS interval widening on the EKG. Extracorporeal elimination measures are ineffective in atropine overdose for toxin removal.

Ecotoxicology Significant intra- and interspecies variation exists among animals surrounding the toxicity of atropine and other belladonna alkaloids. Rabbits, guinea pigs, and birds are resistance to their effects due to internal detoxifying mechanisms. Horses, cattle, and goats are also reportedly resistant to ingested but not injected belladonna. Pigs are susceptible to effects from ingested belladonna compounds. Signs of acute intoxication are similar in all mammalian species.

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Exposure standards and guidelines Published guidelines or occupational standards for atropine exposure are not commonly employed. Exposures are managed with first aid measures and symptomatic and supportive care.

See also: Anticholinergics; Carbamate pesticides; G-series nerve agents; Organophosphorus compounds; Poisoning emergencies in humans

References Bucaretchi F, Prado CC, Branco MM, et al. (2012) Poisoning by illegal rodenticides containing acetylcholinesterase inhibitors (chumbinho): A prospective case series. Clinical Toxicology 50: 44–51. https://doi.org/10.3109/15563650.2011.639715. PMID: 22175788. Chia A, Chua WH, Cheung YB, Wong W, Lingham A, Fong A, and Tan D (2012) Atropine for the treatment of childhood myopia: safety and efficacy of 0.5%, 0.1%, and 0.01% doses (Atropine for the Treatment of Myopia 2). Ophthalmology 119: 347–354. https://doi.org/10.1016/j.ophtha.2011.07.031. PMID: 21963266. Gaire BP and Subedi L (2013) A review on the pharmacological and toxicological aspects of Datura Stramonium L. Journal of Integrative Medicine 11(2): 73–79. https://doi.org/ 10.3736/jintegrmed2013016. PMID: 23506688. Hoefnagel D (1961) Toxic effects of atropine and homatropine eye drops in children. The New England Journal of Medicine 264: 168–171. https://doi.org/10.1056/ NEJM196101262640403. PMID: 13714893. Mercey G, Verdelet T, Renou J, Kliachyna M, Baati R, Nachon F, Jean L, and Renard P (2012) Reactivators of acetylcholinesterase inhibited by organophosphorus nerve agents. Accounts of Chemical Research 45(5): 756–766. https://doi.org/10.1021/ar2002864. PMID: 22360473. Robenshtok E, Luria S, Tashma Z, and Hourvitz A (2002) Adverse reaction to atropine and the treatment of organophosphate intoxication. The Israel Medical Association Journal 4: 535–539. PMID: 12120467.

Relevant website https://www.ncbi.nlm.nih.gov/books/NBK470551/ :StatPearls—atropine toxicity online resource National Library of Medicine (2022)

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Avermectin Mohsen Amin and Navid Mirmohammadsadegh, Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of G.M. Fent, Avermectin, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 342–344, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00099-3.

Chemical profile Introduction Background Uses/occurrence (e.g. industrial, therapeutic – for drugs, side products, and substances that have no use) Ivermectin Selamectin Doramectin Nemadectin Moxidectin Eprinomectin Milbemycin Abamectin Exposure (sources, routes and pathways, media, human exposure, typical levels) and exposure monitoring Toxicokinetics: Absorption, distribution, metabolism, and elimination (ADME) Absorption Distribution Metabolism Elimination Mechanism of toxicity Acute and short-term toxicity (e.g. animal, human; oral, inhalation, dermal) Neurotoxicity Immunotoxicity Reproductive and developmental toxicity Carcinogenicity and genotoxicity Organ toxicity (pulmonary, neurotox, hepatotox, kidney, endocrine disruption, skin, etc.) Interactions Toxicogenomics Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines PubChem URL Conclusion/summary/outlook References Further reading

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Abstract Avermectins are a group of naturally sourced or synthetic compounds used mainly to control nematodes, ticks, mites, lice, grubs in domestic animals and humans. The avermectin derivatives include ivermectin, abamectin, doramectin, eprinomectin, nemadectin, moxidectin, selamectin, and milbemcycin. Ivermectin is the most widely used avermectin derivative. The mechanism of action, toxicity, acceptable daily intake, and maximum residue level have been discussed in this entry.

Keywords Anthelmentic; Avermectins; Insecticidal; Ivermectin; Pharmacokinetics

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Key points

• • •

Avermectins are anthelmentic and insecticidal agents Avermectins are a group of macrocyclic lacton derivatives Toxicity of avermictins have been well studied and acceptable daily intake has been set by regulatory bodies

Chemical profile AVMs chemical profile is presented in Table 1.

Introduction Avermectins (AVMs) are family members of macrocyclic lactone derivatives, which possess strong anthelmintic and insecticidal activities (Pitterna et al., 2009). They were first discovered in 1975 from an actinomycete, Streptomyces avermitilis isolated from a Japanese soil sample (Campbell et al., 1983; Fisher and Mrozik, 1992). Examples of avermectins include ivermectin, abamectin, doramectin, eprinomectin, nemadectin, moxidectin, and selamectin, emamectin benzoate and milbemcycin with chemical structures similar to macrolides and macrocyclic polyene antibiotics. Abamectin (a mixture of 80% avermectin B1a and 20% avermectin B1b) is used as an insecticide, whereas the semisynthetic derivatives of avermectin B1a, emamectin benzoate, and ivermectin, are used as insecticides and antiparasitic agents in humans and animals, respectively (Stevens et al., 2010).

Background The discovery and characterization of the first avermectin family member from a soil sample happened in the late 1970s at the Kitasato Institute, Tokyo, Japan and since then continued to become an important drug in both animal and human health as an antiparasitic agent. Avermectins are fermentation byproducts of Streptomyces avermitilis, an actinomycete isolated from the soil.

Uses/occurrence (e.g. industrial, therapeutic – for drugs, side products, and substances that have no use) Ivermectin Among the family of the AVMs, ivermectin is the most popular and well-studied member. Ivermectin can be administered through different routes in humans and animals because of various pharmaceutical dosage forms available in the market. It has been approved to treat onchocerciasis and strongyloidiasis for human use (El-Saber Batiha et al., 2020). The use of ivermectin is not limited to its approved applications, and the off-label prescription of ivermectin includes ecto- (lice and scabies) and endo-parasitic effects against ascariasis, demodicosis, gnathostomiasis, hookworm-related cutaneous larva migrans, Mansonella ozzardi, Mansonella streptocerca, trichuriasisa, and Wuchereria bancrofti. It is used to treat ectoparasites, prevents heartworm in animals, and is a microfilaricide agent (Plumb, 2018). Besides its parasiticidal activity, ivermectin has been investigated as a potential antiviral drug for the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), during COVID-19 pandemic (Caly et al., 2020). In vitro data showed that the ivermectin dose used for inhibiting replication of SARS-CoV-2 exceeded tolerable dose for humans approved by the US food and drug administration. In another study, Guzzo et al. (Guzzo et al., 2002) showed that 120 mg of ivermectin was safe in humans. However, the peak plasma concentration was significantly lower than the effective concentration of ivermectin that inhibits replication of SARS-CoV-2 as was shown in Caly et al. (2020) study. Yet, the bioavailability of ivermectin in the lungs at effective antiviral concentrations kept hope alive for further investigations (Lespine et al., 2005).

Selamectin Selamectin is an AVM antiparasitic agent applied topically to dogs and cats to treat parasites such as fleas, heartworms, ear mites, scabies, and ticks (Bishop et al., 2000). In cats, fleas, heartworms, ear mites, hookworms, and roundworms are among the parasites treated with selamectin. Notoedric mange, nasal mites, and cordylobiolosis have all been treated with selamectin (Fisher and Shanks, 2008).

Doramectin In ruminants, pigs, and cattle, doramectin has been used as injectable drug (10 mg/mL) or topically (5 mg/mL).

Table 1

The IUPAC name, synonyms, CAS, molecular formula, and chemical structure of Ivermectin, selamectin, moxidectin, eprinomectin, nemadectin, Abamectin, milbemycin, and doramectin. IUPAC Name

Synonyms

CAS

Molecular Formula

Ivermectin

(1R,4S,5’S,6R,6’R,8R,10E,12S,13S,14E,16E,20R,21R,24S)-6’-[(2S )butan-2-yl]-21,24-dihydroxy-12-[(2R,4S,5S,6S)-5-[(2S,4S,5S,6S )5-hydroxy-4-methoxy-6-methyloxan-2-yl] oxy-4-methoxy-6-methyloxan-2-yl]oxy-5’,11,13,22tetramethylspiro[3,7,19-trioxatetracyclo[15.6.1.14,8.020,24] pentacosa-10,14,16,22-tetraene-6,2’-oxane]-2-one

IVERMECTIN Ivermectin B1a 70288-86-7 Dihydroavermectin B1a 22,23-Dihydroavermectin B1a avermectin H2B1a UNII-91Y2202OUW 71827-03-7 70161-11-4 Ivermectin Component B1a

71827-03-7

C48H74O14

Selamectin

(1R,4S,5’S,6R,6’S,8R,10E,12S,13S,14E,16E,20R,21Z,24S )-6’cyclohexyl-24-hydroxy-21-hydroxyimino-12-[(2R,4S,5S,6S )-5hydroxy-4-methoxy-6-methyloxan-2-yl]oxy-5’,11,13,22tetramethylspiro[3,7,19-trioxatetracyclo[15.6.1.14,8.020,24] pentacosa-10,14,16,22-tetraene-6,2’-oxane]-2-one

selamectin 220119-17-5 UNII-A2669OWX9N UK-124,114 Revolution 165108-07-6 A2669OWX9N Stronghold NCGC00095066-01 UK-124114

220119-17-5

C43H63NO11

Doramectin

(1’R,2R,3S,4’S,6S,8’R,10’E,12’S,13’S,14’E,16’E,20’R,21’R,24’S )-2cyclohexyl-21’,24’-dihydroxy-12’-[(2R,4S,5S,6S )-5[(2S,4S,5S,6S)-5-hydroxy-4-methoxy-6-methyloxan-2-yl]oxy4-methoxy-6-methyloxan-2-yl]oxy-3,11’,13’,22’-tetramethylspiro [2,3-dihydropyran-6,6’-3,7,19-trioxatetracyclo[15.6.1.14,8.020,24] pentacosa-10,14,16,22-tetraene]-2’-one

117704-25-3 UNII-KGD7A54H5P Dectomax KGD7A54H5P UK-67,994 Doramectina Doramectine Doramectinum

117704-25-3

C50H74O14

Chemical Structure

Avermectin

Compound

(Continued )

859

(Continued)

860

Table 1

IUPAC Name

Synonyms

CAS

Molecular Formula

Nemadectin

(1R,4S,4’S,5’S,6R,6’S,8R,10E,13R,14E,16E,20R,21R,24S )-4’,21,24trihydroxy-5’,11,13,22-tetramethyl-6’-[(E )-4methylpent-2-en-2-yl]spiro[3,7,19-trioxatetracyclo [15.6.1.14,8.020,24]pentacosa-10,14,16,22-tetraene-6,2’oxane]-2-one

F 28249alpha LL-F 28249alpha LL-F 28249beta LL-F28249gamma

102130-84-7

C36H52O8

Moxidectin

(1R,4S,4’E,5’S,6R,6’S,8R,10E,13R,14E,16E,20R,21R,24S )-21,24dihydroxy-4’-methoxyimino-5’,11,13,22-tetramethyl-6’-[(E )-4methylpent-2-en-2-yl]spiro[3,7,19-trioxatetracyclo [15.6.1.14,8.020,24]pentacosa-10,14,16,22-tetraene-6,2’oxane]-2-one

113507-06-5 ProHeart 6 UNII-NGU5H31YO9 NGU5H31YO9

113507-06-5

C37H53NO8

Eprinomectin

N-[(2S,3R,4R,6S)-6-[(2S,3R,4S,6R )-6[(1’R,2R,3S,4’S,6S,8’R,10’Z,12’S,13’S,14’Z,16’Z,20’R,21’R,24’S )21’,24’-dihydroxy-3,11’,13’,22’-tetramethyl-2’oxo-2-propan-2-ylspiro[2,3-dihydropyran-6,6’-3,7,19trioxatetracyclo[15.6.1.14,8.020,24]pentacosa-10,14,16,22tetraene]-12’-yl]oxy-4-methoxy-2-methyloxan-3-yl] oxy-4-methoxy-2-methyloxan-3-yl]acetamide

Eprinex Avermectin B1, 4”(acetylamino)-4”deoxy-, (4”R)Eprinomectin [USAN:USP: INN] MK 397 ZINC306122586

123997-26-2

C49H73NO14

Chemical Structure

Avermectin

Compound

Milbemycin

(1R,4S,5’S,6R,6’R,8R,10E,13R,14E,16E,20R,24S )6’-ethyl-24-hydroxy-21-hydroxyimino-5’,11,13,22tetramethylspiro[3,7,19-trioxatetracyclo[15.6.1.14,8.020,24] pentacosa-10,14,16,22-tetraene-6,2’-oxane]-2-one

Milbemycin A4 5-oxime UNII-6ZWJ394628 6ZWJ394628 Milbemycin A4 oxime 93074-04-5

Abamectin

(1’R,2R,3S,4’S,6S,8’R,10’E,12’S,13’S,14’E,16’E,20’R,21’R,24’S )-2butan-2-yl-21’,24’-dihydroxy-12’-[(2R,4S,5S,6S )-5[(2S,4S,5S,6S)-5-hydroxy-4-methoxy-6-methyloxan-2-yl] oxy-4-methoxy-6-methyloxan-2-yl]oxy-3,11’,13’,22’tetramethylspiro[2,3-dihydropyran-6,6’-3,7,19-trioxatetracyclo [15.6.1.14,8.020,24]pentacosa-10,14,16,22-tetraene]-2’-one; (1’R,2R,3S,4’S,6S,8’R,10’E,12’S,13’S,14’E,16’E,20’R,21’R,24’S )21’,24’-dihydroxy-12’-[(2R,4S,5S,6S)-5-[(2S,4S,5S,6S )-5hydroxy-4-methoxy-6-methyloxan-2-yl] oxy-4-methoxy-6-methyloxan-2-yl]oxy-3,11’,13’,22’tetramethyl-2-propan-2-ylspiro[2,3-dihydropyran-6,6’3,7,19-trioxatetracyclo[15.6.1.14,8.020,24]pentacosa10,14,16,22-tetraene]-2’-one

Agrimek Vertimec Affirm Avomec Zephyr

93074-04-5

C32H45NO7

C95H142O28

All data from PubChem.

Avermectin 861

862

Avermectin

Nemadectin Nemadectin is given to dogs at doses of 0.2 to 0.6 mg/kg to treat gastrointestinal helminths (Gao et al., 2010).

Moxidectin Moxidectin is another AVM administered orally, topically, and subcutaneous injection at higher doses to prevent heartworms in horses and ruminants (Pérez et al., 2001). Moreover, it can be used to treat heartworms in cats and dogs (Plumb, 2018).

Eprinomectin Eprinomectin is used to treat a variety of gastrointestinal roundworms in cattle. Also, topical administration of eprinomectin can manage psoroptic mange in horses and ear mites in rabbits (Ulutas et al., 2005). There are data showing that eprinomectin can be prescribed to control Toxocara canis in dogs (Kozan et al., 2008).

Milbemycin Milbemycin is another family member of AVMs approved as a once-a-month heartworm preventative (Dirofilaria immitis) and hookworm control (Ancylostoma caninum). Like ivermectin, milbemycin is used to treat systemic demodicosis in dogs, but the treatment costs more than ivermectin therapy. Milbemycin has been shown to effectively prevent Dirofilaria immitis larval infection in cats (Plumb, 2018). It is also provided as a 0.1% otic solution for the treatment of ear mites (Merola and Eubig, 2012).

Abamectin Abamectin, also known as avermectin B1, is a pesticide used to control insects like mites and cockroaches. Also, studies indicate that abamectin has activity against the flour beetle (Tribolium confusum) and the sheep blowfly larva (Lucilia cuprina) (Dybas, 1989).

Exposure (sources, routes and pathways, media, human exposure, typical levels) and exposure monitoring AVMs may be present in animal droppings. Accidental exposure can occur through the respiratory, cutaneous, and oral routes, as well as through a stab to the injection site.

Toxicokinetics: Absorption, distribution, metabolism, and elimination (ADME) Some factors determine the pharmacokinetics of ivermectin. Species, weight, body physiology, nutrition, drug preparation vehicles, and animal delivery routes are only a few variables. However, the route of administration and the vehicle employed in drug preparation are the most critical determinants influencing the drug’s bioavailability and half-life. The oral route is the only approved for ivermectin administration in humans, and parenteral formulations are only approved for veterinary use.

Absorption In healthy volunteers that received 12 mg ivermectin as solutions, tablets, or capsules, the oral solution had almost twice the systemic availability as either of the solid forms. Nonetheless, the absorption rate was identical (Edwards et al., 1988). Moreover, co-administration of ivermectin tablets with orange juice diminished the oral bioavailability of the medication. The reduced bioavailability may be related to the inhibitory effect of fruit juices on drug transporters (Vanapalli et al., 2003).

Distribution Ivermectin is broadly distributed within the body due to its high lipophilicity. The compound is detected in fat, skin, subcutaneous fascia, nodules, and worm fragments. Fat had the highest concentrations and most persistent levels among tissues, while skin, nodular tissues, and worms had similar values, with the lowest concentrations was detected in the subcutaneous fascia (Baraka et al., 1996). In addition, Ivermectin binds tightly to plasma proteins (Klotz et al., 1990).

Metabolism Human liver microsomes majorly metabolize ivermectin by cytochrome P450. The CYP450 3A4 is the major isoform responsible for the metabolism of ivermectin (Zeng et al., 1998).

Avermectin

863

Elimination Studies on the elimination pathway of ivermectin revealed that a significant amount of the medication and its metabolites are excreted in the feces (González Canga et al., 2008).

Mechanism of toxicity The therapeutic effect of AVMs depends on their high-affinity binding to invertebrates’ specific glutamate-gated chloride channels. This binding increases the entrance of chloride ions to cells, causing hyperpolarization and flaccid paralysis of the parasite (Bloomquist, 2003; Chalivendra, 2021). The therapeutic doses used to control infections seem non-toxic in mammals (Srivastava et al., 2020). AVMs have a low affinity for other mammalian ligand-gated channels. They do not usually cross the blood-brain barrier (BBB), and mammalian glutamate-gated chloride channels are found only in the brain and spinal cord. Thus, these chemical compounds are relatively safe for mammals (Omura and Crump, 2014). Other family members may interact with other ligand-gated chloride channels, such as those gated by the neurotransmitter gamma-aminobutyric acid (GABA). Despite the changes in structure, potency, and safety, the process by which AVMs elicit their pharmacology and toxicity is identical (El-Saber Batiha et al., 2020).

Acute and short-term toxicity (e.g. animal, human; oral, inhalation, dermal) Several studies stated that ivermectin, the most extensively used AVM, is generally safe and that most mammalian species tolerate ivermectin exposure with a large margin of safety. However, only a few animals are sensitive to ivermectin toxicity. Usually, toxicity signs occur in animals because of overdose or in ivermectin-sensitive breeds of dogs. Clinical signs of toxicity observed in animal studies. Animal research conducted on rats found ataxia, sluggishness, ptosis. Also, hypersalivation, ataxia, blindness, coma, respiratory failure, tremors, mydriasis, anorexia, and death were observed in dog investigations (Trailovic and Nedeljkovic, 2011). Toxicity in humans is mainly linked to a hypersensitivity reaction (Mazzotti reaction) triggered by dying microfilaria. Some signs are fever, pruritus, arthralgia, myalgia, postural hypotension, edema, lymphadenopathy, headache, sore throat, and stomach discomfort. Rashes, dizziness, seizures, dyspnea, ataxia, and urticaria are other clinical indications that might be encountered (Campillo et al., 2021). Besides mazzotti reaction, some case reports indicate intoxication with AVMs in the medical literature. The neurological, gastrointestinal, and respiratory systems appear to be the most significant clinical symptoms. Patients may experience nausea, vomiting, salivation, diarrhea, and dizziness at first exposure. Aspiration pneumonia, respiratory failure, hypotension, and coma are some of the more severe signs that have been observed. There have also been reports of rhabdomyolysis (Chung et al., 1999). Based on a report, the most severe toxicity occurring in patients who ingested in the range of 67-227 mg/kg and usually taking doses under 40 mg/kg causes either mild or no toxicity (Roberts and Reigart, 2014).

Neurotoxicity Caution should be taken to investigate clinical trials on ivermectin antiviral activities. The BBB in viral infections may be disrupted because of inflammation, allowing ivermectin to cross the BBB and gain access to the central nervous system (CNS) where it can exert neurotoxic effect. Furthermore, ivermectin is metabolized by CYP3A4 that is constantly inhibited by COVID-19 antiviral drugs such as ritonavir and cobicistat. The two drugs have been shown to impair BBB and co-administration of ivermectin and antiviral drugs can cause neurotoxicity (Chaccour et al., 2020).

Immunotoxicity There are limited data available about the immunotoxicity of AVMs, but the recent study indicates that Ivermectin can be immunotoxic to macrophages (Zhang et al., 2022). In addition, AVMs exposure could up-regulate autophagy in a dose-timedependent manner in the pigeon spleen (Liu et al., 2015).

Reproductive and developmental toxicity Documentations reported that selamectin had no adverse effects on reproduction in adult males and females, either in cats or dogs (Novotny et al., 2000; Krautmann et al., 2000). Also, in the study, moxidectin (at three times the therapeutic dose) had no negative impacts on cow reproductive performance (Rae et al., 1994).

864

Avermectin

Carcinogenicity and genotoxicity There are limited data available about the carcinogenicity of AVMs. Based on the risk assessment report of The Food Safety Commission of Japan, Abamectin was observed to be neither carcinogenic nor genotoxic (Japan, 2016).

Organ toxicity (pulmonary, neurotox, hepatotox, kidney, endocrine disruption, skin, etc.) There are only few studies available about the organ toxicity of the AVMs. Researchers suggest that the liver may be harmed due to Ivermectin toxicity (Dong et al., 2020).

Interactions Although no major interaction has been reported, Ivermectin (Systemic) may enhance the anticoagulant effect of vitamin K antagonists (González Canga et al., 2008). Also, data suggest that other medications that inhibit p-glycoprotein should be used with caution while using AVMs (Plumb, 2018).

Toxicogenomics Animals with lower expression of P-glycoprotein or P-glycoprotein deficient species are more susceptible to AVMs toxicity. Therefore, low doses can cause severe toxic reactions (El-Saber Batiha et al., 2020). Also, human subjects who showed severe toxicity had some non-sense mutations in ATP-binding cassette subfamily B member 1 (ABCB1) transporter (also known as MDR1 and P-glycoprotein) genes (Baudou et al., 2020).

Clinical management There are no specific antidotes to treat AVMs intoxication. Thus, supportive care and decontamination must be provided in AVMs intoxication. If intoxication occurs through skin contact, remove skin contamination with soap and water. Also, eyes can be irrigated with clean water or saline. If the medication is ingested, gastrointestinal decontamination must be considered. Emesis, activated charcoal, and a saline cathartic are beneficial at limiting absorption (Roberts and Reigart, 2014). Analgesics and antihistamines are helpful in humans who are experiencing a Mazzotti-like reaction because of microfilaria die-off. Also, glucocorticoids might be needed in extreme situations.

Environmental fate and behavior Most studies about avermectins’ interactions and the environment focus on Ivermectin and Abamectin. Several environmental fate and impacts investigations have been conducted to develop these two compounds as antiparasitic drugs and for Abamectin as a crop protection chemical. They are highly unstable to the light and rapidly undergo photodegradation on the plant, soil surfaces, and dung pat surface. Their high molecular weight, as well as their hydrophobicity, make them immobile in soil. Studies showed that abamectin is not taken up from the soil by plants, nor is it bio concentrated by fish, so Abamectin does not persist or accumulate in the environment. They decompose quickly in soil under aerobic circumstances, with t1/2 of 2–8 weeks for Abamectin in soil and 7–14 days for ivermectin in soil/feces. In water, the half-lives (t1/2) for degradation as thin coatings on surfaces to less bioactive chemicals was less than 12 h and less than 24 h. They have low toxicity to earthworms and birds and no phytotoxicity. Abamectin’s bioavailability in non-target organisms is limited by its instability, poor water solubility, and tight soil binding, preventing it from leaking into groundwater or entering the aquatic environment (Halley et al., 1993). Recent literature findings on the environmental risk assessment of Ivermectin suggest that more risk assessment evaluations may be required (Mancini et al., 2020).

Ecotoxicology A study in aerobic condition on Lufkin fine sandy loam, Houston clay, and coarse sand revealed that AVM is discomposed quickly with t1/2 of 14-28, 28-56, and 56 days respectively, at a concentration of 1-ppm to at least 13 radioactive products (Bull et al., 1984). Then, the primary degradation product of AVM in the soils is an equilibrium mixture (ratio of 1:2.5) of the 8a-hydroxy derivative and the parallel ring-opened aldehyde derivative. The leaching potential of AVM was reported low in three types of soils. Hence unwitting soil surface pollution of AVM does not cause any serious trouble for surface or subterranean water (Bull et al., 1984).

Avermectin

865

Abamectin’s effects on invertebrate populations in artificial ponds were examined. Results showed that Abamectin reduced the community of many aquatic invertebrates (Ali et al., 1997). Among Baetis sp. (Coleoptera larvae and nymphs Hemiptera), immature insects were negatively impacted but recovered within 14 days of treatment in all treated ponds; however, the adult population was inert to Abamectin (Ali et al., 1997). There are some published data on the liver injury of AVM on pigeons (birds) via the mechanism of ultrastructure changes, apoptosis, and oxidative stress in liver cells (Zhu et al., 2013). Birds are mostly unaffected by AVMs; however, they harm fish, aquatic invertebrates, and honeybees. The LD50 and dietary LC50 are 2 g kg−1 and 3102 ppm in bobwhite quail, respectively. Mallard ducks fed up to 12 ppm abamectin showed no deleterious effects on reproduction. In rainbow trout, bluegill sunfish, sheepshead minnow, catfish, and carp, the 96 h LC50 was 3.2, 9.6, 15, 24, and 42 ppb, respectively. Abamectin has a 96-hour LC50 of 0.0016, 430, and 153 mg l-1 in pink shrimp, eastern oysters, and blue crab, respectively. In Daphnia magna, the 48-hour LC50 was 0.34 ppb. Abamectin did not accumulate in bluegill sunfish during a 28-day bioaccumulation investigation (Halley et al., 1993).

Exposure standards and guidelines Based on the FAO/WHO monograph, the acceptable daily intake of ivermectin was 0–10 mg/kg b.w. An ARfD of 200 mg/kg b.w. was established by the Committee. Also, the Committee recommended the following MRLs in cattle tissues: 400 mg/kg for fat, 100 mg/kg for kidney, 800 mg/kg for liver, and 30 mg/kg for muscle (Joe Boison, 2016).

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/Ivermectin https://pubchem.ncbi.nlm.nih.gov/compound/selamectin https://pubchem.ncbi.nlm.nih.gov/compound/moxidectin https://pubchem.ncbi.nlm.nih.gov/compound/9832750 https://pubchem.ncbi.nlm.nih.gov/compound/6450531 https://pubchem.ncbi.nlm.nih.gov/compound/6436124 https://pubchem.ncbi.nlm.nih.gov/compound/91617829 https://pubchem.ncbi.nlm.nih.gov/compound/6435890

Conclusion/summary/outlook Avermectins are anthelmentic and insecticidal agents with well-studied pharmacokinetic and pharmacodynamic mechanisms in humans and animals. Avermectin derivatives include macrocyclic lactone compounds; ivermectin is the most widely used agent which has been proven to be safe and effective at certain acceptable daily intake levels. Exposure standards should be monitored by authorities and updated guidelines may be released periodically. The urgency to discover novel drugs to treat SARS-CoV-2 infection in time of corona pandemic has made the researchers and vaccine manufacturers to investigate possible antiviral activities of ivermectin. Caution should be taken in clinical studies on ivermectin antiviral activities due to its potential deleterious drug-drug interaction with antiviral agents and possible neurotoxicity upon passage through BBB during viral infection.

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Further reading World Health Organization & Joint FAO/WHO Expert Committee on Food Additives (2020) Evaluation of certain veterinary drug residues in food: Eighty-eighth report of the Joint FAO/ WHO Expert Committee on Food Additives. Martin RJ, Robertson AP, and Choudhary S (2021) Ivermectin: An anthelmintic, an insecticide, and much more. Trends in Parasitology 37(1): 48–64. https://doi.org/10.1016/j. pt.2020.10.005 Epub 2020 Nov 11 33189582. PMC7853155.

Azamethiphos Raúl A Alzogaraya,b and Eduardo N Zerbab, aCentro de Investigaciones de Plagas e Insecticidas (UNIDEF-CITEDEF-CONICET-CIPEIN), Villa Martelli, provincia de Buenos Aires, Argentina; bEscuela de Hábitat y Sostenibilidad, Universidad Nacional de San Martín, San Martín, provincia de Buenos Aires, Argentina © 2024 Elsevier Inc. All rights reserved. This is an update of J.R. Richardson, Azamethiphos, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 345–346, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00100-7.

Chemical profile Background Uses Environmental fate and behavior Exposure Toxicokinetics Mechanism of toxicity Human toxicity Animal toxicity Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicity Toxicological interactions Exposure standards and guidelines References Further reading

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Abstract Azamethiphos (CAS: 35575-96-3) is an organophosphorus pesticide used for insect control and as ectoparasiticide in fish farming. It is a neurotoxic compound, which exerts toxicity through the inhibition of acetylcholinesterase. Its acute oral toxicity is low to moderate to rodents; in rats, it is less toxic when applied dermally. Birds are most susceptible to azamethiphos than rodents.

Keywords Azamethiphos; Cholinesterase inhibitors; Ectoparasiticides; Insecticides; Organophosphorus

Chemical profile

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Name: Azamethiphos. IUPAC name: S-[(6-Chloro-2-oxo[1,3]oxazolo[4,5-b]pyridin-3(2H)-yl)methyl] O,O-dimethyl phosphorothioate. Chemical Class: Synthetic organophosphorus insecticide in the phosphorothiolate class. Molecular Formula: C9H10ClN2O5PS Form: Colorless crystals (pure) or beige to gray powder (technical grade). Melting Point: 90  C (ECHA, 2018). Solubility: 1.2 g l−1 in water (ph 7, 20  C) (ECHA, 2018). Chemical Structure:

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Background Azamethiphos (CAS: 35575-96-3) is an organophosphorus pesticide used for insect control and as ectoparasiticide in fish farming. It is a neurotoxic compound, which exerts toxicity through the inhibition of acetylcholinesterase. Its acute oral toxicity is low to moderate to rodents; in rats, it is less toxic when applied dermally. Birds are most susceptible to azamethiphos than rodents.

Uses Azamethiphos is a pesticide used mainly to control flies in animal houses; mosquitoes and cockroaches and among other pest targets (Health and Safety Executive, 2021). In some American and European countries, it is used as ectoparasiticide for controlling sea lice on salmonides farmed in seawater (Pest Management Regulatory Agency, 2016).

Environmental fate and behavior Azamethiphos is not bioaccumulative for the purpose of hazard classification, with a Bioconcentration Factor of 1.56 (Banjare et al., 2021). Its octanol:water partition coefficient, log KOW, is 1.0 at 20  C (Health and Safety Executive, 2021). This value is below the trigger value of 4, and is indicative of a low bioaccumulation potential. It is moderately degraded in water and non-persistent in soil (Health and Safety Executive, 2021). Its hydrolysis half-life is 14 days (at 20  C), with seawater providing a more rapid degradation. In soil, its aerobic half-life is 0.25 days. The extent of complete biodegradation to CO2 (mineralization) was 17% at the end of a 28-days study (Health and Safety Executive, 2021). This result indicates that therefore does not undergo either ready biodegradation. The Groundwater Ubiquity Score (GUS) is an experimentally calculated value that related a pesticide half-life and Koc to predict its ability to contaminate groundwater. The GUS reported for azamethiphos is −1.57, indicating low leachibility (Lewis et al., 2016).

Exposure Dermal, oral, and inhalation routes are all primary exposure pathways (Kaushal et al., 2021).

Toxicokinetics Azamethiphos is well absorbed following oral administration to rats, but much less effective by the dermal route (Committee for Veterinary Medicinal Products, 1999). Following oral administration, it is rapidly excreted in urine and, in a minor amount, in feces (ECHA, 2018). Azamethiphos biotransformation occurs mainly by oxidation, hydrolysis via esterases, and reaction with glutathione (T3DB, 2014). Unlike many other organophosphorus insecticides, it does not undergo bioactivation through the P450 monooxygenase pathway, as it is already in its active oxon form. In rats’ urine, its major metabolite is 3-amino-3-hydroxi-5chloropyridine (Committee for Veterinary Medicinal Products, 1999). Demethylation and glucoronidation are minor routes of azametiphos biotransformation. The simultaneous application with synergists reduced the resistance to azamethiphos in the housefly, suggesting that an increase in the activity of P450 monooxygenases and esterases could be the underlying mechanisms (Saito et al., 1992). No information is available on the metabolism of azamethiphos in humans. An in vitro study of dermal absorption gave a result of 20% using a diluted commercial product (2.5 g l−1) (ECHA, 2018).

Mechanism of toxicity Azamethiphos is an acetylcholinesterase (AChE) inhibitor (Eto, 2003). As result of acetylcholinesterase inhibition, acetylcholine builds up and continues with its neurotransmisor function. This causes nerve impulses to be transmitted continuously and muscle contractions do not stop. As result, intoxicated mammals show salivation and eye-watering, followed by muscle spasms and death (T3DB, 2014). Mutations affecting AChE was reported as the cause of azamethiphos resistance in the sea lice, Lepeophtheirus salmonis, and in the bedbugs, Cimex lectularius and C. hemipterus (Kaur et al., 2015; Komagata et al., 2021).

Human toxicity No human toxicity data are available for azamethiphos (Woodward, 2012). Symptoms of intoxication include abundant salivation and eye-watering (low dose); nausea/vomiting, salivation, sweating, bradycardia, hypotension, collapse, and convulsions (acute dose) (T3DB, 2014). Muscle weakness occurs sometimes and may result in death, if respiratory muscles are affected.

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Animal toxicity The acute oral toxicity of azamethiphos is low to moderate to rodents (Committee for Veterinary Medicinal Products, 1999). Its oral LD50 values are 1040–1180, and 1452 mg kg−1 in rats and mice, respectively. In rats, azamethiphos is less toxic when applied dermally, with an LD50 value greater than 2150 mg kg−1. Its toxicity is greater in birds, with an acute oral LD50 of 91 mg kg−1 in quail. Results from a Local Lymph Node Assay showed that azamethiphos is a skin sensitizer (Health and Safety Executive, 2021). It also produced developmental toxicity in PC-12 cells (Christen et al., 2017). Short and long term repeated-exposure only produced a decrease in the cholinesterase activity in red blood cells of rats and dogs (ECHA, 2018). Azamethiphos was negative in causing delayed neuropathy in hens given two doses at the LD50 level, 21 days apart (Committee for Veterinary Medicinal Products, 1999).

Immunotoxicity Organophosphorus compounds are potential modulators of human immune system (Rajak et al., 2021). They could suppress immunity against viral infections, including SARS-CoV-2. The assessment of short, and long-term (lifetime) animal toxicity tests for the potential of azamethiphos to cause immunotoxicity demonstrated that the level of human exposure is well below the lowest dose at which these effects occur in animals (Pest Management Regulatory Agency, 2016). Beyond humans, azamethiphos (0.1 mg l−1) decreased the phagocytic index following short-term exposure in the blue mussel (Canty et al., 2007).

Reproductive toxicity Azamethiphos did not show to be teratogenic or result in reproductive toxicity in rodent studies (Committee for Veterinary Medicinal Products, 1999).

Genotoxicity Azamethiphos showed mutagenic potential when evaluated in vitro in both bacterial and mouse lymphoma cells studies (ECHA, 2018). However, in vivo tests with rats and mice gave a negative result.

Carcinogenicity Azamethiphos does not appear to be carcinogenic in animals or humans (Lewis et al., 2016; Health and Safety Executive, 2021).

Clinical management For dermal contact, remove contaminated clothing and wash exposed skin immediately. For ocular exposure, hold the eyelid open and wash quickly and gently with clean water for 15–20 min. In the case of inhalation exposure, the victim should be moved to fresh air and seek medical attention immediately. Artificial ventilation is indicated in the case of diminished respiratory function. If exposure is through ingestion, victim should seek medical help immediately. Emesis should not be induced. Initial management involves establishment of adequate ventilation and maintenance of adequate respiratory function. The treatment of acute poisoning, such as the administration of activated charcoal, to prevent or minimize digestive absorption, as well as the indication of atropine sulfate and/or pralidoxime in patients with or without cholinergic symptoms, must be carried out under strict medical indication. These are general recommendations. In any case of azamethiphos exposure, the best course of action is to consult a doctor immediately, even if there are no symptoms of intoxication.

Ecotoxicity Azamethiphos is toxic to several aquatic species. LC50 for the fish Oncorhynchus mykiss was 0.19 mg l−1, while the EC50 for Daphnia magna and the algae Pseudokirchneriella subcapitata were 0.33 mg l−1 and 74 mg l−1, respectively (ECHA, 2018). The LC50 for the American lobster ranges from 0.61 to 3.24 mg l−1, with sensitivity the greatest during spawning and molting seasons (Burridge et al., 2005). Exposure to azamethiphos at 30 ng/l−1 increased locomotor activity in the northern shrimp Pandalus borealis (Bamber et al., 2021).

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Toxicological interactions The classical insecticide synergists piperonyl butoxide, an inhibitor of the monooxygenases, and tributylphosphorotrithioate, an inhibitor of esterases, increased the toxicity of azamethiphos in a susceptible and two organophosphorus-resistant house flies strains (Saito et al., 1992). After topical application, synergism was moderate in the susceptible and one resistant strain (3-5, depending on dose of synergist and route of exposure), but high in the other resistant strain (20-3540). When simultaneously applied on filter paper as binary mixtures, the botanical monoterpenes eugenol, menthol and menthyl acetate increased moderately azamethiphos toxicity in the blood-sucking bug Triatoma infestans (4.5–9.5x) (Reynoso et al., 2020). However, when topically applied, only menthol and menthyl acetate increased azamethiphos toxicity, both of them in a drastical way (49,000 and 15,300x, respectively).

Exposure standards and guidelines The values of Acceptable Daily Intake and Acute Reference Dose for azamethiphos are both the same: 0.0008 mg kg−1 bw day−1 (Pest Management Regulatory Agency, 2016).

See also: Cholinesterase inhibition; Organophosphorus compounds; Pesticides and its toxicity; Veterinary toxicology

References Bamber S, Rundberget JT, Kringstad A, and Bechmann RK (2021) Effects of simulated environmental discharges of the salmon lice pesticides deltamethrin and azamethiphos on the swimming behaviour and survival of adult Northern shrimp (Pandalus borealis). Aquatic Toxicology 240: 105996. Banjare P, Matore B, Singh J, and Roy PP (2021) In silico local QSAR modeling of bioconcentration factor of organophosphate pesticides. In Silico Pharmacology 9: 28. Burridge LE, Haya K, and Waddy SL (2005) Seasonal lethality of the organophosphorus pesticide, azamethiphos to female American lobster (Homarus americanus). Ecotoxicology and Environmental Safety 60: 277–281. Canty M, Hagger JA, Moore RTB, Cooper L, and Galloway TS (2007) Sublethal impact of short term exposure to the organophosphate pesticide azamethiphos in the marine mollusc Mytilus edulis. Marine Pollution Bulletin 54: 396–402. Christen V, Rusconi M, Crettaz P, and Fent K (2017) Developmental neurotoxicity of different pesticides in PC-12 cells in vitro. Toxicology and Applied Pharmacology 325: 25–36. Committee for Veterinary Medicinal Products (1999) Azamethiphos Summary Report (2). London: The European Agency for Evaluation of Medicinal Products. ECHA - European Chemical Agency (2018) CLH report proposal for harmonised classification and labelling. In: International Chemical Identification: Azamethiphos (ISO). Helsinki: ECHA. Eto M (2003) Organophosphorus Insecticides. In: Plimmer JR, Gammon DW, and Ragsdale NA (eds.) Encyclopedia of Agrochemicals. Hoboken: John Wiley & Sons, Inc. Health and Safety Executive (2021) Agency Technical Report on the Classification and Labelling of Azamethiphos (ISO); S-[(6-chloro-2-oxooxazolo[4,5-b]pyridin-3(2H)-yl)methyl] O, O-dimethyl thiophosphorus. Bootle: Health and Safety Executive. Kaur K, Helgesen KO, Bakke MJ, and Horsberg TE (2015) Mechanism behind resistance against the organophosphorus azamethiphos in salmon lice (Lepeophtheirus salmonis). PLoS One. https://doi.org/10.1371/journal.pone.0124220. Kaushal J, Khatri M, and Arya SK (2021) A treatise on organophosphate pesticide pollution: Current strategies and advancements in their environmental degradation and elimination. Ecotoxicology and Environmental Safety 207: 111483. Komagata O, Kasai S, Itokawa K, Minagawa K, Kazuma T, Mizutani K, Muto A, Tanikawa T, Adachi M, Komatsu N, and Tomita T (2021) Common substitution mutation F348Y of acetylcholinesterase gene contributes to organophosphate and carbamate resistance in Cimex lectularius and C. hemipterus. Insect Biochemistry and Molecular Biology 138: 103637. Lewis KA, Tzilivakis J, Warner D, and Green A (2016) An international database for pesticide risk assessments and management. Human and Ecological Risk Assessment 22: 1050–1064. Pest Management Regulatory Agency (2016) Azamethiphos. Ottawa: Health Canada Pest Management Regulatory Agency. Rajak P, Ganguly A, Sarkar S, Mandi M, Dutta M, Podderf S, Khatun S, and Roy S (2021) Immunotoxic role of organophosphates: An unseen risk escalating SARS-CoV-2 pathogenicity. Food and Chemical Toxicology 149: 112007. Reynoso MMN, Seccacini EA, Zerba EN, and Alzogaray RA (2020) Botanical monoterpenes synergise the toxicity of azamethiphos in the vector of Chagas disease, Triatoma infestans (Hemiptera: Reduviidae). Tropical Medicine and International Health 25: 1480–1485. Saito K, Motoyamv N, and Dauterman WC (1992) Effect of synergists on the oral and topical toxicity of azamethiphos to organophosphorus-resistant houseflies (Diptera: Muscidae). Journal of Economic Entomology 85: 1041–1045. T3DB - Toxin and Toxin Target Data Base (2014) Azamethiphos (T3D3794). http://www.t3db.ca. Woodward KN (2012) Veterinary insecticides. In: Mars T (ed.) Mammalian Toxicology of Insecticides. Cambridge: RSC Publishing.

Further reading Garcés DV, Fuentes ME, and Quiñones RA (2020) Effect of azamethiphos on enzymatic activity and metabolic fingerprints of marine microbial communities from the water column. Aquaculture 529: 735650. IRAC-Insecticide Resistance Action Committee (2022) IRAC Mode of Action Classification Scheme. Version 10.3. IRAC. https://irac-online.org. Strachan F and Kennedy CJ (2021) The environmental fate and effects of anti-sea lice chemotherapeutants used in salmon aquaculture. Aquaculture 544: 737079. Urbina MA, Cumillaf JP, Paschke K, and Gebauer P (2019) Effects of pharmaceuticals used to treat salmon lice on non-target species: Evidence from a systematic review. Science of the Total Environment 649: 1124–1136.

Azathioprine Sara Salcedo, Emma Martínez-López, and Antonio J García-Fernández, Area of Toxicology, Department of Health Sciences, University of Murcia, Campus de Espinardo, Murcia, Spain © 2024 Elsevier Inc. All rights reserved. This is an update of S. Espín, A.J. García-Fernández, Azathioprine, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 347-350, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00470-X.

Chemical profile Background Uses/occurrence Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Chronic toxicity Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Organ toxicity (pulmonary, neurotox, hepatotox, kidney, endocrine disruption, skin, etc.) Interactions Toxicogenomics Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines References Further reading

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Abstract Azathioprine (CAS 446-86-6) is an immunosuppressive agent that acts as an antagonist of purine metabolism. Azathioprine (AZA) is used as an anti-inflammatory, immunosuppressant and steroid-sparing agent. Some of its main effects are bone marrow suppression and the occurrence of leukopenia. It appears that AZA at doses of 1 mg/ml is toxic and teratogenic to rat embryos. The U.S. Food and Drug Administration has classified AZA in category D, as a drug that may have a potential risk to the fetus. However, its administration to pregnant women with IBD appears to be beneficial. Azathioprine is classified in Group 1 as “carcinogenic to humans” by the International Agency for Research on Cancer.

Keywords 6-thioguanine; Azathioprine; Carcinogen; Immunosuppressant; Imuran; Steroid-sparing; Teratogen

Chemical profile Purine analog

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Name: Azathioprine Synonyms: Azathioprin; Azothioprine; Azatioprin; Azathiopurine; Azathioprinum; Azamune; Azanin; Azasan; Imuran; Immuran; Imurel; Imurek; Muran; Rorasul; 6- (1-Methyl-4-nitroimidazol-5-yl)thiopurine; 6-((1-Methyl-4-nitro-1H-imidazol5-yl)thio)-1H-purine; 6-(3-methyl-5-nitroimidazol-4-yl)suflanyl-7H-purine. CAS Number: 446-86-6 European Community (EC) Number: 207-175-4 Molecular Formula: C9H7N7O2S Molecular weight: 277.27 g mol−1

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Chemical Structure:

Background Azathioprine (AZA) is an immunosuppressive agent that acts as an antagonist of purine metabolism. Its main active metabolites, 6-mercaptopurine (6-MP) and 6- thioguanine (6-TGN), are the ones that act as anti-inflammatory, immunosuppressive and anticancer agents. They are used to prevent organ rejection and to treat many immunological diseases, such as dermatomyositis and polymyositis, multiple sclerosis, Churg-Strauss syndrome, systemic lupus erythematosus and inflammatory bowel disease, among others (Chavez-Alvarez et al., 2020; Saoji et al., 2022). As well as, to treat disorders of immune regulation and acute lymphoblastic leukemia, elderly-onset IgA vasculitis with nephritis, and also to reduce the risk of audiometric relapse in immune-mediated hearing loss (Mata-Castro et al., 2018; Sugimoto et al., 2021). AZA was first synthesized by Gertrude Elion, William Lange and George Hitchings in 1956. This agent was first used in human transplantation by Calne et al. (1962). It is currently used for its anti-inflammatory and immunosuppressive effects (Greenland et al., 2020).

Uses/occurrence AZA plays an important role as an anti-inflammatory, immunosuppressive and steroid-sparing agent. It is used as a first-line treatment to prevent relapses of neuromyelitis optica spectrum disorder (NMOSD) (Gomes et al., 2021; Zhang et al., 2020). In addition, this agent is often used in conjunction with other different compounds to prevent rejection and maintain graft function (Wagner et al., 2015). Besides, this drug is used as a corticosteroid-sparing agent in neurology (McWilliam and Khan, 2020) and in the treatment of elderly-onset IgA vasculitis with nephritis (Sugimoto et al., 2021). On the other hand, AZA is also utilized in the treatment of disorders of immune regulation and acute lymphoblastic leukemia (Marinaki and Arenas-Hernandez, 2020) and for various dermatological diseases such as atopic dermatitis, photodermatosis and psoriasis, among others (Chavez-Alvarez et al., 2020). In addition, it is applied to reduce the risk of audiometric relapse in immune-mediated hearing loss (Mata-Castro et al., 2018). However, despite its multiple uses, its long-term use has been observed to increase the risk of skin cancer (Karran and Attard, 2008; Leigh et al., 2019).

Exposure and exposure monitoring In medical treatment, AZA is administered by ingestion (daily dose of 0.5 mg kg−1) and intravenous injection (current dose of 1 to 1.5 mg kg−1). Doses of AZA are administered based on clinical response and hematologic tolerability (Harmand and Solassol, 2020). AZA is available as 25, 50, 75 and 100 mg tablets for oral administration and in injectable form as a sodium salt in 100 mg vials. In a treatment, AZA is usually administered at a low dose of 25 to 50 mg/day and then the dose is increased from 25 to 50 mg/day every 1 to 2 weeks, up to 2.0 to 2.5 mg kg−1, with the aim of monitoring to be able to detect the occurrence of adverse effects (Harmand and Solassol, 2020). These tablets should be administered after meals to reduce adverse gastrointestinal effects. In children and adults undergoing renal transplantation, the usual oral dose of AZA is 3–5 mg kg−1/day, which can be reduced to 1 to 3 mg kg−1/day, which would be the maintenance dose (IARC, 2012). In the study by Zhang et al. (2020) AZA was used as first-line treatment to prevent relapses in patients with Neuromyelitis Optica Spectrum Disorder (NMOSD). Patients received an initial dose of 25 mg of oral AZA, which was gradually increased in increments of 25 mg per day until the target daily dose (2–3 mg kg−1) was reached. Moreover, patients with chronic Inflammatory Bowel Disease (IBD) who also had Crohn’s disease (CD) were administered AZA at a low dose (1.0–2.5 mg kg−1/day) for a period ranging from 6 months to 2 years and was very effective. However, many practitioners recommend that patients treated with AZA also receive combination therapy with a corticosteroid to lessen the possible effects (Harmand and Solassol, 2020).

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Toxicokinetics AZA is rapidly metabolized by 80% to 6-mercaptopurine (6-MP), which is converted to active metabolites such as 6-thioguanine (6-TGN) and 6-methylmercaptopurine nucleosides (6-MMP) (Alami et al., 2018; Belizna et al., 2020). AZA is approximately 47% absorbed orally. However, 6-MP is only absorbed by an average of 16% (Belizna et al., 2020). AZA is readily absorbed in the intestine and is catabolized into a variety of oxidized and methylated derivatives. Placental transfer is limited (Hutson et al., 2011), AZA and 6-TGN cross the placenta, whereas 6-MP does not (Saarikoski and Seppälä, 1973). However, 6-TGN was found in similar concentrations in erythrocytes from three mothers (De Boer et al., 2006). After oral administration of 35S-AZA, only 12.6% of the dose is detected in the feces over a 48-h period as unabsorbed material, and 50% in the urine over 24 h (Briggs et al., 1990).

Mechanism of toxicity The toxicity of AZA depends on the patient’s age, genetic variations and drug dose. Metabolization of AZA occurs first because it is transformed into its active form in 6- MP by thiolysis with glutathione (GSH). Next, 6-MP is metabolized to the nucleoside thio-monophosphate (GMP) by the action of the enzymes hypoxanthine-guanine phosphoribosyltransferase (HPRT), guanosine monophosphate synthetase (GMPS), nucleotide diphosphate kinase (DNPK), thiopurine methyltransferase (TPMT) and inosine monophosphate dehydrogenase (IMPDH). This nucleoside is a substrate for the sequential activities of deoxynucleoside kinases and reductases. It is then transformed into nucleotide triphosphate (thio-dGTP), which is incorporated into DNA (Karran and Attard, 2008). It can also be formed into 6-TG, which is the main toxic metabolite that upon inhibition of TPMT increases the bioavailability of 6-MG, thereby increasing its levels and the risk of myelosuppression by inhibiting the cell cycle of lymphocytes (Seidman, 2003; Sparrow et al., 2005; Patel et al., 2006; Wee et al., 2011). It has a half- life of about 5 days in erythrocytes and is able to reach stability with repeated doses of AZA (Gilissen et al., 2004). When AZA is administered at low doses, i.e., 25 to 50 mg day−1 and then the dose is increased from 25 to 50 mg day−1 every 1–2 weeks, up to 2.5 mg kg−1, it has a positive effect on T lymphocytes by acting as an anti- inflammatory. However, when AZA is administered at high doses, i.e., 1.5 times the recommended daily dose of AZA, it has an immunosuppressive and cytotoxic effect (Baumgart and Carding, 2007). Also, AZA can be transformed into inactive metabolites, such as 6-methylmercaptopurine (6-MeMP), 6-methylmercaptopurine ribonucleotide (6-MeMPR), 6-methylthioinosine monophosphate MeTIMP), or as 6-MeTGMP by the enzyme TPMT (Swann et al., 1996). Patients with low TPMT levels are started on a lower dose of AZA to reduce potential risks (Murphy and Atherton, 2002). Some of these include bradycardia, hepatotoxicity, myelosuppression, as well as gastrointestinal symptoms, nausea, fatigue, malaise and others (Jack et al., 2016). AZA is metabolized in the liver and excreted renally, which increases its toxicity in case of renal insufficiency (Wee et al., 2011). This prodrug is widely used as an immunosuppressant in several diseases, such as IBD. In this disease, AZA and 6-MP is effective in the treatment of IBD. Some studies have reported that 3.5% of 173 adult IBD patients developed hepatitis as a consequence of AZA treatment. Hepatotoxicity is an unpredictable side effect of AZA, the molecular and pathogenic mechanisms of which remain unknown (Rietdijk et al., 2001). However, there are several studies that have reported various histopathological findings in AZA-induced hepatotoxicity, such as nodular regenerative hyperplasia, venoocclusive disease, among others (Russmann et al., 2001; Gisbert et al., 2007; Vernier-Massouille et al., 2007). On the other hand, other studies have observed that long-term use of AZA, 6-MP and 6- TGN is more likely to generate skin cancer (Karran and Attard, 2008). One of the most vulnerable patients are those with inflammatory bowel disease and Crohn’s disease (Maddox and Soltani, 2008).

In vitro toxicity data The in vivo complications of AZA occur when the reactive molecules attack indistinctly to the S atom, without having a preferred active site, it does not present a unilateral reaction as in the in vitro case. By having several interactions, active compounds such as 36S- and some reactive groups are presented as by-products of the biological interactions of the compound such as -SH and -NH2 proteins, these reactions occur when 2 g mL−1 is exceeded, in high concentrations exceeding the permitted therapeutic range. In vitro cytotoxicity is in the order of 5 to 10 in concentrations of 1 g mL−1, for bone marrow and lymph node cells, it presents an inhibition in leucine synthesis in human cells in concentrations of 6.8
10−5 M (Sartorelli and Johns, 1974).

Acute and short-term toxicity Acute toxicity studies of AZA are classified in Category II for skin and eye irritation. AZA is also classified in Category IV for acute oral toxicity. The lethal dose for 50% of exposed individuals (LD50) is 400 mg kg−1 in rats and 2500 mg kg−1 in mice. The LD50 by intraperitoneal administration is 310 mg kg−1 in rats and 650 mg kg−1 in mice. The maximum tolerated dose of AZA for five

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consecutive days in mice was 100 mg kg−1 intraperitoneally and 200 mg kg−1 orally. However, the maximum tolerated dose of AZA for five consecutive days in rats LD50 was 100 mg kg−1 and 200 mg kg−1 intraperitoneally and orally (Elion et al., 1961). Also, oral administration of AZA has been studied at high doses in rabbits for 2 weeks. The results of the research showed that oral administration of AZA (10 mg kg−1 day−1) produces reversible thrombocytosis and delayed fatal macrocytic anemia (Al-Safi et al., 2002). In this study, it also evaluated whether AZA administration affected hemoglobin level and white blood cell level and observed that AZA had no effect at high doses (Al-Safi et al., 2002). In studies conducted by Gregoriano et al. (2014) found that the main symptoms associated with acute toxicity were gastrointestinal symptoms, such as nausea, vomiting, abdominal pain and diarrhea. However, acute overdose of AZA for two or more days is associated with abdominal symptoms, such as fever, nausea, swelling of the abdomen, among others. It appears that patients taking more than 1.5 times the recommended daily dose of AZA are more prone to acute toxicity (Mohammadi and Kassim, 2022). In this study no severe symptoms were observed in patients as they were below the LD50, in one patient it was 180 mg kg−1, 33 mg kg−1 in another. One of the methods used in case of overdose is the use of activated charcoal within two hours after ingestion (Gregoriano et al., 2014). AZA is used as a treatment in acute lymphoblastic leukemia (LLA), the most common childhood cancer, and in inflammatory bowel diseases (Guo et al., 2022). For its activation it must form 6-TGN, which are its main active metabolites. These are metabolized and inactivated by the enzyme thiopurine methyltransferase (TPMT) and the enzyme nudix hydrolase 15 (NUDT15). Loss of function of these two enzymes means excessive exposure to 6-TGN, and therefore to suffer increased toxicity side effects, such as myelosuppression (Dean, 2012). To decrease the risk of acute toxicity in LLA, it is recommended that lower than normal doses be used in inward metabolizers of TPMT and reduced doses up to 10 times be used in poor metabolizers of TMPT (Relling et al., 2006; Dean, 2012).

Chronic toxicity In the chronic toxicity study in rats, it was observed that at doses of 60 mg kg−1 body weight (bw) per day and 180 mg kg−1 bw day−1 incorporated into the diet rats exhibited agranulocytic spleens and bone marrows and hemorrhagic lungs (Elion and Hitchings, 1975). Although AZA is used as an anticancer, its chronic treatment presents a high risk of developing squamous cell carcinoma of the skin, as the incorporation of thio-dGTP into DNA and the substitution of guanine for 6-TGN create strong mutagenic DNA damage when additionally exposed to ultraviolet radiation (UVA). An important cellular regulator of the expression of a cytoprotective gene network is Keap1/Nrf2/ARE, which reduces the incorporation of 6-TGN into DNA in both skin and liver. Therebefore it reduces the cancer risk of patients (Kalra et al., 2011). In addition, long-term administration of AZA has also been studied in cats. Five cats were given 2.2 mg kg−1 bw every other day. Their study data showed that two of the five cats had thrombocytopenia and the remaining three had thrombosis. Also, profound neutropenia was observed in all of them, and one of them developed pancytopenia. A bone marrow biopsy was performed to observe the effects of AZA on the bone marrow and it was found that the marrow showed a marked decrease in the myeloid series (Beale et al., 1992).

Immunotoxicity Several studies have analyzed the side effects of AZA administration. Some of its main side effects are related to bone marrow suppression, which manifests as leukopenia in 2.5–10% (Anandi et al., 2020). DNA damage has been observed in mouse bone marrow cells at doses of 1, 2 or 3 mg kg−1 bw and at all exposure times of 24, 48 or 120 h, due to the incorporation of active metabolites of AZA such as 6-TGN, which is incorporated into DNA as a mock metabolite and blocks lymphocyte synthesis (Melo-Bisneto et al., 2021). In addition, lymphocytes and skin epithelium have been observed to incorporate 6-TGN, which increases the risk of developing tumor cells (Nguyen et al., 2009).

Reproductive toxicity Several investigations have been devoted to study the effects of AZA on reproduction in various species. AZA administration has been found to be toxic and teratogenic to rat embryos at a dose of 1 mg/ml (Fazliogullari et al., 2021). Furthermore, it has been observed to induce congenital malformations at doses of 1–20 mg kg−1 bw on days 3–12 of gestation in Wistar rats and Swiss albino mice. On the other hand, a wide variety of skeletal malformations have been observed in rabbits exposed to intragastric doses of 5–15 mg kg−1 bw of AZA. With single doses of 50 mg kg−1 bw of AZA administered in NMRI mice on day 10 of gestation, teratogenicity was observed; NMRI mice exhibited cleft palate in 90%, vertebral malformations in 70%, lower limb malformations in 50% of fetuses, and upper limb malformations in approximately 75% (IARC, 1981). AZA and 6-MP are classified in category D as drugs that may have a potential risk to the fetus. However, their use is considered beneficial during conception and pregnancy, especially in female patients with IBD (Van der Woude et al., 2015). Their administration in pregnant women does not appear to be associated with reduced fertility or adverse events at delivery (Kanis et al., 2017). Studies by Alami et al. (2018) concluded that women who received AZA during the first trimester of gestation was not associated

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with an elevated rate of birth defects. On the other hand, it appears that exposure of pregnant women to AZA is associated with an elevated rate of preterm delivery (Goldstein et al., 2007). However, Alami et al. (2018) states that the high rate of preterm births is likely due to underlying maternal disease. A study in Austria found that infants of mothers receiving AZA did not have an increased incidence of infections (Angelberger et al., 2011), so mothers can breastfeed their infants while taking AZA.

Genotoxicity The genotoxicity of AZA was studied by Melo-Bisneto et al. (2021). In their study they evaluated the genotoxicity of AZA in Drosophila melanogaster, concluding that the use of AZA was associated with a significant increase in genotoxicity in D. melanogaster cells at concentrations of 0.25, 0.5, 0.75 or 1 mg ml−1. Likewise, DNA damage was observed in mouse bone marrow cells at doses of 1, 2 or 3 mg kg−1 bw and at all exposure times of 24, 48 or 120 h. At chronic treatment, at 120 h, greater DNA damage was observed. This study demonstrates that the marked genotoxicity of AZA may be due to the action of its active metabolites, such as 6-TGN, which is incorporated into DNA as a false metabolite and blocks lymphocyte synthesis. Another study that evaluated the genotoxicity of AZA was the one conducted by Cao et al. (2020). In their study, a PIG-A assay (OECD, 2022) was performed on human red blood cells in IBD patients treated with AZA and healthy volunteers to observe possible differences. Their results show that the genotoxic ability of AZA is due to its clastogenicity and not due to gene mutation.

Carcinogenicity The International Agency for Research on Cancer classified AZA as carcinogenic to humans based on several studies in rats and mice (IARC, 2012). AZA is an immunosuppressive agent contributing factors to skin carcinogenesis by altering DNA repair mechanisms, and reducing the elimination of tumor cells, as well as enhancing the entry of cytokinins, which promote tumor development (Leigh et al., 2019; Nguyen et al., 2009). Carcinogenicity was tested by oral administration in rats. AZA was administered in the diet at doses of 0, 3 or 10 mg kg−1 day−1 to groups of 70 male and 70 female Sprague- Dawley rats for 90 and 97 consecutive weeks. Survival of females and males was observed at a dose of 3 mg kg−1 day−1. However, raising the dose to 10 mg kg−1 day−1 reduced their survival, and a marked decrease in body fat could be observed in the rats. Some of the effects derived from AZA exposure were neoplasms of the skin, ear canal and preputial gland. In addition, at 3 mg kg−1 day−1, two mucinous adenocarcinomas in the duodenum were observed in males (IARC, 1981). On the other hand, the carcinogenic effects of AZA were also studied in mice. Six hundred (300 male and 300 female) clinically healthy 21-day-old mice were used and separated into three groups that were orally administered 0, 3 and 10 mg kg−1 day−1 in the diet. AZA significantly reduced the survival of females exposed to 3 mg kg−1 day−1 and of males and females to 10 mg kg−1 day−1. Mucosal pallor was observed, most likely due to anemia, and mice exposed to 10 mg kg−1 day−1 had enlarged lymph nodes and spleens. In summary, both male and female mice showed a dose-related increase in lymphosarcomas, and in male an increased number of squamous cell carcinomas in the preputial area was also observed. Studies carried out by Nguyen et al. (2009) examined the in vivo mutagenicity of long- term AZA treatment by determining the frequency and spectrum of somatic mutation events at the HPRT locus and in T lymphocytes of 119 children and adults with IBD. Their results show that AZA and 6-MP treatment may be associated with the occurrence of somatic mutations at disease-specific loci present in actively replicating tissues, such as bone marrow and activated immune cells and epithelial tissues undergoing repeated cycles of cell repair and regeneration, as both tend to incorporate increased levels of 6- TGN. In addition, AZA-treated lymphocytes and skin epithelium have been observed to incorporate 6-TGN. Furthermore, patients treated with long-term AZA are recommended to use good sun protection all day and all year round, as UVA is very frequent and plays a major role in the risk of developing skin cancer (Leigh et al., 2019).

Organ toxicity (pulmonary, neurotox, hepatotox, kidney, endocrine disruption, skin, etc.) Azathioprine and 6-MP are effective immunomodulators in inducing and maintaining remission in IBD, such as ulcerative colitis (UC) and Crohn’s disease (CD). However, between 10% and 29% of patients treated with these agents are forced to discontinue therapy due to adverse effects that can be severe. In the first 2 weeks, in 5%–10% of patients they can cause an early hypersensitivity reaction and also cause gastrointestinal effects, such as nausea, vomiting and diarrhea (Lees et al., 2008). One of the adverse effects that occurs in IBD patients exposed to these agents is acute pancreatitis. This occurs in 2% to 7% of patients who suffer from it. The factors that determine the risk of pancreatitis are not known, the only thing that is known is that the risk is not associated with the dose (Dubois, 2011; Gearry et al., 2004)). One of the rare but serious side effects is pulmonary toxicity. This toxicity has been studied by Ananthakrishnan et al. (2007) where they describe the symptoms of three patients after administration of these agents within 1 month. On initiation of treatment

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with AZA and 6-MP all patients presented dyspnea, cough and fever. Subsequently, histopathological examination of lung biopsies was performed and showed bronchiolitis obliterans, organizing pneumonia in one patient and usual interstitial pneumonitis in another. Other side effect of AZA administration is bone marrow suppression, which manifests as leukopenia in 2.5–10% (Anandi et al., 2020). In addition, it is known to photosensitizes the skin and causes the production of mutagenic reactive oxygen species. Its chronic treatment is associated with a high risk of developing squamous cell carcinoma of the skin (Jiyad et al., 2016; Kalra et al., 2011).

Interactions AZA has the ability to react quantitatively with the sulfhydryl ion for the formation of 6-MP and 5-mercapto-lmethyl-4-nitroimidazole. This reflects a nucleophilic -SH attack at the 5-imidazole position, as 3S-labeled AZA produces 35S6-mercaptopurine almost quantitatively upon reaction with H2S or glutathione. AZA is also readily cleaved by hydroxyl ions and is rapidly converted to 6-MP in 0.1 N sodium hydroxide, especially upon heating (Sartorelli and Johns, 1974). Regarding drug interactions, AZA dose should be reduced to one-quarter of the original dose when administered with allopurinol, as allopurinol inhibits xanthine oxidase activity, which affects the metabolism of 6-MP. However, it is better not to use these two drugs together. AZA may reduce the anticoagulant effect of warfarin and may alter the effect of certain neuromuscular blocking agents. Administration with medicines that may have a myelosuppressive effect, such as penicillamine, should be avoided. The use of angiotensin-converting enzyme inhibitors to control hypertension has been shown to potentiate the effects of AZA. Aminosalicylate derivatives (sulphasalazine, mesalazine, or olsalazine) inhibit the TPMT enzyme and may potentiate azathioprine toxicity.

Toxicogenomics One of the tools used to evaluate the mutagenic potential of AZA in vivo is the sentinel gene called phosphatidylinositol glycan class A (PIG A- in humans and Pig-a in rodents. This gene is located on the short arm of the X chromosome. It is responsible for encoding the catalytic subunit of the enzyme 1-6-N-acetylglucosaminyltransferase, which is involved in the first stage of the biosynthesis of the anchor molecule glycosylphosphatidylinositol (GPI) (Pacheco-Martínez et al., 2014). This molecule is affected by Pig-a mutations, thus allowing a cellular phenotype that can be identified in erythrocytes by flow cytometry (Miura et al., 2008). Cao et al. (2020) evaluated in vivo mutagenicity of AZA in 36 patients with inflammatory bowel disease (IBD) and 36 healthy volunteers. Data from this study showed that IBD patients respond to frequent PIG-A mutations. Therefore, this gene detects the cumulative genotoxicity of AZA. However, this gene was not related to either AZA exposure or its duration. In Table 1 are reported some examples of gene interaction compiled in CTD (Comparative Toxicogenomics Database).

Clinical management No specific antidote is known for the use of AZA (Mohammadi and Kassim, 2022). However, there are some measures to be taken in case of acute exposure. For example, in case of inhalation, the victim should be removed to fresh air. In case of skin contact, wash with plenty of soap and water and dry thoroughly. In case of eye exposure, immediately flush eyes with water, holding eyelids wide Table 1

Examplesa of the top interacting genes involved in the exposure to asbestos in Homo sapiens (CTD).

Genes

Inter-actions

Examples of interaction

References

TPMT

27

Fabre et al. (2004)

ITPA IGF1 MAPK1 MAPK3 TSC2 RAC1 CXCL8 GCLM GSTA2

7 5 4 4 4 4 3 3 3

TPMT promoter affects the susceptibility to AZA TPMT protein affects the metabolism of AZA ITPA protein affects the susceptibility to AZA IGF1 increases phosphorylation of FOXO1 protein AZA increases phosphorylation of MAPK1 protein AZA increases phosphorylation of MAPK3 protein AZA promotes union MAPK3 and TSC2 proteins AZA decreases activity of RAC1 protein AZA decreases expression of CXCL8 mRNA AZA increases expression of GCLM mRNA GSTA2 gene affects the susceptibility to AZA

a

More information in CTD webpage.

Shipkova et al. (2011) Hernández-Breijo et al. (2013)

Tiede et al. (2003) Andreas et al. (2009) Deferme et al. (2015) Zhang et al. (2010)

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apart. Finally, in case of inhalation, the mouth should be rinsed quickly and under medical supervision. The main symptoms after acute exposure are headaches, nausea and loss of motor skills (ECHA, 2017). One of the methods used in case of acute overdose is the use of activated charcoal within two hours of ingestion (Gregoriano et al., 2014). Recommendations for workers are to wear appropriate protective clothing, gloves and eye and face protection. In addition, direct contact with the product should be avoided and handled under a laboratory hood whenever possible (ECHA, 2017). The respiratory tract should be protected by means of a dust mask with P3 filter. They have to try to be in a ventilated area. In addition, it is also important to collect and store the waste in a safe way. In case of spillage, clean up carefully and wash residues that cannot be recovered with sodium hypochlorite. On the other hand, patients treated with long-term AZA are recommended to use good sun protection all day and all year round, as UVA is very frequent and plays a major role in the risk of developing skin cancer (Leigh et al., 2019). This same study recommends that specialists inform patients about the risk of skin cancer and the importance of protecting themselves.

Environmental fate and behavior AZA is an odorless powder, has a pale-yellow crystal at room temperature. This is insoluble in water (water solubility 0.272 g l−1 at 25  C), very slightly soluble in ethanol and chloroform, sparingly soluble in dilute mineral acids, and soluble in dilute alkaline solutions. It has molecular weight of 277.3 g mol−1, its melting point ranges from 243 to 244  C, the vapor pressure is 2.41
10−12 mmHg at 25  C, and the dissociation constant (pKa) is 8.2. Its octanol/water partition coefficient (Log Kow) is 0.1. AZA reaches its maximum stability at pH 5.5–6.5 but is hydrolyzed to 6-MP in alkaline solutions, especially on warming. In the presence of sulfhydryl compounds such as cysteine hydrolysis to 6-mercaptopurine (6-MP) also occurs (Trissel, 1996).

Ecotoxicology AZA is used as an anticancer drug because of its properties. Due to its high use in clinical treatments, it has been studied in hospital wastewater and found to have a maximum concentration of 187.5 ng/L with a detection frequency of 4.8–100% (Li et al., 2021).

Exposure standards and guidelines The regulatory authorities of the EU and the American Conference of Industrial Hygienists have not established occupational exposure limits for AZA. The healthcare company GlaxoSmithKline established an occupational exposure limit of 3 mg m−3 measured as the 8 h time weighted average limit (TWA). The Working Conditions Regulations in the Netherlands established an occupational exposure limit of 0.005 mg m−3 measured as 8 h TWA.

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CD007746. 122 pp. Wee JS, Marinaki A, and Smith CH (2011) Life threatening myelotoxicity secondary to azathioprine in a patient with atopic eczema and normal thiopurine methyltransferase activity. British Medical Journal 342: d1417. Zhang W, Modén O, and Mannervik B (2010) Differences among allelic variants of human glutathione transferase A2-2 in the activation of azathioprine. Chemico-Biological Interactions 186(2): 110–117. Zhang C, Zhang M, Qiu W, Ma H, Zhang X, Zhu Z, Yang CS, Jia D, Zhang TX, Yuan M, Feng Y, Yang L, Lu W, Yu C, Bennet JL, Shi FD, and TANGO Study Investigators (2020) Safety and efficacy of tocilizumab versus azathioprine in highly relapsing neuromyelitis optica spectrum disorder (TANGO): an open-label, multicentre, randomised, phase 2 trial. Lancet Neurology 19(5): 391–401.

Further reading Elion GB (1972) Significance of azathioprine metabolites. Proceedings of the Royal Society of Medicine 65: 257–260.

Relevant websites https://pubchem.ncbi.nlm.nih.gov/compound/2265 :PubChem. https://sor.epa.gov/sor_internet/registry/substreg/searchandretrieve/advancedsearch/externalSearch.do?p_type¼SRSITN&p_value¼17002858 :USEPA. http://ctdbase.org/detail.go?type¼chem&acc¼D001379 :CTD.

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Azinphos-Methyl Prabhakar Mishra*, Yuvashree Muralidaran, and Shraddha Bijalwan, Department of Biotechnology, School of Applied Sciences, REVA University, Bengaluru, Karnataka, India © 2024 Elsevier Inc. All rights reserved. This is an update of S. Karanth, Azinphos-Methyl, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 351–352, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00101-9.

Introduction Mechanism of toxicity Toxicokinetics Absorption Metabolism Excretion Uses Environmental fate Exposure and exposure monitoring Acute and short-term toxicity Animal Human Inhalation Oral exposure Chronic toxicity Animal Human Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Mutagenicity (germ cell) Development toxicity Clinical management Ecotoxicology Exposure standards and guidelines Sensitization Biomarkers Treatment References Further reading

882 883 883 883 883 883 883 883 883 885 885 885 885 885 885 885 885 885 885 886 886 886 886 886 886 887 887 887 887 887 888

Abstract Any poisonous chemical used to kill insects is referred to as an insecticide. These chemicals are mostly employed to manage pests that infest cultivated plants or to eradicate disease-carrying insects in particular areas. Organophosphorus compounds are phosphorus-containing organic compounds. They are largely employed in pest management as a substitute for chlorinated hydrocarbons, which remain in the environment. Organophosphorus chemicals are widely employed as insecticides across the world, posing significant risks to human health. One of the organophosphate insecticides widely used and now is under phase out condition is Azinphos-methyl. Azinphos-methyl (86-50-0) is an organophosphate insecticide which is colorless brown, white crystalline solid that has been in use to control pests on Brussels sprouts, nursery stock, walnuts, almonds, and pistachios. The Environmental Protection Agency has canceled many of its previous uses, and the handful that remain are being phased away due of the risk of inhaling and acute toxic effects, as well as the potential for damage to mammalian, avian, and aquatic creatures. Azinphos-methyl is an Acetylcholinesterase inhibitor. This article talks about different toxicology studies done on Azinphos- methyl.

Keywords Acetylcholinesterase; Inhibitor; Insecticide; Organophosphates; Toxicology

⁎

Corresponding author.

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00432-2

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Azinphos-Methyl

Introduction Insecticides are chemicals that are used to keep insects under control by killing them or stopping them from participating in undesired or damaging activities. Organophosphorus compounds (OPCs) are organic molecules that include at least one carbon-phosphorus link and are generated from phosphoric acids and their derivatives. Nerve agents, a subset of organophosphorus chemicals, have been manufactured and utilized in conflicts as well as in terrorist actions. By associating with and suppressing the acetylcholinesterase enzyme, these chemicals pose a physiological hazard, resulting in acetylcholine distress (Mukherjee and Gupta, 2020). One of the is Azinphos-methyl. Azinphos-methyl also known as Guthion is brown waxy solid (technical) or white crystalline solid (pure) with molecular weight of 317.3 g mol−1 and chemical Formula of C10H12N3O3PS2. It functions as an acetylcholinesterase (EC 3.1.1.7) inhibitor, a cholinesterase (EC 3.1.1.8) inhibitor, and an agrochemical. Azinphos-methyl belongs to the benzotriazine class where position three has methyl group (dimethoxyphosphorothioylsulfanyl) and 1,2,3-benzotriazine replaced by an oxo group at position 4. According to IUPAC (International Union of Pure and Applied Chemistry) nomenclature, azinphos methyl is written or named as 3-(dimethoxyphosphinothioylsulfanylmethyl)-,2,3-benzotriazin-4-one. Azinphos methyl belongs to or organophosphate group of chemical and is the assigned CAS (Chemical Abstracts Service) number is 86-50-0. Azinphos-methyl is a neurotoxic that was produced during World War II from nerve agents. In 1954, AzM was studied in the field against cotton insects in Mexico, and in 1956, it was approved for use on cotton in the United States. Azinphos-methyl (AZM) was initially registered as an insecticide in the United States in 1959. Because of its health risks to farm workers, pesticide applicators, and aquatic ecosystems, the US Environmental Protection Agency issued a final decision on 16 Nov. 2006, to phase out the remaining uses of AZM by 30 Sep. 2012. Azinphos methyl is produced synthetically i.e., it does not occur naturally. Azinphos methyl decomposes before boiling (boiling point) and melting point is 73–74
C (pure form). AZM is sold under the trade names Gusathion®, Guthion®, and Methyl-Guthion®. Because of the inhalation hazard and acute toxicity they pose, as well as their potential adverse effects on mammalian species, birds, and aquatic organisms, the US Environmental Protection Agency (EPA) has classified all Azinphos-methyl liquids with a concentration greater than 13.5% as Restricted Use Pesticides (RUP). Only certified applicators are permitted to acquire and utilize RUPs. For this substance, the EPA has set a 24-h reentry period. It’s toxicity class I, which means it’s extremely dangerous. The Signal Words DANGER and POISON are shown on products containing Azinphos-methyl. Azinphos-methyl is a broad-spectrum insecticide and is highly persistent. It’s also deadly to snails and slugs, and potentially toxic to mites and ticks. It’s non-systemic, which means it doesn’t move from one part of the plant to another. It’s generally used as a foliar spray to combat leaf-eating insects. It acts as a contact insecticide as well as a stomach toxin. Azinphos-methyl is not carcinogenic or genotoxic. Pregnancy Risk Group B is attributed to Azinphos-methyl and now is consideration under Pregnancy Risk Group C. Azinphos-methyl is not carcinogenic or genotoxic. Because skin contact is predicted to have a substantial role in systemic toxicity, Azinphos-methyl is nevertheless classed as “H”. In the guinea pig, Azinphos-methyl has been found to cause cutaneous sensitization. As a result, the chemical is labeled with the letter “Sh” (Table 1).

Table 1

Azinphos-methyl: Parameters and its details.

S.No

Parameters

Details

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Name CAS number IUPAC Nomenclature Chemical Formula Origin Molecular weight Melting point Boiling point Density Chemical class Physical state Mode of action Formulation Regulatory status

15

Chemical structure

Azinphos-methyl 86-50-0 3-(dimethoxyphosphinothioylsulfanylmethyl)-1,2,3 benzotriazin-4-one C10H12N3O3PS2 Synthetic 317.3 g mol−1 65–68
C (technical); 73–74
C (pure form) Decomposes before boiling 1.44 g cm−3 OP (organophosphate) insecticide Colorless brown, waxy or white crystalline solid Non-systemic, contact and stomach action. Cholinesterase inhibitor Azinphos-methyl is available in emulsifiable liquid, liquid flowable, ULV liquid, and wettable powder formulations Concentration more than 13.5% of azinphos methyl liquid is classified as RUP (Restricted Use Pesticides) by EPA (Environmental Protection Agency)

Azinphos-Methyl

883

Mechanism of toxicity Like other organophosphate insecticides, AZM exerts toxicity by inhibiting the enzyme acetylcholinesterase (AChE). AZM requires bioactivation for its biological action. The parent compound is activated to the potent ‘oxon’ by microsomal mixed-function oxidase enzymes, which in turn elicits toxicity by inhibiting AChE in synapse and neuromuscular junctions. AChE inhibition leads to overstimulation of cholinergic receptors on postsynaptic neurons, muscle cells, and/or end organs and consequent signs and symptoms of cholinergic toxicity.

Toxicokinetics Absorption The intestinal tract, the lungs, the external mucous membranes, and the cutaneous tissue absorb azinphos-methyl quickly (Hartwig, 2020). In rats, oral absorption is estimated to be 90–100%, whereas cutaneous absorption is predicted to be 19% (Hartwig, 2020).

Metabolism Azinphos- methyl is rapidly metabolized in liver and other tissues (Fig. 1). GSH transferase (Glutathione S-transferase) converts azinphos methyl into desmethyl isoazinphos methyl and glutathionyl methyl-benzamide. Another set of enzymes namely MFO (Mixed-function oxidase) converts azinphos methyl into azinphosmethyl oxygen analogue gutoxon and mercaptomethylbenzazimide (by removal of dimethyl thiophosphate—DMTP). Gutoxon can be converted to mercaptomethylbenzazimide by removal of dimethyl phosphate—DMP. Mercaptomethylbenzazimide undergoes hydrolysis and methylation to form benzamide and methylthiomethylbenzazimide. Methylthiomethylbenzazimide is further oxidized by MFO enzyme into sulfinyl (methylsulfinylbenzazimide) and sulfonyl (methylsulfonylbenzamide) in two subsequent steps. Glutathionyl methylbenzamide is converted by D- glutamyltranspeptidase cysteinylmethylbenzazimide to cysteinylmethybenzazimide which is further oxidized by MFO to sulfoxide (cysteinylmethylbenzazimide sulfoxide) and sulfone (cysteinylmethylbenzazimide sulfone) in two subsequent steps. DMP and DMTP are also produced. There was no copy of the original study accessible. As a result, it is impossible to say with certainty if and which metabolites were extracted from the urine, or whether quantification was conducted (World Health Organization (WHO), 2009).

Excretion Dermal absorption was shown to be substantially linked with urinary excretion of the metabolite dimethyl thiophosphate (DMTP) in rats, rabbits, monkeys, and humans. When rats were given a single oral dosage, 99% of the dose was eliminated within 48 h, with 54–66% excreted in the urine and 33–45% excreted in the feces (World Health Organization (WHO), 2009).

Uses AZM is a broad-spectrum, non-systemic insecticide and acaricide commonly used on several fruit, vegetable, and nut crops. It’s poisonous to snails, slugs, mites and ticks. It’s generally used as a foliar spray to combat leaf-eating insects. It is not used in residential and public health pest control. AZM is a broad-spectrum, non-systemic insecticide and acaricide commonly used on a number of fruit, vegetable, and nut crops. It’s poisonous to snails, slugs, mites and ticks. It’s generally used as a foliar spray to combat leaf-eating insects. It is not used in residential and public health pest control.

Environmental fate AZM adsorbs strongly to soil particles, and it has high potential to reach surface water through both spray drift and runoff. In sterile soil, its half-life is almost 1 year. Biodegradation and evaporation are the primary routes of elimination from soil, but it is also degraded by ultraviolet light. Degradation is more rapid at higher temperatures. AZM has a short half-life in surface waters (2 days). Hydrolysis is more prominent under alkaline conditions, but the compound is relatively stable in water below pH 10. The half-life on crops is 3–5 days under normal conditions.

Exposure and exposure monitoring Common routes of AZM exposure include dermal, inhalation, and ingestion. Persistence of AZM is generally low under field conditions. It is immobile in soil, it has low water solubility and low leaching potential, and is unlikely to contaminate groundwater.

884

Azinphos-Methyl

O

O

1.

O

P N

S O

P

CH3

O

N

N

S

SH

O

CH3

CH3

N

N

N MFO

O

GSH transferase

O

O

P N

H3C

O

2.

P

S O

CH3

N

CH3

O CH3

N O

O–

MFO

O

Dimethyl phosphate

GSH transferase

S

P

H3C

OH

3.

CH3

O

4.

N

SH

NH2 OH

HN

O O– O

N

Dimethyl thiophosphate

S

N

O

N

N

O

N

OH

Hydrolysis

D-glutamyltranspeptidase cysteinylglycinase

Methylation

O

7.

6. N

NH

S

CH3

S

N

OH NH2

N

N

N

N

N

N

O

O

O

O 5.

O

NH

MFO

MFO

O

O

O 9.

8. N

S

N

O

CH3

N

S

N

O

N

N

MFO

MFO

OH NH2

O

O

O 11.

O

10. N

S

N

O

O

CH3

N

1. Gutoxon 2. Desmethyl isoazinphos-methyl 3. Mercaptomethylbenzazimide 4. Glutathionyl methylbenzazimide 5. Benzazimide 6. Methylthiomethylbenzazimide 7. Cysteinylmethylbenzazimide 8. Methylsulfinylbenzazimide 9. Cysteinylmethylbenzazimide sulfoxide 10. Methylsulfonylbenzazimide 11. Cysteinylmethylbenzazimide sulfone

Fig. 1 Hypothesized metabolism of Azinphos-methyl in rats (World Health Organization (WHO), 2009).

N

N

S

N

O

OH NH2

Azinphos-Methyl

885

Acute and short-term toxicity Animal Acute toxicity studies in laboratory animals have shown that AZM is highly toxic to mammals. Oral and dermal LD50 values in laboratory rats are 4–16 and 88–220 mg kg−1, respectively.

Human Because of its high acute toxicity, low doses (>1.5 mg day−1) of AZM can lead to severe poisoning. Most common signs and symptoms of acute poisoning include salivation, excessive sweating, stomach pain, vomiting, and diarrhea. Inhalation of dust or aerosol containing AZM can lead to wheezing, tearing of the eyes, blurred vision, and tightness in the chest. Eye contact with concentrated solutions of AZM can be life threatening.

Inhalation For 3 months, male and female win star rats were exposed to azinphos-methyl aerosol at concentrations of 0, 0.195, 1.24, and 4.72 mg m−3 for 6 h per day, 5 days per week. 4.72 mg m−3 was the greatest level of exposure. In this investigation, the NOAEC (no observed adverse effect concentration) was 1.24 mg m−3. However, in terms of the scope of the investigation, the study does not meet today’s standards. Because the inhalation research involved whole-body exposure, it’s safe to presume that extra oral and cutaneous exposure occurred (Hartwig, 2020).

Oral exposure The suppression of AChE activity was the most sensitive harmful impact in rats and dogs, even after repeated oral administration. Hens, on the other hand, were less susceptible. At greater dosages, lower body weights and neurotoxic clinical symptoms were found. Muscarinic symptoms such as diarrhea and salivation were shown to be associated with an 80% reduction in brain AChE activity (World Health Organization (WHO), 2009). Long-term investigations demonstrated NOAELs for critical levels of AChE inhibition ranging from 0.75 to 0.86 mg kg−1 body weight per day in rats and 0.79–0.98 mg kg−1 body weight per day in mice (Hartwig, 2020).

Chronic toxicity Animal Laboratory rats can tolerate a dietary dose of 0.5 mg kg−1 day−1 for 2 months without any adverse effects. Repeated long-term exposure to AZM can lead to memory loss and irritability. In a 1-year trial with dogs, the NOAEL was 0.16 mg kg−1 body weight per day, based on AChE inhibition in the erythrocytes.

Human AChE inhibition caused by AZM can persist for a long time (2–6 weeks). Repeated chronic exposure may therefore result in prolonged AChE inhibition that may lead to flu-like illnesses.

Immunotoxicity A dietary study in rats at doses up to 125 mg kg−1 day−1 for 3 weeks resulted in general toxicological and immunological changes at 125 mg kg−1 day−1 including increased mortality rate, decreased body weight, decreased relative spleen, pituitary, and mesenteric lymph node weights, and unspecified histopathological changes in the thymus, pituitary, adrenal glands, and testes. It is unclear if the immunological changes are due to the direct or indirect effects of AZM.

Reproductive toxicity The high dosage groups multi-generation tests in 0.55 mg kg−1 body weight AChE activity in offspring

showed cholinergic toxicity, lower body weights, and suppression of AChE activities in both rats and other trials with repeated oral administration. For parental toxicity, the NOAEL was and day. Perinatal toxicity had a NOAEL of 0.55 mg kg−1 body weight and day, while lowered brain had a NOAEL of 1.54 mg kg−1 body weight and day. Pregnancy Risk Group B has been assigned to

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Azinphos-Methyl

azinphos-methyl. A dietary reproductive toxicity study in rabbits indicated no effects on litter size, number of stillbirths, sex ratios, fetal weight, fetal development, or pup survival up to 30 days (Hartwig, 2020).

Genotoxicity Azinphos-methyl was tested for genotoxicity in a variety of in vitro and in vivo test systems and found to be non-genotoxic (World Health Organization (WHO), 2009).

Carcinogenicity AZM is classified as a ‘not likely’ human carcinogen.

Mutagenicity (germ cell) Azinphos-methyl was discovered to be non-mutagenic in bacteria and mammalian cells in vitro. The positive outcomes of in vitro research that looked at the clastogenic implications of concurrent cytotoxicity must be weighed against the unfavorable findings of research that looked at the production of micronuclei in mice or chromosomal abnormalities in rat bone marrow. Furthermore, two dominant lethal tests in mice turned out to be negative. As a result, azinphos-methyl has been removed from the list of germ cell mutagens (Hartwig, 2020).

Development toxicity The NOAELs for developmental toxicity in rats were 2 mg kg−1 body weight per day, 6 mg kg−1 body weight per day in rabbits, and 5 mg kg−1 body weight per day in mice; these were the highest dosages studied. The dams’ AChE activity in the brain was not suppressed up to 1 mg kg−1 body weight a per day in rats and 2.5 mg kg−1 body weight per day in rabbits. There are no corresponding studies of AChE activity in mice. On gestation day 20, brain AChE activity in the fetuses was not affected in a developmental toxicity investigation in rats exposed to azinphos-methyl at a dosage of 2 mg kg−1 body weight and day. The NOAELs for effects on brain AChE activity in rat fetuses in the developmental toxicity study (gestation day 20) were 2 mg kg−1 body weight and day and 1.54 mg kg−1 body weight and day in the one-generation study (postnatal day 5). In the two generation trials in rats, pup viability was reduced on postnatal day 5 at 1.48 and 1.54 mg kg−1 body weight for day and above, respectively. NOAELs of 0.48 and 0.55 mg kg−1 body weight for day were therefore determined for perinatal toxicity in pups up to postnatal day 5 (Hartwig, 2020).

Clinical management General decontamination procedures should be immediately initiated in case of AZM exposure. For skin decontamination, the exposed area should be washed with plenty of water or soap, and shampoo can be used during showering. In case of eye contamination, the eyes should be flushed with water repeatedly for several minutes. The contaminated clothing should be removed and the airway cleared. In case of ingestion, vomiting should be induced. Atropine treatment should be initiated immediately to counteract muscarinic effects. Atropine (adults and children >12 years: 2–4 mg; children 12 years: 1–2 g; children 6 h 15–45 min

53–118 h

25%–50%

22 h

1400 mg/m2; however, pulmonary toxicity can occur at lower doses. Early-onset pulmonary toxicity appears within 3 years of therapy (duration: 9 days to 43 months); however, late-onset pulmonary fibrosis has been reported up to 17 years after treatment. Risk factors include smoking, pre-existing respiratory condition(s), sequential or concomitant thoracic irradiation, and the use of other drugs that cause lung damage. Pulmonary function tests should be performed at baseline and throughout treatment. Patients should be advised to immediately report any signs of respiratory complications, and the therapy should be discontinued. Carmustine therapy during childhood may result in asymptomatic lung fibrosis that may become symptomatic later. Unlike the toxicity described above, a condition called BCNU pneumonitis, BCNU lung, or idiopathic pneumonia syndrome, may occur following a single dose or course of therapy. BCNU pneumonitis requires emergency treatment as it is potentially fatal. Patients typically present with fever, cough, dyspnea and pulmonary infiltrates on x-ray. Risk factors include the ones identified previously, as well as female sex. It has been suggested that doses 10% change in organ weight). This incidence data can then be represented as a quantal endpoint, with the BMR as a difference in incidence from the control. This method is not desirable because some information needs to be included during data categorization. A hybrid technique incorporating all the information in the original observations is a superior way to convert the continuous BMR to a quantal BMR. The hybrid method adapts continuous models to continuous data. A BMD for a particular quantal BMR can be generated using the probability information in the continuous dose-response curve and a cutoff value for determining adverse reaction. This result can be directly compared to other BMDs derived from quantal data. The hybrid technique has a disadvantage as it requires the establishment of a background incidence of abnormality or the specification of a degree of response that can be regarded as the cutoff point between normal and abnormal responses (Rezvanfar, 2014).

BMD model evaluation Once a particular data set is determined to be adequate for BMD modeling, and an appropriate BMR is chosen, a group of models which is picked based on the nature of the data will be adjusted to the data. Good BMD model software should provide statistics for measuring model fit, including global and local data fit measures. While statistical evaluation is crucial, scientific judgment is still required when dose-response modeling is performing. An ideal data set would contain information on the shape of dose-response curve, particularly around the BMR. When this occurs, BMD estimates from multiple models and should produce comparable results as long as these models provide a comparable data fit. When the dose distribution is such that there is no information on the shape of the dose-response curve, such as when there is 0% response in the control group and an extreme (e.g., >80%) response in the low-dose group, the BMD model is of limited effectiveness. In some data sets, response rates may plateau or become nonmonotonic in the high-dose area. If such plateauing dominates model fit, resulting in poor fit in the low-dose zone, excluding the high dose(s) from modeling may be acceptable. These are only a few of the many qualitative considerations that must be made in order to obtain a suitable modeling outcome (Rezvanfar, 2014). For BMD estimate, there are some specialized software applications. The U.S. EPA’s “BMDS” is a windows shell software that can estimate reference doses (RfDs), reference concentrations (RfCs), and slope factors (Jensen et al., 2019; U.S. EPA, 2022) for quantal, continuous, and nested data. BMDS development began in 1995, with the first prototype version released in 1997. It was subjected to strict external and public scrutiny in 1998–1999 and quality assurance testing in 1999–2000. The US EPA released BMDS version 1.2 for public usage in September 2000, and additional versions have been released repeatedly since then. The last version, BMDS 3.3, is a Microsoft Excel-based application used for quantitative toxicological dose-response analysis. The software is available for free download from the US EPA’s web site (https://www.epa.gov/bmds/about-bmds). This website also contains support documentation, such as a software user’s manual and a guideline document on the interpretation and (Rezvanfar, 2014) application of BMD modeling and model-source-code. “PROAST” (http://www.rivm.nl/proast) was created by the Dutch RIVM (Bilthoven, The Netherlands) and is based on the statistical software R (Jensen et al., 2019; Rezvanfar, 2014). “MADr-BMD” is a command prompt run software designed for EFSA that handles model averaging. “BMDExpress” is a windows shell software designed to estimate BMD with high throughput. Although the BMDS has the most dose-response models, it currently lacks model-averaging capabilities (Jensen et al., 2019). Bayesian BMD (BBMD) is a web-based BMD system promoting the PyStan library for Markov Chain Monte Carlo simulation, available at https:// benchmarkdose.com (Shao and Andrew, 2018). FastBMD is now presented as a useful device that can process a wide range of transcriptomics data (https://www.fastbmd.ca). It addresses reproducibility by allowing users to download results and generate summary reports after each analytical step. FastBMD employs existing statistical packages to rapidly adopt future modifications because it is implemented in R software (Ewald et al., 2021). However, suitable models may differ in BMD calculations. As a result, the data must be analyzed to establish whether there is a reason to choose certain models, such as an underlying biological basis for selecting a dose-response shape in the region of the BMR or if one of the models suits the data better in the 10% response zone. To promote consistency and repeatability, the US EPA has created a six-step process for BMD analysis (Fig. 2). The six steps in the BMD analysis are as follows: (1) selecting a BMR, (2) selecting

Benchmark dose

943

Fig. 2 US EPA’s benchmark dose analysis framework. Taken from Rezvanfar MA (2014) Benchmark Dose. Encyclopedia of Toxicology. 3rd edn. Elsevier, vol. 2, 402–406.

a set of models, (3) assessing model fit, (4) model selection when BMDLs are divergent, (5) model selection when BMDLs are not distinct, via Akaike’s Information Criterion (AIC), a model selection criterion used to compare models belonging to different classes, and (6) data reporting. The ESFA suggested following steps for BMD method: (1) identifying the dose-response data type, (2) selecting the BMR, (3) analyzing the data’s suitability for dose-response modeling, (4) mentioning prior knowledge regarding the parameter(s) under consideration, (5) using Bayesian model averaging, the most commonly used statistical method, to estimate the BMD and compute its reasonable interval, (6) determining the total BMDL to be used as an RP in order to establish a Health Base Guidance Value or a margins of exposure, (7) reporting BMD analysis. In addition, a BMD analysis report should contain the following information: A) An endpoint summary table; for continuous endpoints, the mean responses and related SDs (or SEMs), as well as sample sizes for each dose level. B) the BMR value and the biological basis for this decision. C) the software that was utilized, along with the version number. D) model fitting parameters and statistical hypotheses. E) a table describing the models and priors utilized for the endpoint(s). F) the BMD estimate(s) and related BMDL-BMDU reasonable interval(s). G) fitted model graphs. H) conclusion respecting the chosen BMDL for use as an RP (EFSA Scientific Committee, 2022).

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Conclusion The BMD methodology is likely to become much more broadly used and accepted in the current years as scientific understanding grows and BMD approaches are incorporated into several regulatory testing guidance documents on the health risk assessment process. According to the BMD concept supporters, its application is recommended to improve decision-making as a supplement or replacement for the NOAEL strategy. The ESFA Scientific Committee strongly advises the BMD technique, specifically model averaging. The SC strongly believes that current toxicity test guidelines should be reconsidered to optimize the study design for determining the RP for establishing the Health-Based Guidance Value. The scientific committee recommended BMD instructions for human data analysis, advised experts in the Scientific Panels and EFSA Units to continue training in dose-response modeling and the use of BMD software, and strongly reiterated the necessity for present toxicity test recommendations to be revised. As a result, the BMD technique represents a possible enhancement in the pharmaceutical development process’s dose setting and safety evaluation phases.

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Benfluralin M Noruzia and M Sharifzadehb, aFaculty of Pharmacy, Pharmaceutical Sciences Research Center (PSRC), Tehran University of Medical Sciences (TUMS), Tehran, Iran; bFaculty of Pharmacy, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of Y.R. Rodriguez, Benfluralin, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 407–410, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01166-0.

Chemical profile Background Use Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity Acute toxicity Chronic toxicity Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Exposure standards and guidelines References

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Abstract Benfluralin is a preemergent herbicide used for nonagricultural and agricultural applications. Benfluralin has mobility in soil; subsequently, leach from soil processed with benfluralin into surface water is probable; however, groundwater is unlikely to be contaminated. The acute toxicity of Benfluralin is low but classified as a dermal sensitizer agent. Renal and Hepatic damage has been noticed in chronic animal experiments. This herbicide has been determined to be dangerous for aquatic life; however, the acute toxicity of Benfluralin to birds and honey bees is low.

Keywords Benefin; Benfluralin; Carcinogenicity; Preemergent herbicides

Key points

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Introduction of Benfluralin: To provide information about background, uses, half-life, and relevant metabolites in the environment. Benfluralin toxicokinetic and mechanism of toxicity : To give more information about its kinetic (especially metabolism pathway) and related toxicity mechanisms. Evaluation of Benfluralin toxicity (acute and chronic, reproduction and developmental, genotoxicity, carcinogenicity, and immunotoxicity): To describe toxic effects because of short and long-term exposure and particular organs toxicity. Benfluralin Ecotoxicity: To demonstrate in detail its toxic effect on microorganisms, fish, insects, wildlife, and plants. Benfluralin exposure guideline: To explain about risk assessment of occupational exposure.

Chemical profile

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Name: Benfluralin. CAS Number: 1861-40-1. Synonyms: Balan, Benefin, Balfin, Benafine, Benefex, Benalan. Molecular Formula: C13H16F3N3O4

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Chemical Structure:

Background In the United States, Benfluralin has been approved as a preemergent herbicide for the past 52 years. In 2004, the EPA assessed Benfluralin-containing pesticides for re-registration. It was determined whether the herbicide posed an unacceptable environmental and human health risk. Re-registration of pesticides containing Benfluralin as an active ingredient was made possible by the RED document, which determined that new label standards had been completed (USEPA, 2004a; USEPA, 2004b).

Use Preemergent herbicides, such as Benfluralin, are approved for use on both commercial and residential properties to combat the growth of dicot (an embryonic plant with two seed leaves) and monocot (a plant embryo with only one seed leaf ) species. There are four different formulation types: water spread granules and emulsifiable concentrates. 42 final products and a water-soluble granule are generated from Benfluralin. Treatment of band and golf course (granules and fertilizer mixed), soil-integrated procedure, and irrigation systems can all be used. Lettuce, alfalfa, birds foot trefoil, vineyards, and non-bearing fruit and nut trees are the most common agricultural applications for Benfluralin. The most common Benfluralin uses are lettuce, chicory, and alfalfa (USEPA, 2004a; Rodriguez, 2014) Almost 56 billion dollars was spent on pesticides at the producer level around the world in 2012. Pesticide expenditures in the United States in 2012 were close to 9 billion dollars. Approximately half of the expenditure was spent on herbicides between 2008 and 2012, roughly 450,000 dollars (Atwood and Paisley-jones, 2017). There are several instances of non-agricultural consumption, such as golf areas, grass, Christmas tree colonies, ornamentals, and hedgerow fence. The use of Benfluralin in broccoli, tobacco, onions, and pasture/range has also been announced (Rodriguez, 2014).

Environmental fate and behavior here is an immediate release of Benfluralin into the environment. The Henry’s law constant for Benfluralin has been calculated at 2.91
10−4 atm/mol/m3 obtained from a vapor pressure of 6.6 10–5 mmHg. Benfluralin water solubility has been 0.1 mg/1, octanol/water partition coefficient (Kow) is 5.29, and soil organic carbon (Koc) ranges from 9840 to 11,660. Benfluralin is volatilized from moist soil and water surfaces; thus, it is typically formulated and applied to minimize volatilization. According to Koc, Benfluralin has decreased mobility in soil and may adsorb to suspended materials and accumulate in the water (USEPA, 2004a). As demonstrated by its 5% breakdown in 28-day ready biodegradability tests and its hydrolytic stability in pH 4, 7, and 9 sterile buffer solutions, benfluralin is considered “not rapidly degradable” (ECHA, 2021). When Benfluralin is exposed to aerobic or anaerobic soil conditions, oxidation or reduction occurs, resulting in environmental degradation. The rate of Benfluralin degradation in the soil is determined by factors like temperature, moisture content, and organic carbon matter. Benfluralin has a moderate half-life of 22–79 days through aerobic soil type, indicating a slow degradation process, and has a half-life of 12 days in anaerobic soil type. Based on some Studies, Photodecomposition may occur with residual Benfluralin on the soil. In soil, photolysis has a half-life of 12.5 days, whereas, in water, it lasts between 5.5 and 9.9 h. Benfluralin has an estimated half-life in the air of fewer than 12 h, indicating that air persistence is minimal. 2,6-dinitro-N-ethyl-4-trifluoro-methylbenzeneamine, N-(N-butyl)-2,6-dinitro4-trifluoromethylbenzeneamine, and 4-nitro-2-propyl-6-trifluoromethyl-1H-benzimidazole are considerable Benfluralin degradation products. Benfluralin is unlikely to percolate into groundwater depending on the Koc. Even though it is possible that contaminated Benfluralin soil leach into surface water. Benfluralin has the potential to accumulate in submarine species due to its high Kow. According to Submarine studies, the daily depuration rate for an entire fish is 0.54 (USEPA, 2004b).

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Exposure and exposure monitoring Benfluralin is directly released into the environment. Environmental contamination may also occur due to the manufacturing and application of Benfluralin. Inhalation and dermal contact during all application steps of Benfluralin caused exposure at work. When applied to Ornamental Grass and Grass-like Plants, there is the potential for widespread public contact with contaminated water sources and dermal exposure (USEPA, 2004b). Benfluralin is detected at a lower concentration in surface water than predicted by the models, according to the available monitoring data. Benfluralin is detected at a lower concentration in surface water than predicted by the models, according to the available monitoring data. Parent Benfluralin has been monitored by the U.S. Geological Survey (USGS) as part of the NAWQA program. Benfluralin was found in 92 of the more than 5000 samples analyzed, with the highest value of 0.097 ppb. None of the detections are cause for alarm. Agricultural pesticide use of Benfluralin has decreased since 1992, according to the National Water-Quality Assessment (USEPA, 2004b; NAWQA, 2021). No Benfluralin was identified in any of the 3115 Fruit and Vegetable samples analyzed by the USDA’s PDP 2020 Annual Summary (USDA, 2022). Benfluralin showed moderate to high persistence in soil laboratory incubations. Under aerobic conditions in the dark, secondary metabolites, including 2,6-dinitro-4-(trifluoromethyl) phenol, known as B12, showed modest to moderate persistence. B12 was formerly thought to be a metabolite of groundwater. The rate of B12 breakdown in soil depends on pH. It was discovered that Benfluralin had a different metabolic pathway due to anaerobic soil conditions. Benfluralin diamine (B36) and ethyl-propyl-benzimidazole were the main breakdown byproducts. Member States may need to conduct an environmental risk analysis of anaerobic Benfluralin metabolites for crops other than those acceptable to address prolonged soil anaerobic conditions. Benfluralin degradation does not occur mainly via soil photolysis (EFSA et al., 2019).

Toxicokinetics Benfluralin was rapidly absorbed, reaching maximal plasmatic concentrations in 5–10 h at the smaller dose and 24 h at the higher dose within a relatively short period. Benfluralin had broad tissue distribution, especially liver and fat. Throughout the 48-h investigation, tissue concentration percentages decreased significantly. It was found that the liver contained about 0.1% of the dose. Values of RBCs ranged from 2.4 to four times greater than plasmatic concentrations. Tissue residue levels were higher in females than males in both doses. Benfluralin, which accounted for 35% of the total dose in the metabolism research, was the most prevalent molecule in the feces. Benfluralin was dealkylated. According to comparative metabolic research, human, mouse, rat, dog, and rabbit liver microsomes created more than 5% of the starting substrate concentration. Only human liver microsomes produced two metabolites. Males had a plasmatic half-life of approximately 55 h, whereas females had a plasmatic half-life of roughly 62 h. After 48 h, the majority of the radioactivity had been removed. In urine and feces, males excreted 14.9% and 78.9% at the low and high doses on day 7. At the smaller dose, 22.6% of the substance was excreted by females, while 71% was found in their urine. At the higher dose, 19.9% of the drug was excreted, and 64.6% was in their urine. In male subjects, biliary excretion ranged from 7% to 8% at both doses (ECHA, 2021).

Mechanism of toxicity The liver is one of the most well-known organs targeted by this drug. In the 3-month subchronic toxicity study, rats in the control group had considerably higher p-nitroanisole O-demethylase activity in their livers. In the rat liver, the results suggested that mixed function oxidases should be produced. While in plants, Benfluralin interferes with microtubule production, disturbing cell division and ultimately disorienting microfibrils, whether or not this has any connection to the toxicity mechanism in mammals is unknown (Rodriguez, 2014).

Acute toxicity Benfluralin causes low short-term toxicity when exposure routes are inhalation and ingestion. The acute dermal and oral LD50 in male and female rats were more than 5000 mg/kg. The LC50 is expected to be greater than 2.16 mg/L. According to OECD TG 404 and CLP Regulation 1272/2008, New Zealand White rabbits were subjected to two skin irritation studies; Benfluralin induced moderate skin irritation in both studies. In non-moistened form, erythema remained in 3/6 rabbits at the end of the study period, while scaliness was discovered in 5/6 of the animals after the study in moistened form. Irritation reactions should take into account the extent to which skin lesions can be reversed. Benfluralin is classified as Skin Irit. 2; H315. The potential for Benfluralin to cause skin sensitization (95.8%) was investigated in two studies, according to OECD TG 406. Benfluralin is a skin sensitizer classified as Skin Sens. 1; H317 (ECHA, 2021). Benfluralin is not the most irritating eye irritant. However, in a short-term investigation on rabbits, Benfluralin caused only minimal ocular irritation and was quickly reversed. Acute Benfluralin eye exposure is categorized as Toxicity Category III. The drug was not found to have any phototoxic or photogenotoxic properties. No data are available on the acute or subchronic neurotoxic or immunotoxic effects of Benfluralin on humans (EFSA et al., 2019).

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Chronic toxicity Hepatic and renal damages have been observed in chronic animal experiments with Benfluralin. The 2-year rat research shows that the NOAEL is 0.5 mg/kg daily. Hepatocellular hypertrophy was found to have a greater prevalence in large doses than in smaller doses. In addition, substantial levels of hemosiderin pigment were found in the liver, spleen, and intestines. Benfluralin is harmful to the thyroid in high dosages. Thyroid toxicity was observed in long-term studies involving the F344 rat model receiving doses higher than 136.3 mg/kg daily. According to the available information, this herbicide appears to have a secondary effect on thyroid tumors rather than having a direct effect on them. Benfluralin does not appear to be an endocrine disruptor based on the evidence that is currently available for human health. The human subchronic neurotoxicity data on Benfluralin is lacking and insufficient (Rodriguez, 2014; ECHA, 2021; EFSA et al., 2019).

Immunotoxicity Benfluralin is not known to cause contact dermatitis in humans due to a known allergic reaction. It was discovered that a 61-year-old man had allergic contact dermatitis due to contact with the pesticide mixture. The patient has tested a variety of pesticides and herbicides. In addition to benefin, oxadiazon, and diazinon, the individual was exposed to prodiamine, carbaryl, triadimefon, and the herbicides triadimefon and dithiopyr as well as chlorothalonil, mecoprop, and trifluralin. There is a wide range of exposure and frequency, ranging from months to 20 years. Non-irritating amounts of analytical grade benefin and four other comparable compounds were applied to the patient’s skin as part of a North American Contact Dermatitis Group patch test. There were positive reactions from the patient to the benefin and two additional medications. The same herbicides were patch tested on 12 healthy volunteers who served as the case study controls, and none showed any adverse responses. The case study investigators could not identify which herbicide had induced the initial allergic reaction (Rodriguez, 2014).

Reproductive and developmental toxicity Reproductive toxicity of Benfluralin has been examined in two-generation rat studies, a multi-generational experiment, and various developmental experiments in rats and rabbits. Implantations, live fetuses, and the postnatal survival index at the lowest dose studied (1000 ppm) were reproductive endpoints. Male and female fertility indices and pre-coital intervals were unaffected by the therapy in either F0 or F1 generation. The sex ratio at each sampling point in every generation was unaffected by the treatment (ECHA, 2021). In a two-generation reproductive toxicity study in rats with a parental NOAEL of 5.5 mg/kg per day, there was low body weight gain, relatively high liver and kidney weight, and increased pup mortality (reduced body weight gain and increased liver and kidney weight). The rat and rabbit developmental toxicity research, found NOAEL of 50 mg/kg daily based on the rabbit developmental abnormalities research identified at paternal toxic amounts (EFSA et al., 2019). More study is required to complete its reproductive toxicity and endocrine disruption profiles.

Genotoxicity Benfluralin genotoxicity has been established through well-established in vitro and in vivo tests. Ames tests, mouse lymphoma cells, and micronucleus assays found no evidence of mutagenicity or clastogenicity in different batches of Benfluralin. Chromosomal aberration assay was unacceptable in unscheduled DNA synthesis on CHO cells. Benfluralin was administered to rats, and two bone marrow micronucleus tests were successful. Somatic cell mutagenicity of Benfluralin has yet to be established in the available research. No evidence suggests that Benfluralin causes germ cell mutations. Despite this, EBNA levels in the tested batches were below the current permissible limit (0.1 mg/kg) in genotoxicity tests. The genotoxicity of batches of Benfluralin with more than 0.085 mg/kg EBNA has not been evaluated. Benfluralin (at the current 0.1 mg EBNA/kg specification) has no genotoxic potential, and the EBNA impurity must be ruled out (ECHA, 2021).

Carcinogenicity However, there is insufficient evidence to attribute human carcinogenic risk to the substances examined by the Cancer Assessment Review Committee (CARC). An animal carcinogenicity investigation in B6C3F1 mice provided the basis for these findings. Mice were orally fed 0, 7, 42, and 224 mg/kg/day Benfluralin for 2 years. A statistically insignificant rise in the combined hepatic adenomas/ carcinomas was noticed in the 224 mg/kg group. Benfluralin, according to the CARC, is linked to liver cancers. Trifluralin is categorized as a group 3 carcinogen by the International Agency for Research on Cancer (IARC), even though the IARC has not classified Benfluralin (Rodriguez, 2014). “Suggestive evidence of carcinogenicity,” that is insufficient to evaluate the possibility of

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human cancer, has been assigned to Benfluralin by the U.S. EPA. Previous animal studies found that liver tumors observed in the chronic rodent studies and thyroid cancer in rodents were thought to be an unrelated mechanism to humans (Strupp et al., 2020a; Strupp et al., 2020b).

Clinical management Humans have been exposed to Benfluralin in both non-occupational and occupational settings, with 47 documented cases. Three of them had to be taken to the hospital for treatment of pain or itching of the skin. Only minor irritation or discomfort resulted from exposures that did not require medical attention. Hospitalization was not required in any of the exposure cases. Benfluralin may cause skin and eye irritation when used as directed. Benfluralin decontamination measures should be taken if exposure to it has occurred. The afflicted region should be rinsed as soon as the exposed person removes his or her contaminated clothing. The most efficient treatment in accidental consumption situations is probably activated charcoal. Diarrhea and vomiting are more likely to occur if a smaller amount is consumed. There have been no reported human deaths as a result of Benfluralin exposure (Rodriguez, 2014).

Ecotoxicology Humans have been exposed to Benfluralin in both non-occupational and occupational settings, with 47 documented cases. Three of them had to be taken to the hospital for treatment of pain or itching of the skin. Only minor irritation or discomfort resulted from exposures that did not require medical Benfluralin acute toxicity to birds was found to be low. Long-term bird risk assessment specialists agreed on 6.7 mg/kg daily. NOAEL 5.5 mg/kg per day was determined based on a Long-term risk assessment, which mammalian toxicology specialists also confirmed. In one investigation using Oncorhynchus mykiss (Rainbow trout), According to 96-h fish toxicity, the LC50 was 0.081 mg/L. Four different fish species, including Rainbow trout O. mykiss, Bluegill sunfish Lepomis macrochirus, Sheepshead Minnow Cyprinodon variegatus, and Carp Cyprinus carpio, were subjected to benfluralin for 96 h. 96-h LC50 of Cyprinodon variegatus was more than 0.027 mg/L. The Daphnia magna acute toxicity study followed OECD TG 202 protocols and recommendations. 48-h EC50 of Benfluralin was more than 0.034 mg/L. 96-h Mysid shrimp Mysidopsis bahia LC50 was 0.04 mg/L. Based on frond density EC50 >0.032 mg/L and EC50 ¼ 0.017 mg/L were found in a 7-day semi-static Lemna gibba toxicity test (ECHA, 2021). Benfluralin has been deemed extremely dangerous for aquatic life. According to the rapporteur Member State (RMS) calculations, the lower confidence limit (LC10) was approved to be used as a risk assessment factor (1.3 mg/L). Fish, daphnids, and algae had acute endpoints for metabolite 358R. The acute risk of 358R was modest. Benfluralin metabolites had 10 times more chronic toxicity risk to fish, daphnids, and aquatic plants. 371R, 372R, and 379R assumed 10-times more significant toxicity risk than Benfluralin. Risk evaluation of Benfluralin covered 379R and 358R risks, although 371R and 372R were high-risk. The evaluation of long-term risks to birds and mammals and secondary poisoning risks for animals and birds who eat earthworms is undetermined. The short-term risk of Benfluralin to honeybee was minimal. Dermal and oral exposure pose minimal acute danger to honeybees; however, long-term risk to larvae/adult honeybees was not determined. The danger of contaminated water exposure is not considered. Some spices like Poecilus cupreus, Arthropod species Typhlodromus pyri, and Chrysoperla carnea had a low probability of being affected. Soil fauna, microbes, and non-target plants were found to have low toxicity potential. According to specialists, a significant probability of biological wastewater treatment is unlikely because even at an extremely high concentration, no effects greater than 50% were found, and indeed exposure will most expectedly be minor for Benfluralin use, even if the study is incorrect (EFSA et al., 2019).

Exposure standards and guidelines The United States has well-established tolerances for Benfluralin residues on plants. Benfluralin does not have a Codex maximum residue limit. The 40 CFR x180.208 tolerances, 0.05 ppm, have not been changed and are currently being reviewed for re-enrollment. Short- and long-term dietary consumptions were below the acceptable daily intake (ADI) and acute reference dose (ARfD). Even though lettuce residue levels are not high enough to warrant a consumer risk, the metabolic pattern of primary crops has not been established, and the suggested risk assessment residue definition in plants must be considered Provisionally. Inhalation risk assessments for non-cancer occupational instructors were conducted, and the extent of concern was low. Post-application exposure is possible, but evaluations have not yet been completed (Rodriguez, 2014; EFSA et al., 2019).

References Atwood D and Paisley-jones C (2017) Pesticides Industry Sales and Usage: 2008–2012 Market Estimates. Washington, DC: US Environmental Protection Agency. 20460, 2017-01. EFSA, Arena M, Auteri D, Brancato A, Bura L, Carrasco Cabrera L, Chaideftou E, Chiusolo A, Court Marques D, and Crivellente F (2019) Peer review of the pesticide risk assessment of the active substance benfluralin. EFSA Journal 17: e05842. NAWQA (2021) Estimated Annual Agricultural Pesticide Use Pesticide Use Maps [Accessed].

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Rodriguez YR (2014) Benfluralin, Encyclopedia of Toxicology, 3rd edn. Elsevier. Strupp C, Quesnot N, Richert L, Moore J, Bomann WH, and Singh P (2020a) Weight of evidence and human relevance evaluation of the benfluralin mode of action in rodents (Part I): Liver carcinogenesis. Regulatory Toxicology and Pharmacology 117: 104758. ECHA (2021) Annex 1: Background document to the Opinion proposing harmonised classification and labelling at EU level of benfluralin (ISO); N-butyl-N-ethyla,a,a-trifluoro-2,6-dinitro-p-toluidine [Online]. Committee for Risk Assessment (RAC). [Accessed]. USDA (2022) Pesticide Data 2020 Program Annual Summary. Agricultural Marketing Service (AMS) [Accessed]. USEPA (2004a) R.E.D. FACTS: Benfluralin. Prevention, Pesticides And Toxic Substances. [Accessed]. USEPA (2004b) Reregistration Eligibility Decision for Benfluralin. [Accessed]. Strupp C, Quesnot N, Weber-parmentier C, Richert L, Bomann WH, and Singh P (2020b) Weight of Evidence and Human Relevance Evaluation of the Benfluralin Mode of Action in Rats (Part II): Thyroid carcinogenesis. Regulatory Toxicology and Pharmacology 117: 104736.

Relevant websites http://www.epa.gov :U.S. Environmental Protection Agency. Search for Benfluralin. https://pubchem.ncbi.nlm.nih.gov :PubChem. Search for Benfluralin.

Benomyl Hosna MohammadSadeghi, Ida Adeli, and Behnaz Bameri, Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of MA Pearson, GW Miller, Benomyl, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 411–412, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00102-0.

Chemical profile Background (significance/history) Uses Environmental fate and behavior Exposure routes and pathways Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Animals Humans Chronic toxicity Animals Humans Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicity Exposure standard and guidelines References

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Abstract Benomyl (a fungicidal compound) was recalled today in many countries, but not everywhere, Due to the development of resistance in many crops. Benomyl is of low toxicity to mammals and its main irritation is thought to be skin rashes or contact dermatitis. Since it may own some teratogenic characteristics in humans, all contact with this chemical should be reduced to the lowest possible level. Benomyl hydrolyzes to methyl 2-benzimidazole carbamate (carbendazim or MBC) within a very short time and excretes in the urine and feces. This agent might cause testicular dysfunctions in males, increase liver and ovarian tumors and play a significant role in the pathogenesis of Parkinson’s disease.

Keywords Benomyl; Benzimidazole; CAS 17804-35-2; Fungicide

Chemical profile

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Name: Benomyl Chemical Abstracts Service Registry Number: CAS 17804-35-2. Synonyms: Benlate, Fundazol, Agrocit, Uzgen, Fungochrom, Arilate, Benlate 50, Arbortrine, Fundasol, Fungicide D-1991, Benomyl-Imex, Kribenomyl, Benlate 50, Benomyl 50W, Methyl 1-(butylcarbamoyl)-2-benzimidazolylcarbamate Molecular Formula: C14H18N4O3

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Chemical Structure:

Background (significance/history) Benomyl is a broad-spectrum benzimidazole systemic fungicide, first introduced in 1968 by DuPont. Benomyl is a white crystalline solid with a faint, acrid odor that is usually produced through carbendazim’s reaction with butyl isocyanate. This chemical is widely used in agriculture and even in public health or houses to prevent the harmful actions of fungi, and it is stable in food, water, and soil. Noteworthy, the production of benomyl was prohibited in Europe and United States in 2003 due to its harmful effects on humans and animals and parasitic resistances. However, this chemical is still being used in developing countries (Kara et al., 2020; Mehtap et al., 2021; Jang et al., 2016; Müller et al., 2000; NIOSH, n.d.).

Uses Benomyl was first introduced to the market as a fungicide and pesticide to control a wide range of diseases of fruit, nuts, vegetables, mushrooms, field crops, ornamentals, turf, and trees. The significant uses of benomyl for the control of fungal diseases on fruits, vegetables, and ornamentals are pre-harvest applications. Benomyl is also used in textile processing and paint pigment manufacture. The active ingredient of benomyl has an inhibitory effect on mite populations too. It is marketed mainly as a 50% wettable powder (PubChem, n.d.; InChem, n.d.).

Environmental fate and behavior Benomyl, like most pesticides, is present in food and environmental samples, including wastewaters, at low concentrations since it is used for agricultural purposes (Guzzella et al., 2019). If released to air, a vapor pressure of 3.7  10−9 mmHg indicates benomyl will exist solely in the particulate phase in the ambient atmosphere. If Benomyl is released to soil, it will demonstrate slight mobility and volatilization; so leaching out from moist and dry soil surfaces is not expected to be an important fate. Biodegradation is also not reported to be a necessary environmental fate process as benomyl hydrolyzes rapidly to methyl 2-benzimidazole carbamate (carbendazim or MBC) and butyl isocyanate in moist soil and water. The half-life of benomyl is 2 h and 19 h in water and soil, respectively. Monitoring data indicate that the general population may be exposed to benomyl via ingestion of food products containing benomyl (PubChem, n.d.). The 2014 EU monitoring program on pesticide residues on food reports carbendazim presence in about 3% of the food products analyzed from 28 countries. Therefore, the European monitoring program lists carbendazim among the substances to be monitored to assess consumer exposure to pesticide residues (Guzzella et al., 2019).

Exposure routes and pathways Occupational exposure to benomyl may occur through inhalation of dust particles and dermal contact at workplaces. Monitoring data indicate that the general population may be exposed to benomyl via ingestion of food products containing Benomyl (PubChem, n.d.).

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Toxicokinetics Benomyl is readily absorbed in animal experiments after oral and inhalation exposure, but much less by dermal exposure. Absorbed benomyl is rapidly metabolized and excreted in the urine and feces of rats & dogs as methyl-5-hydroxy-2-benzimidazole-carbamate (5HBC). Additional data on dog and rat tissues feeding over 2 years supports that benomyl and its metabolites do not accumulate in animal tissues (PubChem, n.d.).

Mechanism of toxicity Benomyl is a xenobiotic, exerting its toxicity through a redox-related mechanism (Kara et al., 2020). Its mechanism of action is related to the inhibition of fungal growth via disruption of tubulin polymerization. These cellular structures, tubulins, are present in all eukaryotic cells and are involved in several vital functions such as intracellular transports and cell division (Zhou et al., 2016). Benomyl has been found to bind to fungal tubulin but not to porcine brain tubulin, indicating that mammalian tubulin has no or at least affinity for benomyl (PubChem, n.d.). Under bioactivation, benomyl is converted to S-methyl N-butyldithiocarbamate sulfoxide (MBT-SO), a potent aldehyde dehydrogenase (ALDH) inhibitor, and ALDH, the primary enzyme metabolizing the toxic dopamine metabolite (3,4-dihydroxyphenylacetaldehyde (DOPAL)). Thus, a potential mechanism for increased Parkinson’s disease risk from benomyl exposure is ALDH inhibition with the formation or functioning of microtubules (Casida et al., 2014).

Acute and short-term toxicity Animals Benomyl has a low toxicity rate, and its routine exposures do not cause signs and symptoms of cholinergic stimulation. Although, administration of a high dose of benomyl could lead to signs and symptoms of cholinergic overload. The LD50 of this compound in rabbits and rats is more than 10,000 mg/kg via dermal and oral routes. In rats, benomyl has been shown to cause temporary and mild conjunctival irritations (Von Burg and Liu, 1993).

Humans Benomyl contact with the skin may cause mild irritations, eczema, and contact dermatitis. Rare eye irritations have been reported, and it seems that benomyl can only temporarily cause inflammation in the eyes. Given the low vapor pressure of benomyl, it is unlikely that humans could be at risk of its inhalation exposures. However, benomyl-containing dust might be responsible for some respiratory tract irritations. The worst form of intoxication with benomyl is associated with its ingestions. Depending on the ingested dose, signs vary from salivation, sweating, lassitude, muscular incoordination, nausea and vomiting, abdominal cramps to even central nervous system (CNS) depression (Von Burg and Liu, 1993). Moreover, benomyl rapidly degrades to carbendazim or methyl 2-benzimidazole carbamate (MBC), also of toxicological concern. Both benomyl and MBC are considered possible human carcinogens (BenomylFactSheet, n.d.). Carbendazim is thought to be a more potent microtubule assembly inhibitor than benomyl (Kim et al., 2009).

Chronic toxicity Animals Repetitive dosing studies of benomyl indicated teratogenicity, oncogenicity, reproductive toxicity, and adverse effects on the liver. Teratogenic effects following benomyl administration during pregnancy included microphthalmia, hydrocephaly, and encephaloceles, and, in mice, skeletal and visceral anomalies were observed. Hepatotoxicity was observed in mice, rats, and dogs following long-term repetitive dosing with benomyl and/or carbendazim (Von Burg and Liu, 1993).

Humans Benomyl induces a significant increase in ROS levels and severe DNA damage at all examined concentrations. Furthermore, it also induces apoptosis, showing that it exerts its oxidative stress-promoted action via the apoptotic pathway. Benomyl is a toxic agent that promotes the appearance of liver tumors and brain malformations, whereas it shows selective toxicity to dopaminergic neurons. It is known that benomyl is a toxic agent against the nervous system (Kara et al., 2020). As mentioned above, Benomyl produces MBC, and both of them could induce liver toxicity, developmental toxicity (such as fetal eye and brain malformations and increased mortality), and reproductive (testicular) effects (Casida et al., 2014). Besides, several studies have indicated the possible

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association between benomyl occupational exposures and Parkinson’s disease. In Japanese women who used to work in greenhouses where benomyl had been previously consumed as a fumigant, symptoms, and signs of contact dermatitis have also been reported (Von Burg and Liu, 1993).

Immunotoxicity There is not much information concerning benomyl’s immunotoxic effects.

Reproductive toxicity Exposure to benomyl during the pubertal or post-pubertal period in male Sprague-Dawley rats might cause a decrease in epididymal sperm count, vas deferens sperm concentration, testicular or epididymal weight, or testicular lesion (Carter et al., 1984). Seminiferous epithelial sloughing was reported due to the administration of benomyl to rats (Aire, 2005). Furthermore, both female and male rat offsprings born from parents exposed to benomyl show developmental toxicity (Lu et al., 2004). As well, benomyl may damage the reproductive system in the male earthworm Eisenia fetida. In human, low to moderate doses of benomyl leads to sloughing of germ cells, dividing germ cells, and abnormal development of the head of the elongating spermatids. Higher doses result in obstruction of efferent ducts. This blockage causes seminiferous tubular atrophy and infertility (Hess and Nakai, 2000).

Genotoxicity There is some evidence proving the genotoxicity of benomyl, such as benomyl exerting an effect on somatic cells by inhibiting tubulin polymerization and causing numerous changes in chromosomes. As well, benomyl causes DNA strand breaks and chromosomal aberrations. Genotoxic effect and chromosome changes were respectively reported in rat hepatocyte culture and mouse oocyte (Kara et al., 2020). In a study on mice, benomyl led to metaphase I oocyte arrest and aneuploidy and a rise in hyperploid and aneuploidy oocytes’ prevalence. Also, polyploidy metaphase II oocytes were detected in the group treated with benomyl (Mailhes and Aardema, 1992). In another study, benomyl induced chromosomal aberrations, sister chromatid exchange, and aneuploidy in human lymphocytes (Amer et al., 2003; Bianchi-Santamaria et al., 1997). Moreover, micro-nuclei and aneugenic impact were in bone marrow, and aneuploidy in oocytes of mice were observed (Amer et al., 2003).

Carcinogenicity Benomyl has been shown to trigger liver tumor emergence (Kara et al., 2020). Benomyl actuates hepatocellular carcinoma and neoplasm in mice (Ramırez-Mares et al., 1999).

Clinical management In case of eye contamination, remove contact lenses if present. Flush eyes with water or typical saline solution for 20–30 min. Avoid using ointments, oils, or any medication without a physician’s instruction. Take the victim to the hospital even if there is no symptom like redness and irritation. If the skin has been exposed to benomyl, immediately wash skin with water while removing and isolating clothes. Wash skin with water and soap carefully, and be prepared to take the victim to the hospital in case of appearance of symptoms such as redness and irritation. If inhalation of the compound occurs, leave the polluted place and take deep breathes in the fresh air. Victims should immediately be taken to the hospital even symptoms, including wheezing, shortness of breath, coughing, or burning feeling in the mouth, throat, or chest, do not exist. If the exposure happens by ingestion, do not induce vomiting at all. If the victim is conscious and non-convulsive, give them a mixture of activated charcoal in water. Otherwise, lay the victim on their side with their head in a position lower than the body and make sure their airway is open. Immediately take the victim to the hospital (Carter et al., 1984).

Ecotoxicity Benomyl is slight to highly toxic to aquatic organisms and moderately toxic to birds. Reports have been shown benomyl is highly toxic to two fish species: Lebistes reticulatus and Salmo gairdneri. As well, reduced populations of earthworms have been reported in benomyl-treated orchards, as low, chronic exposures are lethal to earthworms (Canton, 1976).

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Exposure standard and guidelines The legal airborne permissible exposure limit in the average over an 8-h work shift is 5 and 15 mg/m3, respectively, for total dust and a respirable fraction (Sorour and Larink, 2001); However, some references recommend a health-based occupational exposure limit of 1 mg/m3. Furthermore, the acceptable dose for each day’s intake is 0.03 mg/kg.

References Aire T (2005) Short-term effects of carbendazim on the gross and microscopic features of the testes of Japanese quails (Coturnix coturnix japonica). Anatomy and Embryology 210(1): 43–49. Amer SM, Donya SM, and Aly FA (2003) Genotoxicity of benomyl and its residues in somatic and germ cells of mice fed on treated stored wheat grains. Archives of Toxicology 77(12): 712–721. BenomylFactSheet (n.d.) Available from: https://archive.epa.gov/pesticides/reregistration/web/html/benomyl_fs.html. Bianchi-Santamaria A, Gobbi M, Cembran M, and Arnaboldi A (1997) Human lymphocyte micronucleus genotoxicity test with mixtures of phytochemicals in environmental concentrations. Mutation Research, Genetic Toxicology and Environmental Mutagenesis 388(1): 27–32. Canton J (1976) The toxicity of benomyl, thiophanate-methyl, and BCM to four freshwater organisms. Bulletin of Environmental Contamination and Toxicology 16(2): 214–218. Carter SD, Hein JF, Rehnberg GL, and Laskey JW (1984) Effect of benomyl on the reproductive development of male rats. Journal of Toxicology and Environmental Health, Part A Current Issues. 13(1): 53–68. Casida JE, Ford B, Jinsmaa Y, Sullivan P, Cooney A, and Goldstein DS (2014) Benomyl, aldehyde dehydrogenase, DOPAL, and the catecholaldehyde hypothesis for the pathogenesis of Parkinson’s disease. Chemical Research in Toxicology 27(8): 1359–1361. Guzzella L, Casatta N, Dahchour A, Baggiani C, and Pozzoni F (2019) Molecularly imprinted polymers for the detection of benomyl residues in water and soil samples. Journal of Environmental Science and Health, Part B. 54(8): 702–708. Hess RA and Nakai M (2000) Histopathology of the male reproductive system induced by the fungicide benomyl. Histology and Histopathology 15(1): 207–224. InChem (n.d.) Benomyl—InChem. Available from: https://inchem.org/documents/jmpr/jmpmono/v073pr04.htm. Jang Y, Lee AY, Kim J-E, Jeong S-H, Kim JS, and Cho M-H (2016) Benomyl-induced effects of ORMDL3 overexpression via oxidative stress in human bronchial epithelial cells. Food and Chemical Toxicology 98: 100–106. Kara M, Oztas E, Ramazanogulları R, Kouretas D, Nepka C, Tsatsakis AM, et al. (2020) Benomyl, a benzimidazole fungicide, induces oxidative stress and apoptosis in neural cells. Toxicology Reports 7: 501–509. Kim DJ, Seok SH, Baek MW, Lee HY, Na YR, Park SH, et al. (2009) Benomyl induction of brain aromatase and toxic effects in the zebrafish embryo. Journal of Applied Toxicology 29(4): 289–294. Lu S-Y, Liao J-W, Kuo M-L, Wang S-C, Hwang J-S, and Ueng T-H (2004) Endocrine-disrupting activity in carbendazim-induced reproductive and developmental toxicity in rats. Journal of Toxicology and Environmental Health, Part A. 67(19): 1501–1515. Mailhes JB and Aardema MJ (1992) Benomyl-induced aneuploidy in mouse oocytes. Mutagenesis 7(4): 303–309. Mehtap K, Ezgi Ö, Tugce B, Fatma KE, and Gul O (2021) Benomyl induced oxidative stress related DNA damage and apoptosis in H9c2 cardiomyoblast cells. Toxicology In Vitro 75: 105180. Müller F, Ackermann P, and Margot P (2000) Fungicides, Agricultural, 2. Individual Fungicides. In: Ullmann’s Encyclopedia of Industrial Chemistry. Wiley. NIOSH (n.d.) Benomyl—NIOSH. Available from: https://www.cdc.gov/niosh/npg/npgd0048.html. PubChem (n.d.) Benomyl—PubChem. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Benomyl. Ramırez-Mares M, Fatell S, Villa-Trevino S, and de Mejı´a EG (1999) Protection of extract from leaves of Ardisia compressa against benomyl-induced cytotoxicity and genotoxicity in cultured rat hepatocytes. Toxicology In Vitro 13(6): 889–896. Sorour J and Larink O (2001) Toxic effects of benomyl on the ultrastructure during spermatogenesis of the earthworm Eisenia fetida. Ecotoxicology and Environmental Safety 50(3): 180–188. Von Burg R and Liu D (1993) Toxicology update. Journal of Applied Toxicology 13: 435. https://toxnet.nlm.nih.gov—Hazardous Substances Data Bank. Zhou Y, Xu J, Zhu Y, Duan Y, and Zhou M (2016) Mechanism of action of the benzimidazole fungicide on Fusarium graminearum: Interfering with polymerization of monomeric tubulin but not polymerized microtubule. Phytopathology 106(8): 807–813.

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Benz[a]anthracene Joshua P Gray, U.S. Coast Guard Academy, New London, CT, United States © 2024 Elsevier Inc. All rights reserved. This is an update of J.P. Gray, G.J. Hall, Benz[a]anthracene, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 413–414, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00247-5.

Chemical profile Background Uses/occurrence Exposure Toxicokinetics (absorption, distribution, metabolism, and excretion - ADME) Mechanism of toxicity Genotoxicity Carcinogenicity Interactions Clinical management Ecotoxicology Exposure standards and guidelines Other PubChem URL Comptox EPA URL References Further reading

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Abstract Benz[a]anthracene, a prototypical polycyclic aromatic hydrocarbon, is bioactivated by phase 1 metabolism to form a carcinogen. The formation of a diol epoxide in its bay region contributes to its carcinogenicity, allowing conjugation with intracellular molecules including DNA. Exposure predominantly occurs through smoking, second-hand smoke, air polluted with combustion products, or food and water contaminated with combustion products. It is primarily elminated by glucuronidation and sulfation followed by biliary excretion.

Keywords Bay region; Bioactivation; Cytochrome P450; Polycyclic aromatic hydrocarbon; Procarcinogen

Chemical profile

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Name: Benz[a]anthracene Synonyms: 1,2-Benz[a]anthracene; 1,2-Benzanthracene; 1,2-Benzanthrene; 1,2-Benzoanthracene; 2,3-Benzophenanthrene; BA; B[a]A; Benzanthracene; Benzanthrene; Benz[a]anthracene; Benzo[b]phenanthrene; Benzoanthracene; Tetraphene; Naphthanthracene Chemical Abstracts Service Registry Number: 56-55-3 Molecular Formula: C18H12 Chemical Structure:

Background Benz[a]anthracene is a prototypical polycyclic aromatic hydrocarbon (Skupinska et al., 2004). A product of combustion, exposures occur through smoking, transportation, or in occupational settings such as coke ovens (Menzie et al., 1992). The molecule is

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bioactivated through metabolism within the Bay region to form a reactive intermediate capable of forming DNA adducts (Jacob et al., 1979, 1983). It is also a persistent environmental pollutant.

Uses/occurrence Benz[a]anthracene is primarily used in research. It is also one of the most abundant polycyclic aromatic hydrocarbons in the environment.

Exposure Incomplete combustion of fuels results in inhalational exposure which is common in certain industrial and transportation settings, through smoking, and cooking. Dietary exposure occurs when foods come into contact with incomplete combustion products during cooking or through consumption of animals that have eaten PAH’s (Ramesh et al., 2004).

Toxicokinetics (absorption, distribution, metabolism, and excretion - ADME) Most of the ADME data for BaA is derived from general data for the entire polycyclic aromatic hydrocarbon class of chemicals (Ramesh et al., 2004). The primary route of exposure is pulmonary through elution from carbon particles associate with incomplete combustion (Creasia et al., 1976). PAH’s are also consumed in cooked food exposed to incomplete combustion products. After absorption, PAH’s circulate in the lymph and blood. Mixed-function oxidase catalyzes epoxidation within the Bay region (Yang and Chou, 1980). Epoxide hydrolase then catalyzes the formation of diols which subsequently form dihydrodiol epoxides which are highly reactive. Glucuronidation and sulfation are the primary phase II metabolic pathways (Hu and Wells, 1994). 1-hydroxypyrene, a metabolite of the PAH pyrene, is a sensitive indicator in urine for exposure to PAH’s (Alexandrie et al., 2000; Tsai et al., 2004). Hepatobiliary excretion typically occurs within 2 days of exposure (Bartosek et al., 1984).

Mechanism of toxicity Benz[a]anthracene is a procarcinogen bioactivated by cytochrome P450s to form oxides and dihydrodiols which subsequently form diol epoxides that are highly rective. Non-carcinogenic effects include immunosuppression, hematopoietic suppression, and reproductive and neurological toxicity typically at higher doses than those that induce mutagenesis (Blanton et al., 1986; Zhao, 1990; Szczeklik et al., 1994). Benz[a]anthracene is a potent inducer of the AhR pathway (Nebert et al., 2004).

Genotoxicity Metabolism of benz[a]anthracene within the bay region creates a high reactive diol epoxide capable of forming DNA adducts (Jerina et al., 1980). The epoxides undergo nucleophilic attack by sites in the DNA via an SN2 reaction or by SN1 reaction after formation of a carbonium ion (Guillen et al., 1997). A second mechanism of DNA damage involves formation of radical cations that cause depurinated DNA adducts.

Carcinogenicity Epidemiological studies have associated environmental exposure to polycyclic aromatic hydrocarbons such as benz[a]anthracene with a variety of cancer types, including lung, breast, esophageal, pancreatic, gastric, colorectal, bladder, skin, prostate, and cervical cancers (reviewed in Ramesh et al., 2004). Polymorphisms in CYP1A1 are associated with higher levels of the metabolite 1-hydroxypyrene and are hypothesized to contribute to carcinogenicity (Adonis et al., 2003; Alexandrie et al., 2000).

Interactions As benz[a]anthracene is a potent inducer of the AhR, which regulates the expression of CYP1A1 and other cytochrome P450 enzymes, it has the potential for many interactions.

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Clinical management Upon exposure to any PAH, recommendations include washing contaminated skin with soap and water and flushing contaminated eyes with water or saline. Charcoal administration has been recommended but there is no evidence of its efficacy.

Ecotoxicology Many polycyclic aromatic hydrocarbons accumulate in the aquatic environment where they may induce toxicity to marine organisms (Honda and Suzuki, 2020).

Exposure standards and guidelines The OSHA PEL for PAH’s in the workplace is 0.2 mg/m3. The Environmental Protection Agency’s standard for benz[a]anthracene in water is 0.0001 mg/L. Benz[a]anthracene is a ‘probable human carcinogen’ as classified by the EPA.

Other Benz[a]anthracene exposure in the environment occurs together with other polycyclic aromatic hydrocarbons as a mixture, making attribution of pathological outcomes difficult. This compound is one of many PAH’s with similar mechanisms of action due to the presence of a Bay region.

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/Benz_a_anthracene#section¼Metabolism-Metabolites

Comptox EPA URL https://comptox.epa.gov/dashboard/dsstoxdb/results?search¼DTXSID5023902

References Adonis M, Martinez V, Riquelme R, Ancic P, Gonzalez G, Tapia R, Castro M, Lucas D, Berthou F, and Gil L (2003) Susceptibility and exposure biomarkers in people exposed to PAHs from diesel exhaust. Toxicology Letters 144: 3–15. Alexandrie AK, Warholm M, Carstensen U, Axmon A, Hagmar L, Levin JO, Ostman C, and Rannug A (2000) CYP1A1 and GSTM1 polymorphisms affect urinary 1-hydroxypyrene levels after PAH exposure. Carcinogenesis 21: 669–676. Bartosek I, Guaitani A, Modica R, Fiume M, and Urso R (1984) Comparative kinetics of oral benz(a)anthracene, chrysene and triphenylene in rats: Study with hydrocarbon mixtures. Toxicology Letters 23: 333–339. Blanton RH, Lyte M, Myers MJ, and Bick PH (1986) Immunomodulation by polyaromatic hydrocarbons in mice and murine cells. Cancer Research 46: 2735–2739. Creasia DA, Poggenburg JK Jr., and Nettesheim P (1976) Elution of benzo[alpha]pyrene from carbon particles in the respiratory tract of mice. Journal of Toxicology and Environmental Health 1: 967–975. Guillen MD, Sopelana P, and Partearroyo MA (1997) Food as a source of polycyclic aromatic carcinogens. Reviews on Environmental Health 12: 133–146. Honda M and Suzuki N (2020) Toxicities of polycyclic aromatic hydrocarbons for aquatic animals. International Journal of Environmental Research and Public Health 17. Hu Z and Wells PG (1994) Modulation of benzo[a]pyrene bioactivation by glucuronidation in lymphocytes and hepatic microsomes from rats with a hereditary deficiency in bilirubin UDPglucuronosyltransferase. Toxicology and Applied Pharmacology 127: 306–313. Jacob J, Grimmer G, and Schmoldt A (1979) Profiles of polycyclic aromatic hydrocarbon metabolites after treatment with various inducers of microsomal rat liver monoxygenases (author’s transl). Hoppe-Seyler’s Zeitschrift für Physiologische Chemie 360: 1525–1534. Jacob J, Schmoldt A, Raab G, Hamann M, and Grimmer G (1983) Induction of specific monooxygenases by isosteric heterocyclic compounds of benz[a]anthracene, benzo[c] phenanthrene and chrysene. Cancer Letters 20: 341–348. Jerina DM, Sayer JM, Thakker DR, Yagi H, Levin W, Wood AW, and Conney AH (1980) Carcinogenicity of polycyclic aromatic hydrocarbons: The bay-region theory. In: Pullman B, Ts’o POP, and Gelboin H (eds.) Carcinogenesis: Fundamental Mechanisms and Environmental Effects. The Jerusalem Symposia on Quantum Chemistry and Biochemistry, pp. 1–12. Dordrecht: Springer. Menzie CA, Potocki BB, and Santodonato J (1992) Exposure to carcinogenic PAHs in the environment. Environmental Science & Technology 26: 1278–1284. Nebert DW, Dalton TP, Okey AB, and Gonzalez FJ (2004) Role of aryl hydrocarbon receptor-mediated induction of the CYP1 enzymes in environmental toxicity and cancer. The Journal of Biological Chemistry 279: 23847–23850. Ramesh A, Walker SA, Hood DB, Guillen MD, Schneider K, and Weyand EH (2004) Bioavailability and risk assessment of orally ingested polycyclic aromatic hydrocarbons. International Journal of Toxicology 23: 301–333. Skupinska K, Misiewicz I, and Kasprzycka-Guttman T (2004) Polycyclic aromatic hydrocarbons: Physicochemical properties, environmental appearance and impact on living organisms. Acta Poloniae Pharmaceutica 61: 233–240.

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Szczeklik A, Szczeklik J, Galuszka Z, Musial J, Kolarzyk E, and Targosz D (1994) Humoral immunosuppression in men exposed to polycyclic aromatic hydrocarbons and related carcinogens in polluted environments. Environmental Health Perspectives 102: 302–304. Tsai PJ, Shih TS, Chen HL, Lee WJ, Lai CH, and Liou SH (2004) Urinary 1-hydroxypyrene as an indicator for assessing the exposures of booth attendants of a highway toll station to polycyclic aromatic hydrocarbons. Environmental Science & Technology 38: 56–61. Yang SK and Chou MW (1980) Metabolism of the bay-region trans-1,2-dihydrodiol of benz[a]anthracene in rat liver microsomes occurs primarily at the 3,4-double bond. Carcinogenesis 1: 803–806. Zhao XL (1990) Effects of benzo(a)pyrene on the humoral immunity of mice exposed by single intraperitoneal injection. Zhonghua Yu Fang Yi Xue Za Zhi 24: 220–222.

Further reading https://journals.sagepub.com/doi/10.1080/10915810490517063#. USEPA (2007) Provisional Peer Reviewed Toxicity Values for Benz[a]anthracene. https://cfpub.epa.gov/ncea/pprtv/documents/Benzaanthracene.pdf. Haber LT, Pecquet AM, Vincent MJ, and White LM (2022) The long goodbye: Finally moving on from the relative potency approach to a mixtures approach for polycyclic aromatic hydrocarbons (PAHs). International Journal of Environmental Research and Public Health 19: 9490. Available at: https://doi.org/10.3390/ijerph19159490.

Benzene Charles C Barton, JUUL Inc., Washington, DC, United States © 2024 Elsevier Inc. All rights reserved. This is a reproduction of C. Barton, Benzene, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 415–418, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00364-X, with revisions made by the Editor.

Chemical profile Uses Exposure routes and pathways Toxicokinetics Mechanism of toxicity Acute and short-term toxicity (or exposure) Animal Human Chronic toxicity (or exposure) Animal Human Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Environmental fate Ecotoxicology Exposure standards and guidelines PubChem URL CompTox URL Addendum References Further reading

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Abstract Benzene (71-43-2) is a colorless liquid hydrocarbon with a sweet odor. It evaporates into the air very quickly and dissolves slightly in water. It is highly flammable. Benzene is a widely used chemical formed from both natural processes and human activities. It ranks in the top 20 chemicals for production volume. Breathing benzene can cause drowsiness, dizziness, and unconsciousness; long-term benzene exposure causes effects on the bone marrow and can cause anemia and leukemia.

Keywords Acute nonlymphocytic leukemia (ANLL); Benzene; BTEX; Chronic lymphocytic leukemia (CLL); Chronic nonlymphocytic leukemia (CNLL); Hydrocarbon; Myelodysplastic syndrome (MDS); Solvent; Tobacco smoke

Chemical profile

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Name: Benzene Chemical Abstracts Service Registry Number: 71-43-2 Synonyms: Cyclohexatriene, Benzol, Coal naphtha, Benzole, Phenyl hydride Chemical/Pharmaceutical/Other Class: Aromatic hydrocarbon Molecular Formula: C6H6 Chemical Structure:

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Uses Benzene is one of the world’s major commodity chemicals. Its primary use (85% of production) is as an intermediate in the production of other chemicals, predominantly styrene (for Styrofoam and other plastics), cumene (for various resins), and cyclohexane (for nylon and other synthetic fibers). Smaller amounts of benzene are used to make some types of rubbers, lubricants, dyes, drugs, synthetic detergents, insecticides, fumigants, solvents, paint removers, and gasoline. Benzene is a natural component of crude and refined petroleum. The mandatory decrease in lead alkyls in gasoline has led to an increase in the aromatic hydrocarbon content of gasoline to maintain high octane levels and antiknock properties. In the United States, gasoline typically contains less than 2% benzene by volume, but in other countries, the benzene concentration may be as high as 5%. Benzene is also a by-product of the coking process during steel production. Benzene ranks in the top 20 chemicals for production volume in the United States. Because of its lipophilic nature, benzene is an excellent solvent. Its use in paints, thinners, inks, adhesives, and rubbers, however, is decreasing and now accounts for less than 2% of current benzene production. Benzene was also an important component of many industrial cleaning and degreasing formulations, but now has been replaced mostly by toluene, chlorinated solvents, or mineral spirits. Although benzene is no longer added in significant quantities to most commercial products, traces of it may still be present as a contaminant.

Exposure routes and pathways Benzene is widespread in the environment. The major sources of benzene exposure are tobacco smoke, automobile service stations, exhaust from motor vehicles, and industrial emissions. Vapors (or gases) from products that contain benzene, such as glues, paints, furniture wax, and detergents, can also be a source of exposure. Airborne benzene is usually produced by processes associated with chemical manufacturing or the gasoline industry, including gasoline bulk-loading and discharging facilities and combustion engines (e.g., automobiles, lawnmowers, and snowblowers). Benzene is a component of both indoor and outdoor air pollution. Benzene levels measured in ambient outdoor air have a global average of 6 mg m−3 (range 2–9 mg m−3). In almost all cases, benzene levels inside residences or offices are higher than levels outside and still higher in homes with attached garages and those occupied by smokers. Seasonal variations also affect benzene levels, with higher levels found in the fall and winter when buildings are less well ventilated. People living around hazardous waste sites, petroleum-refining operations, petrochemical manufacturing sites, or gas stations may be exposed to higher levels of benzene in air. In addition to being inhaled, airborne benzene is absorbed across intact skin in experimental animals. For most people, the level of exposure to benzene through food, beverages, or drinking water is not as high as their exposure through air. Leakage from underground storage tanks and seepage from landfills or improper disposal of hazardous wastes have resulted in benzene contamination of groundwater used for drinking. Effluent from industries is also a source of groundwater contamination. In addition to being ingested, benzene in water can also be absorbed through wet skin and inhaled as it volatilizes during showering, laundering, or cooking. Typical drinking water contains less than 0.1 ppb benzene. Benzene has been detected in bottled water, liquor, and food. Cigarette smoke is another common source of personal and environmental benzene exposure, representing about half of the benzene to which the general population is exposed. Persons who smoke one pack of cigarettes a day inhale a daily dose of approximately 1 mg of benzene, about 3–4% of the amount inhaled daily by a worker exposed at the current occupational permissible exposure limit (PEL). Nonsmokers who live with smokers and who are passively exposed to environmental tobacco smoke typically experience 50% greater exposure to benzene than do nonsmokers who live in a smoke-free environment.

Toxicokinetics Benzene is lipid soluble and highly volatile at room temperature. As such, benzene readily crosses the alveolar membranes and is taken up by circulating blood in pulmonary vessels. Benzene is rapidly absorbed through the lungs; approximately 50% of the benzene inhaled in the air is absorbed. Benzene can also be readily absorbed from the gastrointestinal tract. Over 90% of ingested benzene is absorbed through the gastrointestinal tract. Benzene is poorly absorbed dermally. Absorbed benzene is rapidly distributed throughout the body and tends to accumulate in fatty tissues. Circulating benzene is preferentially taken up by lipid-rich tissues such as adipose and nervous tissue. Benzene has also been detected in the bone marrow, liver, kidneys, lungs, and spleen. The liver serves an important function in benzene metabolism, which results in the production of several reactive metabolites. The major end products of benzene metabolism include phenol (hydroxybenzene), catechol (1,2-dihydroxybenzene), and quinol (1,4-dihydroxybenzene). These metabolic products are subsequently conjugated with inorganic sulfate and glucuronic acid before being excreted in the urine. A small fraction of the catechol derived from benzene metabolism is oxidized to hydroxyhydroquinol or transformed to muconic acids. At low exposure levels, benzene is rapidly metabolized and excreted predominantly as conjugated urinary metabolites. At higher exposure levels, metabolic pathways appear to become saturated, and a large portion of an absorbed dose of benzene is excreted as a parent compound in exhaled air.

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Mechanism of toxicity Benzene can be irritating to mucus membranes. Dermal exposures can defat the skin’s keratin layer and can result in erythema, vesiculation, and dry, scaly dermatitis. Acute exposures to high concentrations can produce pulmonary irritation and edema, and gastrointestinal irritation (if ingested). Chronic exposure to benzene produces bone-marrow depression. Experimental evidence indicates that benzene’s bone-marrow toxicity is mediated by one or more of its metabolites. For example, inhibition of benzene metabolism by administration of toluene or partial hepatectomy protects bone marrow against benzene damage. Benzene metabolites, such as 1,2-dihydroxybenzene (catechol), 1,4-dihydroxybenzene (quinol), and 1,2,4-trihydroxybenzene (hydroxyhydroquinol), have been shown to inhibit cell mitosis. The mechanism of toxicity appears to be comprising five key events: (1) metabolism of benzene to a benzene oxide metabolite in the liver; (2) interaction of the benzene metabolite with target cells in the bone marrow; (3) formation of initiated, mutated bone-marrow target cells; (4) selective clonal proliferation of these mutated cells; and (5) formation of the neoplasm (leukemia). There are major uncertainties to the nature of the toxic metabolites and their mechanism of toxicity. Any hypothesis of benzene toxicity must account for the role of hepatic metabolism and the selective toxicity of benzene in the bone marrow. A puzzling aspect of benzene toxicology is its lack of hepatotoxicity.

Acute and short-term toxicity (or exposure) Animal Benzene can cause severe eye irritation and moderate skin irritation. When given orally, benzene is moderately toxic. The oral LD50 in rats and mice is 3400 and 4700 mg kg−1, respectively. The median lethal dose through inhalation has been evaluated in rats, mice, dogs, and cats. In these laboratory species, the LC50 ranges from 31,887 mg m−3 in mice to 170,000 mg m−3 in cats.

Human The acute effects of benzene exposure generally differ markedly from the chronic effects. Acute exposure to high doses of benzene in air (at concentrations in excess of 3000 ppm) causes symptoms typical of organic solvent intoxication. Symptoms may progress from excitation, euphoria, headache, and vertigo, in mild cases, to central nervous system depression, confusion, seizures, coma, and death from respiratory failure in severe cases. The rate of recovery depends on the initial exposure time and concentration, but, following severe intoxication, the symptoms may persist for weeks.

Chronic toxicity (or exposure) Animal The effects of lifetime exposure to benzene have also been evaluated in laboratory animals. A number of animal studies have demonstrated that benzene exposure can induce bone-marrow damage, changes in circulating blood cells, developmental and reproductive effects, alterations of the immune response, and cancer. With respect to chronic toxicity, hematological changes appear to be the most sensitive indicator. Although human epidemiological studies provide the bulk of the evidence that benzene is a human carcinogen, many experimental animal studies, both inhalation and oral, also support the evidence that exposure to benzene increases the risk of cancer in multiple organ systems, including the hematopoietic system, oral and nasal cavities, liver, forestomach, preputial gland, lung, ovary, and mammary gland.

Human The major toxicological manifestation of chronic benzene exposure in humans is bone-marrow depression. Clinical manifestations include anemia, leucopenia, and thrombocytopenia. In severe cases, bone-marrow aplasia develops. Later stages of toxicity are manifested by pancytopenia and aplastic anemia. Death may result from aplastic anemia or from leukemia. The US Environmental Protection Agency (EPA) and International Agency for Research on Cancer classify benzene as a known human carcinogen. This classification was given to benzene in view of strong epidemiological and experimental evidence. Epidemiologic studies and case studies provide clear evidence of a causal association between exposure to benzene and acute nonlymphocytic leukemia (ANLL) and also suggest evidence of chronic nonlymphocytic leukemia (CNLL) and chronic lymphocytic leukemia (CLL). Other neoplastic conditions that are associated with an increased risk in humans are hematologic neoplasms, blood disorders such as preleukemia and aplastic anemia, Hodgkin’s lymphoma, and myelodysplastic syndrome (MDS). These human data are supported by animal studies. The experimental animal data add to the argument that exposure to benzene increases the risk of cancer in multiple species at multiple organ sites (hematopoietic, oral and nasal, liver, forestomach, preputial gland, lung, ovary, and mammary gland). It is likely that these responses are due to interactions of the metabolites of benzene with DNA. Recent evidence supports the viewpoint that there are likely multiple mechanistic pathways leading to cancer and, in particular, to leukemogenesis from exposure to benzene.

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Immunotoxicity Damage to both the humoral and cellular components of the immune system has been known to occur in humans following inhalation. This is manifested by decreased levels of antibodies, leukocytes, and B-lymphocytes. Benzene-induced immunological effects are probably a reflection of bone-marrow toxicity. Animal studies have also shown that benzene decreases circulating leukocytes and decreases the ability of lymphoid tissue to produce the mature lymphocytes necessary to form antibodies. Animals exhibited lymphoid depletion of the thymus and spleen and hyperplasia of the bone marrow. This has been demonstrated in animals exposed for acute, intermediate, or chronic periods via the inhalation route.

Reproductive toxicity Benzene diffuses across the placenta and is toxic to the fetus in the presence of maternal toxicity. However, benzene is not considered to be a teratogen. Data on the reproductive effects of benzene in animals have been negative. Data on the reproductive effects of occupational exposure to benzene suggest that it may impair fertility in women. However, the findings are inconclusive and limited.

Genotoxicity Based on studies of the biochemical effects of benzene and its metabolites, four specific genotoxic mechanisms have been postulated: (1) DNA-reactive benzene metabolites forming adducts or cross-links; (2) oxidative DNA damage; (3) damage to components of the mitotic apparatus; and (4) topoisomerase II inhibition. Animal studies using radiolabeled benzene found a low level of radiolabel in isolated DNA with no preferential binding in target tissues of neoplasia. Adducts were not identified under dosage conditions that produce neoplasms in animals. These findings suggest that DNA-adduct formation may not be a significant mechanism for benzene-induced neoplasia. The evaluation of other genotoxicity test results revealed that benzene and its metabolites did not produce reverse mutations in Salmonella typhimurium but were clastogenic and aneugenic, producing micronuclei, chromosomal aberrations, sister chromatid exchanges, and DNA strand breaks. Studies support a mode of action that involves clastogenicity rather than mutagenicity secondary to DNA-adduct formation. Although unstable DNA adducts, oxidative DNA damage, or spindle poisoning could contribute to the overall toxic effects and cannot be ruled out, the genotoxic effects produced by benzene and its metabolites are most consistent with inhibition of topoisomerase II or ribonucleotide reductase inhibition.

Carcinogenicity The carcinogenicity of benzene has been documented in exposed workers. Epidemiological studies and case reports provide evidence of a causal relationship between occupational exposure to benzene and benzene-containing solvents and the occurrence of acute myelogenous leukemia (AML). Benzene has also been associated with ANLL in humans, and aplastic anemia may be an early indicator of developing ANLL in some cases. The US EPA classifies benzene as a known human carcinogen (Category A carcinogen) for all routes of exposure based on convincing human evidence as well as supporting evidence from animal studies. According to EPA’s Integrated Risk Information System (IRIS), “Epidemiologic studies and case studies provide clear evidence of a causal association between exposure to benzene and ANLL and also suggest evidence for CNLL and CLL. Other neoplastic conditions that are associated with an increased risk in humans are hematologic neoplasms, blood disorders such as preleukemia and aplastic anemia, Hodgkin’s lymphoma, and MDS. These human data are supported by animal studies. The experimental animal data add to the argument that exposure to benzene increases the risk of cancer in multiple species at multiple organ sites (hematopoietic, oral and nasal, liver, forestomach, preputial gland, lung, ovary, and mammary gland). It is likely that these responses are due to interactions of the metabolites of benzene with DNA. Recent evidence supports the viewpoint that there are likely multiple mechanistic pathways leading to cancer and, in particular, to leukemogenesis from exposure to benzene.” The United Nations’ International Agency for Research on Cancer classifies benzene as a Group 1 carcinogen (carcinogenic to humans) because there is sufficient evidence in humans for the carcinogenicity of benzene, specifically AML and ANLL.

Clinical management The victim should be removed from the contaminated atmosphere. Contaminated clothing should be removed and the affected area should be washed with soap and water. There is no antidote for benzene. Treatment is symptomatic and supportive. Most exposed persons recover fully. Persons who have experienced serious symptoms may need to be hospitalized. In cases of ingestion, vomiting should not be induced. Benzene or organic solvents containing benzene can cause acute hemorrhagic pneumonitis if aspirated into the lungs. Activated charcoal can be given to minimize absorption from the gastrointestinal tract. Charcoal can be given in a slurry or mixed with sorbitol or a saline cathartic. The recommended doses of activated charcoal are 30–100 g for adults, 15–30 g for children, and 1 or 2 g kg−1 for infants. The indicated doses can be prepared in a slurry by mixing charcoal in a diluent at a rate of 10 g charcoal per 80 mL of diluent.

Benzene

965

Environmental fate Benzene enters the air, water, and soil as a result of industrial processes, emissions from burning coal and oil, tobacco smoke, gasoline exhaust, and gasoline leaks, and from natural sources, including volcanoes and forest fires. Benzene in the atmosphere chemically degrades in only a few days. Benzene released to soil or waterways is subject to volatilization, photooxidation, and biodegradation. Benzene has a short half-life in surface water because it is so volatile. If benzene is released to soil, it will be subject to rapid volatilization near the surface. That which does not evaporate will be highly to very highly mobile in soil and may leach to groundwater. It may be subject to biodegradation in shallow, aerobic groundwater, but probably not under anaerobic conditions. If benzene is released to water, it will be subject to rapid volatilization. It will not be expected to significantly adsorb to sediment, bioconcentrate in aquatic organisms, or hydrolyze. It may be subject to biodegradation based on a reported biodegradation half-life of 16 days in an aerobic river die-away test. In a marine ecosystem, biodegradation occurred in 2 days after an acclimation period of 2 days and 2 weeks in the summer and spring, respectively, whereas no degradation occurred in winter. If benzene is released to the atmosphere, it will exist predominantly in the vapor phase. Gas-phase benzene will not be subject to direct photolysis but it will react with photochemically produced hydroxyl radicals with a half-life of 13.4 days, calculated using an experimental rate constant for the reaction. The reaction time in polluted atmospheres that contain nitrogen oxides or sulfur dioxide is accelerated, with the half-life being reported as 4–6 h. Products of photooxidation include phenol, nitrophenols, nitrobenzene, formic acid, and peroxyacetyl nitrate. Benzene is fairly soluble in water and is removed from the atmosphere in rain.

Ecotoxicology With the exception of accidental spillage of petroleum products, the routine levels of environmental benzene exposure are not associated with risk to fish and wildlife. Reasons for a reduced concern of the environmental risk include (1) the lack of bioaccumulation and biomagnification, (2) the low persistence due to its high volatility from surface waters and soil, and (3) the rapid photooxidation of airborne benzene and its biodegradation in soil and water. Studies have shown that high levels of benzene are toxic to aquatic life under controlled conditions. The US EPA ECOTOX database reports that Ceriodaphnia and Daphnia species are the most sensitive freshwater organisms following acute (48 h) exposure to benzene, with respective EC50 values of 130 and 400 ppb. Most organisms, however, can tolerate acute concentrations higher than this (in the 1–10 mg L−1 range). Following chronic exposures (4–7 day exposures), fish are relatively unaffected at concentrations up to 5 mg L−1 (at higher concentrations fish start to show adverse narcotic effects).

Exposure standards and guidelines The odor threshold for benzene is 30 ppm, but the current American Conference of Governmental Industrial Hygienists threshold limit value considered safe for occupational exposure (8 h day−1) is 0.5 ppm, with a short-term exposure limit (STEL) of 2.5 ppm. The Occupational Safety and Health Administration PEL is 1 ppm, with an STEL of 5 ppm. The National Institute for Occupational Safety and Health recommends an exposure limit (recommended exposure limit) of 0.1 ppm with an STEL of 1 ppm. EPA’s maximum contaminant level for benzene in drinking water is 5 ppb. EPA classifies benzene as a Category “A” carcinogen (“known” human carcinogen).

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/241

CompTox URL https://comptox.epa.gov/dashboard/dsstoxdb/results?search¼benzene

Addendum Based on the previous evaluations by IARC, NIOSH, NTP and OSHA, NCI (2022) confirmed that exposure to benzene increases the risk of developing leukemia and other blood disorders and updated the information on human exposure. Workers in industries that produce or use benzene may be exposed to the highest levels, although federal and state regulations have reduced benzene exposures in recent decades. In addition, limits on the amount of benzene allowed in gasoline have contributed to reduced exposures.

966

Benzene

Mainstream cigarette smoke is another source of benzene exposure, accounting for about half of the total U.S. population exposure to this chemical. Among smokers, 90 percent of benzene exposures come from smoking. Benzene may also be found in glues, adhesives, cleaning products, and paint strippers. Outdoor air contains low levels of benzene from secondhand tobacco smoke, gasoline fumes, motor vehicle exhaust, and industrial emissions. As a result of several restriction, esp. setting a limit for benzene in gasoline the concentrations in ambient air decreased significantly. For example, during the 16-year period of 1994-2009 average benzene concentrations at 22 monitoring sites in the US decreased form about 2.5 mg/m3 to less than 1.5 mg/m3 (EPA, 2010). In addition to the limit values reported above regulated benzene levels in the US are as follows: Although the EPA limits concentrations of benzene in drinking water to 5 ppb, some states or agencies may have lower limits (ACS, 2023). The US Food and Drug Administration (FDA) sets a limit of 5 ppb in bottled water. The Consumer Product Safety Commission (CPSC) considers any product containing 5% or more by weight of benzene to be hazardous, requiring special labeling. The EPA limits the percentage of benzene allowed in gasoline to a yearly average of 0.62% by volume, with a maximum of 1.3% (EPA, 2023). In Europe the European Chemicals Agency (ECHA, 2018) proposed an OEL as 8 hr average for benzene of 0.16 mg/m3 (0.05 ppm). Based on the mode of action it is assumed that there is a threshold for the carcinogenic effect of benzene. The indicated OEL value is considered to fully protect against any carcinogenic risk. The opinion specifically focuses on the carcinogenicity and the mode of action of benzene and concludes as follows:

• • • •

A mode-of-action-based threshold for chromosomal damage (aneugenicity and clastogenicity) in workers can, in the view of RAC, be used to establish an OEL for carcinogenicity; The limit will avoid exposures that induce chromosomal damage in workers, no significant residual cancer risk is considered and avoids other adverse effects; The leading genotoxic effects, aneugenicity and clastogenicity, are considered to be of secondary nature, i.e., acting indirectly and to follow a non-linear threshold- mechanism. Primary DNA reactivity of benzene and/or its metabolites seems of little importance.

See also: Blood; Carcinogen-DNA adduct formation and DNA repair; Solvents

References ACS American Cancer Society (2023) Cancer.Org | 1.800.227.2345, Benzene and Cancer Risk. https://www.cancer.org. ECHA (2018) Committee of Risk Assessment (RAC) Opinion on the Scientific Evaluation of Occupational Exposure Limits for Benzene. ECHA/RAC/0-000000-1412-86-187/F. EPA (2010) United States Environmental Protection Agency. Data from the Air Quality System. Accessed 2010. http://www.epa.gov/ttn/airs/airsaqs/ EPA (2023) United States Environmental Protection Agency. Gasoline Standards/US EPA. NCI (2022) National Cancer Institute. Benzene—Cancer-Causing Substances—NCI.

Further reading Galbraith D, Gross SA, and Paustenbach D (2010) Benzene and human health: A historical review and appraisal of associations with various diseases. Critical Reviews in Toxicology 40(Suppl. 2): 1–46. IARC (International Agency for Research on Cancer) (2012) Benzene, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. vol. 100F. Lyon, France: World Health Organization. Loomis D, Guyton KZ, Grosse Y, El Ghissassi F, Bouvard V, Benbrahim-Tallaa L, Guha N, Vilahur N, Mattock H, Straif K, and International Agency for Research on Cancer Monograph Working Group (2017) Carcinogenicity of benzene. The Lancet Oncology 18(12): 1574–1575. NIOSH (National Institute of Occupational Safety and Health) (2010) Benzene, NIOSH Pocket Guide to Chemical Hazards. Atlanta, GA: Centers for Disease Control and Prevention. Last accessed April 4, 2023. NTP (National Toxicology Program) (2021) Benzene, Report on Carcinogens, 15th edn. Triangle Park, NC: National Institute of Environmental Health and Safety. Last accessed April 4, 2023. Occupational Safety and Health Administration. Benzene, Safety and Health Topics. Washington, DC: U.S. Department of Labor. Last accessed April 4, 2023. Rushton E, Boogaard PJ, Ostapenkaite V, and Williams SD (2020) Derivation of an occupational exposure limit for benzene using epidemiological study quality assessment tools. Toxicology Letters 334: 117–144.

Relevant websites https://wwwn.cdc.gov/TSP/ToxProfiles. https://pubmed.ncbi.nlm.nih.gov/36767288/. https://pubmed.ncbi.nlm.nih.gov/36880454/.

ENCYCLOPEDIA OF TOXICOLOGY

DEDICATION Dedicated to the remediation of planet earth’s largely human-generated centuries of environmental degradation and its resultant climate crisis, and also to the toxicologists who, via scientific research, application, and communication play a critical role in helping ameliorate and forestall further damage to the natural world and to the health of populations and individuals. Equally dedicated to the elimination of strife, injustice, conflict, and divisiveness among the world’s citizens and to the brave people and movements striving to create a peaceful and lawful realm where freedom is presumed, diversity is valued, and equal opportunity is affirmed. And to my mom, Yetty, who celebrated her 95th birthday in 2023.

ENCYCLOPEDIA OF TOXICOLOGY FOURTH EDITION

EDITOR IN CHIEF Philip Wexler Independent Toxicology Information Specialist and U.S. National Library of Medicine (retired)

VOLUME 2

ASSOCIATE EDITORS Mohammad Abdollahi Tehran University of Medical Sciences (TUMS), Tehran, Iran

Shayne Gad Gad Consulting Services, Raleigh, NC, USA

Helmut Greim Technical University of Munich, Freising-Weihenstephan, Germany

Mary Gulumian North West University, Water Research Unit, South Africa

Evangelia I. Iatrou Laboratory of Toxicology and Forensic Chemistry, Medical School, University of Crete, Heraklion, Crete, Greece

Diana Miguez Latitud - LATU Foundation, Technological Laboratory of Uruguay (LATU), Montevideo, Uruguay

Asish Mohapatra Health Risk Assessment and Toxicology Specialist, Environmental Health Program, Health Canada, Calgary, Alberta, Canada

Sidhartha D. Ray Department of Pharmaceutical & Biomedical Sciences, Touro University College of Pharmacy, NY, USA

Jose Tarazona European Food Safety Authority, Parma, Italy and Spanish National Environmental Health Centre (CNSA)Instituto de Salud Carlos III. Ministry of Science and Innovation. Madrid, Spain

Aristidis Tsatsakis Laboratory of Toxicology and Forensic Chemistry, Medical School, University of Crete, Heraklion, Crete, Greece

Timothy Wiegand University of Rochester Medical Center and Strong Memorial Hospital, Rochester, NY, USA

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge MA 02139, United States Copyright © 2024 Elsevier Inc. All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers may always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-824315-2 For information on all publications visit our website at http://store.elsevier.com

Publisher: Oliver Walter Acquisitions Editors: Clodagh Holland-Borosh and Blerina Osmanaj Content Project Managers: Pamela Sadhukhan and Greetal Carolyn Associate Content Project Managers: Nandhini Mahendran and N. Kiruthigadevi Designer: Miles Hitchen

CONTENTS OF VOLUME 2 List of Contributors for Volume 2

xi

Editor Biographies

xix

Foreword

xxv

Preface Benzidine

xxvii 1

Aniqa Niha, Diana Genis, and Sidhartha D Ray

Benzo(a)pyrene

9

Joshua P Gray

Benzyl alcohol

17

Nancy A Ibrahim

Benzyl benzoate

23

Sofia Angela P Federico, Amelia B Hizon-Fradejas, Jeb Reece H Grabato, and Elmer-Rico E Mojica

Beryllium

27

Shayne C Gad

Beta-blockers

33

Daniel L Overbeek

Betapropiolactone

39

Swapnaa Balaji, Rabin Neupane, Amit K Tiwari, and Sidhartha D Ray

Bifenthrin

47

Arindam Basu Sarkar and Rahul Khupse

Biguanides

53

Mina Rena Chapler and Sidhartha D Ray

Bio warfare and terrorism: Toxins and other mid-spectrum agents

63

Leila Etemad, Mahdi Balali-Mood, and Mohammad Moshiri

Bioaccumulation

77

Katarzyna Chojnacka and Marcin Mikulewicz

Biocides

85

Virginia Rodrí guez Unamuno, Erik van de Plassche, and Leon van der Wal

Biocompatibility

91

Samantha E Gad and Shayne C Gad

Biofuels

99

Linda G Roberts and Thomas Smagala

Biological products in medicine

117

Marzieh Daniali, Taraneh Mousavi, and Mohammad Abdollahi

v

vi

Contents of Volume 2

Biomarkers, human health

133

Solange Costa and Filipa Esteves

Biomonitoring

141

Carla Costa and João Paulo Teixeira

Bioremediation

145

Eric A Seagren

Biotransformation/metabolism

161

Natalia Guevara, Marta Vázquez, and Pietro Fagiolino

Bis(chloromethyl) ether

181

Jisha Reji and Sidhartha D Ray

Bis (2-methoxyethyl) ether

185

Rajsumeet Macwan and Sidhartha D Ray

Bismuth

193

Shayne C Gad

Bisphenol A

197

Helmut Greim

Bleach

205

K Hahn and JA Weber

Blister agents

209

Vahid Pourbarkhordar, Mahdi Balali-Mood, Leila Etemad, and Mohammad Moshiri

Blood

225

Timothy J Wiegand and Militza Moreno

Boric acid

235

Timothy J Wiegand

Boron

241

Vera Bulakhova and Sidhartha D Ray

Botulinum toxin

249

HR Watson and Steven A Burr

British anti-lewsite (BAL)

255

David Guirguis and Sidhartha D Ray

Brodifacoum

261

Alek Q Adkins

Bromacil and its lithium salt

267

Arindam Basu Sarkar and Rahul Khupse

Bromadiolone

273

Jose V Tarazona

Bromethalin

279

Isabel Navas, Emma Pereira, and Antonio J Garcí a-Fernández

Bromine

287

Michael Olshansky, Tate Pumphrey, and Nilank Shah

Bromobenzene

295

David Guirguis and Sidhartha D Ray

Bromoform

301

Manish Verma and Sidhartha D Ray

Bromotrichloromethane Tejas S Lahoti and Rupangi S Patel

307

Contents of Volume 2

Busulfan

vii 313

Rahul Khupse and Arindam Basu Sarkar

1,3-Butadiene

317

Frank Faulhammer

Butane

325

Hermann M Bolt

Butter yellow

329

Kashyap N Thakore

Button batteries

333

Farzaneh Kefayati and Maryam Armandeh

Butyl acrylate

337

Sandra R Murphy and Elizabeth K Hunt

Butylamines

343

Maria Chiara Astuto and Catalina Manieu

Butylated hydroxyanisole

353

Ayesha Rahman Ahmed and Samiha Ahmed

Butylated hydroxytoluene

359

Ayesha Rahman Ahmed

Butyl ether

365

Heriberto Robles

Butyl nitrite

369

Kashyap N Thakore

Butyraldehyde

373

Raghunandan Yendapally and Sushma Ramsinghani

Butyric acid

379

Timothy J Wiegand

Butyronitrile

387

Amelia B Hizon-Fradejas, Jeb Reece H Grabato, Sofia Angela P Federico, and Elmer-Rico E Mojica

Butyrophenones

393

Alicia P DeFalco

BZ (3-quinuclidinyl benzilate) a psychotomimetic agent

403

Omid Mehrpour and Samaneh Nakhaee

Cadmium

411

Shayne C Gad

Caffeine

417

Matthew Lambrych

Calcium channel blockers

427

Shayne C Gad

Calomel

433

Kashyap N Thakore

Camphor

437

Alek Q Adkins

Canadian Centre for Occupational Health and Safety (CCOHS)

443

Janet Mannella

Cancer potency factor Azhar Hussain, Fred F Farris, and Sidhartha D Ray

447

viii

Contents of Volume 2

Candidate list of substances of very high concern (SVHC), reach

455

Marí a J Ramos-Peralonso

Cannabinoids

461

Arijeta Kaba and Sidhartha D Ray

Captafol

473

Iva Srdanovic, Azhar Hussain, and Sidhartha D Ray

Captan

479

Ida Adeli, Hosna MohammadSadeghi, and Behnaz Bameri

Carbamate pesticides

485

Horacio Heinzen and Marí a Verónica Cesio

Carbamazepine

493

Anca Oana Docea, Valentina Patricia Predoi, Daniela Calina, and Andreea Letitia Arsene

Carbaryl

499

Leona D Scanlan and Svetlana E Koshlukova

Carbofuran

513

Siong Fong Sim and Jocephine Jonip

Carbon dioxide

527

Iva Srdanovic and Sidhartha D Ray

Carbon disulfide

535

Madiha Khalid, Fatemeh Matin Moradkhan, and Zahra Bayrami

Carbon monoxide

547

Michael Keenan and Christine Stork

Carbon tetrabromide

551

Kashyap N Thakore

Carbon tetrachloride

555

Eugenio Vilanova, Eva del Rí o, Carmen Estevan, Jorge Estévez, and Miguel A Sogorb

Carbonyl Sulfide

561

Marí a del Prado Mí guez-Santiyán, Ana Lourdes Oropesa-Jiménez, and Francisco Soler-Rodrí guez

Carboxylesterases

571

Somayeh Mojtabavi and Mohsen Amin

Carcinogen classification schemes

579

Helmut Greim

Carcinogen-DNA adduct formation and DNA repair

589

Madiha Khalid and Mohammad Abdollahi

Carcinogenesis

597

Catarina V Jota Baptista, Ana I Faustino-Rocha, Fernanda Seixas, and Paula A Oliveira

Cardiovascular system

619

EC Bowdridge, E DeVallance, KL Garner, JA Griffith, PA Stapleton, S Hussain, and TR Nurkiewicz

Careers in toxicology

641

Mary Beth Genter

Catecholamines

649

Bracha Gurwitz and Sidhartha D Ray

CCA-treated wood

657

Ayesha Rahman Ahmed

Cell cycle Aarthi Nivasini Mahesh, Karanpreet Singh Bhatia, and Shruti Bhatt

667

Contents of Volume 2

Cell phones

ix 675

Mahshid Ataei and Mohammad Abdollahi

Cell proliferation

685

Iva Srdanovic, Ningning Yang, and Sidhartha D Ray

Centipedes

701

Timothy J Wiegand

Cephalosporins

705

Shayne C Gad

Cerium

711

Shayne C Gad

Cesium

715

Shayne C Gad

Charcoal

719

Sara Mostafalou and Perham Mohammadi

Chemical hazard communication and safety data sheets

725

Marí a J Ramos-Peralonso

Chemical safety assessment and reporting tool (Chesar), REACH

735

Marí a J Ramos-Peralonso

Chemical specific adjustment factor: A shift from default/refined toward hybrid uncertainty

741

Seyed Mojtaba Daghighi, Maryam Baeeri, and Hamid Rashidi Nodeh

Chemical toxicity of per- and poly-fluorinated alkyl substances (PFAS)

747

Noah Peter Christian

Chemical warfare

757

Steven A Burr

Chemical warfare agents and delivery systems

761

Alicia P DeFalco

Chemical warfare delivery systems

769

Steven A Burr

Chemicals alternatives assessments

777

Margaret H Whittaker

Chemicals in consumer products

785

L Molander

Chemicals of Environmental Concern

789

OI Kalantzi and AA Kanelli

Chernobyl

795

RK Chesser and BE Rodgers

Children's Environmental Health

803

Kristie Trousdale

Chloral hydrate

815

Emily Kershner and Michelle Troendle

Chlorambucil

819

Maria Chiara Astuto and Catalina Manieu

Chloramphenicol

825

Besen Sanga and Madan K Kharel

Chlordane Puttappa R Dodmane and Svetlana E Koshlukova

831

x

Contents of Volume 2

Chlordecone

843

Nilank Shah, Hayeon Chung, and Kaylin Huitsing

Chlordimeform

851

Lucio G Costa

Chlorfenvinphos

855

Mitra Geier and Svetlana E Koshlukova

Chlorination byproducts

865

Toni Mikhael and Sidhartha D Ray

Chlorine

873

Dhatrika Uggumudi and Sidhartha D Ray

Chlorine dioxide

883

Vicente M Gómez-López

Chloroacetic acid

889

Kenneth R Still

Chlorobenzene

893

Jannatul Arshad, Jannatul Ferdous, and Sidhartha D Ray

Chlorobenzilate

901

Atoosa Karimi Babaahmadi and Maryam Armandeh

Chlorodibenzofurans (CDFs)

905

Amelia B Hizon-Fradejas, Jeb Reece H Grabato, Sofia Angela P Federico, and Elmer-Rico E Mojica

Chloroethane

911

Atoosa Karimi Babaahmadi and Maryam Armandeh

Chlorofluorocarbons

917

WT Tsai

Chloroform

921

Eugenio Vilanova, Carmen Estevan, Miguel A Sogorb, and Jorge Estévez

Chloromethane (methyl chloride)

929

Kenneth R Still

Chlorophenols

935

Murali Badanthadka

Chlorophenoxy herbicides

943

Fred F Farris

Chloropicrin

953

Kanchita Patel and Sidhartha D Ray

Chloroprene

961

Elmira Meghrazi Ahadi, Seyedeh Azin Mirmotahari, and Mehdi Khoobi

2-Chloropropionitrile

969

Seyed Iman Mirnezami and Mehdi Khoobi

Chloroquine/hydroxychloroquine

973

Ahmad Naeem, Michael Gualano, Angy Ahmed, Mahwish Qureshi, and Sidhartha D Ray

Chlorothalonil

981

Priya Raman and Neha Bhavnani

Chlorpheniramine

989

Samaneh Nakhaee and Omid Mehrpour

Chlorpromazine Sophia Anagnostis, Nimrat Khehra, and Mayur S Parmar

995

LIST OF CONTRIBUTORS FOR VOLUME 2 Mohammad Abdollahi Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Ida Adeli Toxicology and Disease Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Alek Q Adkins University of Pittsburgh Medical Center, Pittsburgh, PA, United States Elmira Meghrazi Ahadi Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Angy Ahmed Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Ayesha Rahman Ahmed Department of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, WA, United States Samiha Ahmed Department of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, WA, United States Mohsen Amin Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran

Sophia Anagnostis Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Fort Lauderdale, FL, United States Maryam Armandeh Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Andreea Letitia Arsene Department of Microbiology, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Jannatul Arshad Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Maria Chiara Astuto European Food Safety Authority, Parma, Italy Mahshid Ataei Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Atoosa Karimi Babaahmadi Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Murali Badanthadka NUCARE Nitte (Deemed to be University), NGSM Institute of Pharmaceutical Sciences (NGSMIPS),

xi

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List of Contributors for Volume 2

Department of Nitte University Centre for Animal Research and Experimentation (NUCARE), Deralakatte, Mangalore, India Maryam Baeeri Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran Swapnaa Balaji Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, Toledo, OH, United States Mahdi Balali-Mood Medical Toxicology and Drug Abuse Research Center, Birjand University of Medical Sciences, Birjand, Iran; Medical Toxicology Research Centre, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Behnaz Bameri Toxicology and Disease Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Arindam Basu Sarkar Department of Pharmaceutical Sciences, College of Pharmacy, University of Findlay, Findlay, OH, United States Zahra Bayrami Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Karanpreet Singh Bhatia Department of Pharmacy, National University of Singapore, Singapore, Singapore Shruti Bhatt Department of Pharmacy, National University of Singapore, Singapore, Singapore Neha Bhavnani Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH, United States Hermann M Bolt Leibniz Research Centre for Working Environment and Human Factors (IfADo) at TU Dortmund, Dortmund, Germany

EC Bowdridge West Virginia University School of Medicine, Morgantown, WV, United States; Center for Inhalation Toxicology (iTOX), Morgantown, WV, United States Vera Bulakhova Department of Pharmaceutical and Biomedical Sciences, Touro Univeristy College of Pharmacy, New York, NY, United States Steven A Burr Peninsula Medical School, University of Plymouth, Plymouth, United Kingdom Daniela Calina Department of Clinical Pharmacy, University of Medicine and Pharmacy of Craiova, Craiova, Romania María Verónica Cesio Grupo de Análisis de Contaminantes Traza (GACT), Facultad de Quí mica, Universidad de la República, Montevideo, Uruguay Mina Rena Chapler Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States RK Chesser Texas Tech University, Lubbock, TX, USA Katarzyna Chojnacka Department of Advanced Material Technologies, Faculty of Chemistry, Wroclaw University of Science and Technology, Wrocław, Poland Noah Peter Christian Leidos, Incorporated, San Diego, CA, United States Hayeon Chung Touro College of Osteopathic Medicine, Middletown, NY, United States Carla Costa Environmental Health Department, National Institute of Health, Porto, Portugal; EPIUnit, Institute of Public Health, University of Porto, Porto, Portugal; Laboratory for Integrative and Translational Research in Population Health (ITR), Porto, Portugal Lucio G Costa Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA, United States Solange Costa Environmental Health Department, National Institute of Health, Porto, Portugal; EPIUnit-Instituto de Saúde Publica da Universidade do Porto, Porto, Portugal

List of Contributors for Volume 2

Seyed Mojtaba Daghighi Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran Marzieh Daniali Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran; Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran Alicia P DeFalco South College School of Pharmacy, Knoxville, TN, United States Eva del Río Instituto de Bioingenierí a, Unidad de Toxicologí a y Seguridad Quí mica, Universidad Miguel Hernández de Elche, Elche, Spain E DeVallance West Virginia University School of Medicine, Morgantown, WV, United States; Center for Inhalation Toxicology (iTOX), Morgantown, WV, United States Anca Oana Docea Department of Toxicology, University of Medicine and Pharmacy of Craiova, Craiova, Romania Puttappa R Dodmane Department of Pesticide Regulation, California Environmental Protection Agency, Sacramento, CA, United States Carmen Estevan Instituto de Bioingenierí a, Unidad de Toxicologí a y Seguridad Quí mica, Universidad Miguel Hernández de Elche, Elche, Spain Filipa Esteves Environmental Health Department, National Institute of Health, Porto, Portugal; EPIUnit-Instituto de Saúde Publica da Universidade do Porto, Porto, Portugal Jorge Estévez Instituto de Bioingenierí a, Unidad de Toxicologí a y Seguridad Quí mica, Universidad Miguel Hernández de Elche, Elche, Spain Leila Etemad Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran Pietro Fagiolino Facultad de Quí mica, Universidad de la República. Montevideo, Uruguay

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Fred F Farris School of Pharmacy, West Coast University, Los Angeles, CA, United States Frank Faulhammer BASF SE, Ludwigshafen am Rhein, Germany Ana I Faustino-Rocha Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Inov4Agro, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal; Department of Zootechnics, School of Sciences and Technology, University of Évora, Évora, Portugal; Comprehensive Health Research Center (CHRC), University of Évora, Évora, Portugal Sofia Angela P Federico Institute of Chemistry, University of the Philippines Los Baños, College, Laguna, Philippines Jannatul Ferdous Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Samantha E Gad Gad Consulting Services, Raleigh, NC, United States Shayne C Gad Gad Consulting Services, Raleigh, NC, United States Antonio J García-Fernández Toxicology and Risk Assessment Group, IMIB-Pascual Parrilla, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain KL Garner West Virginia University School of Medicine, Morgantown, WV, United States; Center for Inhalation Toxicology (iTOX), Morgantown, WV, United States Mitra Geier Department of Pesticide Regulation, California Environmental Protection Agency, Sacramento, CA, United States Diana Genis Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Mary Beth Genter University of Cincinnati, Cincinnati, OH, United States Vicente M Gómez-López Universidad Católica San Antonio de Murcia (UCAM), HiTech, Murcia, Spain Jeb Reece H Grabato Institute of Chemistry, University of the Philippines Los Baños, College, Laguna, Philippines

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List of Contributors for Volume 2

Joshua P Gray Department of Chemical & Environmental Sciences, U.S. Coast Guard Academy, New London, CT, United States Helmut Greim Technical University of Munich, Munich, Germany JA Griffith West Virginia University School of Medicine, Morgantown, WV, United States; Center for Inhalation Toxicology (iTOX), Morgantown, WV, United States Michael Gualano Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Natalia Guevara Facultad de Quí mica, Universidad de la República. Montevideo, Uruguay David Guirguis Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Bracha Gurwitz Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States K Hahn Missouri Poison Center at SSM Health Cardinal Glennon Children's Hospital, St. Louis, MO, United States Horacio Heinzen Grupo de Análisis de Contaminantes Traza (GACT), Facultad de Quí mica, Universidad de la República, Montevideo, Uruguay Amelia B Hizon-Fradejas Institute of Chemistry, University of the Philippines Los Baños, College, Laguna, Philippines Kaylin Huitsing Touro College of Osteopathic Medicine, Middletown, NY, United States Elizabeth K Hunt BAMM, Williamsburg, VA, United States Azhar Hussain Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States S Hussain West Virginia University School of Medicine, Morgantown, WV, United States; Center for Inhalation Toxicology (iTOX), Morgantown, WV, United States

Nancy A Ibrahim Research Scientist III, New Jersey Public Health Environmental and Chemistry Laboratory, Ewing, NJ, United States Jocephine Jonip Chemsain Konsultant Sdn Bhd, Kuching, Sarawak, Malaysia Catarina V Jota Baptista Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Inov4Agro, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal; Department of Zootechnics, School of Sciences and Technology, University of Évora, Évora, Portugal Arijeta Kaba Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States OI Kalantzi University of the Aegean, Mytilene, Greece AA Kanelli University of the Aegean, Mytilene, Greece Michael Keenan Department of Emergency Medicine, Upstate Medical University Upstate NY Poison Center, Syracuse, NY, United States Farzaneh Kefayati Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Emily Kershner Department of Emergency Medicine, Division of Medical Toxicology, Virginia Commonwealth University Health System, Richmond, VA, United States Madiha Khalid Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran Madan K Kharel School of Pharmacy and Health Professions, University of Maryland Eastern Shore, Princess Anne, MD, United States

List of Contributors for Volume 2

Nimrat Khehra Saint James School of Medicine, Arnos Vale, Saint Vincent and the Grenadines Mehdi Khoobi Deparatment of Pharmaceutical Biomaterials and Medical Biomaterials Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; Department of Radiopharmacy, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Rahul Khupse Department of Pharmaceutical Sciences, College of Pharmacy, University of Findlay, Findlay, OH, United States Svetlana E Koshlukova Department of Pesticide Regulation, California Environmental Protection Agency, Sacramento, CA, United States Tejas S Lahoti Drug Safety Research and Evaluation, Takeda Development Center Americas, Inc., San Diego, CA, United States Matthew Lambrych Department of Emergency Medicine, University of Rochester, Rochester, NY, United States Rajsumeet Macwan Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Aarthi Nivasini Mahesh Department of Pharmacy, National University of Singapore, Singapore, Singapore Catalina Manieu European Food Safety Authority, Parma, Italy Janet Mannella Canadian Centre for Occupational Health and Safety, Hamilton, ON, Canada Omid Mehrpour Data Science Institute, Southern Methodist University, Dallas, TX, United States María del Prado Míguez-Santiyán Universidad de Extremadura, Veterinary Faculty, Department of Animal Health, Laboratory of Toxicology, Cáceres, Spain Toni Mikhael Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States

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Marcin Mikulewicz Division of Facial Abnormalities, Department of Dentofacial Orthopaedics and Orthodontics, Medical University of Wroclaw, Wrocław, Poland Seyedeh Azin Mirmotahari Deparatment of Pharmaceutical Biomaterials and Medical Biomaterials Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Seyed Iman Mirnezami Department of Toxicology & Pharmacology, Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran Perham Mohammadi Department of Physiology and Pharmacology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran Hosna MohammadSadeghi Toxicology and Disease Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Elmer-Rico E Mojica Department of Chemistry and Physical Sciences, Pace University, New York, NY, United States Somayeh Mojtabavi Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran L Molander Stockholm University, Stockholm, Sweden Fatemeh Matin Moradkhan Faculty of Science, University of British Columbia, Vancouver, BC, Canada Militza Moreno Department of Public Health Sciences, Strong Memorial Hospital, University of Rochester Medical Center, Rochester, NY, United States Mohammad Moshiri Medical Toxicology Research Centre, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Clinical Toxicology and Poisoning, Imam Reza (p) Hospital, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Sara Mostafalou Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran

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List of Contributors for Volume 2

Taraneh Mousavi Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran; Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran Sandra R Murphy Consultant, Kennett Square, PA, United States Ahmad Naeem Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Samaneh Nakhaee Medical Toxicology and Drug Abuse Research Center (MTDRC), Birjand University of Medical Sciences, Birjand, Iran Isabel Navas Toxicology and Risk Assessment Group, IMIB-Pascual Parrilla, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain Rabin Neupane Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, Toledo, OH, United States Aniqa Niha Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Hamid Rashidi Nodeh Food Technology and Agricultural Products Research Center, Standard Research Institute (SRI), Karaj, Iran TR Nurkiewicz West Virginia University School of Medicine, Morgantown, WV, United States; Center for Inhalation Toxicology (iTOX), Morgantown, WV, United States Paula A Oliveira Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Inov4Agro, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal; Department of Veterinary Sciences, UTAD, Vila Real, Portugal

Daniel L Overbeek University of Rochester School of Medicine and Dentistry, Rochester, NY, United States Mayur S Parmar Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Clearwater, FL, United States Kanchita Patel Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Rupangi S Patel Pharmacy Department, Albertsons Companies, Inc., San Diego, CA, United States Emma Pereira Toxicology and Risk Assessment Group, IMIB-Pascual Parrilla, Faculty of Veterinary Medicine, University of Murcia, Murcia, Spain Vahid Pourbarkhordar Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Valentina Patricia Predoi Faculty of Pharmacy, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania Tate Pumphrey Touro College of Osteopathic Medicine, Middletown, NY, United States Mahwish Qureshi Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States Priya Raman Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH, United States María J Ramos-Peralonso Occupational Risk Prevention and in Social Communication and Health, Madrid, Spain

Michael Olshansky Touro College of Osteopathic Medicine, Middletown, NY, United States

Sushma Ramsinghani University of the Incarnate Word Feik School of Pharmacy, San Antonio, TX, United States

Ana Lourdes Oropesa-Jiménez Universidad de Extremadura, Veterinary Faculty, Department of Animal Health, Laboratory of Toxicology, Cáceres, Spain

Sidhartha D Ray Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States

List of Contributors for Volume 2

Jisha Reji Touro University College of Osteopathic Medicine, New York, NY, United States

Kenneth R Still Occupational Toxicology Associates, Inc., Lake Oswego, OR, United States

Linda G Roberts NapaTox Consulting LLC, Napa, CA, United States

Christine Stork Department of Emergency Medicine, Upstate Medical University Upstate NY Poison Center, Syracuse, NY, United States

Heriberto Robles Enviro-Tox Services, Inc., Irvine, CA, United States BE Rodgers Texas Tech University, Lubbock, TX, USA Besen Sanga School of Pharmacy and Health Professions, University of Maryland Eastern Shore, Princess Anne, MD, United States Leona D Scanlan Office of Environmental Health Hazard Assessment, California Environmental Protection Agency, Sacramento, CA, United States Eric A Seagren Department of Civil, Environmental, and Geospatial Engineering, Michigan Technological University, Houghton, MI, United States Fernanda Seixas Department of Veterinary Sciences, UTAD, Vila Real, Portugal; Veterinary and Animal Research Center (CECAV), UTAD, Vila Real, Portugal Nilank Shah Department of Medical Physiology and Pharmacology, Touro College of Osteopathic Medicine, Middletown, NY, United States Siong Fong Sim Faculty of Resource Science & Technology, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia Thomas Smagala Chevron Technical Center, San Ramon, CA, United States Miguel A Sogorb Instituto de Bioingenierí a, Unidad de Toxicologí a y Seguridad Quí mica, Universidad Miguel Hernández de Elche, Elche, Spain Francisco Soler-Rodríguez Universidad de Extremadura, Veterinary Faculty, Department of Animal Health, Laboratory of Toxicology, Cáceres, Spain Iva Srdanovic Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States PA Stapleton Rutgers University-Ernest Mario School of Pharmacy University, Piscataway, NJ, United States

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Jose V Tarazona Risk Assessment Unit, National Centre for Environmental Health, Instituto de Salud Carlos III, Madrid, Spain João Paulo Teixeira Environmental Health Department, National Institute of Health, Porto, Portugal; EPIUnit, Institute of Public Health, University of Porto, Porto, Portugal; Laboratory for Integrative and Translational Research in Population Health (ITR), Porto, Portugal Kashyap N Thakore California Department of Public Health, Richmond, CA, United States Amit K Tiwari Department of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, Toledo, OH, United States Michelle Troendle Department of Emergency Medicine, Division of Medical Toxicology, Virginia Commonwealth University Health System, Richmond, VA, United States Kristie Trousdale Children's Environmental Health Network, Washington, DC, United States WT Tsai National Pingtung University of Science and Technology, Pingtung, Taiwan Dhatrika Uggumudi Department of Pharmacology and Toxicology, Wright State University, Dayton, OH, United States Virginia Rodríguez Unamuno European Chemicals Agency, Directorate Hazard Assessment, Helsinki, Finland Erik van de Plassche European Chemicals Agency, Directorate Risk Management, Helsinki, Finland Leon van der Wal Organisation for Economic Cooperation and Development, Environment Directorate, Paris, France Marta Vázquez Facultad de Quí mica, Universidad de la República. Montevideo, Uruguay

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List of Contributors for Volume 2

Manish Verma Touro College of Osteopathic Medicine, New York, NY, United States Eugenio Vilanova Instituto de Bioingenierí a, Unidad de Toxicologí a y Seguridad Quí mica, Universidad Miguel Hernández de Elche, Elche, Spain HR Watson Peninsula Medical and Dental Schools, University of Plymouth, Plymouth, United Kingdom JA Weber Missouri Poison Center at SSM Health Cardinal Glennon Children's Hospital, St. Louis, MO, United States

Margaret H Whittaker ToxServices LLC, Washington, DC, United States Timothy J Wiegand University of Rochester Medical Center and Strong Memorial Hospital, Rochester, NY, USA Ningning Yang Lake Erie College of Osteopathic Medicine, Bradenton, FL, United States Raghunandan Yendapally University of the Incarnate Word Feik School of Pharmacy, San Antonio, TX, United States

EDITOR BIOGRAPHIES Editor-in-Chief

Philip Wexler has published, taught, and otherwise lectured extensively in the United States and abroad in the disciplines of toxicology and toxico-informatics. He is the Editor-in-Chief of four editions of the Encyclopedia of Toxicology (Elsevier. 4th edition 2023) and five editions of Information Resources in Toxicology (Elsevier. 5th edition 2020), as well as Chemicals, Environment, Health: A Global Management Perspective (CRC Press/Taylor and Francis, 2011). He has served as an Associate Editor for Toxicology Information and Resources for Elsevier’s journal, Toxicology and in that capacity, edited special issues on Digital Information and Tools. He is also overseeing and editing an ongoing monographic series on toxicology history. He is one of the senior editors of the Taylor and Francis journal, Global Security: Health, Science, and Policy. A longtime member of the U.S. Society of Toxicology (SOT), he has served as Chair of its World Wide Web Advisory Team and president of its Ethical, Legal, and Social Issues Specialty Section as well as a member of the Education and Communications Work Group of the U.S. CDC/ATSDR’s National Conversation on Public Health and Chemical Exposure. He is a trustee of the Toxicology Education Foundation (TEF). Mr. Wexler is retired from a distinguished U.S. Government career with the National Library of Medicine’s (NLM) former Toxicology and Environmental Health Information Program where he participated in and served as team leader for a spectrum of toxicology databases and initiatives. He is a recipient of SOT’s Public Communication Award, the NLM Regents Award for Scholarly or Technical Achievement, and the Distinguished Technical Communication Award of the Washington chapter of the Society for Technical Communication. During his toxicological downtime, Mr. Wexler writes poetry, with five collections to his credit, and recreationally works in mosaics.

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Editor Biographies

Associate Editors Mohammad Abdollahi is a highly respected Toxicopharmacological scientist who has made significant contributions to Mechanistic Toxicology. He also focused on Drug and Poison Information Centers and co-established the Tehran Center. He helped TUMS in establishing Digital Journals management system along with the digital library. He was elected and worked for more than 10 years as the Director of the Toxicology Board and the National Society. Through his research, he has identified biomarkers of diseases caused by environmental toxicants. MA’s groundbreaking work has earned him numerous national and international awards, including the prestigious IAS-COMSTECH 2005 and Clarivate Highly Cited 2021 awards and prestigious medals from IFIA for his inventions. His collaborations with various departments on innovative projects, such as the OPCW, WHO, COPE, CINVU/COMSTECH/OIC, UNOG-UNEP-Chemicals (SAICM), and the IAS, are remarkable. Additionally, MA has been recognized as the Best Leader Awardee of Medicine in 2022 and 2023 reported by Research.com. In addition to his research, MA also served as the Associate Editor of the 3rd edition of the “Encyclopedia of Toxicology,” a highly regarded reference book in the field. The Editor-in-Chief of the 3rd and 4th editions is Philip Wexler, a renowned toxicologist, who worked at the National Library of Medicine (NLM) and has written several books on toxicology. The 4th edition of Encyclopedia of Toxicology addresses the challenges of a rapidly changing world. With its comprehensive coverage, it will undoubtedly become the go-to resource for all queries in this field. More information is noted at https://orcid.org/0000-0003-0123-1209. Shayne C. Gad, Ph.D., D.A.B.T., BS Chemistry/Biology Whittier College, Ph.D. Pharmacology/Toxicology University of Texas, 1977, was past president of American College of Toxicology and Roundtable of Toxicology Consultants. He is an expert in neurotoxicology, inhalation toxicology, biocompatibility assessment, statistics and risk assessment, and biopharmaceutical and medical devices safety assessment and development. He successfully prepared and filed 129 IND’s, and was principal of GAD Consulting Services since 1993. He had published 53 books, 75 chapters, 387 abstracts and presentations, and more than 400 sections in large works and encyclopedias. He was a member of SOT, ACT, STP, Safety Pharmacological Society American Statistical; association teacher and adjunct professor at USC and University of Addis Ababa; and has taught courses globally.

Helmut Greim, born in 1935, has studied medicine at the universities of Freiburg and Berlin, Germany. Thereafter, he had research positions in Biochemistry and Pharmacology at the Free University of Berlin and of the Institute of Toxicology, University of Tübingen, 1970–73 as Visiting Research Associate Professor of Pathology, Mount Sinai School of Medicine and Visiting Fellow of Pharmacology, Yale University. After 2 years back in Tübingen, he was appointed as Director of the Institute of Toxicology of the GSF, a federal Research Institute in Munich, in 1975. In 1982, he became Professor and Director of the Institute of Toxicology and Environmental Hygiene, Technical University Munich. He retired from these positions in 2003. His research experience was in the fields of drug metabolism, toxicokinetics, mechanisms of carcinogenic agents, and in vitro test systems. Besides many publications and contributions to textbooks, he has published three German (1996, 2017, 2022), two English (2008, 2019), and one Italian (2000) textbooks in Toxicology (Wiley) and “The cellular response to the genotoxic insult: the question of threshold for genotoxic carcinogens” (the Royal Society of Chemistry, London, 2013). He has organized several workshops on fiber and particle toxicology and on benzene toxicity and as chair of MAK and SCHER of DG SANCO (EU Commission), member of SCOEL and RAC of ECHA, and has been involved in the risk assessment of chemicals.

Editor Biographies

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Mary Gulumian was the Head of the Toxicology and Biochemistry Section at the National Institute for Occupational Health (NIOH) and thereafter Head of Toxicology Research Projects, at the NIOH. Presently, she holds an honorary Professorial post in the Hematology and Molecular Medicine Department, the University of the Witwatersrand where she has presented courses on Health Risk Assessment and supervised postgraduate students. She has also been appointed as an Extraordinary Professor at the North-West University where she organized workshops on Health Risk assessment. She is the founder member and the past President of the Society for Free Radical Society of South Africa (SFRR-SA), the founder member and President of the Toxicology Society of South Africa (TOXSA), and the founder member and President of the Society of Risk Analysis (SRA)-Africa. Her research interests include hazard identification and communication as well as elucidation of mechanisms of toxicity of micro and nano particles. She received funding from local (DSI and MHSC) and international agencies (EU projects) to conduct research on the topic. She has authored and co-authored numerous scientific publications on this topic and made a great number of keynote and invited presentations at local and overseas conferences. She has also provided expert consultation to industry and government departments on the toxicity of chemicals in the working and ambient environments. Professor Gulumian is a member of the Editorial Board of the Journal, Human and Experimental Toxicology (HET), Toxicology, and Particle and Fibre Toxicology (PFT), and also the Associate Editor of the Journal, Inhalation Toxicology. Evangelia Iatrou was born in Heraklion, Greece, in 1986. In 2008, she graduated from the Department of the Environment, School of the Environment, University of the Aegean. In 2009, she got her master’s degree in environmental and Ecological Engineering at the University of the Aegean. Her master thesis concerned the “Pesticides photodegradation study and assessment of their combined toxicity in Lemna minor.” She has worked in the Laboratory of Toxicology and Forensic Sciences, Medical School, University of Crete for the period 2009–10. Afterward, she turned back at University of Aegean, and she made her doctoral thesis in Environmental Sciences, titled “Study of the fate of antimicrobial substances in artificial wetland systems planted with the Lemna minor organism and investigation of the possibilities of utilization of the produced biomass.” During her Ph.D., she provided lectures as ancillary work for the following courses: Ecotoxicology, Analytical Chemistry, and Wastewater Management. Since then (2017), she is a postdoctoral researcher in Laboratory of Toxicology and Forensic Sciences, Medical School, University of Crete. Evangelia Iatrou has published original articles in peer-reviewed international journals, abstracts, and presentations in national and international congresses. She has participated in national and international congresses and in educational EUROTOX Advanced Course at year 2013. She is also a reviewer in the scientific journals Toxicology Reports and Ecotoxicology and Environmental Safety. Furthermore, Evangelia Iatrou is an adult educator in Second Chance Schools, teaching Environmental Education. She has organized various speeches and awareness raising campaigns on environmental issues in the context of school activities. Diana Míguez is a Principal Research Scientist and Water Program Director at Latitud—LATU Foundation, Technological Laboratory of Uruguay (LATU) since 2017. Pharmaceutical chemist (UDELAR, 1989) and Ph.D. in Water Sciences, Cranfield University, UK, 2014. Thesis: Integrated Risk Assessment of Endocrine Disruptors in the Uruguay River. Dr. Míguez possesses more than 30 years of experience in Analytical Chemistry, Water Science and Technology, Environmental Toxicology, Ecotoxicology, Medical Geology, Sustainability, and Risk Assessment fields. She held prior posts in pharmaceutical and food analyses, and since 1991, she is the head of the Water and Chemicals Department, and senior specialist at LATU.

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Editor Biographies

Asish Mohapatra is a toxicologist and a human health risk assessment specialist with Health Canada. He has a masters’ degree in Life Sciences, a predoctoral degree in Environmental Sciences (chemical toxicology), and a graduate certificate in environmental management from the University of Calgary, and certificates in case works in toxicology and public health risk analysis from Harvard University. His 25 years of work experience includes toxicology and human health risk assessment (HHRA) of industrial chemicals, mines and radiological contaminated sites, chemical fate, transport and transformation and HHRA implications, chemical class specific expertise include metals/metalloids, petroleum hydrocarbons, fluorinated and chlorinated chemicals toxicology, computational systems toxicology, elimination kinetics and persistence of effects and exposure analysis, mechanism of action, mode of action and adverse outcome pathways framework and knowledgebase development and applications; physiological-based pharmacokinetic models review and applications; Emerging informatics tools and new approaches and methodologies (NAMs) developments in collaborative computational toxicology and opensource platforms; toxico-genomics and epigenomics applications; Data fusion tools and methodological applications in statistics, toxicology and risk assessments; climate change health risk assessments, cumulative exposure (chemical and nonchemical stressors) and risk analysis; permafrost affected soils and active layer dynamics. Major reference publication includes Information Resources in Toxicology (2019, fifth edition). Asish is one of the Co-Editor-in-Chief for the journal—Global Security: Health, Science and Policy and a founding member of the OpenTox Association (a global community of practice promoting collaborations and opensource tools and frameworks in support of knowledgebase development, applications and publications). Sidhartha D. Ray, Ph.D., FACN, Past Acting chair, currently serves as a senior professor of Pharmaceutical and Biomedical Sciences at The Touro University College of Pharmacy, New York. Prior to this, he was at the AMS College of Pharmacy of Long Island University, NY, for 18 years and as the founding chair of Pharmaceutical Science department at Manchester University College of Pharmacy at Indiana. Dr. Ray’s academic career spans over 40 years in pharmacy, teaching, research, and service. His research focuses on pharmacology, molecular toxicology, adverse drug reactions, side effects of a number of therapeutically used drugs and environmental chemicals. His service contributions are reflected by his election into multiple international professional organizations, such as the SOT, AACP, ACN, ASPET, and SFRBM. Dr. Ray serves as the Editor-in-Chief of “Side Effects of Drugs Annual” (Elsevier since 2014), and as Associate Editor of “Archives of Toxicology” and OMCL. He is a “Fellow of the American College of Nutrition” since 1999. He has won Teaching Excellence Award (2005 & 2023), Lifetime Scholarly Achievement Award (2008), “Wall-of-Fame” honor (2011), the Society of Toxicology’s “Educator of the Year” national award in 2013, “Outstanding Scholar award” (2014), and Senior Toxicologist award from ASIOA/SOT (2015). In his lifetime, he has mentored numerous PharmD, MS, MD, and PhD students and colleagues in health and life sciences. His 200+ publications have garnered him 8000 Google scholar citations. His key mantra to success has been instilling a “Lifelong Learning” mindset in his mentees. Visit URL: www.sidhartharay.com/ for more details. José V. Tarazona, Doctor in Veterinary Medicine, Ph.D. in Toxicology, Full Member of the Spanish Royal Academy of Veterinary Sciences. Current affiliation: Research Professor and Head of the Risk Assessment Unit at the National Environmental Health Centre, Instituto de Salud Carlos III, the Spanish public research institution on health under the Ministry of Science and Innovation. Main involvements are in EU projects, particularly in the research partnership PARC, collaborating in several activities and leading the project “Quantify effects of PPP and other stressors through landscape risk assessment informing on environmental impacts.” The activities cover human and environmental risk assessments, focusing on environmental pollutants and pesticides,

Editor Biographies

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and the evolution of the risk assessment paradigm for incorporating New Approach Methodologies (NAMs), contributing to international actions through APCRA, ILMERAC as co-chairing the NAMs Working Group, the European Parliament and the European Partnership for Alternative Approaches to Animal Testing. Previous affiliation: Head of the Pesticides Unit and Senior Scientific Officer at the Scientific Committee and Emerging Risk Unit, European Food Safety Authority, Parma, Italy. Involved in regulatory risk assessments, chairing the EFSA Working Group on Nanomaterials and the EFSA Nanonetwork, coordinating several Scientific Committee Working Groups and EFSA internal projects focused on NAMs. Aristidis Tsatsakis is the Director of the Laboratory of Toxicology and Forensic Sciences of the University of Crete and the University Hospital of Crete, Greece. Furthermore, he is the initiator, founder, scientific director, and head of the spin-off company of the University of Crete, ToxPlus S.A. He has more than 1500 international publications (books, articles, and conference presentations) and holds several patents. He is the coordinator of several European HORIZON projects and has organized several international conferences as chairman. He served as president of the European Federation of European Societies of Toxicology (EUROTOX) from 2014 to 2016. He is the editor-in-chief of the Public Health Toxicology journal and has served as editor of several leading international journals. He has been awarded Honorary Doctorate and Professor in many universities and institutes around the world as well as member of the Academy of Toxicological Sciences of the United States, the World Academy of Sciences and honorary member of many Toxicological Societies such as Bulgaria and Slovakia. In 2018, he was nominated Honorary President of the European Institute of Nutritional Medicine and Honorary Member of EUROTOX. In 2020 and 2021, he was recognized as Highly Cited Researcher in the field of Pharmacology—Toxicology of Biomedical Sciences taking the top position in the list of the most influential researchers, for the field of Toxicology, worldwide. In 2022, he was awarded the EUROTOX Merit Award 2022 by EUROTOX for his significant contribution to the advancement of the field of Toxicology. Recently, Prof. Tsatsakis was elected as a member of the Academia Europaea recognizing his outstanding achievements as a researcher. Timothy J. Wiegand, MD, FACMT, FAACT, DFASAM, holds Board Certification in Medical Toxicology and Addiction Medicine. He completed Toxicology and Clinical Pharmacology fellowship training at the University of California, San Francisco, in 2006 and is currently the Director of Addiction Toxicology at the University of Rochester Medical Center and an Associate Professor of Emergency Medicine in the Department of Emergency Medicine at Strong Memorial Hospital in Rochester, New York, United States. He is also Medical Director of Huther-Doyle Chemical Dependency program in Rochester, New York, and a consultant toxicologist for the SUNY Upstate Poison Center in Syracuse, New York. Dr. Wiegand founded the Toxicology Consult Service for the University of Rochester Medical Center hospitals and affiliate system in 2010 and subsequently the Addiction Medicine consultation-liaison service for the hospital system. Dr. Wiegand has served as Fellowship Director for the URMC Combined Addiction Medicine Fellowship program, housed in Emergency Medicine. He is also the Fellowship Director for the Medical Toxicology Fellowship program which is anticipated to start in July 2023. Dr. Wiegand has served on the Board of Directors for the American College of Medical Toxicology (ACMT), and he is currently serving on the Executive Council of the American Society of Addiction Medicine (ASAM) Board of Directors as Vice President. Dr. Wiegand has authored numerous chapters in textbooks and papers in peer-reviewed journals and is an associate editor of the Encyclopedia of Toxicology, 4th edition.

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FOREWORD As is true of the terrain of every scientific discipline, new toxicological knowledge is uncovered continuously. Like ascending a mountain, features faintly visible come into clarity the higher we rise. Toxicologists are driven to ponder and reveal answers to the mysteries underlying the actions of potentially toxic agents and weighing risks versus benefits in their application in medicine and other fields. Over four editions now, the Encyclopedia of Toxicology has been helping to map that climb by expanding and distilling into readily digestible entries a more and more extensive view of the current landscape of toxicology. Toxicology continues to be rooted in the health and welfare of the human species. A recent emphasis on environmental toxicology is driven by population growth and industry. Toxicology advances as new technologies and techniques are discovered and implemented. These changes can be rapid and, in many cases, alter the traditional path and pace up the mountain. The concept of “alternative” or “new approach methodologies” is complex and the views of those who will use the information generated by such novel tests will only be properly informed by careful evaluation of outcomes. Big data, artificial intelligence, and complex exposures are a few of the specific present-day issues we are grappling with. There is a need for rigorous, open, and reproducible science as we journey up the mountain. The Encyclopedia is one of an armamentarium of tools helping us chart the course to the mountain’s ever-receding crest because, in truth, mistaken sightings are not uncommon and ultimate goals are elusive. This new 4th edition of the Encyclopedia of Toxicology is an important link between condensed dictionary definitions and detailed research papers, reviews and monographs, and is devoted to wide-ranging aspects of the field, both narrow and broad. In addition to its value to the toxicology community, it will play an important role for nontoxicologists’ gathering background knowledge on one topic or another, or simply curious about the role of poisons in science and society. It is my privilege to play a supporting role in scaling the toxicology mountain by contributing this Foreword to the most recently expanded, updated, and welcome 4th edition of the Encyclopedia of Toxicology. A Wallace Hayes, Ph.D., DABT, FATS, FRSB, FACFE, ERT, University of South Florida College of Public Health, Tampa, FL, United States Michigan State University Center for Integrative Toxicology, East Lansing, MI, United States

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PREFACE Ten years have elapsed since the Encyclopedia of Toxicology’s previous edition and the world of toxicology, as the world itself, has not stood still. On a macro level, human-generated global pollution, despite activist and government pleas to curtail its release, and a handful of good faith national efforts, continues virtually unabated. Greenhouse gas emissions, much of which result from the burning of fossil fuels such as coal, oil, vehicular gasoline, and natural gas, blanket the Earth and trap the sun’s heat. Climate change is at a crisis stage with atmospheric and ocean heat, severe storms, drought, a rising ocean, and food insecurity all on the increase. Heat and drought have been among the drivers of an increase in the risk and extent of wildfires, such as in the western United States recently. Indeed, the United Nations’ Intergovernmental Panel on Climate Change (IPCC)’s most recent synthesis report on climate change, released in March 2023, warns of a dire future unless people individually and governments broadly take swift action. Over the years, our exposure to microplastics and other micromaterials has become ubiquitous. More specifically, we find ourselves increasingly exposed to PFAS (per- and polyfluoroalkyl substances), antimicrobials, flame retardants, bisphenols and phthalates, solvents, and metals. Our usage, storage, and disposal habits must change. Drug overdose deaths, driven by synthetic opioids, fentanyl in particular, some prescribed but frequently manufactured illicitly, have attained record levels. The rise has been a result of both intentional and accidental exposures. The U.S. Centers for Disease Control and Prevention’s (CDC) National Center for Health Statistics data show a provisional predicted value count of more than 107,689 drug overdose deaths for the 12-month period ending October 2022. A concerted effort is required to stem the tide of this epidemic, including securing the borders. The passage of the Omnibus Spending Bill in late 2022 included increased federal funding for state opioid response grants to make medications such as buprenorphine and naltrexone more readily available to all who need them. Apart from the opioid crisis, more research, prevention, and treatment resources need to be devoted to the abuse of prescription and over-the-counter drugs, including their synergistic effects with other substances. The sudden appearance of COVID-19 on the world scene at the start of 2020 upturned lives of people everywhere and continues to do so in one way or another. Interestingly, this infectious disease has had several toxicological implications. A broad spectrum of treatments and prevention measures were employed globally. They ranged from repurposed treatments with proven safety profiles to inadequately tested new technologies or clearly bogus and dangerous recommendations such as drinking or injecting bleach. Given the potential for other novel disease outbreaks, a greater emphasis on the toxicological profiling of a wide range of antivirals needs to be initiated well in advance. In addition to new and wider exposures to drugs and other chemicals, people are being subjected to increasing levels of nonionizing radiation, particularly as generated by cell phones, radar, power lines, Wi-Fi (as in routers), as well as any number of existing and yet to be developed smart devices. More toxicological research is imperative to determine and minimize potentially adverse effects. With tensions and hostilities rising within and among nations more than in decades, threats and pursuit of military warfare are increasing. Such conflicts increase the potential for the use of chemical, biological, and nuclear weapons. Even engaging in traditional battles raises the toxicological stakes, as munitions raining down upon “enemy” territory bring, in addition to casualties, swaths of environmental devastation, impacting the water and food supply and other human necessities. Individual targeted attacks in which poisoning is a component of the espionage game is another tactic, ancient in origin, which some governments seem to have never outgrown, to silence dissidents and enemies.

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High-dose mammalian toxicity testing continues to be a mainstay of identifying toxic agents and will not disappear overnight. Nonetheless, in the name of animal welfare, expense, and limits in interpretation when extrapolating to humans, they have already begun to diminish. The 3 Rs mantra (replacement, reduction, and refinement) has much to commend it and is adhered to by many laboratories. Zebrafish have become the latest animal model darling of toxicity testing, especially in high-throughput early screening. In vitro testing is a mature alternative methodology to testing in animals and is in widespread use. In silico testing uses computer technology as a platform for assessment and read-across is an approach to evaluate a substance’s toxicity based on data gathered from structurally similar compounds. New Approach Methodologies (NAM) for toxicological evaluation are now pervasive in the United States and elsewhere. The International Cooperation on Alternative Test Methods (ICATM) is a multinational organization whose primary goals are to establish cooperation in validation studies, peer reviews, and new alternative test methods and strategies. Two specific toxicology research frontiers, which are certain to experience growth in the coming years, are multiple exposures and epigenetics. Current risk assessments focus largely on individual substances. However, in order to be relevant to real-world scenarios, more attention will be paid to determining risk from combined exposures to chemical, biological, and radiological agents. Epigenetics is the study of the way cells control gene activity without changing the DNA sequence. Its significance for toxicology is that the broad environment, including pollutants and potentially even diet, can alter the epigenome, affecting the way DNA sequences are read. Edition by edition, the Encyclopedia, to encompass the growth in the field, has grown from 749 entries in its first 1998 edition to over 1200 today. Some 75 new entries have been added since the third edition. These include a potpourri of subjects such as COVID-19, in silico toxicology, imaging, microbiome, micro- and nano-plastics, telomeres, data fusion applications, organ-on-a chip, wildfires, gender differences, nurdles, burn pits, toxicology in the ICU, medical devices, nuclear warfare agents, monoclonal antibodies, 3D printers, and Novichok. Virtually every other entry, whether representing a specific agent or class of chemicals, method/ technology/tool, or other broad topic, except for a very limited number of entries, such as those of historical import, have been updated to reflect current research and thinking. Given all the research and applications that have been advanced to strengthen the scientific underpinnings of toxicology, some principles remain ironclad. First articulated in 1538, Paracelsus’ dictum, Alle Dinge sind Gift, und nichts ist ohne Gift; allein die Dosis macht, dass ein Ding kein Gift ist (All things are poison, and nothing is without poison; the dosage alone makes it so a thing is not a poison more colloquially expressed as The dose makes the poison) has remained a succinct and unwavering summation of at least one aspect of toxicology. Similarly, the four widely acknowledged steps of risk assessment (1. hazard identification, 2. hazard characterization, 3. exposure assessment, and 4. risk characterization) have withstood the test of time. The idea of toxicity has infiltrated the psyche of individuals and society as never before, and the term and its cognates have become ever more prevalent buzzwords and are routinely applied to circumstances that have little if anything to do with science. It has become a ready verbal tool to connote negativity of almost any sort. Thus, we hear talk of toxic behaviors, toxic situations, toxic relationships, toxic people, etc. In fact, toxic was the Oxford English Dictionary’s 2018 Word of the Year and has not lost its sparkle and overuse since then. We are no longer in an era when a major Encyclopedia can be a one-person undertaking. Pliny the Elder may have successfully compiled his Naturalis Historia single-handedly in the first century AD, but those days are long gone. That said, I would like first and foremost to thank my 11 Associate Editors, paragons of toxicological wisdom, without whose expertise and commitment, I could not have completed even the first few entries under the letter, A. On the publisher side, Elsevier’s Blerina Osmanaj, the Encyclopedia’ s Acquisitions Editor, as well as Paula Davies and Pamela Sadhukhan, its Content Project Managers played prominent roles. Other Elsevier staff with less visible roles have also done their part to bring the book to fruition and are due an equally well-deserved shout-out. Finally, I am grateful to Prof. A. Wallace Hayes, an exemplar of the scholarly research toxicologist, whom I have known and worked with over decades, for his kind Foreword. Wally’s own extensive toxicology text, while not an Encyclopedia per se, is no less encyclopedic and informative than this book, and makes for a good companion piece. My Associate Editors and I would like to pay tribute to the enduring relevance of toxicology as a scientific discipline impacting our everyday lives. As with any science, it will continue to evolve. On a lighter note, maybe someday toxicologists will eventually reach a consensus on whether moderate amounts of coffee and wine are good for us or not. And with Consumer Reports recently finding that many brands of dark chocolate bars vary widely in their lead and cadmium compositions, when will we ever be safe? As for mercury in fish?—don’t get me started! Encyclopedias are not revised all that often but no doubt, a fifth edition of this one will be warranted before long. Will I, personally, dive in? Time will tell. Meanwhile, Happy Reading in this Fourth! Philip Wexler Editor-in-Chief

Benzidine Aniqa Niha, Diana Genis, and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of C.V. Rao, Benzidine, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 419–422, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00249-9.

Chemical profile Background Uses Environmental fate and behavior Partition behavior in water, sediment, and soil Environmental persistency (degradation/speciation) Bioaccumulation and biomagnification Exposure and exposure monitoring Routes and pathways Human exposure Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive toxicity Genotoxicity Human genotoxicity Carcinogenicity Clinical management Conclusion References Further reading

2 2 3 3 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 7 7 7 8

Abstract Benzidine (CAS# 92-87-5) is a synthetic biphenyl diamine used as an intermediate in the production of azo dyes, sulfur dyes, fast color salts, naphthol, and other dye compounds. It has been classified as a human carcinogen (Class A) based on sufficient evidence of carcinogenicity from studies in humans. Benzidine may induces cancer via numerous molecular mechanisms such as upregulation of p53, increasing the expression of bladder cancer stem cell markers, inducing the epithelial-mesenchymal transition of human bladder cancer cells, and triggering cell proliferation and the formation of urothelial carcinomas via activation of the PI3K/Akt signal pathway. Commonest routes of exposure to benzidine are inhalation, ingestion, and dermal contact and several case reports and cohort studies conducted in the U.S. and worldwide several locations. A strong association between occupational exposure to benzidine with urinary-bladder cancer and bladder-cancer related deaths were reported. The risk of incidence and death from bladder cancer remains higher even 20 years after the last exposure to benzidine in those who worked >5 years. Due to its carcinogenicity, the use of benzidine has been limited in the U.S. since the mid-1970s and additional epidemiological data suggest a decreased incidence of urinary-bladder cancer since measures to limit benzidine exposure were instituted. Animal studies have shown that administration of benzidine via oral, subcutaneous, and intraperitoneal injection can lead to numerous types of cancers and tumors, establishing the carcinogenicity of benzidine. Benzidine is metabolized to an aromatic amine by intestinal microflora or liver azo-reductase. LD50 doses are: mouse orally ¼ 214 mg/kg, mouse ip ¼ 110 mg/kg and rat orally ¼ 309 mg/kg.

Keywords Arylamides; Arylamines; Azo dyes; Azo-reductase; Benzidine; Bladder cancer; Carcinogenic; DNA damage; Intestinal microflora; Mutagenic; N-hydroxyarylamides; N-hydroxyarylamines; P53

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00714-4

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Benzidine

Key points

• • • •

Benzidine-based dyes were used primarily to color textiles, leather, paper products, petroleum industry, rubber, plastics, wood, soap, fur, and hair-dye industries. However, the production was phased out in 1970s. Occupational exposure to benzidine results in an increased risk of bladder cancer, according to studies of workers in different geographic locations. Benzidine is rapidly absorbed through the skin in solid and vapor forms. It is also quickly absorbed through the lungs on inhalation and from the GI tract by consuming contaminated water and food. Although benzidine exposure has been associated with cancer in the liver, kidney, central nervous system, oral cavity, larynx, esophagus, bile duct, gallbladder, stomach, and pancreas in some studies, it is most closely associated with urinary-bladder cancer.

Abbreviations BCSCs DHHS EMT ERK5 IARC MAPK NAT2 OSHA PCNA

Bladder cancer stem cells Department of Health and Human Services Epithelial-mesenchymal transition Extracellular regulated protein kinases 5 International Agency for Research on Cancer Mitogen-activated protein kinase N-acetyl transferase gene The Occupational Safety and Health Administration Proliferating cell nuclear antigen

Chemical profile

• • • • •

Name: Benzidine Chemical Abstracts Service Registry Number: 92-87-5 Synonyms: (1,10 -Biphenyl)-4,40 -diamine; 4,40 -Bianiline; 4,40 -Biphenyldiamine; 4,40 -Biphenylenediamine; 4,40 -Diamino-1,10 biphenyl Molecular Formula: C12H12N2 Chemical Structure: H

H

N

N

H

H

Structure of Benzidine

Background Benzidine is a manufactured biphenyl diamine that exists at room temperature as a grayish-red, yellowish or white colored crystalline powder which darkens upon exposure to air and light. While benzidine is only slightly soluble in cold water, its solubility increases with hot water and it is readily soluble in less-polar solvents such as diethyl ether and ethanol. Structurally, benzidine is manufactured as a synthetic aromatic hydrocarbon with two benzene rings covalently bonded to one another at 1,1,

Benzidine Table 1

3

Physical and chemical properties of benzidine.

Property

Information

Molecular weight Density Melting point Boiling point Log Kow Water solubility Vapor pressure Vapor density relative to air Dissociation constant (pKa)

184.24 1.250 at 20  C/4  C 120  C 401  C 1.34 322 mg/L at 25  C 7  10−7 mmHg at 25  C 6.36 4.3⁎ (pKa1) and 3.3 (pKa2)

Source: PubChem.

with substituted amino groups at 4,40 . Nitrobenzene is used to synthesize benzidine via a two-step process: nitrobenzene is converted to 1,2-diphenylhydrazine where iron powder may be used as the reducing agent, and consequently, the hydrazine is treated with mineral acids to induce a rearrangement reaction producing 4,40 -benzidine. In the environment, benzidine is found in either its ‘free’ state (as an organic base) or as a salt (benzidine dihydrochloride or benzidine sulfate) (USEPA, 2022; NTP, 2021; NCI, n.d.) (Table 1). Ingestion, inhalation, and skin absorption of benzidine are toxic and its combustion produces toxic oxides of nitrogen. Ingestion of this compound can cause cyanosis, headache, mental confusion, nausea, and vomiting. In vivo, benzidine covalently binds with DNA in the liver of mice and rats. Other benzidine-induced genomic changes in vivo include micronuclei formation, sister chromatid exchanges, DNA strand breaks, and unscheduled DNA synthesis in the cells of rodents. Upregulation of the p53 protein, a DNA damage-sensor protein, in benzidine-treated cells suggests the induction of the p53 DNA damage signaling pathway. This compound is mutagenic to plants and bacteria. Induction of bladder cancer remains the most prominent effect of benzidine. Cancer stem cells are suggested to be the main cause of the initiation, progression, and recurrence of tumors. A study conducted using bladder cancer cell lines has shown that benzidine promotes the stemness of bladder cancer stem cells (BCSCs) by increasing the expression of BCSC markers, promoting the sphere-forming ability of these cells, and activating the Sonic hedgehog pathway. Epithelial-mesenchymal transition (EMT) is a major pathophysiological process in the progression of bladder cancer and further studies have shown that benzidine is able to induce the EMT of human bladder cancer cells via the activation of the extracellular regulated protein kinases 5 (ERK5) pathway. Another molecular mechanism that is utilized by benzidine for the formation of urothelial carcinomas is the activation of the PI3K/Akt signal pathway which promotes benzidine-triggered cell proliferation (Xiang et al., 2007; Ching et al., 2011; Letašiová et al., 2012; Wang et al., 2021). Most studies in diverse geographic locations describe benzidine exposure primarily due to working in a dyestuff industry. Data from these studies have also unequivocally demonstrated co-exposure to 2-naphthylamine along with benzidine precipitating bladder cancer in humans. Benzidine has been classified as a human carcinogen (Class A) based on sufficient ev-idence of carcinogenicity from studies in humans. Most humans are exposed to this compound in occupational settings, and it causes urinary-bladder cancer and bladder-cancer related deaths; the longer the exposure, the greater the risk of developing bladder cancer. Furthermore, the incidence and mortality as a result of bladder cancer increased among workers exposed to benzidine, benzidine, and dichlorobenzidine, but not among workers exposed to dichlorobenzidine alone. The risk of incidence and death from bladder cancer remains elevated more than 20 years after the last exposure to benzidine in those who worked greater than 5 years.

Uses Benzidine is used as an intermediate in the production of azo dyes, sulfur dyes, fast color salts, naphthol, and other dye compounds. Some of its other uses include the detection of blood, rubber-compounding agents, and the manufacture of plastic films. Benzidine has not been marketed or sold in the United States since the mid-1970s and U.S. dye companies no longer manufacture benzidine-based dyes. However, a small amount of benzidine may still be manufactured or imported for scientific research in the United States, but in some countries, it is still being manufactured. To date, more than 250 benzidine-based dyes have been reported. These dyes are primarily used for dyeing textiles, paper, and leather products.

Environmental fate and behavior Benzidine can be released into the environment through waste streams in the form of liquid waste and sludges. However, since benzidine is only used captively in the United States, its direct release into the environment is unlikely. Benzidine may also be released into the environment due to spillage during transport. In the air, benzidine is found bound to suspended particles or as a vapor. Particulate benzidine may be brought back to the earth’s surface by rain or gravity.

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Benzidine

Partition behavior in water, sediment, and soil Very small amounts of benzidine dissolve in water at moderate environmental temperatures. When released into water benzidine is able to adsorb to suspended solids and sediment based on the Koc values. The Koc values determine the tendency of a chemical to bind to soils, corrected for soil organic carbon content. Most benzidine is strongly attached to soil particles, so it does not easily leach into underground water from the waste dumps. When released into waterways, it sinks to the bottom and becomes part of the bottom sludge (Donaldson and Nyman, 2006).

Environmental persistency (degradation/speciation) Benzidine is slowly destroyed in the environment by light (photolysis via absorption of UV light), other chemicals, and microorganisms. Benzidine vapor is degraded in the atmosphere by reacting with hydroxyl radicals that are photochemically produced. Benzidine particles are removed from the atmosphere by wet and dry deposition. Due to its microbial toxicity, benzidine is resistant to biodegradation at high concentrations. Benzidine can also be degraded in aerated water samples upon exposure to UV light suggesting that photolysis in sunlit surface waters may occur.

Bioaccumulation and biomagnification Although accumulation in the food chain is unlikely, moderate levels of benzidine have been measured in fish suggesting water life may take up and store very small amounts of benzidine (ATSDR, 2011).

Exposure and exposure monitoring Routes and pathways The primary routes of potential human exposure to benzidine are inhalation, ingestion, and dermal contact. The general population is not likely to be exposed to benzidine through contaminated air, water, soil, or food (ATSDR, 2011).

Human exposure The highest risk of exposure to benzidine is in occupational settings via dermal contact and inhalation of benzidine vapors or dust. Exposure may also be heightened in populations living near former benzidine or benzidine-based dyes manufacturing or disposal sites through contaminated drinking water and inhaling contaminated air or dust. Ingestion of benzidine-contaminated soil can also result in exposure, especially in children. Since benzidine has no uses in agriculture or food production, exposure via ingestion of contaminated food is unlikely although impurities in certain food dyes can be transformed into benzidine once inside the body. Exposure monitoring can be conducted by collecting blood and urine samples to test for benzidine and benzidine products. Urine samples should be collected within 2 weeks of the last exposure whereas blood samples can be collected within 4 months of the last exposure. Tests can also be conducted to detect benzidine binding to DNA. However, these tests cannot determine if the individual will face harmful effects at a later period (Hemstreet and Wang, 2004; ATSDR, 2011).

Toxicokinetics Benzidine is rapidly absorbed through the skin in solid and vapor forms. It is also quickly absorbed through the lungs on inhalation and from the gastrointestinal tract by consuming contaminated water and food. Generally, it will take only a few hours for most of the benzidine to get into the body through the lungs and intestine. Breathing, eating, or drinking benzidine-based dyes may also expose a person to benzidine because the intestinal microflora can break down these dyes into benzidine. It is a lipophilic substance, hence easily stored in fat tissues, and it firmly binds to cell receptors. Benzidine is metabolized to an aromatic amine by intestinal microflora or liver azo-reductase. The liver is the chief organ of metabolism where benzidine is converted to more reactive, toxic, and mutagenic (carcinogenic) N-hydroxyarylamides and N-hydroxylamine is considered to be a proximate carcinogen. N-Hydroxylamides are converted to the ultimate carcinogens through conjugation with sulfuric, acetic, or glucuronic acids. N-Acetoxyarylamines are also produced as metabolites and are highly reactive mutagens and carcinogens. Glutathione transferase plays an important role in the elimination of reactive metabolites of benzidine. Sulfonation, carboxylation, deamination, or substitution of an ethyl alcohol or an acetyl group for the hydrogen in the amino groups leads to a decrease in the mutagenicity of benzidine metabolites as well as to easy elimination, primarily through urine and feces.

Benzidine

5

Mechanism of toxicity Benzidine is metabolized to highly toxic, reactive metabolites, such as N-hydroxyarylamides and N-hydroxyarylamines, which act as procarcinogens and are more mutagenic than parent compounds. The metabolites act as DNA adducts and bind to cell receptors. Some of the benzidine derivatives are strong mutagens. The metabolites on conjugation with sulfuric, acetic, and glucuronic acids form ultimate carcinogens. Benzidine is metabolized by cytochrome P450 enzymes (via N-oxidation) to form electrophilic compounds that can bind cova-lently to DNA. Benzidine caused mutations in bacteria and plants but gave conflicting results in cultured rodent cells. It also caused many other types of genetic damage in various test systems, including yeast, cultured human and other mamma-lian cells, and rodents exposed in vivo. The damage included mi-totic gene conversion (in yeast), micronucleus formation, DNA strand breaks, unscheduled DNA synthesis, cell transformation, chromo-somal aberrations, sister chromatid exchange, and aneuploidy. Workers exposed to benzidine and or benzidine-based dyes had higher levels of chromosomal aberrations in their white blood cells than did unexposed workers. A study done in normal human urothelial cell lines showed that lipoxygenase (LOX) can co-oxidize benzidine. Thus, benzidine (BZ) induced oxidative stress in these cells by increasing reactive oxygen species (ROS) and malondialdehyde levels significantly in the 100 and 200 mmol/L-BZ-treated groups and decreased the level of the antioxidant reduced glutathione significantly at 200 mmol/L BZ. In addition, the activity of catalase and superoxide dismutase gradually decreased with BZ levels over 50 mmol/L. Benzidine can be activated by 5-LOX to produce ROS and oxidative stress, which may be associated with benzidine-induced bladder cancer (Carreón et al., 2006).

Acute and short-term toxicity Animal No data available on acute animal toxicity.

Human Symptoms of acute ingestion exposure include cyanosis, headache, mental confusion, nausea, and vertigo. Dermal exposure may cause a burning sensation, skin rashes, and allergies. Contact may irritate the eyes and skin, and inhalation may irritate the nose and throat.

Chronic toxicity Animal There is sufficient evidence from animal studies that benzidine is a carcinogen. When administered in the diet, benzidine-induced bladder cancer in dogs, multiple mammary carcinomas in rats and liver cell tumors in hamsters of both sexes. When administered by the subcutaneous route to mice of both sexes, it induced malignant tumors of the Zymbal gland (ear) and hepatocellular carcinoma; hepatomas, malignant tumors of the Zymbal gland, and local sarcomas in male rats; and malignant tumors of the Zymbal gland, mammary adenocarcinomas, and amyloid leukemia in female rats. When administered by intraperitoneal injection, benzidine induced Zymbal gland adenomas and carcinomas, and malignant mammary tumors in female rats. The lethal dose in dogs is 400 mg/kg by the subcutaneous route and 200 mg/kg by the oral route. Dyes made from benzidine, such as Direct Blue 6, Direct Black 38, and Direct Brown 95 have been shown to cause cancer in animals. The Department of Health and Human Services (DHHS) has determined that Direct Black 38 and Direct Blue 6 cause cancer in animals, and the International Agency for Research on Cancer (IARC) has also determined that Direct Black 38, Direct Blue 6, and Direct Brown 95 cause cancer in animals (NTP, 2021; NCI, n.d.; USEPA, 2022).

Human Benzidine can cause cancer in humans. This has been shown in studies of workers who were exposed for many years to levels much higher than the general population would be. An IARC study on dye industry workers reported that there is a direct correlation between the incidence of bladder cancer in occupationally benzidine-exposed workers and the incidence of this cancer decreasing in workers after a reduction in occupational exposure. In addition, a study comparing 1745 cases of lung cancer showed a significantly increased lung cancer risk among workers in dye and manufacturing industries who were directly exposed to benzidine. This suggests occupational exposure to benzidine may lead to lung cancer diagnosis as an occupational disease (Tomioka et al., 2016). Some evidence indicates that dyes made from benzidine, such as Direct Blue 6, Direct Black 38, and Direct Brown 95 may cause cancer in humans. Benzidine poisoning causes vomiting, nausea, hemolysis, liver and kidney damage, and hematuria (bloody urine). Benzidine is considered to be acutely toxic to humans by ingestion, with an estimated oral lethal dose of between 50 and 500 mg/kg for a 70 kg person. Acetylated benzidine metabolites such as N-acetoxyarylamines are known to cause bladder cancer in dye industry workers. A recent study completed in 2021 following 488 workers at the last benzidine manufacturing facility in the

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USA concluded that the risk of incidence and death from bladder cancer remain elevated more than 20 years after last exposure to benzidine in those who worked greater than 5 years. This elevation was shown in workers exposed to benzidine and not shown among workers exposed only to dichlorobenzidine.

Immunotoxicity In a study performed on mice, benzidine was found to be immunosuppressive after subchronic exposure. Suppression of cell-mediated immunity occurred at subtumorigenic doses, where data showed suppressed lymphoproliferative and delayed hypersensitivity responses, decreased host resistance (to infection with Listeria). This may indicate that the development of cancer could be facilitated by the ability of benzidine to alter the immune response. In vitro studies suggested that alterations in metabolites of the arachidonic acid/lipoxygenase pathway were responsible for the immune alterations, where arachidonic acid metabolism and the mitogen response in lymphocytes were inhibited.

Reproductive toxicity According to the CDC, it is not known whether exposure to benzidine will result in birth defects or other developmental effects in people.

Genotoxicity Mutagenic potential of benzidine and its analogs were repeatedly tested using strains TA98 and TA100 in the presence and absence of Aroclor 1254-induced rat S9 mix in Ames Salmonella/microsome assay. 3,30 -Dichlorobenzidine-2HCl and 4,40 dinitro-2-biphenylamine were directly mutagenic to TA98, while 4,40 -dinitro-2-biphenylamine was directly mutagenic to both TA98 and TA100 in the absence of S9 mix. 2-Aminobiphenyl, 3-aminobiphenyl, and 3,30 -5,50 -tetramethylbenzidine were not mutagenic in either strains in the presence or absence of S9. In the presence of S9 mix, 4-aminobiphenyl, benzidine, 3, 30 -dichlorobenzidine-2HCl, 3,30 -dimethoxybenzidine, 3,30 -4, 40 -tetraaminobiphenyl, o-tolidine, N, N-N0 , N0 -tetramethylbenzidine, and 4,40 -dinitro-2-biphenylamine were mutagenic to TA98; 4-aminobiphenyl, 3,30 -dichlorobenzidine-2HCl, 3, 30 -dimethoxybenzidine, and 4,40 -dinitro-2-biphenylamine were mutagenic to TA100. Some of the benzidine derivatives, such as, 3,3,5,5-tetramethylbenzidine, 3,3-dimethylbenzidine (O-tolidine), and N, N-diacetylbenzidine were not mutagenic. Incorporation of the free radical and metal scavengers and antioxidants reduced the mutagenic responses, whereas heat-inactivated catalase and SOD had no effect. Induction of lipid peroxidation in the presence of S9 mix was observed in several instances, which suggested that benzidine derivatives induce mutations through the induction of ROS (NTP, 2021; NCI, n.d.; ACGIH, 2008).

Human genotoxicity Cytogenetic effects of occupational exposure to benzidine and benzidine-based dyes (Direct Black 38 and Direct Blue 6) were studied in workers at a manufacturing plant in Bulgaria having a recognized high risk of occupational cancer. Twenty-three workers exposed for a mean of 15 years were compared with 30 controls presumed to have no exposure. A tenfold increase in chromosomal aberrations (polyploidy) was observed in the circulating lymphocytes of exposed workers when compared with controls. The highest frequency of aberrant lymphocytes was associated with the highest airborne dust concentrations of benzidine (0.42–0.86 mg/m3) or benzidine-based dyes (7.8–32.3 mg/m3), with the highest mean level of benzidine in urine (1.8–2.3 mg/L) (NTP, 2021; NCI, n.d.; Carreón et al., 2006).

Carcinogenicity Benzidine is a confirmed human carcinogen (Class A). Though not universal, a synergistically elevated rate of bladder cancer in workers exposed to benzidine in cigarette smokers has been found. It is hypothesized that smokers have a nearly 31-fold higher risk of developing bladder cancer compared to an 11-fold risk of developing bladder cancer among nonsmoking coworkers. This was based on a large cohort of 3322 Japanese workers exposed to benzidine and/or b-naphthylamine for 22 years and the control cohort of 13.5 years; the control consisted of 177 male unexposed patients with bladder cancer. Among employees exposed to benzidine and/or b-naphthylamine, 244 workers were found to have suffered from and consequently died of cancer of genitourinary organs (primary cancer). Of these, 11 developed secondary cancers of the liver, gall bladder, bile duct, large intestine, or lung. Susceptibility to bladder cancer in humans is linked to the slow acetylator type of the polymorphic NAT2 (N-acetyl transferase gene) (Wang and King, 2007; Weistenhofer et al., 2008; Rosenman and Reilly, 2004).

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According to a study performed on two human bladder cell lines, T24 and EJ, the administration of Benzidine leads to the activation of the ERK5 and activator protein 1 (AP-1) proteins. This potentiates the mitogen-activated protein kinase (MAPK) pathway and results in benzidine-induced EMT (Epithelial-Mesenchymal Transition), which is a critical pathophysiological process in bladder cancer progression. Significantly, benzidine-induced EMT and ERK5 activation were completely suppressed by XMD8-92 and siRNAs specific to ERK5, indicating a potential target and therapeutic significance of ERK5 in benzidine-induced bladder cancer. A similar study found that benzidine increased the proliferation of human bladder cancer T24 cells, triggered the transition of the cells from G1 to S phase, elevated the expression of cyclin D1 and proliferating cell nuclear antigen (PCNA) and decreased p21 expression (Sun et al., 2016, 2018).

Clinical management There is no antidote for benzidine poisoning. Since it produces reactive metabolites, the administration of free radical scavengers would alleviate the toxicity. A complex of benzidine metabolites with copper and hydrochloride is known to decrease its mutagenic effects. Due to the involvement of the ERK5 pathway, ERK5 activation blockers may be a therapeutically significant target to prevent benzidine-induced bladder cancer. Furthermore, a study utilizing human bladder cancer cells showed that treatment with ERK1/2 inhibitor U0126 or curcumin effectively abrogated benzidine-triggered cell proliferation and ERK1/2/AP-1 activation. Thus, curcumin, a yellow pigment found in turmeric which has previously been used clinically for chemoprevention and treatment of cancer, in low concentrations has been found to play a protective role in benzidine-induced ERK pathway activation and proliferation of bladder cancer cells. This may represent a new and therapeutically significant strategy in the chemoprevention of benzidine-associated bladder cancer (Sun et al., 2018; Ding et al., 2019).

Conclusion Benzidine, a toxic, mutagenic, and carcinogenic aromatic amine, has been experimentally proven to cause acute and chronic toxicity, immunotoxicity, genotoxicity, and more, in both animal and human studies. Humans may become exposed to benzidine through occupational settings via dermal contact and inhalation of benzidine vapors or dust. The incidence of exposure in the US has dramatically decreased due to the banning of its production for commercial sale by OSHA in 1973. Thus, the production of benzidine-based dyes in leather, textile, and paper industries is no longer allowed and is no longer available as consumer products. New studies characterizing the pathways involved in benzidine-induced bladder cancer have led to novel prospects for therapeutic targets, treatments, and clinical management after exposure to the carcinogen.

References ACGIH (2008) American Conference of Governmental Industrial Hygienists TLVs and BEIs. Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices. Cincinnati, OH. 13. ATSDR (2011) Public Health Statement for Benzidine. https://wwwn.cdc.gov/TSP/PHS/PHS.aspx?phsid¼567&toxid¼105. Carreón T, LeMasters GK, Ruder AM, and Schulte PA (2006) The genetic and environmental factors involved in benzidine metabolism and bladder carcinogenesis in exposed workers. Frontiers in Bioscience 11: 2889–2902. Ching CS, Hseu YC, Sung JC, et al. (2011) Induction of DNA damage signaling genes in benzidine-treated HepG2 cells. Environmental and Molecular Mutagenesis 52(8): 664–672. Ding D, Liu Z, Zhao L, et al. (2019) Role of PI3K/Akt pathway in Benzidine-induced proliferation in SV-40 immortalized human uroepithelial cell. Translational Cancer Research 8(4): 1301–1310. https://doi.org/10.21037/tcr.2019.07.14. Donaldson FP and Nyman MC (2006) Short-term interactions of aniline and benzidine with three soils in both natural and artificial matrices. Chemosphere 65(5): 854–862. Hemstreet GP and Wang W (2004) Genotypic and phenotypic biomarker profiles for individual risk assessment and cancer detection (lessons from bladder cancer risk assessment in symptomatic patients and workers exposed to benzidine). Frontiers in Bioscience-Landmark 9: 2671–2679. Letašiová S, Medve’ová A, Šovcíková A, et al. (2012) Bladder cancer, a review of the environmental risk factors. Environmental Health 11(Suppl 1): S11. https://doi.org/ 10.1186/1476-069X-11-S1-S11. NCI (n.d.) https://www.cancer.gov/about-cancer/causes-prevention/risk/substances/benzidine NTP: National Toxicology Program (2021) Benzidine and Dyes Metabolized to Benzidine, Report on Carcinogens, 15th edn, Triangle Park, NC: National Institute of Environmental Health and Safety.https://ntp.niehs.nih.gov/ntp/roc/content/profiles/benzidineanddyes.pdf. Rosenman KD and Reilly MJ (2004) Cancer mortality and incidence among a cohort of benzidine and dichlorobenzidine dye manufacturing workers. American Journal of Industrial Medicine 46: 505–512. Sun X, Deng QF, Liang ZF, et al. (2016) Curcumin reverses benzidine-induced cell proliferation by suppressing ERK1/2 pathway in human bladder cancer T24 cells. Experimental and Toxicologic Pathology 68(4): 215–222. https://doi.org/10.1016/j.etp.2015.12.003. Sun X, Zhang T, Deng Q, et al. (2018) Benzidine induces epithelial-mesenchymal transition of human bladder cancer cells through activation of ERK5 pathway. Molecules and Cells 41(3): 188–197. https://doi.org/10.14348/molcells.2018.2113. Tomioka K, Saeki K, Obayashi K, and Kurumatani N (2016) Risk of lung cancer in workers exposed to benzidine and/or beta-naphthylamine: A systematic review and meta-analysis. Journal of Epidemiology 26(9): 447–458. https://doi.org/10.2188/jea.JE20150233. USEPA (2022) Fact Sheet: Benzidine-Based Chemical Substances. https://www.epa.gov/assessing-and-managing-chemicals-under-tsca/fact-sheet-benzidine-based-chemical-substances. Wang CY and King CM (2007) N-Acetyltransferases and the susceptibility to benzidine-induced bladder carcinogenesis. International Journal of Cancer 120(11): 2523–2524. Wang D, et al. (2021) Benzidine promotes the stemness of bladder cancer stem cells via activation of the sonic hedgehog pathway. Oncology Letters 21(2): 146. https://doi.org/ 10.3892/ol.2020.12407. Weistenhofer W, et al. (2008) N-acetyltransferase-2 and medical history in bladder cancer cases with a suspected occupational disease (BK 1301) in Germany. Journal of Toxicology and Environmental Health. Part A 71(13–14): 906–910. Xiang CQ, Shen CL, Wu ZR, et al. (2007) Detection of mutant p53 protein in workers occupationally exposed to benzidine. Journal of Occupational Health 49(4): 279–284.

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Further reading Alves de Lima RO, Bazo AP, Salvadori DM, et al. (2007) Mutagenic and carcinogenic potential of a textile azo dye processing plant effluent that impacts a drinking water source. Mutation Research 626(1–2): 53–60. Huang Y, Huang S, Wu Y, et al. (2019) Lipoxygenase protein expression and its effect on oxidative stress caused by benzidine in normal human urothelial cell lines. International Journal of Toxicology 38(2): 121–128. Kobylewski S and Jacobson MF (2012) Toxicology of food dyes. International Journal of Occupational and Environmental Health 18(3): 220–246. Luster MI, Tucker AN, Hayes HT, et al. (1985) Immunosuppressive effects of benzidine in mice: Evidence of alterations in arachidonic acid metabolism. Journal of Immunology 135(4): 2754–2761. PMID: 2993415. Makena P and Chung KT (2007) Evidence that 4-aminobiphenyl, benzidine, and benzidine congeners produce genotoxicity through reactive oxygen species. Environmental and Molecular Mutagenesis 48(5): 404–413. Millerick-May ML, Wang L, Rice C, and Rosenman KD (2021) Ongoing risk of bladder cancer among former workers at the last benzidine manufacturing facility in the USA. Occupational and Environmental Medicine 78(9): 625–631. https://doi.org/10.1136/oemed-2020-106431. Ohsako S and Deguchi T (1990) Cloning and expression of cDNA for polymorphic and monomorphic arylamine N-acetyltransferases from human liver. Journal of Biological Chemistry 265: 4630–4634. Park J, Shin KS, and Kim Y (2010) Occupational reproductive function abnormalities and bladder cancer in Korea. Journal of Korean Medical Science 25(Suppl): S41–S45. Nj.gov (n.d.) Right to Know Hazardous Substance Fact Sheet. https://nj.gov/health/eoh/rtkweb/documents/fs/0204.pdf. Yang M (2011) A current global view of environmental and occupational cancers. Journal of Environmental Science and Health. Part C, Environmental Carcinogenesis & Ecotoxicology Reviews 29(3): 223–249.

Relevant websites https://ntp.niehs.nih.gov/ntp/roc/content/profiles/benzidineanddyesmetabolized.pdf :NTP/NIH/NIEHS. http://www.atsdr.cdc.gov :Agency for Toxic Substances and Disease Registry. Toxicological Profile for Benzidine. http://toxnet.nlm.nih.gov/cgi-bin/sis/search/a?dbs+hsdb:@term+@DOCNO+948 :TOXNET: Benzidine: Specialized Information Services-NLM. http://monographs.iarc.fr/index.php :IARC.

Benzo(a)pyrene Joshua P Gray, Department of Chemical & Environmental Sciences, U.S. Coast Guard Academy, New London, CT, United States © 2024 Elsevier Inc. All rights reserved. This is an update of J.P. Gray, Benzo(a)pyrene, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 423–428, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00250-5.

Chemical profile Background Uses/occurrence Exposure Toxicokinetics (absorption, distribution, metabolism, excretion) Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity (e.g. animal, human; oral, inhalation, dermal) Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Toxicogenomics Environmental fate and behavior Ecotoxicology Exposure standards and guidelines References Further reading

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Abstract Benzo(a)pyrene is a polycyclic aromatic hydrocarbon produced by incomplete combustion of fuels. The most common route of human exposure is through tobacco smoke. It is bioactivated to produce a highly reactive epoxide molecule that can chemically react with biological molecules including DNA to form adducts. The presence of a “bay region” within benzo(a) pyrene and similar polycyclic aromatic hydrocarbons enhances its capacity for inducing DNA damage through this process. As such, benzo(a)pyrene is carcinogenic and is associated with lung cancer and other types of cancer. This chemical also induces immunosuppression together with reproductive and developmental toxicities.

Keywords Bay region; Bioactivation; Cytochrome P450; Polycyclic aromatic hydrocarbon; Procarcinogen

Key points

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BaP is a product of combustion; and human exposure typically occurs through tobacco smoke, other airborne combustion product, or foods cooked over charcoal or broiled. The primary routes of exposure to BaP are via oral and inhalation pathways. Dermal absorption also occurs readily in the presence of lipophilic solvents. BaP is present in fossil fuels, crude oils, shale oils, and coal tars. It is also emitted with gases and fly ash from active volcanoes. BaP-induced DNA adducts lead to carcinogenicity. It is also a reproductive and developmental toxicant.

Chemical profile

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Name: Benzo(a)pyrene Synonyms: BAP; BaP; B(a)P; BP; 3,4-Benzopyrene; 6,7-Benzopyrene; 3,4-Benzpyrene; 3,4-Benz(a)pyrene; Benzo[d,e, f]chrysene; and many more. CAS Number: 50-32-8 Molecular Formula: C20H12

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.01032-0

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Benzo(a )pyrene Chemical Structure

Image source: https://comptox.epa.gov/dashboard/chemical/details/DTXSID2020139

Background Benzo(a)pyrene is the prototypical polcyclic aromatic hydrocarbon (PAH), one of the most-studied compounds in its class. Classic studies in toxicology using BaP led to the discovery of the inducible phase 1 and 2 xenobiotic metabolizing enzymes including the first cytochrome P450 oxidoreductase (Conney et al., 1957; Wattenberg et al., 1962). Subsequent discoveries included bioactivation (Grover and Sims, 1968; Sims and Grover, 1968), which induces carcinogenesis via DNA adduct formation. BaP is a product of combustion; and human exposure typically occurs through tobacco smoke, other airborne combustion product, or foods cooked over charcoal or broiled (Ramesh et al., 2004; Hecht, 1999).

Uses/occurrence There is no commercial use for BaP. It is primarily used as a model PAH molecule for research.

Exposure The primary routes of exposure to BaP are oral and inhalational. Oral exposure primarily through foods that are broiled or cooked by incomplete combustion of fuels. For example, smoked fish has high levels of many PAH’s (Ramesh et al., 2004). The levels of PAH’s in other foods is well-documented (Ramesh et al., 2004). However, levels of PAH exposure are generally low via this route. The average human diet includes 0.008 mg/kg/day, which is well below the benchmark dose level (BMDL10) of 0.122 mg/kg/day (Benford et al., 2010). Inhalational exposure is more common, either through tobacco smoke or other combustion products. Tobacco smoke contains many carcinogens, including BaP (Hecht, 1999). BaP is present in fossil fuels, crude oils, shale oils, and coal tars, and is emitted with gases and fly ash from active volcanoes. Occupational exposure is a significant risk; an assessment of workers exposed to PAH’s in Europe found that exposure levels were greater than the maximal risk level for 30% of exposure groups (Petit et al., 2019).

Toxicokinetics (absorption, distribution, metabolism, excretion) Oral absorption of BaP is low but increases with lipophilicity and in the presence of oils in the gastrointestinal (GI) tract. Dermal absorption also occurs readily in the presence of lipophilic solvents (Bourgart et al., 2019). Inhalational exposure is common in smokers and those exposed to occupational smoke. PAHs are widely distributed in tissues regardless of route of exposure (Weyand and Bevan, 1986). Metabolism is widely varied, and includes the formation of epoxides, dihydrodiols, phenols, quinones, and combinations, some of which are bioactivated to become mutagenic products (Gao et al., 2018). Conjugation to glucuronides and sulfate esters occurs via phase II metabolism, which are subsequently excreted (Miller and Ramos, 2001). Hepatobiliary excretion is the primary elimination route of PAHs in animals within 2 days of exposure. Specifically for BaP, following GI absorption (33% is absorbed), levels can peak in the blood at 6 h after exposure. A toxicokinetic study found only 3–6% of the parent compound in the serum following oral dosing at 30 min post-exposure, suggesting extensive first pass metabolism (Vermillion Maier et al., 2022). ABC transport proteins in the small intestine reabsorb BaP metabolites, which serves to attenuate further BaP absorption via competition (Buesen et al., 2002). The liver and lung are the most susceptible to adduct formation by BaP metabolites, correlating with their high levels of cytochrome P450 enzyme activity and role in detoxification of BaP. BaP is biodistributed to body fat, other fatty tissues, placenta, and bile. Like other PAH’s, BaP contains a ‘bay region’ created by three fused benzene rings known to contribute to carcinogenicity in a variety of compounds (Lehr et al., 1985). BaP is bioactivated through phase I metabolism by cytochrome P450 1A1 and/or 1A2. It is first metabolized to (+) benzo(a)pyrene 7,8-oxide, next by epoxide hydrolase to (−) benzo(a)pyrene 7,8-dihydrodiol, and finally by cytochrome P450 1A1 and/or 1A2 to (+) benzo(a)pyrene 7,8-dihydrodiol-9,10-epoxide (Alexandrov et al., 2010; Shiizaki et al., 2017) (Fig. 1). This epoxide, which is resistant to epoxidases, subsequently chemically reacts with DNA producing multiple adducts, the most important of which are benzo(a)pyrene-7,8-diol-9,10-epoxide-N(2)-deoxyguanosine (BPDE-dG) and -deoxyadenosine

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Fig. 1 Bioactivation of BaP to (−) benzo(a)pyrene 7,8-dihydrodiol-9,10-epoxide. Image generated by the author using ChemDraw, Joshua Gray, public domain.

(Alexandrov et al., 2010). These and other adducts have been associated with lung cancer and other types of cancer. Other phase 1 metabolic products are BaP 3,6-quinone and BaP 4,5-oxide (Nebert et al., 2013). Other enzymes may also participate in Phase I bioactivation, including oxidoreductases, which may reduce any benefit found with inhibition of CYP1A1 (Barnes et al., 2018; Shiizaki et al., 2017). The terminal products of phase 1 metabolism can also be detoxified by phase 2 metabolism through conjugation reactions (Talalay, 2000). Phase 2 metabolism primarily occurs by glucuronidation (Olson et al., 2011), although several studies have shown that glutathione depletion increases DNA damage induced by BaP (Maier et al., 2002; Lim et al., 2013). Sulfate conjugation is a third phase 2 conjugation pathway (Marinkovic et al., 2013). Polymorphisms in cytochrome P450 enzymes or in DNA repair enzymes may increase the risk of toxicity from BaP (Bukowska et al., 2022).

Mechanism of toxicity Several studies with knockout animals have demonstrated the importance of bioactivation in BaP’s mechanism of toxicity. While CYP1A1 knockout mice were protected from BaP-mediated liver toxicity, they cleared BaP much more slowly leading to greater formation of BaP-DNA adducts (Uno et al., 2001; Alexandrov et al., 2010). Metabolism in one tissue protects against toxicity in another. For example, CYP1A1 expression within the gastrointestinal tract protects against BaP-mediated toxicity (Nebert et al., 2013). Indeed, targeted knockout of CYP1A1 in the GI tract renders mice more sensitive to BaP, suggesting a protective role for this enzyme despite its role in bioactivation of BaP perhaps due in part to alternative bioactivation pathways (Nebert et al., 2013).

In vitro toxicity data Acute and short-term toxicity (e.g. animal, human; oral, inhalation, dermal ) The LD50 dose in rat (subcutaneous) is 50 mg/kg and in mouse (intraperitoneal) is 250 mg/kg (Druckrey et al., 1967; Epstein et al., 1972).

Immunotoxicity BaP and related PAH’s are well-known immunosuppressants (reviewed in Zaccaria and McClure, 2013). Mechanistically, BaP induces immunosuppression through bioactivation and subsequent induction of DNA damage in T- and B-lymphocytes (Sul et al., 2003). Humoral immunosuppression is prevalent in occupational exposures such as coke oven workers and cold-rolling mill workers (Szczeklik et al., 1994).

Reproductive and developmental toxicity BaP is a developmental toxicant; it acts as an endocrine disruptor for the ovary (Craig et al., 2011). More generally, PAH’s can also induce ovarian failure and infertility in progeny of humans with occupational exposures (Beranger et al., 2012). Indeed, BaP

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prenatal exposure depleted ovarian germ cells and increased ovarian/oviductal mutations in the progeny of rodents, causing absence of follicles, cell packs, and epithelial tubular structures (Luderer et al., 2019). In vitro, preantral follicles treated with BaP showed inhibited follicular growth, decreased estrogen production, and decreased anti-Mullerian hormone (Neal et al., 2010). BaP can also affect the developing fetus, as it effectively crosses the placenta and creates placental DNA adducts (Mathiesen et al., 2009; Karttunen et al., 2010). Fetal erythrocytes are decreased in number as well as deficits in organ formation occurred in rats orally exposed to BaP (da Silva Moreira et al., 2021). Neural tube defects were observed in developing mice maternally exposed to BaP via intraperitoneal injection (Lin et al., 2018). Spermatogonial stem cell mutations also occur following BaP exposure (O’brien et al., 2016a). Transgenerational toxicity was observed in the offspring of male rats treated with environmentally-relevant levels of BaP, leading to decreased body weight, reduction in the anogenital distance of males, altered testicular histology, and altered ovary and uterus histology (Jorge et al., 2021).

Genotoxicity Many bioactivated metabolites of BaP readily form DNA adducts. Benzo(a)pyrene-7,8-diol-9,10-epoxide-N(2)-deoxyguanosine is a major adduct associated with lung cancer (Alexandrov et al., 2010). A similar adduct forms through reaction with the N(6) position of adenine, producing Benzo(a)pyrene-7,8-diol-9,10-epoxide-N(6)-deoxyadenosine (Alexandrov et al., 2010). The mutations induced by BaP are different in sperm versus bone marrow, perhaps due to differences in error-prone DNA polymerases between those tissues (O’brien et al., 2016b).

Carcinogenicity BaP-induced DNA adducts lead to carcinogenicity. Checkpoint kinase 1 (chk1) is induced in response to BaP and air particulate matter fractions in HepG2 cells (Jarvis et al., 2013). Cotreatment with BaP and arsenic caused synergistic increases in DNA damage in Hepa-1 cells through a CYP1A1-dependent mechanism (Maier et al., 2002). These adducts may impair DNA repair pathways; BaP blocks the DNA mismatch repair pathway in the ZR75-1 cell line for example (Chen et al., 2013).

Organ toxicity In addition to ovarian toxicity (discussed in section “Reproductive and Developmental Toxicity”), BaP targets multiple other organs. Mechanistically this occurs when BaP is bioactivated at the target organ. Indeed, when phase I bioactivation was inhibited in a GI-specific fashion, toxicity occurred elsewhere, suggesting phase I metabolism in one location can protect another part of the organism (Nebert et al., 2013). Neurotoxicity is induced by BaP, which causes learning and memory deficits in rats, including altered behavior in the Morris water maze test (Cheng et al., 2013; Wang et al., 2018). Neonatal exposure causes neurological deficits that continue into adulthood (Chen et al., 2012). Mechanistically BaP binds to the AhR which alters transcription of N-methyl-D-aspartate glutamate receptor which causes a loss in neuronal activity and reduced learning and memory (Chepelev et al., 2015). BaP also induces immunotoxicity through bone marrow suppression. Rats orally exposed to BaP showed dose-dependent decreases in thymus weight, reduced spleen and bone marrow cell counts, and red and white blood cell counts (De Jong et al., 1999).

Toxicogenomics BaP was used as a model chemical to evaluate whether the toxicogenomics approach could recapitulate known toxic mechanisms using a cell line system (Moffat et al., 2015). The TK6 lymphoblastoid cell line demonstrated a similar response to that observed in animal tissues, and the authors concluded that even in data-poor scenarios, a toxicogenomics-only approach would be useful. Transcriptomes of embryonic mice which were maternally exposed to BaP showed induction of bioactivation genes to a higher extent in ovaries than testes, consistent with the known reproductive toxicities of BaP (Lim et al., 2022).

Environmental fate and behavior The primary sources of BaP are incomplete combustion of wood, gas, fuels, industrial coal, and forest fires (Bukowska et al., 2022). BaP’s extrapolated vapor pressure is 5.49E-9 at 25C indicating it exists in the atmosphere only as bound to particulate matter (Murray et al., 1974). Particulate-phase BaP is usually removed from the atmosphere by wet or dry deposition. If released to soil, BaP is expected to have very low to no mobility due to Koc values from 3760 to 1.3E6 (Krauss and Wilcke, 2001; Symons et al., 1988). Volatilization from moist soil surfaces is not expected to be an important fate process based on a

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Henry’s Law constant of 4.57E-7 atm m3 mol−1 (Ten Hulscher et al., 1992). The stability of BaP in soil is expected to vary depending on the nature of compounds accompanying it and the nature and previous history of the soil; biodegradation half-lives of 309 and 229 days were observed in Kidman and McLaurin sandy loam soils, respectively (Park et al., 1990). BaP is expected to adsorb to suspended solids and sediment based on the measured Koc values, when released into water (Ten Hulscher et al., 1992). Biodegradation of BaP is possible in aquatic systems. Volatilization from water surfaces is not expected to be an important fate process based on this compound’s Henry’s Law constant. Hydrolysis is not expected to be an important environmental fate process since this compound lacks functional groups that hydrolyze under environmental conditions (Lyman et al., 1990). The bioavailability of BaP bound to soil is conservatively estimated by the EPA using the default meal oral relative bioavailability factor value; actual bioavailability can be as much as 8 times different than that using a conservative estimation (Forsberg et al., 2021). This risk assessment methodology might be used to better assess resource allocation for environmental cleanup.

Ecotoxicology BaP is a persistent organic pollutant. Ecotoxicity values have been determined for several species, including Neanthes arenaceodentata (LC50 of >1.0 mg/L for 96 h) (Verschueren, 1996), Daphnia pulix (LC50 of 0.005 mg/L for 96 h), Daphnia magna juveniles (EC50 of 40 mg/L for 24 h) (Wernersson and Dave, 1997), and Daphnia magna neonates (29.3 mg/L for 24 h) (Ha and Choi, 2009). Data for more species are available in the Hazardous Substances Data Bank.

Exposure standards and guidelines Exposure to BaP primarily occurs through diet or smoking. Monitoring data indicate that the general population may be exposed to BaP via inhalation of ambient air, ingestion of food and drinking water, smoking of tobacco, and cooking processes that produce smoke. The benchmark dose level (BMDL10) for BaP is 0.122 mg/kg/day (Benford et al., 2010). The Permissible Exposure Limit as determined by the Occupational Safety and Health Administration is 0.2 mg/m3 for 8 h. The National Institute for Occupational Safety & Health Recommended Exposure Limit is 0.1 mg/m3 for 10 h. The Chronic Oral Reference dose is 3.00E-4 mg/kg day and the Chronic Inhalation Reference Concentration is 2E-6 mg/m3.

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Lim J, Lawson GW, Nakamura BN, Ortiz L, Hur JA, Kavanagh TJ, and Luderer U (2013) Glutathione-deficient mice have increased sensitivity to transplacental benzo[a]pyrene-induced premature ovarian failure and ovarian tumorigenesis. Cancer Research 73: 908–917. Lim J, Ramesh A, Shioda T, Leon Parada K, and Luderer U (2022) Sex differences in embryonic gonad transcriptomes and benzo[a]pyrene metabolite levels after transplacental exposure. Endocrinology 163. Lin S, Ren A, Wang L, Huang Y, Wang Y, Wang C, and Greene ND (2018) Oxidative stress and apoptosis in benzo[a]pyrene-induced neural tube defects. Free Radical Biology & Medicine 116: 149–158. Luderer U, Meier MJ, Lawson GW, Beal MA, Yauk CL, and Marchetti F (2019) In utero exposure to benzo[a]pyrene induces ovarian mutations at doses that deplete ovarian follicles in mice. Environmental and Molecular Mutagenesis 60: 410–420. Lyman WJ, Reehl WF, and Rosenblatt DH (1990) Handbook of Chemical Property Estimation Methods: Environmental Behavior of Organic Compounds. United States. Maier A, Schumann BL, Chang X, Talaska G, and Puga A (2002) Arsenic co-exposure potentiates benzo[a]pyrene genotoxicity. Mutation Research 517: 101–111. Marinkovic N, Pasalic D, and Potocki S (2013) Polymorphisms of genes involved in polycyclic aromatic hydrocarbons’ biotransformation and atherosclerosis. Biochemia Medica 23: 255–265. Mathiesen L, Rytting E, Mose T, and Knudsen LE (2009) Transport of benzo[alpha]pyrene in the dually perfused human placenta perfusion model: effect of albumin in the perfusion medium. Basic & Clinical Pharmacology & Toxicology 105: 181–187. Miller KP and Ramos KS (2001) Impact of cellular metabolism on the biological effects of benzo[a]pyrene and related hydrocarbons. Drug Metabolism Reviews 33: 1–35. Moffat I, Chepelev N, Labib S, Bourdon-Lacombe J, Kuo B, Buick JK, Lemieux F, Williams A, Halappanavar S, Malik A, Luijten M, Aubrecht J, Hyduke DR, Fornace AJ Jr., Swartz CD, Recio L, and Yauk CL (2015) Comparison of toxicogenomics and traditional approaches to inform mode of action and points of departure in human health risk assessment of benzo [a]pyrene in drinking water. Critical Reviews in Toxicology 45: 1–43. Murray JJ, Pottie RF, and Pupp C (1974) The vapor pressures and enthalpies of sublimation of five polycyclic aromatic hydrocarbons. Canadian Journal of Chemistry 52: 557–563. Neal MS, Mulligan Tuttle AM, Casper RF, Lagunov A, and Foster WG (2010) Aryl hydrocarbon receptor antagonists attenuate the deleterious effects of benzo[a]pyrene on isolated rat follicle development. Reproductive Biomedicine Online 21: 100–108. Nebert DW, Shi Z, Galvez-Peralta M, Uno S, and Dragin N (2013) Oral benzo[a]pyrene: understanding pharmacokinetics, detoxication, and consequences--Cyp1 knockout mouse lines as a paradigm. Molecular Pharmacology 84: 304–313. O’brien JM, Beal MA, Yauk CL, and Marchetti F (2016a) Benzo(a)pyrene Is Mutagenic in Mouse Spermatogonial Stem Cells and Dividing Spermatogonia. Toxicological Sciences 152: 363–371. O’brien JM, Beal MA, Yauk CL, and Marchetti F (2016b) Next generation sequencing of benzo(a)pyrene-induced lacZ mutants identifies a germ cell-specific mutation spectrum. Scientific Reports 6: 36743. Olson KC, Sun D, Chen G, Sharma AK, Amin S, Ropson IJ, Spratt TE, and Lazarus P (2011) Characterization of dibenzo[a,l]pyrene-trans-11,12-diol (dibenzo[def,p]chrysene) glucuronidation by UDP-glucuronosyltransferases. Chemical Research in Toxicology 24: 1549–1559. Park KS, Sims RC, Dupont RR, Doucette WJ, and Matthews JE (1990) Fate of PAH compounds in two soil types: Influence of volatilization, abiotic loss and biological activity. Environmental Toxicology and Chemistry 9: 187–195. Petit P, Maitre A, Persoons R, and Bicout DJ (2019) Lung cancer risk assessment for workers exposed to polycyclic aromatic hydrocarbons in various industries. Environment International 124: 109–120. Ramesh A, Walker SA, Hood DB, Guillen MD, Schneider K, and Weyand EH (2004) Bioavailability and risk assessment of orally ingested polycyclic aromatic hydrocarbons. International Journal of Toxicology 23: 301–333. Shiizaki K, Kawanishi M, and Yagi T (2017) Modulation of benzo[a]pyrene-DNA adduct formation by CYP1 inducer and inhibitor. Genes and Environment 39: 14. Sims P and Grover PL (1968) Quantitative aspects of the metabolism of 7, 12-dimethylbenz[a]anthracene by liver homogenates from animals of different age, sex and species. Biochemical Pharmacology 17: 1751–1758. Sul D, Oh E, Im H, Yang M, Kim CW, and Lee E (2003) DNA damage in T- and B-lymphocytes and granulocytes in emission inspection and incineration workers exposed to polycyclic aromatic hydrocarbons. Mutation Research 538: 109–119. Symons BD, Sims RC, and Greeney WJ (1988) Fate and transport of organics in soil: model predictions and experimental results. Journal of the Water Pollution Control Federation 60: 1684–1693. Szczeklik A, Szczeklik J, Galuszka Z, Musial J, Kolarzyk E, and Targosz D (1994) Humoral immunosuppression in men exposed to polycyclic aromatic hydrocarbons and related carcinogens in polluted environments. Environmental Health Perspectives 102: 302–304. Talalay P (2000) Chemoprotection against cancer by induction of phase 2 enzymes. BioFactors 12: 5–11. Ten Hulscher TEM, van der Velde LE, and Bruggeman WA (1992) Temperature dependence of Henry’s Law constants for selected chlorobenzenes, polychlorinated biphenyls and polycyclic aromatic hydrocarbons. Environmental Toxicology and Chemistry 11: 1595–1603. Uno S, Dalton TP, Shertzer HG, Genter MB, Warshawsky D, Talaska G, and Nebert DW (2001) Benzo[a]pyrene-induced toxicity: paradoxical protection in Cyp1a1(−/−) knockout mice having increased hepatic BaP-DNA adduct levels. Biochemical and Biophysical Research Communications 289: 1049–1056. Vermillion Maier ML, Siddens LK, Pennington JM, Uesugi SL, Anderson KA, Tidwell LG, Tilton SC, Ognibene TJ, Turteltaub KW, Smith JN, and Williams DE (2022) Benzo[a]pyrene (BaP) metabolites predominant in human plasma following escalating oral micro-dosing with [(14)C]-BaP. Environment International 159: 107045. Verschueren K (1996) Handbook of Environmental Data on Organic Chemicals, 3rd edn. New York, NY: Van Nostrand Rehinhold Co. 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Zaccaria KJ and Mcclure PR (2013) Using immunotoxicity information to improve cancer risk assessment for polycyclic aromatic hydrocarbon mixtures. International Journal of Toxicology 32: 236–250.

Further reading U.S. Environmental Protection Agency (2017) Toxicological Review of Benzo[a]pyrene [CASRN 50–32-8], Integrated Risk Information System National Center for Environmental Assessment Office of Research and Development. Washington, DC: U.S. Environmental Protection Agency. Available at: https://iris.epa.gov/static/pdfs/0136tr.pdf. Accessed 23 March 2023. International Agency for Research on Cancer (IARC), World Health Organization (2018) Benzo[a]pyrene, Monograph 100F. Available at: https://monographs.iarc.who.int/wp-content/ uploads/2018/06/mono100F-14.pdf. Accessed 23 March 2023. Public Health England (2018) Polycyclic Aromatic Hydrocarbons (Benzo[a]pyrene) Toxicological Overview. Available at: https://assets.publishing.service.gov.uk/government/uploads/ system/uploads/attachment_data/file/737017/PAH_TO_PHE_240818.pdf. Accessed 23 March 2023. Health Canada (2016) Guidelines for Canadian Drinking Water Quality: Guideline Technical Document — Benzo[a]pyrene. Ottawa, Ontario: Water and Air Quality Bureau, Healthy Environments and Consumer Safety Branch, Health Canada. (Catalogue No H144-35/2016E-PDF). Available at https://www.canada.ca/content/dam/phac-aspc/documents/ services/publications/healthy-living/guidelines-canadian-drinking-water-quality-guideline-technical-document-benzo-alpha-pyrene/pub-eng.pdf. Accessed 23 March 2023.

Relevant websites https://pubchem.ncbi.nlm.nih.gov/compound/Benzo_a_pyrene :PUBCHEM URL for Benzo[a]pyrene https://comptox.epa.gov/dashboard/chemical/details/DTXSID2020139 :U.S. Environmental Protection Agency COMPTOX Dashboard resource for Benzo[a]pyrene

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Benzyl alcohol Nancy A Ibrahim, Research Scientist III, New Jersey Public Health Environmental and Chemistry Laboratory, Ewing, NJ, United States © 2024 Elsevier Inc. All rights reserved. This is an update of G.B. Corcoran, S.D. Ray, Benzyl Alcohol, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 429–432, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00251-7.

Chemical profile Introduction Uses Environmental behavior, fate, routes, and pathways Exposure and exposure monitoring Toxicokinetics Mechanisms of action Acute and short-term toxicity Chronic toxicity Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Exposure standards and guidelines Conclusion References Further reading

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Abstract Benzyl alcohol (BA) is an aromatic alcohol used as a solvent in the production of fragrances/perfumes, paints and adhesives. BA has been widely used in human medicine as an antimicrobial preservative and as a local anesthetic and antipruritic. Benzyl alcohol (5%) in various formulations has been approved by USFDA for treatment of head lice. No adverse effects of benzyl alcohol were seen in chronic exposure studies using rats and mice. The non-immunological reactions of the chemical are not a concern when limited body exposure, frequency of use, and duration of use are considered. Because of the wide variety of product types in which benzyl alcohol can be used, it is likely that the substance may be absorbed into the body by inhalation of its vapor. In adults, benzyl alcohol is oxidized to benzoic acid, conjugated in the liver with glycine, and excreted in the urine as hippuric acid. Infants have limited ability to metabolize and excrete benzyl alcohol (BA exposed neonates show gasping syndrome). Human subjects eliminated 75 to 85% of the dose in the urine as hippuric acid within 6 h after taking 1.5 g of benzyl alcohol orally. LD50 doses for this compound have been reported in several species: 950 mg kg−1 body weight in mouse when administered subcutaneously; 1.23 g to 3.12 g when rats were dosed orally. The main treatment for benzyl alcohol toxicity is discontinuation of the exposure and supportive care. There is no known antidote for benzyl alcohol poisoning.

Keywords Benzoic acid; Benzyl alcohol; Food flavor; Fragrance; Hippuric acid

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Benzyl alcohol, its metabolite benzoic acid and its salts are used as fragrance ingredients, preservatives, and viscosity-decreasing agent in a wide range of cosmetic formulations. Most recent safety data on these agents suggests that benzyl alcohol, benzoic acid and its salts are safe in the present practices. No adverse effects of BA were seen in chronic exposure animal studies using rats and mice. Benzoic Acid is an aromatic acid used in a wide variety of cosmetics as a pH adjuster and preservative. Sodium Benzoate, the sodium salt of Benzoic Acid is also used in similar manner. BA is metabolized to Benzoic Acid, which reacts with glycine and excreted as hippuric acid in the human body. ADI were established by the World Health Organization at 5 mg kg−1 for Benzyl Alcohol, Benzoic Acid, and Sodium Benzoate. Benzoic Acid and Sodium Benzoate have earned GRAS status in foods according to the USFDA.

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Chemical profile

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Name: Benzyl Alcohol Chemical Abstracts Service Registry Number: 100-51-6 Synonyms: Benzoyl alcohol; (Hydroxymethyl)benzene; Benzenecarbinol; Benzenemethanol; Hydroxytoluene; Methanol, phenyl-; Phenylcarbinol; Phenylmethyl alcohol; Phenylmethanol; alpha-Hydroxytoluene Molecular Formula: C7H8O Chemical Structure: H O

Introduction Benzyl alcohol is one of the simplest alcohols that finds applications in a variety of industries, especially the fragrance and coating industry. At room temperature, it is a colorless liquid with a mild aromatic smell, making it ideal for a wide variety of applications for chemical synthesis for pharmaceuticals and personal care. The safety of benzyl alcohol was reported in one study (Johnson et al., 2017). Benzyl alcohol is manufactured by the hydrolysis of benzyl chloride in the presence of soda ash. The alcohol is produced naturally in a range of fruits and plants including honey, apricots, mushrooms, cocoa, and cranberries as well as in the essential oils of plants like hyacinth, ylang-ylang, and jasmine. There are no human carcinogenicity data for benzyl alcohol.

Uses Although available for some years as an over-the-counter health product, benzyl alcohol was approved in 2003 by the Food and Drug Administration (FDA) as a new prescription drug for the treatment of head lice. Unlike typical pediculicides such as permethrin and lindane which act through a neurotoxic mode of action, benzyl alcohol is thought to operate via a unique mechanism involving physical pulmonary asphyxiation. The presence of benzyl alcohol in such a wide range of consumer products is explained by its bacteriostatic and antiseptic properties in conjunction with its comparatively modest toxicity. Outside of its natural occurrence in foods (apricots, cranberries, and cocoa), manufactured benzyl alcohol is used as a flavor-enhancing solvent, and can be used in baked goods, liqueurs and wines. In commercially manufactured cosmetics and skin products, benzyl alcohol is frequently used as a preservative due to its ability to kill microbes – especially parasites. In 1998, the FDA reported benzyl alcohol to be present in 322 cosmetic formulations belonging to 43 cosmetic-product categories. Additionally, benzyl alcohol is used in photographic development. Aside from developing color movie films, as a solvent, benzyl alcohol is a component of inks, paints and epoxy resin coatings. It is an indirect food additive for use as a component of resinous and polymeric coatings.

Environmental behavior, fate, routes, and pathways From industrial production to consumer products, benzyl alcohol is present in the environment and is steadily released through commercial and household waste streams. This chemical was an early object of chemists striving for greener synthetic approaches involving mixed catalysts for oxidation. It is released into the atmosphere as a vapor in its entirety due to its high vapor pressure, where it is lost by degradation involving reaction with hydroxyl radicals at a half-life of about 2 days. Benzyl alcohol is expected to have quite high mobility based upon its soil to water partition coefficient, and a projected soil half-life of about 13 days.

Exposure and exposure monitoring The principal routes of exposure to benzyl alcohol are via inhalation and dermal (skin) contact where benzyl alcohol is produced or used. Individuals encounter their most common exposure to this compound via dermal contact with consumer products, and to a lesser extent via inhalation of ambient air. Prolonged or excessive inhalation can result in headache, nausea, diarrhea, or respiratory stimulation followed by respiratory and muscular paralysis. Benzyl alcohol can be absorbed through skin with anesthetic or irritant effect.

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Toxicokinetics Adults readily oxidize benzyl alcohol to benzoic acid. This is followed by the conjugation of benzoic acid with glycine in the liver to form hippuric acid. The latter is excreted in the urine. The immature metabolic capacities of infants result in limited ability to metabolize the benzyl alcohol metabolite benzoic acid to its primary excreted metabolite hippuric acid resulting in increased benzoic acid concentrations in preterm infants. These concentrations are further increased in preterm babies over term babies due to an increased ability to metabolize benzyl alcohol to benzoic acid. The relative inability to convert benzoic acid to hippuric acid may be related to glycine deficiency. Accumulation of benzoic acid in preterm infants has led to cases of severe toxicity and death. In dogs administered 52 or 105 mg kg−1 iv doses of benzyl alcohol in saline, plasma half-life was about 1.5 h. Benzyl alcohol rapidly disappeared from the injection site following intramuscular administration in rats, with a disappearance half-life estimated to be less than 10 min. In non-human primate studies, the Rhesus monkey demonstrates high absorption (56–80%) of topically administered benzyl alcohol over 24 h, but only when the administration site is occluded. Rabbits receiving 1 g of subcutaneous benzyl alcohol eliminate 300–400 mg of hippuric acid. In humans, benzyl alcohol is readily absorbed from the gastrointestinal tract following oral ingestion. Percutaneous absorption of benzyl alcohol during its use to treat pediculosis is reported to be limited and low, although application of higher concentrations of the alcohol can result in substantial uptake. Levels of benzyl alcohol from 5 to 500 mg per 10 ml plasma are reported in uremic patients on hemodialysis. No drug interaction studies have been reported to date with benzyl alcohol.

Mechanisms of action Benzyl alcohol is thought as treatment for head lice by inhibiting the lice from closing their respiratory spiracles. This is thought to operate via a unique mechanism involving physical pulmonary obstruction of respiratory spiracles by the solvent and subsequently resulting in asphyxiation of the lice. Benzyl alcohol is a local anesthetic and produces metabolic acidosis. The latter action can be attributed to direct acidification and fixed anion effects of the metabolite benzoic acid and potentially to secondary lactic acid production due to inhibition of cellular metabolism. Weak local anesthetic effects have been related to membrane fluidization.

Acute and short-term toxicity Benzyl alcohol has been studied across a large number of animal species including mice, rat, cat, dog, rabbit, and chicken. The oral LD50 values of benzyl alcohol in seven rat studies range from 1230 to over 3100 mg kg−1. These levels of observed effects have been categorized as moderately toxic in some comparative toxicity scales. In rats, clinical signs and symptoms of toxicity include increased respiration, tremors, half-closed eyes, lethargy, ataxia, prostration, and ultimately coma. Depression occurs within 10–15 min, followed by death from 1 h to as late as 4 days. Signs in mice include depression with animal deaths within 18 h. Subcutaneous benzyl alcohol produces respiratory stimulation, followed by respiratory and muscular paralysis, convulsion, and central nervous system (CNS) depression. Interestingly, intravenous administration of benzyl alcohol to rat, cat, and dog produces a decrease in arterial blood pressure, which is not seen in the dog after oral administration. The general toxicity of lower dose benzyl alcohol exposures in humans places it among Class I agents that cause reversible effects which are generally not life-threatening. Common effects are irritation and mild CNS depression. Toxic effects associated with higher exposures include vasodilatation, hypotension, convulsions, paralysis, and respiratory failure. Serious problems have occurred with this alcohol when present as a preservative in fluids administered to neonates. Benzyl alcohol was responsible for a number of deaths stemming from metabolic acidosis that progressed to respiratory distress and gasping respirations. Some infants developed CNS dysfunction, including developmental delay, convulsions, intracranial hemorrhage, and hypotension followed by cardiovascular collapse. Neonatal toxicity has been attributed to the immaturity of the benzoic acid detoxification process in premature newborns. Ocular exposure to dilute solutions of benzyl alcohol can cause slight irritation and local anesthesia. Use of benzyl alcohol in consumer products has resulted in a relatively low incidence of contact dermatitis characterized by urticaria, angioedema, erythema, and pruritus. Finally, the comparatively more favorable safety properties of benzyl alcohol have resulted in it being advocated in 2013 as a less-toxic alternative for paint stripping products containing dichloromethane. LD50 doses for benzyl alcohol in several rodent models are listed below: LD50 Mouse sc 950 mg kg−1 LD50 Mouse iv 400–800 mg kg−1

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LD50 Rat oral 1230–3120 mg kg−1 LC100 Rat inhalation 200–300 ppm per 8 h LC50 Rat inhalation 74 mg l−1 per 4 h LC50 Rat inhalation 1000 ppm per 8 h LD50 Rat iv 53 mg kg−1 LD50 Rat iv 314 mg kg−1 LD50 Rabbit oral 1940 mg kg−1 LD50 Rabbit dermal 2000 mg kg−1 LD50 Guinea pig dermal 400–800 mg kg−1 Source: NLM-HSDB.

Chronic toxicity Benzyl alcohol was the subject of chronic animal studies conducted by the National Toxicology Program (NTP). Limited signs and symptoms of chronic oral toxicity were noted for benzyl alcohol when given to rats and mice in doses of 200–400 mg kg−1 over a 2 year-period. According to NTP, there was no evidence of carcinogenic activity. However, reviewing the study, the EPA considered the increased incidence of adrenal cortex adenoma in high-dose male mice to be ‘equivocal evidence of carcinogenic activity rather than negative’ (NTP, 1989; US EPA, 2008). No other relevant repeated-dose toxicity studies were located. Chronic studies evaluating the toxicity of benzyl alcohol following inhalation exposure were unavailable.

Immunotoxicity Benzyl alcohol is toxic by all routes of administration. Immunotoxicity has been consistently reported to be the most sensitive indicator of non-cancer toxicity in both humans and experimental animals.

Reproductive toxicity No human studies on fertility is available for benzyl alcohol. As for rodent-studies, histopathological examinations of reproductive tissues are of high sensitivity for the evaluation of reproductive toxicity. Histopathological changes on the reproductive organs in these studies are indicative of effects on fertility, whereas the absence of such effects gives evidence that a substance does not influence fertility.

Genotoxicity A number of studies have demonstrated that benzyl alcohol is not mutagenic in the bacterial reverse mutation assay for Salmonella typhimurium strains TA92, TA94, TA98, TA100, TA1535, TA1537 or TA1538, both in the presence or absence of exogenous metabolic activation (NTP, 1989). Benzyl alcohol has also shown to be negative in the sex-linked recessive lethal assay and the replicative DNA synthesis assay in rodents. Although negative in the mouse lymphoma assay with metabolic activation, benzyl alcohol is positive in this assay without metabolic activation at levels that also produce significant cell death. Oppositely, benzyl alcohol noticeably increases the rate of sister chromatid exchange in Chinese hamster ovary cells in the presence, but not the absence of the S9 fraction (Carvalho et al., 2012).

Carcinogenicity The National Toxicology Program (NTP) has conducted carcinogenesis studies of technical-grade benzyl alcohol (99% pure) by administering the chemical by gavage in corn oil vehicle. Groups of F344/N rats and B6C3F1 mice were administered the test chemical for 16 days, 13 weeks, or 2 years. Benzyl alcohol produced no evidence of carcinogenic activity in male or female F344/N rats dosed with 200 or 400 mg kg−1 (NTP. 1989). No evidence of carcinogenic activity was also demonstrated for 2 years in male or female B6C3F1 mice dosed with 100 or 200 mg kg−1. There are no human carcinogenicity data for benzyl alcohol.

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Clinical management There is no proven antidote for benzyl alcohol poisoning. Following standard airway, breathing and circulation support as appropriate, treatments for significant benzyl alcohol exposure are vague and include discontinuation of the exposure. Management of inhalation exposure can include increased ventilation, fresh air, and rest. In cases of dermal exposure, protective gloves are recommended during removal of contaminated clothing and repeated rinsing with water. Ocular contact resulting in redness should prompt rinsing with water for several minutes, and immediate removal of contact lenses if present. Individuals who are suspected of acute benzyl alcohol ingestion and demonstrate serious symptoms including diarrhea, nausea, and/or vomiting, should be promptly referred for advanced medical attention, as should those exposed via other routes (Nair, 2001). Hemodialysis may enhance the elimination of benzyl alcohol and its metabolites and may also be a useful adjunct in correcting severe metabolic acidosis.

Exposure standards and guidelines Studies characterizing safe exposure limits to benzyl alcohol are not as available. One of the few published values for airborne exposure limits comes from the Occupational Alliance for Risk Science, which sets the workplace environmental exposure level (WEEL) for 8-h time weighted average (TWA) at 10 ppm. This WEEL was developed and maintained by the American Industrial Hygiene Association until 2012. The American Conference of Governmental Industrial Hygienists has no set threshold limit value for benzyl alcohol. The Research Institute for Fragrance Materials (RIFM) classifies benzyl alcohol potency as a skin sensitizer as weak, based on animal data including a local lymph node assay value of >12,500. They report the lowest observable effect level value of 8858 mg cm−2. The RIFM Expert Panel established the no expected sensitization induction level (NESIL) at 5900 mg cm−2. The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area concluded that human and animal studies have yielded insufficient information to set a maximum workplace concentration value, placing it in Category II B (International Fragrance Association, 2008). More recently, the German Working Group on Indoor Air Guidelines of the Federal Environment Agency has issued indoor air hazard guide value of 4 mg m−3 and a health precaution guide value of 0.4 mg m−3 (Bundesgesundheitshlatt et al., 2010).

Conclusion Benzyl alcohol (BA) is a popular compound in food, flavor and fragrance industries. Because of its anti-bacterial and anti-fungal properties in addition to serving as a flavor enhancer, it is used in the manufacture of soaps, topical creams, skin lotions, shampoos, and facial cleansers. BA is often used as a general solvent for inks, waxes, shellacs, paints, lacquers, and epoxy resin coatings. BA can induce severe allergic contact dermatitis in some people. In 2009, 5% BA (Ulesfia®) was approved by USFDA for the treatment of head lice. BA is a naturally found phytochemical in fruits and teas, and a variety of essential oils (jasmine, hyacinth and ylang-ylang). Because of its low toxicity profile, it has earned GRAS status from USFDA.

References Bundesgesundheitshlatt, Gesundheitsforschung, and Gesundheitsschutz (2010) Indoor air guide values for benzyl alcohol. Bundesgesundheitsbl 53(9): 984–987. https://doi.org/ 10.1007/s00103-010-1123-y. Carvalho CM, Menezes PF, Letenski GC, et al. (2012) In vitro induction of apoptosis, necrosis and genotoxicity by cosmetic preservatives: Application of flow cytometry as a complementary analysis by NRU. International Journal of Cosmetic Science 34(2): 176–182. IFRA (International Fragrance Association) (2008) Use Level Survey. December 2008. Johnson W, Bergfeld WF, Belsito DV, et al. (2017) Safety Assessment of Benzyl Alcohol, Benzoic Acid and its Salts, and Benzyl Benzoate. International Journal of Toxicology 36(3_suppl): 5S–30S. https://doi.org/10.1177/1091581817728996. Nair B (2001) Final report on the safety assessment of benzyl alcohol, benzoic acid, and sodium benzoate. International Journal of Toxicology 20(Suppl. 3): 23–50. NTP (1989) Toxicology and carcinogenesis studies of benzyl alcohol (CAS No. 100-51-6) in F344/N rats and B6C3F1 mice (Gavage studies). National Toxicological Program 343: 1–158. US EPA (2008) Non-confidential production volume information submitted by companies for chemicals under the 1986–2002. Inventory Update Rule (IUR). Benzenemethanol (100-51-6). http://www.epa.gov/oppt/cdr/tools/data/2002-vol.html.

Further reading Masuck I, Hutzler C, and Luch A (2011) Estimation of dermal and oral exposure of children to scented toys: Analysis of the migration of fragrance allergens by dynamic headspace GC-MS. Journal of Separation Science 34(19): 2686–2696. Scognamiglio J, Jones L, Vitale D, Letizia CS, and Api AM (2012) Fragrance material review on benzyl alcohol. Food and Chemical Toxicology 50(Suppl. 2): S140–S160. Shehab N, Lewis CL, Streetman DD, and Donn SM (2009) Exposure to the pharmaceutical excipients benzyl alcohol and propylene glycol among critically ill neonates. Pediatric Critical Care Medicine 10(2): 256–259.

Relevant websites http://www.osha.gov/ :Occupational Safety and Health Administration. http://toxnet.nlm.nih.gov/ :Toxnet (Toxicology Data Network): search under Toxline for Benzyl Alcohol. http://chem.sis.nlm.nih.gov/chemidplus :US National Library of Medicine: ChemIDplus Advanced: Search for: Benzyl Alcohol.

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Benzyl benzoate Sofia Angela P Federicoa, Amelia B Hizon-Fradejasa, Jeb Reece H Grabatoa, and Elmer-Rico E Mojicab, aInstitute of Chemistry, University of the Philippines Los Baños, College, Laguna, Philippines; bDepartment of Chemistry and Physical Sciences, Pace University, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of M.A. Pearson, G.W. Miller, Benzyl Benzoate, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 433–434, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00103-2.

Chemical profile Background Uses Environmental fate and behavior Exposure routes and pathways Toxicokinetics Acute and short-term toxicity Animals Humans Chronic toxicity Animals Humans Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Exposure standards and guidelines References Further reading

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Abstract Benzyl benzoate is an organic compound with a molecular weight of 212.24 g/mol and chemical formula C6H5CH2O2CC6H5. Its several uses include being an insecticide, plasticizer, a fixative in fragrances, a food additive, and a solvent. Exposure to benzyl benzoate can be through dermal contact, inhalation of its aerosol or dust, or ingestion of food. Non-allergic contact dermatitis is the most common skin reaction by fragrance compounds containing benzyl benzoate. At high concentrations, it has been found to possess estrogenic properties and to stimulate growth of human breast cells. Oral LD50 values fall within 1700 mg/g in rats.

Keywords Benzoic acid; Dermatitis; Estrogen; Food additive; Hippuric acid; Plasticizer; Scabicide; Solvent

Chemical profile

• • • • •

Name: Benzyl Benzoate Chemical Abstracts Service Registry Number: CAS 120–51-4 Synonyms: Ascabin, Benylate, Benzyl alcohol benzoic ester, Scabide, Vanzoate Molecular Formula: C14H12O2 Chemical Structure:

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Background Benzyl benzoate is an organic compound with a molecular weight of 212.24 g/mol and chemical formula C6H5CH2O2CC6H5. It is made from the condensation of benzoic acid and benzyl alcohol. It is a colorless oily liquid or a white solid, insoluble in water, miscible with alcohol (95%), chloroform, ether, oils, acetone, benzene, methanol, and petroleum ether. It has a pleasant aromatic odor and burning taste. It has a boiling point of 323–324  C and a melting point of 19  C. This natural product has been isolated from plant species of the genus Polyalthia. It is an active substance in Peruvian and Tulu balsams as well as essential oil. It is also synthetically produced and formulated as a liquid, emulsion, and lotion.

Uses Benzyl benzoate is used to address scabies and lice and is used pediculicide. When adsorbed by lice and mites, it acts on their nervous system, thereby destroying them. For scabies, permethrin or malathion as topical cream or lotion is commonly selected. Its primary use in humans is as main ingredient in repellents of ticks, chiggers, and mosquitoes. Benzyl benzoate is a usual additive in body care, cosmetics, and food. It can be utilized as a plasticizer in cellulose and other polymers. It can also be integrated into fragrances as a fixative, a food additive, and as a solvent. It protects crops, prevents insect attack of woolen products, and disinfect arriving aircraft in aviation industry.

Environmental fate and behavior More than 60% benzyl benzoate residues were left at 2 and 7 days after treatment when tested in water. Benzyl benzoate has low water distribution and a high soil distribution.

Exposure routes and pathways Benzyl benzoate can be adsorbed in the body either by dermal contact, inhalation of its aerosol or dust, or ingestion of food. Its media of exposure is through air, water and soil. Humans can be exposed to benzyl benzoate if overused as a topical scabicide. From contact with toys, migration of benzyl benzoate was found out from oral and skin exposure in children. It has the highest migration rate among tested compounds with exposure level at the maximum of 22.2 mg/ kg bw day−1.

Toxicokinetics It was found out in one investigation that 54% of the percutaneously applied dosage permeated the human skin. Upon absorption, it is metabolized into benzoic acid and benzyl alcohol which in conjugation with glycine produces hippuric acid, and in reaction with glucuronic acid yields benzoylglucuronic acid (Page, 2008). Depending on the species and dose, both conjugates are excreted in urine in varying proportions. Free benzoic acid may segregate acetyl coenzyme A in the body, impeding cholinergic signaling.

Acute and short-term toxicity Animals Oral LD50 values fall within 1700 mg/g in rats. Koçkaya and Kiliç Suloglu (2011) studied the maternal and fetal toxicity of benzyl benzoate in pregnant rats after oral dose of 25 and 100 mg/kg. Changes in biochemical parameters and placental and skeletal measurements and differences in immunolocalization of VEGF were shown in treatment groups, indicating that benzyl benzoate and its metabolites can transport to the placenta and can enter the fetuses. The rate (mL/g/h) of oxygen consumption was reported by Vishnu (2019) following the method of Sakr and Lail to assess the acute toxicity on the fish Labeo rohita, During 1, 2, 3, and 4 h of exposure, the lethal concentration of benzyl benzoate was revealed to be decreasing with increasing period of exposure. The symptoms of toxicity in 4 h LC50 value of benzyl benzoate appeared to be a sudden change in fish behavior with erected swimming movement, loss of equilibrium and skin sensation. Cats are sensitive to 2240 mg/kg oral LD50 benzyl benzoate. In dogs, oral LD50 values range over 22,440 m/kg. Moreover, benzyl benzoate applied to the skin was not toxic to pigs, sheep, heifers, or horses. Signs of toxicity to benzyl benzoate include nausea, vomiting, diarrhea, salivation, hair growth, progressive incoordination, central nervous system agitation, tremor, convulsions, progressive hindlimb paralysis, weakness, dyspnea and death. Fatal skin administration in cats caused excessive salivation, seizures at the treatment site, systemic tremor, muscle incoordination, hindlimb paralysis, seizures, and premortal respiratory failure.

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Humans Non-allergic contact dermatitis can be caused as most common skin reaction by fragrance compounds containing benzyl benzoate. A16-year old boy being treated for primary hypogonadism by an intramuscular injection of a depot testosterone undecanoate manifested anaphylaxis. A low number of convulsion events have been testified as serious reactions to benzyl benzoate. The death of a 7-year-old child was recorded due to an overdose of benzyl benzoate-based scabicide. In beverages, benzoates can be reduced to benzyl alcohol and toluene in orange juice which is a risk of toxicity (Bocharova et al., 2017).

Chronic toxicity Animals In a study by Kılıç Sülo glu et al. (2022), two varying doses of benzyl benzoate with 25 mg/ kg body weight and 100 mg/ kg body weight were orally treated to 5-week old male rats for 90 days. It was concluded that there are statistically significant differences in terms of monocyte, neutrophil, lymphocyte %, and serum AST levels in control and benzyl benzoate treatment groups. Immunolocalization of histochemical markers were altered between control and treatment groups.

Humans To date, the chronic effects of benzyl benzoate on humans are not fully understood. Based on the available human studies summary information available from PUBCHEM database, direct contact to benzyl benzoate may cause skin irritation. Liquid form of the chemical is irritating to eyes on direct contact. There was no evidence of reported adverse health effects on pregnancy outcome as a result of topical benzyl benzoate lotion application. Benzyl benzoate gave estrogenic responses in a human breast cancer cell line in culture. For additional information, refer to PUBCHEM chemical profile for benzyl benzoate.

Reproductive toxicity Benzyl benzoate induces increased fetal resorption in pregnant rats which could imply an endocrine endpoint, the estrogenic response of human MCF7 breast cancer cells in vitro.

Genotoxicity There is very little information on the genotoxicity of benzyl benzoate. It was reported that the expression of the ERE-CAT reporter gene was triggered by benzyl benzoate at high concentrations and the endogenous pS2 gene in human MCF7 breast cells (Charles and Darbre, 2009).

Carcinogenicity Screening of tobacco smoke components through Ames test showed that benzyl benzoate was not mutagenic.

Clinical management If dermally exposed, it is advised to remove the contaminated clothing and wash the affected skin with soap and water. Once inhaled, the patient should be transferred to fresh air as first aid. In case of contact with eyes, rinse with water for at least 15 min with the eyelids open. If swallowed, induction of vomiting is not recommended. If patient is conscious, rinse mouth with plenty of water, drink about 500 ml of water and see a doctor.

Ecotoxicology Benzyl benzoate is comparatively toxic to brine shrimp, zebra fish (LC50, 3.9 mg l−1), and bluegill sunfish (LC50, 2.5 mg l−1).

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Exposure standards and guidelines In 2016, EFSA ANS Panel re-evaluated the safety of benzoic acid and benzoates (E210–231) and established a group Acceptable Daily Intake (ADI) of 5 mg/kg bw per day (expressed a benzoic acid). However, a specific ADI of benzyl benzoate has not been established. The EFSA Panel on Food Additives and Flavorings (FAF) re-evaluated Benzyl Alcohol in 2019 and established an ADI of 4 mg benzyl alcohol/kg bw per day based on a NOAEL of 400 mg/kg bw per day from the carcinogenicity study in rats, based on no effect in the highest dose tested (EFSA, 2019). Benzyl alcohol was evaluated by JECFA in 1980. A group acceptable daily intake (ADI) (for benzoic acid together with benzyl alcohol) of 0–5 mg/kg body weight (bw) was recommended.

References Bocharova O, Reshta S, and Eshtokin V (2017) Toluene and benzyl alcohol formation in fruit juices containing benzoates. Journal of Food Processing and Preservation 41(4): e13054. Charles AK and Darbre PD (2009) Oestrogenic activity of benzyl salicylate, benzyl benzoate and butylphenylmethylpropional (Lilial) in MCF7 human breast cancer cells in vitro. Journal of Applied Toxicology 29(5): 422–434. https://doi.org/10.1002/jat.1429. PMID: 19338011. EFSA Panel on Food Additives and Flavourings (FAF), Younes M, Aquilina G, Castle L, Engel KH, Fowler P, Fürst P, Gürtler R, Gundert-Remy U, Husøy T, and Mennes W (2019) Re-evaluation of benzyl alcohol (E 1519) as food additive. EFSA Journal 17(10): e05876. https://doi.org/10.2903/j.efsa.2019.5876. (Accessed Feb 11, 2023). Kılıç Süloglu A, Koçkaya EA, and Selmanoglu G (2022) Toxicity of benzyl benzoate as a food additive and pharmaceutical agent. Toxicology and Industrial Health 38(4): 221–233. https://doi.org/10.1177/07482337221086133 Epub 2022 Mar 25 PMID: 35332820. Koçkaya EA and Kiliç Suloglu A (2011) Developmental toxicity of benzyl benzoate in rats after maternal exposure throughout pregnancy. Environmental Toxicology. https://doi.org/ 10.1002/tox.20771. PMID: 21922633. Page SW (2008) Benzyl Benzoate, Antiparasitic drugs (chapter 10). In: Maddison JE, Page SW, and Church DB (eds.) Small Animal Clinical Pharmacology, 2nd edn, 226–227. Vishnu M (2019) Evaluation of Phyoto and Aquatic Toxicity of Neonicotinoid Insecticide Additives - Benzyl Benzoate and N-Cyclohexyl Benzothiazole-2-Sulfenamide and their Metabolites produced by Pseudomonas desmolyticum NCIM 2112. Research Journal of Biotechnology 14(10).

Further reading CDC; International Chemical Safety Cards (ICSC) (2012) Atlanta, GA: Centers for Disease Prevention & Control. National Institute for Occupational Safety & Health (NIOSH). Ed Info Div. Available from, as of Oct 3, 2018. https://www.cdc.gov/niosh/ipcs/default.html Johnson W, Bergfeld WF, Belsito DV, Hill RA, Klaassen C, Liebler D, Marks DC, Shank JG, Slaga RC, Snyder TJ, and P.l W. Andersen, F. A. (2017) Safety assessment of benzyl alcohol, benzoic acid and its salts, and benzyl benzoate. International Journal of Toxicology 36(3_suppl): 5S–30S. https://doi.org/10.1177/1091581817728996. Keystone JS, Kozarsky PE, Freedman DO, and Connor BA (2013) Travel Medicine. Elsevier Health Sciences, p. 58. ISBN 978-1-4557-1076-8. Kwon YS, Park CB, Lee SM, Zee S, Kim GE, Kim YJ, Sim HJ, Kim JH, and Seo JS (2022) Proteomic analysis of zebrafish (Danio rerio) embryos exposed to benzyl benzoate. Environmental Science and Pollution Research 1–12. O’Neil MJ (ed.) (2013) The Merck Index - An Encyclopedia of Chemicals, Drugs, and Biologicals, p. 199. Cambridge, UK: Royal Society of Chemistry. World Health Organization, Stuart MC, Kouimtzi M, and Hill S (2009) In: Stuart MC, Kouimtzi M, and Hill SR (eds.) WHO Model Formulary 2008. World Health Organization. https://apps. who.int/iris/handle/10665/44053.

Relevant websites http://www.fao.org/food/food-safety-quality/scientific-advice/jecfa/en/ :Food and Agriculture Organisation (Chemical Risk and The Joint FAO/WHO Expert Committee on Food Additives (JECFA) https://www.mayoclinic.org/drugs-supplements/benzyl-benzoate-topical-route :Mayo Clinic - Benzyl Benzoate (Topical Route) Proper Use https://pubchem.ncbi.nlm.nih.gov/compound/Benzyl-benzoate :Benzyl Benzoate (Pubchem)

Beryllium Shayne C Gad, Gad Consulting Services, Raleigh, NC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.C. Gad, Beryllium, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 435–437, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00820-4.

Chemical profile Background Uses/occurrence Exposure Toxicokinetics (ADME) Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines Other PubChem URL References Further reading

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Abstract Beryllium, CAS#: 7440-41-7, was discovered as an element in 1797. Beryllium is an important industrial metal because of its material properties; that is, it is lighter than aluminum and six times stronger than steel. Its use in metallurgy and electrical components were largely developed in the 1920s. The primary exposure pathway for beryllium is inhalation, but beryllium is not well absorbed by any route. The major toxicological effects of beryllium are on the lung. Beryllium is extremely toxic to warm water fish in soft water, and the degree of toxicity decreases with increasing water hardness. ACGIH classifies beryllium as a suspected human carcinogen.

Keywords Beryl; Berylliosis; CBD (Chronic Beryllium Disease); Chelation; Hypercalcemia

Chemical profile

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Valence States: 0, +2 Name: Beryllium Synonyms: Glucinum, Glucinium Chemical Abstracts Service Registry Number: 7440-41-7 Molecular Formula: Be Chemical Structure:

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Background Although known through the mineral beryl, Be3Al2(SiO3)6, for millennia, beryllium was discovered as an element in 1797 by Louis-Nicolas Vauquelin, although it was not isolated as a metal until Friedrich Wöhler and Antoine Bussy independently succeeded in this venture independently by reacting potassium metal with beryllium chloride in a platinum crucible, yielding beryllium metal and potassium chloride. Its use in metallurgy and electrical components were largely developed in the 1920s (Welch, 2012).

Uses/occurrence Beryllium is an important industrial metal because of its material properties; that is, it is lighter than aluminum and six times stronger than steel. Often alloyed with other metals such as copper, beryllium is a key component of materials used in the aerospace and electronics industries. Beryllium has a small neutron cross-section, which makes it useful in the production of nuclear weapons and in sealed neutron sources. Specifically, beryllium is used in nuclear reactors as a neutron reflector or moderator, and in the aerospace industry in inertial guidance systems; beryllium alloys (consisting of copper or aluminum) are also used in structural material. Beryllium oxide is used as an additive in glass, ceramics, and plastics and as a catalyst in organic reactions. In the past, beryllium was widely used in the manufacture of fluorescent lights and neon signs. Alloyed with copper, aluminum, or nickel, beryllium imparts excellent electrical and thermal conductivity (Welch, 2012).

Exposure The primary exposure pathway for beryllium is inhalation. Inhalation, ingestion, and dermal contact are possible exposure pathways in workplace settings. Exposure to small amounts of beryllium occurs with ingestion of some foods and drinking water. Beryllium enters the air, water, and soil as a result of natural and human activities. Emissions from burning coal and oil increase beryllium levels in air. Beryllium enters waterways from the wearing away of rocks and soil. Most of the synthetic beryllium that enters waterways comes when industry dumps wastewater and when beryllium dust in the air from industrial activities settles over water. Beryllium, as a chemical component, occurs naturally in soil; however, disposal of coal ash, incinerator ash, and industrial wastes may increase the concentration of beryllium in soil. In air, beryllium compounds are present mostly as fine dust particles. The dust eventually settles over land and water (Kriebel et al., 1988).

Toxicokinetics (ADME) Beryllium is not well absorbed by any route. Oral absorption of beryllium is 1–10 years

> 10 years to lifetime

Allowable Daily intake

120 mg

20 mg

10 mg

1.5 mg

Pohanish and Sittig (2017).

Beryllium

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Other Emeralds are a beryllium compound, beryl, Be3Al2(SiO3)6, which is colored green by the presence of trace quantities of chromium or vanadium.

PubChem URL Beryllium | Be - PubChem (nih.gov).

See also: Metals; Respiratory toxicology

References Chang LW (ed.) (1996) Toxicology of Metals, pp. 929–930. Boca Raton, FL: Lewis Publishers. Coast Guard US (1999) Chemical Hazard Response Information System (CHRIS) - Hazardous Chemical Data. Commandant Instruction 16465.12C. Washington, D.C.: U.S. Government Printing Office. Gordon T and Bowser D (2003) Beryllium: Genotoxicity and carcinogenicity. Mutation Research 533: 99–105. Kreiss K (2011) Beryllium: A paradigm for occupational lung disease and its prevention. Occupational and Environmental Medicine 68(11): 787–788. Kriebel D, Brain JD, Sprince NL, and Kazemi H (1988) The pulmonary toxicity of beryllium. The American Review of Respiratory Disease 137: 464–473. Muller C, Salehi F, Mazer B, Bouchard M, Adam-Poupart A, Chevalier G, Truchon G, Lambert J, and Zayed J (2011) Immunotoxicity of 3 chemical forms of beryllium following inhalation exposure. International Journal of Toxicology 30(5): 538–545. Pawlas N and Palczynski CM (2022) Beryllium. In: Nordberg GF and Costa M (eds.) Handbook on the Toxicology of Metals, 5th edn., pp. 635–653. New York: Elsevier. Pohanish RP and Sittig M (2017) Sittig’s Handbook of Toxic and Hazardous Chemicals and Carcinogens, 7th edn. William Andrew is an imprint of Elsevier. Taylor TP, Ding M, Ehler DS, et al. (2003) Beryllium in the environment: A review. Journal of Environmental Science and Health, Part A: Environmental Science and Engineering & Toxic and Hazardous Substance Control 38: 439–469. Welch LS (2012) In: Bingham E, Cohrssen B, and Powell CH (eds.) Patty’s toxicology, 5th edn. New York: John Wiley & Sons, Inc.

Further reading Williams WJ (1988) Beryllium disease. Postgraduate Medical Journal 64: 511–516.

Relevant websites Beryllium | Toxicological Profile | ATSDR (cdc.gov) :ATSDR CDC Toxicological Profile for Beryllium http://www.epa.gov/ttn/atw/hlthef/berylliu.html :Environmental Protection Agency, Air Toxics website: search Beryllium Compounds http://minerals.usgs.gov/minerals/pubs/commodity/beryllium/ :US Geological Survey, Minerals Information: Beryllium http://www.cdc.gov/niosh/topics/beryllium/ :Centers for Disease Control, Workplace Safety and Health Topics: Beryllium

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Beta-blockers Daniel L Overbeek, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of V. Dissanayake, M. Wahl, Beta-Blockers, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 438–441, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00698-9.

Chemical profile Background Uses Environmental fate and behavior Exposure routes and pathways Toxicokinetics Mechanism of toxicity Acute toxicity Immunotoxicity Reproductive toxicity Genotoxicity and carcinogenicity Clinical management Conclusion References

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Abstract Sir James Black synthesized propranolol, the first clinically important beta-blocker, in the 1960s, and half a century later, beta-blockers play an integral role in the management of hypertensive and ischemic heart disease. However, these agents are high risk in the setting of poisoning and overdose, with severe toxicity primarily resulting from cardiac dysfunction. Supportive measures including airway management, fluids, and vasopressors are the foundation for treatment of overdose. Glucagon is a targeted therapy that aims to promote cardiac contractility via increased calcium release by increasing cyclic adenosine monophosphate levels. High dose insulin therapy is used to supplement cardiac contractility. Lipid emulsion has shown promise as rescue therapy for lipophilic beta-blockers such as propranolol, and extracorporeal cardiac support methods such as ECMO have also been used in cases of refractory cardiogenic shock.

Keywords Beta-adrenergic antagonist; Beta-blocker; Bradyarrhythmias; Glucagon; Hypotension; Lipid emulsion; Overdose; Poisoning; Toxicity; ECMO

Key points

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Beta-blockers are one of the most used cardiac medications in clinical practice Acute overdose can induce severe cardiac toxicity with hypotension, bradycardia, shock and death The mainstays of management of acute overdose are vasopressor agents, glucagon/high dose insulin therapy, with the consideration for external mechanical cardiac support in severe cases Extracorporeal elimination methods such as hemodialysis are only indicated for a small number of beta blockers, including atenolol and sotalol

Chemical profile

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Name: Beta-Blockers Synonyms: Acebutolol (Chemical Abstracts Service Registry Number (CAS) 37517-30-9), Atenolol (CAS 29122-68-7), Betaxolol (CAS 63659-19-8), Bisoprolol (CAS 66722-44-9), Carteolol (CAS 51781-21-6), Esmolol (CAS 81161-17-3), Labetalol (CAS 32780-64-6), Metoprolol (CAS 37350-58-6), Nadolol (CAS 42200-33-9), Nebivolol (CAS 99200-09-6), Penbutolol (CAS 38363-32-5), Pindolol (CAS 13523-86-9), Propranolol (CAS 318-98-9), Sotalol (CAS 959-24-0), Timolol (CAS 26921-17-5).

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Background Sir James Black synthesized the first clinically useful beta-blocker after being inspired to find an agent that would lower heart rate in addition to blood pressure. At that time, phenoxybenzamine was the commonly utilized medication for hypertension, but the unfortunate side effect of tachycardia limited its use. Sir Black revolutionized the treatment of hypertension with his discovery, one of the most important pharmacological contributions of the twentieth century (Stapleton, 1997). As such, beta-blocker use has skyrocketed and concomitantly beta-blockers have now become a significant cause of serious poisonings in both accidental and intentional overdoses. Liver and renal dysfunction can increase the risk of toxicity, making older populations also vulnerable to unintentional poisoning. It is vital that beta-blocker toxicity is considered in patients presenting with hemodynamic compromise.

Uses In therapeutic settings, the use of beta-blockers is intended to lower heart rate and blood pressure. Their indications include the treatment of hypertensive heart disease, ischemic heart disease, supraventricular arrhythmias, structural heart disease, hyperadrenergic conditions, glaucoma, and migraine headache (Frishman and Saunders, 2011). Selected beta-blockers also have unique indications such as propranolol for public speaking anxiety.

Environmental fate and behavior A few studies have examined the risk of environmental exposure of beta-blockers. Beta-blockers have been found in water environments, but can be removed at wastewater treatment facilities via multiple processes including adsorption and biodegradation. Beta-blockers are also susceptible to photolysis, causing them to be broken down in aquatic environments (Yi et al., 2020).

Exposure routes and pathways Oral exposure is the most common, including both intentional and accidental overdoses. Accidental toxicity also occurs secondary to decreased clearance in the setting of renal or hepatic injury. Esmolol, labetalol, metoprolol, and propranolol are all available for parenteral administration; therefore, toxicity can occur via this additional route. Beta-blockers are also administered as ocular medications and systemic toxicity has also been described via administration by this seemingly innocuous route (Taniguchi and Kitazawa, 1997).

Toxicokinetics Beta-blockers have notable variation in their oral bioavailability and kinetics. Generally, oral bioavailability is correlated with the lipid-solubility of the beta-blocker. From the most lipid-soluble beta-blocker, propranolol, to the least, atenolol, oral bioavailability ranges from 25% to 100%. The rate of absorption is rapid for non-sustained release preparations. There are sustained release preparations for carvedilol, metoprolol, and propranolol, and these are more slowly absorbed with delayed and prolonged clinical effects following overdose. Protein-bound drugs are poorly excreted by the kidneys, and can accumulate in those with liver failure, whereas less protein-bound and less lipophilic drugs are more likely to be excreted by the kidneys. Most of the beta-blockers have hepatic metabolism of at least 50%. However, atenolol, nadolol, and sotalol are principally excreted unchanged in the urine. Esmolol, although water soluble, is the most rapidly metabolized of the beta-blockers via esterases in the cytosol of red blood cells. Both renal and fecal elimination occurs with beta-blockers. Elimination half-life ranges from 0.15 (esmolol) to 32 h (nebivolol) (Nelson et al., 2019).

Mechanism of toxicity Beta-blocker toxicity is directly related to the pharmacologic effects, including their beta-selectivity profiles. These agents block the effects of catecholamines such as epinephrine and norepinephrine on the beta-1 and beta-2 receptors. Beta-1 receptors are primarily located in the heart and kidneys while beta-2 receptors are primarily located in the airway and vasculature. Severe toxicity is most often due to antagonism of the cardiac beta-1 receptors, but varies depending on the agent’s beta selectivity. For example, propranolol lacks selectivity, whereas labetalol and carvedilol are known to have both alpha-1 and beta-receptor blockade. Acebutolol, atenolol, betaxolol, bisoprolol, esmolol, metoprolol, and nebivolol are beta-1 selective antagonists (Baker, 2005). Some beta-blockers such as propranolol also have sodium-channel blocking effects, with alternative toxic effects. Nebivolol has an additional feature of nitric oxide activity that results in enhancement of vasodilation and can lead to toxicity in overdose as well. In large overdose, the receptor selectivity is often lost, so effects can vary even with agents which are receptor selective at therapeutic dosing.

Beta-blockers

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Acute toxicity The primary clinical effects observed in beta-blocker toxicity are cardiovascular in nature. Direct cardiac effects include bradycardia (sinus, atrioventricular node, ventricular), all degrees of atrioventricular block, bundle branch blocks, and asystole. Ventricular arrhythmias may occur secondary to bradycardia (Love et al., 2002). Hypotension occurs and is due to decreased cardiac output and/or vasodilation. Central nervous system effects of these drugs including lethargy, coma, and seizures are usually secondary to toxicity from lipophilic beta-blockers, such as propranolol and penbutolol. Bronchospasm can occur secondary to beta-2 blockade. Hypoglycemia can also occur and is more common in younger patients and unintentional exposures in toddlers. Beta-blockers are also known to worsen decompensated heart failure, exacerbate peripheral vascular resistance, and blunt the warning symptoms of hypoglycemia. In overdose, beta blockers with binding effects on fast sodium channels including propranolol can cause cardiac toxicity from fast sodium channel blockade that can result in QRS prolongation, seizures, and death. Pindolol has demonstrated some sympathomimetic activity with mild tachycardia and hypertension in overdose. Torsades de pointes has been associated with chronic toxicity from sotalol due to effects on delayed potassium channels responsible for repolarization, prolonging the action potential and the QT interval (Dancey et al., 1997).

Immunotoxicity There is no evidence that beta-blockers currently used cause immunotoxicity. Practolol, which was withdrawn from the market in 1975, was thought to cause an oculo-mucocutaneous syndrome that resulted in a kerato-conjunctivitis, psoriasis-like skin condition and effusions of the pleura and pericardium, but this was never definitively determined (Amos, 1979).

Reproductive toxicity Currently, some beta blockers are used for the management of hypertension in pregnancy, including primarily labetalol. However, many of these medications have limited data regarding safety. Many studies may be confounded by the chronic condition necessitating the beta blockers, including hypertension, which can be associated with many fetal abnormalities. Acebutolol, pindolol, and sotalol were classified as pregnancy category B medications when used early in pregnancy. These medications have not been found to cause harm in animal studies but have not been fully studied in pregnant humans. Category C medications have been shown to cause harm in animal studies but have not been studied in humans, or medications that have not been evaluated in animal studies. The following beta-blockers belonged to category C: betaxolol, bisoprolol, carteolol, carvedilol, esmolol, labetalol, levatol, levobunolol, metipranolol, metoprolol, nebivolol, penbutolol, propranolol, and timolol. Most references, including the American College of Obstetrics and Gynecology, include labetalol in their recommendations for the management of hypertension in pregnancy (Vidaeff et al., 2019; Magee et al., 2000). In late pregnancy, there are concerns about possible complications related to neonatal hypoglycemia or bradycardia, and the risk to benefit relationship will need to be considered by the healthcare provider. Highly protein-bound beta-blockers, such as acebutolol, atenolol, metoprolol, nadolol, and sotalol are capable of being excreted in breast milk. Atenolol and acebutolol have resulted in bradycardia, cyanosis, and hypotension in the newborn infant. One study found that the majority of beta-blockers do not result in significant levels in the breast-fed infant, except for atenolol and labetalol. Beta-blockers with lower milk to plasma ratios can be considered as alternatives for nursing mothers (Gupta, 2017).

Genotoxicity and carcinogenicity Multiple studies have been conducted on the beta-blocker class to determine potential mutagenicity, and there is no evidence that they cause genotoxicity or carcinogenicity (Okine et al., 1983).

Clinical management Advanced life-support measures should be instituted as necessary. A baseline 12-lead electrocardiogram should be obtained. Continuous cardiac and blood pressure monitoring should be initiated. Airway protection should be addressed if the patient is obtunded with signs of airway compromise. Gastric decontamination procedures should be initiated based on the history of the ingestion and the patient’s neurologic status. Consider charcoal for recent ingestions at a 10:1 dosing ratio for known ingestions (gram of charcoal per gram of medication). If the expected dose of activated charcoal is too high to administer, the patient’s mental status is problematic, or it is not certain when the patient ingested the medication, charcoal should not be administered as it can easily be aspirated. Whole bowel irrigation may be

36

Beta-blockers

useful following ingestions of sustained release preparations as long as these patients are not hemodynamically compromised and in the setting of airway protection. Due to the similarities between their clinical presentations, it is commonly difficult to determine whether a patient’s clinical picture is due to beta-blocker overdose or calcium channel-blocker toxicity. Evaluating the patient’s glucose level may be helpful in distinguishing the former from the latter, since calcium channel blockers inhibit insulin release and clinically demonstrate insulin resistance and hyperglycemia. Drug levels are not useful in the acute management of beta blocker toxicity (Love, 1994). Initially hypotension can be managed with crystalloid resuscitation, but if shock persists positive chronotropic and ionotropic agents as well as vasopressor agents should be considered. In some cases, low systemic vascular resistance predominates, and vasoconstrictors such as norepinephrine should be used. In the setting of depressed cardiac output, norepinephrine or epinephrine may overcome the beta-blockade, and other positive ionotropic agents such as dobutamine or isoproterenol can be considered. Bradycardia can be tolerated if total cardiac output maintains systemic perfusion. However, in severe overdoses, profound and refractory shock can persist through the use of these agents, and alternate therapies can be needed. Glucagon has been effective in increasing myocardial contractility in beta-blocker toxicity, most well described in animal studies (Bailey, 2003; Peterson et al., 1984). Glucagon stimulates production of cyclic adenosine monophosphate, which enhances contraction by activating sarcoplasmic reticulum calcium release (Lvoff and Wilcken, 1972). Glucagon metabolism additionally releases arachidonic acid, which also aids cardiac contractility. Initial intravenous doses of 3–5 mg have been used in adults, and if no response results, 6–10 mg (total dose) is usually administered. If there is improvement in hemodynamic parameters, an hourly infusion of the response dose of glucagon is continued. The initial pediatric dose is 50 mg kg−1. One challenge with the use of glucagon is that individual hospital stores often limit the possible duration of this therapy if not replenished. If cardiogenic shock is resistant to traditional measures, high dose insulin euglycemia therapy has been used to enhance cardiac contractility. Initial insulin boluses of 1 mg/kg and infusions starting at 1 mg/kg/h are given with supplemental dextrose to maintain euglycemia. Infusion dosing can be increased to at least 10 mg/kg/h. This insulin therapy increases cardiac contractility with minimal effects on heart rate. Complications of this therapy include hypoglycemia and fluid overload (as the infusions of insulin and dextrose will come with significant additional fluid resuscitation) (Engebretsen et al., 2011). For patients who fail all other therapies, the patient should be evaluated for extracorporeal cardiac support if available. Veno-arterial extracorporeal membrane oxygenation pumps support organ perfusion while bridging to metabolism of the xenobiotic. In overdoses of certain beta-blockers, including atenolol and sotalol, hemodialysis or hemoperfusion may be effective in increasing clearance, but maintaining perfusion remains the highest priority (Bouchard et al., 2021). More recently, intravenous lipid emulsion has been found to be potentially helpful in the critically ill patient suffering from lipophilic beta-blocker toxicity. It is believed that lipid infusion is able to provide a lipid sink that retains circulating drug in the lipemic portion of blood, preventing it from causing more toxicity and allowing more time for drug metabolism. If the patient continues to do poorly after implementation of all discussed treatment strategies, it is appropriate to consider this rescue therapy for it has shown promise in experimental animal models and has been successful in the resuscitation of select human cases (Cave and Harvey, 2009; Harvey and Cave, 2008).

Conclusion The discovery of beta-blockers added a critical tool for the management of cardiac diseases, but their toxicities can be severe. The wide range of pharmacokinetics and pharmacodynamics leads to significant variation in the clinical presentations of toxicity in acute overdose. Mild cases can be managed with fluids and observations, but severe cases often necessitate pharmacologic cardiac support with vasopressor agents, glucagon or high dose insulin therapies. Refractory cases may be candidates for extracorporeal mechanical cardiac support.

See also: Angiotensin converting enzyme (ACE) inhibitors; Alpha-1 adrenergic receptor antagonists; Digitalis glycosides; Calcium channel blockers; Cardiovascular system

References Amos HE (1979) Immunological aspects of practolol toxicity. International Journal of Immunopharmacology 1(1): 9–16. https://doi.org/10.1016/0192-0561(79)90025-0. Bailey B (2003) Glucagon in beta-blocker and calcium channel blocker overdoses: A systematic review. Journal of Toxicology. Clinical Toxicology 41(5): 595–602. https://doi.org/ 10.1081/CLT-120023761. Baker JG (2005) The selectivity of b-adrenoceptor antagonists at the human b1, b2 and b3 adrenoceptors. British Journal of Pharmacology 144(3): 317–322. https://doi.org/ 10.1038/SJ.BJP.0706048. Bouchard J, Shepherd G, Hoffman RS, et al. (2021) Extracorporeal treatment for poisoning to beta-adrenergic antagonists: systematic review and recommendations from the EXTRIP workgroup. Critical Care 25(1). https://doi.org/10.1186/S13054-021-03585-7. Cave G and Harvey M (2009) Intravenous lipid emulsion as antidote beyond local anesthetic toxicity: A systematic review. Academic Emergency Medicine 16(9): 815–824. https://doi. org/10.1111/J.1553-2712.2009.00499.X.

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Dancey D, Wulffhart Z, and Mcewan P (1997) Sotalol-induced torsades de pointes in patients with renal failure. The Canadian Journal of Cardiology 13(1): 55–58. https://europepmc. org/article/med/9039065. Accessed November 28, 2022. Engebretsen KM, Kaczmarek KM, Morgan J, and Holger JS (2011) High-dose insulin therapy in beta-blocker and calcium channel-blocker poisoning. Clinical Toxicology 49(4): 277–283. https://doi.org/10.3109/15563650.2011.582471. Frishman WH and Saunders E (2011) b-Adrenergic blockers. Journal of Clinical Hypertension 13(9): 649–653. https://doi.org/10.1111/J.1751-7176.2011.00515.X. Gupta RC (2017) Transfer of drugs and xenobiotics through milk. In: Reproductive and Developmental Toxicology, pp. 65–66. Elsevier. Harvey MG and Cave GR (2008) Intralipid infusion ameliorates propranolol-induced hypotension in rabbits. Journal of Medical Toxicology 4(2): 71. https://doi.org/10.1007/ BF03160958. Love JN (1994) Beta-blocker toxicity: a clinical diagnosis. The American Journal of Emergency Medicine 12(3): 356–357. https://doi.org/10.1016/0735-6757(94)90160-0. Love JN, Enlow B, Howell JM, Klein-Schwartz W, and Litovitz TL (2002) Electrocardiographic changes associated with b-blocker toxicity. Annals of Emergency Medicine 40(6): 603–610. https://doi.org/10.1067/mem.2002.129829. Lvoff R and Wilcken DE (1972) Glucagon in heart failure and in cardiogenic shock. Circulation 45(3): 534–542. https://doi.org/10.1161/01.CIR.45.3.534. Magee LA, Elran E, Bull SB, Logan A, and Koren G (2000) Risks and benefits of b-receptor blockers for pregnancy hypertension: Overview of the randomized trials. European Journal of Obstetrics, Gynecology, and Reproductive Biology 88(1): 15–26. https://doi.org/10.1016/S0301-2115(99)00113-X. Nelson LS, Howland MA, Lewin NA, Smith SW, Goldfrank LR, and Hoffman RS (2019) Goldfrank’s Toxicologic Emergencies, 11th edn. McGraw Hill. Okine LKN, Ioannides C, and Parke DV (1983) Studies on the possible mutagenicity of b-adrenergic blocker drugs. Toxicology Letters 16(3–4): 167–174. https://doi.org/ 10.1016/0378-4274(83)90175-3. Peterson CD, Leeder JS, and Sterner S (1984) Glucagon therapy for beta-blocker overdose. Drug Intelligence & Clinical Pharmacy 18(5): 394–398. https://doi.org/ 10.1177/106002808401800507. Stapleton MP (1997) Sir James Black and propranolol; The role of the basic sciences in the history of cardiovascular pharmacology. Texas Heart Institute Journal 24: 336–342. Taniguchi T and Kitazawa Y (1997) The potential systemic effect of topically applied beta-blockers in glaucoma therapy. Current Opinion in Ophthalmology 8(20): 55–58. https://doi. org/10.1097/00055735-199704000-00010. PMID: 10168358. Vidaeff A, Espinoza J, Simhan H, and Pettker CM (2019) ACOG practice bulletin no. 203: Chronic hypertension in pregnancy. Obstetrics & Gynecology 133(1): E26–E50. https://doi. org/10.1097/AOG.0000000000003020. Yi M, Sheng Q, Sui Q, and Lu H (2020) b-blockers in the environment: Distribution, transformation, and ecotoxicity. Environmental Pollution 266(Pt 2). https://doi.org/10.1016/J. ENVPOL.2020.115269.

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Betapropiolactone Swapnaa Balajia, Rabin Neupanea, Amit K Tiwaria, and Sidhartha D Rayb, aDepartment of Pharmacology and Experimental Therapeutics, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, Toledo, OH, United States; bDepartment of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of A. de Peyster, Beta-Propiolactone, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 442–445, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01168-4.

Chemical profile Background Uses Environmental fate and behavior Properties Exposure potential for humans and the environment Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Humans Animals Chronic toxicity Reproductive toxicity Genotoxicity Carcinogenicity Cancer studies in experimental animals Inhalation exposure Cancer studies in human Clinical management Ecotoxicology Exposure standards and guidelines Regulations Environmental Protection Agency (EPA) Clean Air Act Mine Safety and Health Administration (MSHA, Dept. of Labor) Occupational Safety and Health Administration (OSHA, Dept. of Labor) Guidelines American Conference of Governmental Industrial Hygienists (ACGIH) National Institute for Occupational Safety and Health (NIOSH, CDC, HHS) Conclusion References

40 40 41 41 41 41 42 42 42 42 42 43 43 43 43 44 44 44 44 44 45 45 45 45 45 45 45 45 45 45 45

Abstract Beta-propiolactone (BPL) is commonly used to inactivate reagent viruses during production of vaccines, because it likely inactivates viral infectivity by modifying viral protein. BPL was used for the sterilization of blood plasma, vaccines, tissue grafts, surgical instruments, and enzymes, and as an antimicrobial agent. BPL is a colorless liquid with a sweet odor, unstable at room temperature but stable at 5
C in glass containers. BPL vapor has been experimented with ethylene oxide for effectiveness as a sterilizing agent for influenza virus, and COVID virus vaccines. Its use as a sterilant was at one time extended to many other products. The main metabolite of BPL is lactic acid, and its main hydrolysis product is hydracrylic acid, both of which are excreted rapidly. BPL does not absorb or distribute appreciably after oral, or inhalation exposures based on the fact that tissue types of tumors seen in cancer bioassays are those experiencing first contact. Oral/gastrointestinal exposures resulted in increased combined incidence of benign and malignant tumors of the forestomach, and inhalation exposures caused nasal cancers. Toxicity to kidney and liver has been reported in animal studies but only after intravenous administration. BPL is sometimes classified as a direct-acting alkylating agent capable of attaching to DNA and forming DNA adducts. Glutathione has been proposed as a natural scavenger of BPL, and its presence in the organism likely protect from BPL’s genotoxicity, mutagenicity, and even carcinogenicity. This property probably accounts for its mutagenicity in a wide variety of in vitro and in vivo test systems (both somatic and germ cells). The NTP’s 2011 Report on Carcinogens considers BPL to be a reasonably anticipated human carcinogen based on sufficient evidence of carcinogenicity from studies in experimental animals. Potential for BPL exposure of the general population is becoming increasingly limited, although it is currently an FDA approved indirect additive used in food contact substances.

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00769-7

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Betapropiolactone Keywords Adenomatous polyps; Alkylating agent; Betapropiolactone; Carcinogenicity; Chemically reactive; Genotoxic; Hydracrylic acid; Mutagenic; Sarcoma; Sterilizing agent

Key points

• • • • • •

b-Propiolactone (BPL) is a lactone compound with a four-membered ring. It exists as a colorless liquid with a pungent, slightly sweet odor at room temperature. Although itis unstable at room temperature but stable at 5
C in glass containers. Its instability and the ability to react with other molecules in the vicinity is responsible for both its toxicity and its usefulness. BPL was manufactured in large quantities around 1950s through the mid-1970s, when it was widely used in chemical synthesis in reacting with other molecules to generate new entities. It is also used in research laboratories. Because it is no longer used as a sterilant in medical procedures or in food, the potential for exposure to the general population is minimal. The National Occupational Hazard Survey estimated that 575 workers potentially were exposed to BPL (Pubchem Beta-Prpiolactone, n.d.), and no more recent exposure estimates were found in the literature (Pubchem, n.d). It is currently FDA approved for its use as an indirect additive used in food contact substances. There has been an upsurge in the interest of using BPL to inactivate influenza and COVID19 virus in the recent years. Its use as a sterilant was at one time extended to many other products (Li et al., 2020; Izda et al., 2021; Allen and Murphy, 1960; Sasaki et al., 2016).

Chemical profile

• • • • •

Name: Betapropiolactone CAS Registry Number: 57-57-8 Synonyms: Propiolactone; 1,3-Propiolactone; 2-Oxetanone; 3-Hydroxypropionic acid lactone; 3-Propanolide Molecular Formula: C3H4O2 Chemical Structure:

Background Betapropiolactone (BPL) is a colorless liquid with a mild sweet odor. It may occur naturally, but no clear documentation of its occurrence in nature was found, and it must be synthesized for commercial purposes. BPL is unstable at room temperature but stable when stored at 5
C in glass containers. Its tendency to be unstable and react with other molecules in the vicinity is responsible for both its toxicity and its usefulness. Significant commercial production of BPL took place during the late 1950s through the mid-1970s, when it was widely used in chemical synthesis in reacting with other molecules to produce new chemicals. All lactones are characterized by a ring structure consisting of two or more carbon atoms – as can be seen from its structure, it has three in its ring – and a single oxygen, coupled with an adjacent ketone. The fewer the carbons in the ring, the more ‘strained’ is the ring structure and it becomes unstable and reactive. When the ring bonds break, the BPL molecules attach to other nearby molecules. Recent literature suggests that glutathione may serve as an efficient natural scavenger of BPL. Therefore, intracellular glutathione in the organisms may provide protection to the DNA and thus prevent BPL’s genotoxicity, mutagenicity, and possibly even carcinogenicity (Drugbank). BPL has a boiling point of 162
C with a low molecular weight (72.1). As early as the 1960s, BPL vapor was tested along with ethylene oxide for effectiveness as a sterilizing agent for influenza virus vaccines. Its use as a sterilant was at one time extended to many other products. The Hazardous Substance Database notes that BPL was first produced commercially in the United States in 1958, and the volume produced was significant through the early 1970s; for example, approximately 22 million kilograms (48.5 million pounds) were made in 1972. Production in the United States dropped to 454 kg (1000 lb) in 1975. A 2011 National Toxicology Program report stated that in 2009 beta-propiolactone was produced by one European manufacturer and was available from many suppliers. However, specific details about current production, imports, exports, or annual amounts used world-wide remains unknown. BPL has been replaced in newer chemical synthesis methods, and its use as a sterilant has been minimized over the years. Because of its carcinogenic potential and many other toxic effects, its use remains limited and risks to the general public remains insignificant (NTP Report, 2011; Fan et al., 2017; Španinger and Bren, 2020).

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Uses Once a commercially important industrial chemical in the United States, more than 85% of the beta-propiolactone produced was used to manufacture acrylic acid and esters. Preparation of commercial acrylates involved reacting beta-propiolactone with ethylene cyanohydrin. The instability of beta-propiolactone, coupled with the discovery of other more desirable chemical synthesis methods, led to its replacement by other starting materials in newer manufacturing methods. A variety of sources report that beta-propiolactone has also been used for disinfecting blood plasma, human and veterinary vaccines, tissue grafts, surgical instruments, enzymes, nutrient broth, and milk; as a vapor-phase disinfectant in enclosed spaces; and as a sporicide against vegetative bacteria, pathogenic fungi, and viruses. BPL has also been used to inactivate viruses for use in vaccines for animals and humans (Ita, 2021; Parker et al., 1975; Scheidler et al., 1998; Report on Carcinogens, Fifteenth Edition, 2021). It has also been used by shipboard military personnel as a disinfectant/decontaminant. It is sometimes also used in laboratory research. Because it is no longer used as a sterilant in medical procedures or in food, the potential for exposure of the general population is increasingly limited.

Environmental fate and behavior It is soluble in water (370 g/l−1 at 25
C) and miscible in other common organic solvents including acetone, chloroform, diethyl ether, and ethanol (Log Kow 0.462). Hydrolysis occurs in water where the half-life in aqueous media at 25
C is approximately 3.5 h. If released to soil, relatively rapid hydrolysis can be expected to occur in the presence of moisture. Significant evaporation may occur from dry surfaces. With a vapor pressure of 3.4 mmHg at 25
C, it can also vaporize into the air as temperature rises. If released to the atmosphere, BPL is expected to exist in the gas phase, where it may be relatively more persistent in the absence of moisture than it is in aqueous media. The half-life for the reaction with photochemically produced hydroxyl radicals was estimated to be a relatively slow rate of 45 days in the atmosphere.

Properties b-Propiolactone is a lactone compound with a four-membered ring. It exists as a colorless liquid with a pungent, slightly sweet odor at room temperature. It is soluble in water, miscible with alcohol, acetone, ether, and chloroform, and probably miscible with most polar organic solvents and lipids (HSDB, 2009). It is unstable at room temperature, but stable when stored at 5
C in glass containers. Propiolactone is highly protein bound and shows almost 2-fold more when compared to DNA and RNA. It is completely hydrolyzed after 3 h of being in an aqueous solution and this time can be even faster in the presence of cells or organic media. When in water, the lactone ring opens at the alkyl and acyl bonds.3 The degradation products of propiolactone are not toxic. Half-life of BPL is 24 h–30 days) C-permanent (>30 days)

Surface device

Intact skin

A B C A B C A B C A B C A B C A B C A B C A B C

Mucosal membrane

External communicating device

Breached or compromised surface Blood path, indirect Tissue/bone/dentina Circulating blood

Implant device

Tissue/bone Blood

Chronic toxicity

Carcinogenicity

Reproductive/ Developmental

Biodegradable

O O O

O

O

O

O

O

O

O

O

O

X ¼ ISO evaluation tests for consideration. O ¼ These additional evaluation tests should be addressed in the submission, either by inclusion of the testing or a rationale for its omission. a Tissue includes tissue fluids and subcutaneous spaces. From Biocompatibility. S.E. Gad. Encyclopedia of Toxicology (3rd edn.), 2014, pp 464-468, http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/ GuidanceDocuments/UCM348890.pdf.

period of time (undefined by FDA). Manufacturers of devices should choose a color additive listed for use in foods, drugs, or cosmetics as a starting point, but keep in mind that these may not be appropriate for devices. The color listing regulation may permit the use of the color additives or may place limitations on its use; PMA applicants must demonstrate their safety. Color additives listed for use in medical devices are provided in 21 CFR 73 (Color additives exempt from batch certification) and 21 CFR 74 (Color additives subject to batch certification). Other color additives require significant additional testing or data. FDA considers the addition of color, flavor, or any chemical to a medical glove to be a significant change that should have a new 510(k) submission (21 CFR 807.81(a)(3)). The applicant should provide full characterization and chemical identity of the color, flavor, or scent additives. They may submit a 510(k) submission for a modification to an existing glove as a “Special 510(k).” Color additive and flavor additive regulations are in 21 CFR parts 70 to 82 and 21 CFR part 172, Subpart F, respectively.

Nanomaterials A special concern that has arisien in the last 20 years are nanoparticles. ISO has created a specific guidance for their assessment (Freitas Jr., 2003).

Combination products A combination product is a product consisting of two or more regulated components (drug/biologic/device, etc.) that are combined as a single entity or is a product labeled for use with a separate device or biologic where both are required to achieve the intended use, indication, or effectiveness. Intercenter agreements have been made within FDA to review and oversee these categories. More information can be found at FDA website for the CBER (Center for Biologics Evaluation and Research) and CDRH (Center for

Biocompatibility

97

Devices and Radiological Health) Intercenter agreement, and the CDER (Center for Drug Evaluation and Research) and CDRH Intercenter agreement.

In vitro diagnostic (IVD) products These are medical devices that analyze human body fluids, such as blood or urine, to provide information for the diagnosis, prevention, or treatment of a disease. Classification for these devices can be found within regulations: 21 CFR 862, 21 CFR 864, and 21 CFR 866. These do not require any biocompatibility testing as they have no direct or indirect patient contact.

Radiation-emitting products Electronic product radiation means any ionizing or nonionizing electromagnetic or particulate radiation, or any sonic, infrasonic, or ultrasonic wave, that is emitted from an electronic production because of the operation of an electronic circuit in such product. If a medical device emits electronic product radiation, additional requirements apply through the Radiation Control for Health and Safety Act. Additional information concerning radiation-emitting products can be found at the FDA website.

Phthalates Within the past few decades, specific additives to some plasticizers (such as DEHP and bis-phenol-A) have been identified as toxic to animals and thus have come under scrutiny. The impact of these formulation aids to human health has not been fully characterized. Regardless, an effort is underway to reformulate device materials without them. Fibrosarcomas are another example of a biocompatibility measure that has been noted in animal and not yet determined if it will translate into humans (Gad and Gad-McDonald, 2016; Gad, 2020). Known as the Oppenheimer effect, smooth materials with a minimum surface area, and implantation time in rats produce an increased occurrence of hard, tumor like masses. The same material when implanted in a different configuration will not produce the same response. Similar to this are anaplastic large cell lymphomas associated with textured breast implants (Gad, 2020).

Latex testing: Testing for skin sensitization to chemicals The labeling may include special claims regarding reduced potential chemical sensitization in a 510(k), such as:

• •

reduced potential for sensitizing users to rubber chemical additives, or. reduced potential for causing reaction in individuals sensitized to rubber chemical additives.

The applicant should support these claims by data from human testing. Additional guidance on testing for skin sensitization to chemicals in latex products is available in the guidance document listed in the Relevant Websites section.

References Black J (2005) Biological performance of materials. In: Fundamentals of Biocompatibility, 4th edn. Boca Raton, FL: CRC Press. Chu CC, von Fraunhofer JA, and Greisler HP (1997) Wound Closure Biomaterials and Devices. Washington, DC: CRC Press. Freitas RA Jr. (2003) Nanomedicine. Biocompatibility. vol. IIA. Georgetown, TX: Landes Bioscience. FDA (2020) (ISO 10993-1) Biological Evaluation of Medical Devices - Part 1: Evaluation and Testing Within a Risk Management Process. Fries RC (1998) Medical Device Quality Assurance and Regulatory Compliance. New York: Marcel Dekker, Inc. Gad SC (2009) Safety Evaluation of Medical Devices, 3rd edn. CRC Press: Boca Raton, FL. Gad SC (2020) Integrated Safety and Risk Assessment for Medical Devices and combination Products. Berlin: Springer. Gad SC and Gad-McDonald SE (2016) Biomaterials, Medical Devices and Combination Products: Biocompatibility Testing and Safety Assessments. Boca Raton, FL: CRC Press. Greco RS (1994) Implantation Biology. Boca Raton, FL: CRC Press. Guelcher SA and Hollinger JO (2006) An Introduction to Biomaterials. Boca Raton, FL: Taylor and Francis. Heller MA (2002) Guide to Medical Device Regulation. vols. 1 & 2. Washington, DC: Thompson Publishing Company. ISO-10993-4 (2017) ISO 10993-4:2017—Biological Evaluation of medical Devices—Part 4: Selection of Tests for Interactions With Blood. Available at https://www.iso.org/standard/ 63448.html Kammula RG and Morris JM (2001) Considerations for the biocompatibility evaluation of medical devices. MDDI 23: 82–92. Murphy W, Black J, and Hastings (eds.) (2016) Handbook of Biomaterials Properties, 2nd edn. Basel: Springer. Silvio LD (2009) Cellular Response to Biomaterials. Cambridge, England: Woodhead Publishing Limited and CRC Press. von Recum AF (ed.) (1998) Handbook of Biomaterials Evaluation, 2nd edn. Ann Arbor, MI: Taylor & Francis. ISO-18562 (2017) available online at: ISO—SO 18562-4:2017—Biocompatibility Evaluation of Breathing Gas Pathways in Healthcare Applications—Part 4: Tests For Leachables in Condensate.

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Further reading ISO 10993-1 (2018) ISO 10993-1:2018—Biological Evaluation of Medical Devices—Part 1: Evaluation and Testing Within a Risk Management Process. Available at https://www.iso. org/standard/68936.html. Johanson JD (2021) In: Mahter V and Lepoittoven JP (eds.) Contact Dermatitis, 16th edn. Fsael: Springer. ODE (1995) FDA Blue Book Memo 95–1. CDRH. Rutner BD, Hoffman AC, Schoen FJ, and Lemons JE (2004) Biomaterials Science, 2nd edn. San Diego: Academic Press.

Relevant websites http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/ucm073792.htm :Premarket Notification [510(k)] Submissions for Testing for Skin Sensitization to Chemicals in Natural Rubber Products. http://www.fda.gov :US Food and Drug Administration (FDA) website. See index pages for ‘Required Biocompatibility Training and Toxicology Profiles for Evaluation of Medical Devices, Blue Book Memo, G95–1. May 1, 1995’. ‘US FDA. Special Considerations. Biocompatibility.’ More information can be found at the website for the CBER (Center for Biologics Evaluation and Research) and CDRH (Center for Devices and Radiological Health) Intercenter agreement, and the CDER (Center for Drug Evaluation and Research) and CDRH Intercenter agreement. http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/UCM348890.pdf :Use of International Standard ISO-10993, “Biological Evaluation of Medical Devices Part 1: Evaluation and Testing” Draft Guidance for Industry and Food and Drug Administration Staff Document issued on: April 23, 2013. http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/UCM348890.pdf :Food and Drug Administration.

Biofuels Linda G Robertsa and Thomas Smagalab, aNapaTox Consulting LLC, Napa, CA, United States; bChevron Technical Center, San Ramon, CA, United States © 2024 Elsevier Inc. All rights reserved. This is an update of L.G. Roberts, T.J. Patterson, Biofuels, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 469-475, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01054-X.

Chemical profile Introduction and general background for major types of biofuels Uses of biofuels Biodiesel Synonyms CAS numbers Molecular formula Chemical structure Background Uses/occurrence Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Chronic toxicity Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Toxicogenomics Clinical management Environmental fate and behavior Ecotoxicity Exposure standards and guidelines Other PubChem URLs Hydrotreated renewable fuels Synonyms CAS numbers Molecular formula Chemical structure Background Uses/occurrence Exposure Toxicokinetics Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Chronic toxicity Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Toxicogenomics Clinical management Environmental fate and behavior

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Ecotoxicity Exposure standards and guidelines Other PubChem URLs Conclusion References Further reading

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Abstract Biofuels are produced from biomass-based renewable resources including plants, animal by-products, or microorganisms. Bio-derived fuels include alcohols, oils reacted to form fatty acid methyl esters or saturated hydrocarbons, and paraffinic hydrocarbons synthesized from gas or alcohol. Materials in current use have very low aromatics. Final products must meet engine performance requirements for gasoline (naphtha range), jet fuel (kerosene range), or diesel (gas oil range) engines. Naphtha materials are more readily inhaled and may produce central nervous system (CNS) effects; all may pose an aspiration risk if ingested, may cause skin defatting or irritation. Naphtha materials should biodegrade in the environment.

Keywords Biodiesel; Biofuels; Biomass-to-liquid (BTL); Combustion exhaust emissions; Fatty acid methyl esters; Fischer-Tropsch (F-T); Gas oil; Gas-to-liquid (GTL); Hydrotreated renewable fuels; Naphtha; Renewable diesel; Renewable hydrocarbon fuels; Synthetic paraffinic kerosene

Key points

• • •

Biofuels are fuels produced from biomass-based renewable resources such as plants, animal, or microbial matter. Biofuels must be compatible with engine performance requirements, similar to conventional petroleum fuels. Similar to petroleum fuels, both the liquid transportation biofuel and the combustion exhaust emissions should be considered for effects to health and the environment, and a key consideration may be toxicity relative to conventional fuel counterparts.

Chemical profile Varied. Biofuels are synthesized to meet engine performance requirements; chemical profiles may vary according to feedstock and manufacturing procedures.

• • •

Name: Biofuels CAS numbers: Multiple Chemical structures: Varied

Introduction and general background for major types of biofuels Biofuels are transportation fuels produced from biomass-based renewable resources such as plants, animal by-products, or microorganisms, although peanut oil was first used as a diesel fuel approximately a century ago. Currently, these fuels are usually blended with petroleum fuels, but in some circumstances, they may be used on their own. In order to function in an engine, a biofuel must be compatible with the performance requirements of existing engine technology, which as of today requires compatibility with conventional petroleum fuels. Thus, for many biofuels, there may be considerable physicochemical overlap between the characteristic properties of a conventional petroleum-derived fuel and its biofuel counterpart. Biofuels may be generally grouped into the following categories: (1) those produced by fermentation of biomass into alcohols; (2) oils separated from biomass and reacted to form fatty acid methyl esters (FAMEs); (3) saturated hydrocarbons produced from a biogenic gas such as methane and synthesized to form a renewable hydrocarbon fuel in the naphtha, kerosene, or gas oil (diesel) range; (4) oils separated from biomass and hydrogenated to produce hydrocarbons, primarily non-aromatic, within the carbon range and performance characteristics of conventional petroleum fuels. With the exception of individual alcohols such as ethanol or butanol, and biomass-derived individual hydrocarbons such as 2,6,10-trimethyldodecane (farnesane) (Alleman et al., 2020), biofuels in commercial use are substances of variable composition (UVCB: Unknown or Variable composition, Complex reaction products or Biological materials), similar to their conventional gasoline, jet/aviation fuels, and diesel fuel counterparts.

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One of the most common biofuels currently in widespread use is ethanol. Ethanol is typically derived from corn or sugar cane. Ethanol is a separate entry in the encyclopedia; readers are recommended to review the toxicity of ethanol there. Descriptions of toxicology programs to assess the health effects of gasoline:ethanol blends may be found in this chapter’s Further Reading (Smith et al., 2017; Bushnell et al., 2015) and Useful Websites. It is important to understand that the term “biofuels” may have varying definitions across different agencies and resources, may be applied to fuels developed for a wide range of engine performance requirements, and also that the biomass feedstock as well as the production processes will impact the composition of the final fuel product. Thus, assessing the potential toxicity of “biofuels” is a broad topic. Biofuel technology is also an innovative field, as both engine design (and thus engine requirements) and biofuel production methods continue to evolve. This encyclopedia entry will focus on biofuels with the greatest current commercial viability: biodiesel and hydrotreated renewable fuels. As is true for petroleum derived conventional fuels, the physical and chemical properties of biofuels may provide insight into environmental behavior, exposure concerns, and health effects. Similar to conventional gasoline, for example, the chemical constituents of highly volatile biofuels are expected to partition into air from water, soil surfaces, and open containers, and branched hydrocarbon structures may be more difficult to biodegrade by microorganisms. For volatile constituents, the potential exposure through inhalation will be greater than for less volatile, longer-chain, oily constituents which are more likely to have longer skin contact and for which dermal absorption may be of primary interest. Also, as with conventional petroleum fuels, concerns arise for both the potential effects to health and the environment of the evaporative emissions and the liquid fuel, as well as from effects of the combustion exhaust emissions.

Uses of biofuels The major current uses of biofuels are as liquid transportation fuels, although use as a heating fuel may also occur. An essential consideration for use is compatibility with existing technology for engine design, fuel performance and handling requirements. Biodiesel is compatible with existing diesel (compression ignition) technology and has come into common use as a blend in petroleum-derived diesel. The application of hydrotreated renewable fuels is dependent on the carbon range for the renewable fuel; for example, hydrotreated renewable fuel comprised of hydrocarbons in the range of approximately 10 to 22 carbons in length (C10 - C22) could be compatible with diesel engines (Chevron, 2007).

Biodiesel Synonyms Fatty acid methyl esters (FAMEs); fatty acids, vegetable-oil, Me esters. Examples of additional feedstock-based synonyms include soybean oil methyl ester, tallow methyl ester, waste or used cooking oil methyl ester.

CAS numbers There are multiple biodiesel CAS numbers. These are frequently identified by feedstock. Several are shown below. CAS 68990-52-3: vegetable fatty acid methyl esters CAS 67762-38-3: fatty acids, C16-18 and C18-unsatd., methyl esters CAS 67784-80-9: soybean methyl esters (SME) CAS 73891-99-3: rapeseed oil methyl esters (RME) CAS 61788-61-2: tallow methyl esters (TME) CAS 91051-34-2: palm methyl esters (PME)

Molecular formula Variable, dependent upon the biomass feedstock used for production. The molecular formula for a methyl ester is RCO2CH3, where R represents a fatty acid.

Chemical structure Multiple fatty acid methyl ester structures, dependent upon the biomass feedstock used for production.

Background Biodiesel is fuel composed of mono-alkyl esters of long chain fatty acids derived from biomass-based lipids such as vegetable oils or animal fats. It is typically produced by a base- catalyzed transesterification between the lipid and an alcohol, usually methanol, to produce FAMEs. The carbon range distribution will mirror the carbon lengths of the fatty acids in the feedstock. FAMEs are described

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by their carbon length and number of unsaturated bonds. Feedstock composition, both of plant and animal origin, may vary quantitatively but are qualitatively consistent; the primary fatty acids for several common feedstocks are illustrated below (Knothe et al., 2010):

• • •

Soybean oil: 2.3–13.3% C16:0; 17.7–30.8% C18:1; 49–57.1% C18:2 Palm oil: 44.1% C16:0; 39% C18:1; 10..6% C18:2 Tallow (beef ): 29.5% C16:0, 26% C18:0; 34.9% C18:1

Fatty acid composition will influence fuel performance. As an example, unsaturated bonds improve cold flow properties, enabling use in cold weather, but increase susceptibility to oxidation. When blended with conventional petroleum-derived diesel (frequently referred herein as “diesel”), the proportion of the blend that is biodiesel is indicated by a number following “B;” i.e., B20 is a 20:80 biodiesel:diesel fuel blend. The exhaust emissions from combustion of biodiesel or biodiesel:diesel blended fuels differ from the combustion of petroleum diesel alone (Larcombe et al., 2015; Bünger et al., 2012). The higher oxygen content of biodiesel can lead to more complete combustion of the fuel, reducing the emission of carbon monoxide, unburned hydrocarbons, aromatic and sulfur-containing compounds, and particulate matter (PM). Biodiesel emissions, though, may have higher levels of nitric oxides and ultrafine PM (PM0.1) materials, as well as aldehydes, acrolein, and acetone (Bünger et al., 2012; Selley et al., 2019). Studies that attempt to compare the toxicity of exhaust emissions may differ in their findings due to variations in engine technology and the engine load used for running the test (Bünger et al., 2012). It may also be due to changes to conventional diesel fuel such as the reduction of sulfur content that has occurred over time, and the feedstock used to manufacture biodiesel, for example rapeseed and soy (Larcombe et al., 2015; Landwehr et al., 2019). These differences can impact on the physical and chemical parameters of the emissions (Selley et al., 2019). Biodiesel exhaust, for example, is reported to have reduced overall emissions of particulate matter, but a greater proportion of the ultra-fine particles (PM0.1) likely to reach deep lung tissue. Although dose metrics in health studies have more generally been based upon the mass of PM of respirable size, usually PM0.1 or PM2.5, particle surface area has been reported to be the more useful metric for dose-response assessment (Stoeger et al., 2006; Sager and Castranova, 2009).

Uses/occurrence The primary use of biodiesel is as a transportation fuel or fuel blend. FAMEs are also used in washing and cleaning products, cosmetics, and personal care and hair care products (ECHA REACH dossier, 2021a, b). Biodiesel is listed by US EPA on the National Contingency Plan for use in oil spill environmental cleanup.

Exposure and exposure monitoring Due to the low volatility of biodiesel, inhalation exposure is considered a minimal pathway; occupational monitoring may be unnecessary unless oil mists are generated. Organic vapor respirators are recommended or required to prevent potential inhalation of mists that might result in chemical pneumonitis (US EPA, 2007). Skin contact may result in penetration of the stratum corneum (Wertz and Downing, 1990). Biodiesel combustion products include respirable ultra-fine particulates (PM0.1), nitrogen oxides, carbon monoxides, and hydrocarbons including aldehydes and polycyclic aromatic compounds (PAHs).

Toxicokinetics Ingested FAMEs are hydrolyzed to their free fatty acids which can subsequently be absorbed from the intestine and metabolized in the liver, similarly to dietary fatty acids. Further oxidation occurs to form carbon dioxide and water via breakdown into 2-carbon fragments used by the body for energy and incorporation into tissues (Mattson and Volpenhein, 1972). Fatty acids may be absorbed from the intestine and stored in fat deposits in the body. 14C-palmitate methyl ester, a major constituent of FAMEs derived from animal fats, palm and coconut oils, is distributed to tissue lipids in rats, with lower retention in younger rats that have lower body fat than mature adults. Topically applied FAME can penetrate epidermis and enter into epidermal lipid metabolism (Wertz and Downing, 1990).

Mechanism of toxicity Biodiesel has limited toxicity. Some fatty acids are dietary requirements; however, diets consisting solely of nonessential fatty acids are reported to lead to symptoms of fat deficiency in rats. Ingestion of high levels of nonessential fatty acids appears to interfere with the

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absorption and/or incorporation of essential fatty acids into lipid tissues and produce symptoms of fatty acid deficiency (Alfin-Slater et al., 1965). The oily nature of biodiesel can be a physical fouling hazard to aquatic invertebrates and waterfowl if biodiesel is spilled into wetlands. Mechanisms of toxicity by exhaust emissions have been actively studied due to the known concerns of mutagenicity, carcinogenicity, and respiratory toxicity of conventional petroleum diesel exhaust products and the public health impact of air pollution from mobile sources. Biodiesel exhaust emissions can induce responses similar to emissions from diesel exhaust. Repeated exposure to exhaust emissions produced oxidative stress and inflammation in mice, with increased concentrations of macrophages found in lungs, and increased expression of TNF-a, NFKB, Nrf2, and HO-1 proteins (Selley et al., 2019). Dose-responsive increases occurred in pulmonary macrophages and evidence of inflammatory pulmonary lesions in the alveoli. PM from various fuels have been shown to induce airway inflammation through activation of toll-like receptors (TLR), cytokine secretion, and by activating cytosolic aryl hydrocarbon receptors (AhRs), contributing to cytotoxicity and mutagenicity (Selley et al., 2019; Hawley et al., 2014). Human pneumocytes were used to study the role of transient receptor potential ankyrin 1 (TRPA1) ion channel, involved in calcium homeostasis and cytokine secretion, to inflammatory responses to biodiesel exhaust. IL-8 secretion was decreased when a TRPA1 antagonist was present in the assay, suggesting a role for TRPA1 in the inflammatory cascade (Jaramillo et al., 2017). In THP-monocyte-derived macrophages, a human leukemia monocytic cell line, particulate matter from a soy methyl ester B20 blend exhaust induced greater toxicity than particulate matter from the combustion of petroleum fuel based upon quantification of reactive oxygen species (Fukagawa et al., 2013). Combustion products of biodiesel can up regulate CYP1A1 expression in human bronchial epithelial cells, similar to diesel combustion exposure (Hawley et al., 2014).

In vitro toxicity data In vitro assays have been used to assess the potential of biodiesel fuel to produce genetic toxicity. No increase in chromosomal aberrations or mutations in mammalian cells or mutations in bacteria were observed following exposure to the biodiesel fuel itself (NTP, 2021a, 2021b). Additional in vitro assessments have been performed with the exhaust emissions of biodiesel or with extracts of exhaust emissions. The majority of this research has focused upon the potential for mutagenicity, cytotoxicity, and pro-inflammatory responses, particularly in pulmonary cells. These studies have generally shown that, relative to petroleum diesel exhaust, mutagenicity is reduced but cytotoxicity and inflammatory markers may be increased (Møller et al., 2020). Biodiesel exhaust extracts produced cell death and release of inflammatory markers in primary human epithelial cells obtained from young children; the exhaust from combustion of waste cooking oil, canola, and tallow FAMEs produced the strongest effects (Landwehr et al., 2019).

Acute and short-term toxicity Existing data indicate that biodiesel produced from many different feedstocks is not hazardous (ECHA REACH dossier, 2021a, b; US EPA, 2007). Soy, sunflower, and canola methyl esters are classified as nonhazardous (acute oral LD50 > 5 g kg−1), similar to petroleum diesel. Oral toxicity testing of individual FAMEs (laureate, C12:0; palmitate, C16:0; stearate, C18:0) also support the conclusion that acute exposure is not hazardous. Dermal exposure is similarly considered nonhazardous: soy methyl ester, rapeseed methyl ester, and butyl methyl ester were each not hazardous (acute dermal LD50 > 2 or 5 g kg−1). Essentially no vapors are generated from biodiesel at ambient temperatures, and therefore neat (pure) biodiesel should not pose a health hazard from inhalation unless used in a manner that produces significant respirable mist. The low viscosity of biodiesel provides the potential for the fuel to enter the respiratory tract and may result in a chemical pneumonitis, a potentially fatal condition (Craan, 1996). Test results with FAMEs suggest that biodiesel is only slightly irritating to eyes or skin (US EPA, 2007). Slight, temporary conjunctivitis and chemosis were the only findings in eye irritancy testing, and slight redness to the skin also resolved within one or 2 days (ECHA REACH dossier, 2021a, b). Although several individual FAMEs such as methyl laurate, methyl palmitate, and methyl stearate produced irritation on rabbit skin, human volunteers exposed to FAMEs of various carbon lengths (C8-10, C12-14, C16-18) displayed virtually no irritation. Repeated exposure could potentially cause skin defatting. Biodiesel is unlikely to cause allergic skin reactions; no skin sensitization occurred in guinea pig testing (ECHA REACH dossier, 2021a, b). Tallow methyl ester and methyl laurate were negative in guinea pig sensitization studies. Soy methyl ester was reported as positive in a guinea pig skin sensitization study; however, the erythema observed in this study at 24 h post-challenge exposure nearly dissipated by 48 h, which is atypical for an allergic reaction but common for mild irritation. Soy as a food product can produce allergic reactions in sensitive individuals. In small-scale human testing (n ¼ 25–68 subjects), neither soy methyl ester nor palm methyl ester produced allergic reactions; thus, there is generally no classification for skin sensitization for biodiesel. Subchronic toxicity testing suggests minimal health effects from FAMEs in biodiesel. No effects to general health, blood chemistry, organ weights or histopathology were produced in an oral subchronic toxicity screening study conducted with C16-18 and C18 unsaturated fatty acid methyl esters (ECHA REACH Dossier, 2021a). Four-week ingestion of canola, soy, or fish oil methyl

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esters at doses up to 500 mg kg−1 d−1 produced liver hypertrophy but few other changes (reduced thymus weight from soy methyl ester). Four-week ingestion of FAMEs derived from soy, canola, or animal frying oil decreased serum free fatty acids and increased hepatic acyl-CoA oxidase activity (Poon et al., 2009). A longer, 12-week feeding study in rats of methyl oleate, with dose levels of up to 3500 mg kg−1 d−1, produced only a reduction in female body weight. Male rats fed 100 mg d−1 per animal, equivalent to approximately 300–500 mg kg−1 d−1, for 12 weeks exhibited decreased body weight while females were virtually unaffected. Biodiesel prepared from Jatropha seed oil produced no effects in rats orally dosed for 30 days at up to 2000 mg kg−1 d−1 (Siriarchavatana et al., 2010). Methyl esters of fatty acids produced from edible fats and oils are approved for some uses as direct food additives or as a supplemental source of fat for animal feed (21CFR172.225, 1992; 21CFR573.640, 2008). Several of the fatty acids within the approved FAMEs are essential fatty acids. The ingestion of nonessential fatty acids appears to interfere with the absorption or incorporation of essential fatty acids into lipid tissues to produce symptoms of fatty acid deficiency (Alfin-Slater et al., 1965). The possibility of fatty acid deficiency occurring through occupational exposure or even accidental exposure should be extremely unlikely, given common dietary ingestion of fatty acids. Exhaust emissions produced from an engine running on soy methyl ester biodiesel did not produce systemic toxicity in F344 rats (Finch et al., 2002). Exposure levels were approximately 0.04, 0.2, and 0.5 mg particulates per cubic meter. The study was conducted as part of a Tier 2 testing program under the Clean Air Act. Biologically significant effects were limited to high-exposure female rats and consisted of increased lung weight, increased alveolar macrophage content, and evidence of multifocal bronchiolar metaplasia. The increases in lung weight and alveolar macrophage content were considered to be normal responses to repeated inhalation of particulate matter. The authors reported no effects upon other organ systems, including bone marrow micronuclei, fertility, or prenatal development. Impaired clearance of PM, resulting in longer retention of particulates, was observed in the lungs of mice exposed to biodiesel exhaust (Yanamala et al., 2013). Up-regulation of biomarkers of inflammation, such as cytokines, chemokines, and growth factors, occurred in the lungs of mice exposed to either biodiesel or petroleum diesel exhaust particulate matter; the response was greater following exposure to biodiesel. Relative to petroleum diesel exhaust exposure, markers of tissue damage in the lung (lactate dehydrogenase) and inflammation in the lung and liver (myeloperoxidase) were more pronounced in response to 4-week exposure to biodiesel exhaust, with a reduction of glutathione. Both exposures increased concentrations of inflammatory cytokines IL-6, IL-10, IL-12p70, IFN, TNF-a, and MCP-1 (Shvedova et al., 2013). The authors concluded that tissue damage, oxidative stress, inflammation, and cytokine responses were more pronounced in mice exposed to biodiesel exhaust than petroleum diesel exhaust.

Chronic toxicity No long-term testing with biodiesel has been conducted, possibly due to the limited effects observed in subchronic laboratory studies. Methyl esters of higher fatty acids, including methyl esters of myristate (C14:0), palmitate (C16:0), stearate (C18:0), oleate (C18:1) and linoleate (C18:2) are accepted by the US Food and Drug Administration as a supplementary source of fat for animal feed under 21CFR573.640 (2008).

Immunotoxicity No immunotoxicity tests were found for biodiesel fuel. Biodiesel and blended fuel exhaust emissions have been tested in vivo and in vitro for comparison of inflammatory responses to those produced by petroleum diesel emissions. Results in most studies have shown more pronounced inflammatory responses from exposure to biodiesel exhaust than from exposure to petroleum diesel, although this is not consistently observed. Biodiesel exhaust induced greater production of pro-inflammatory responses both in vivo and in vitro than diesel exhaust emissions; concentrations of inflammatory mediators IL-6, IP-10, G-CSF were higher in human cell lines for bronchial epithelial cells and macrophages as well as bronchoalveolar lavage fluid of mice exposed to B20 than in those exposed solely to petroleum diesel (Fukagawa et al., 2013). Organic extracts of soy methyl ester biodiesel particulate matter increased the release of pro-inflammatory cytokines, IL-8 and IL-6, by respiratory epithelial cells; the biodiesel particulate matter elicited a greater response than petroleum diesel particulate matter (Swanson et al., 2009). In contrast, exposure to exhaust (PM2.5) from soy methyl ester biodiesel was compared to that of a B20 blend and to petroleum diesel, with the biodiesel and blended exhaust emission producing lesser inflammatory responses in mice than that of petroleum diesel (Gavett et al., 2015).

Reproductive and developmental toxicity Limited information is available for the assessment of biodiesel fuel effects upon prenatal development or reproduction. C16-18 and C18 unsaturated methyl esters produced no harm to fertility, reproductive endpoints, or offspring size or survival in a combined subchronic/reproductive toxicity screen in rats (ECHA REACH dossier, 2021a); the no adverse effect level for general, reproductive, and offspring toxicity was 1000 mg kg−1 d−1. A wider carbon range of FAMEs, C6-24 and C6-24 unsaturated, was

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tested in rats for potential to harm prenatal development or maternal health during gestation (ECHA REACH dossier, 2021b). Exposure by oral gavage daily, gestation day 5–19, produced no findings of maternal or developmental toxicity; the no adverse effect level was 1000 mg kg−1 d−1. A 12-week feeding study in rats administered methyl oleate, 100 mg kg−1 d−1, produced no effects to reproductive organs. Several oils used as biodiesel feedstocks have been tested for reproductive or developmental toxicity potential. High dietary levels of vegetable and animal fats, 20% in ad libitum feed versus a 5% fat control diet, resulted in earlier vaginal opening in weaning rats, but no differences in basal or surge levels of prolactin, luteinizing hormone, or estradiol as adults (Sylvester et al., 1986). Biodiesel exhaust (B100 soy-derived, 0.5 mg particulates m−3 d−1) did not cause developmental toxicity in rats (Finch et al., 2002). Prenatal mortality, fetal body weight, and the incidence of malformations were unaffected.

Genotoxicity Genetic toxicity studies indicate that biodiesel fuel is not genotoxic, although the exhaust emissions from combustion of biodiesel may be mutagenic. Methyl soyate was negative in the Ames bacterial reverse mutation assay, with and without metabolic activation (NTP, 2021a), as well as negative for clastogenicity in an in vivo mouse bone marrow study (NTP, 2021b). FAMEs ranging from C12 to C19, both saturated and unsaturated, were not clastogenic in the mouse bone marrow chromosome aberration test (Renner, 1986); longer chain FAMEs decreased the chromosome breakage effects of busulfan, i.e. were anti-clastogenic. There are numerous mutagenicity assessments of biodiesel exhaust, many comparing the mutagenicity of the exhaust from biodiesel with that from petroleum diesel. Although there are inconsistencies regarding the mutagenic potency of biodiesel exhaust relative to the exhaust of diesel, many of these studies demonstrate some degree of mutagenicity, most often associated with the particulate fraction (Selley et al., 2019). Mutagenic activity in bacterial assays appears to be associated with the concentration of polycyclic aromatic compounds in the exhaust; the chromatographic profiles of DNA adducts formed from biodiesel or diesel exhaust were consistent with those found from PAH and nitro-PAH (Ross et al., 2015). The soluble organic fraction of exhaust from combustion of rapeseed methyl ester biodiesel was found to be mutagenic in the Ames assay with TA98 and TA100, but less potent for mutagenicity than the exhaust from petroleum diesel combustion, which the authors hypothesized to be due to much lower emissions of polycyclic aromatic compounds. The soluble organic fraction of rapeseed methyl ester biodiesel exhaust from an idling engine was more cytotoxic than petroleum diesel exhaust in L929 mouse fibroblasts, but in contrast not when the engine test was run at rated power load (Bünger et al., 2000). The frequency of micronucleus formation in human bronchial epithelial BEAS-2B cells was similar for organic extracts of exhaust particle emissions from rapeseed methyl ester biodiesel, petroleum diesel, and a B30 blend (Cervena et al., 2017). Not all results indicate that biodiesel exhaust is genotoxic. In vivo exposure to exhaust emissions from soy methyl ester biodiesel were reported to be negative for bone marrow micronuclei and sister chromatid exchange in peripheral blood lymphocytes of F344 rats (Finch et al., 2002). Subchronic exposure of F344 rats to exhaust emissions from a rapeseed methyl ester B20 blend combusted in a modern Euro 5 light diesel vehicle for one or 4 weeks produced no indications of genotoxicity in the Comet assay (Magnusson et al., 2017).

Carcinogenicity Limited testing information is available on the potential carcinogenicity of biodiesel fuels. Methyl oleate did not increase the number of tumor-bearing mice in a two-year initiation promotion study, although the total number of forestomach papillomas increased (ECHA REACH dossier, 2021a). Methyl stearate was not carcinogenic after subcutaneous exposure for 28 weeks with treatment groups followed post-exposure for a total study period of 2 years (Swern et al., 1970). Methyl linoleate, by gavage, did not enhance the carcinogenicity of N-methyl-N-nitro-N-nitrosoguanidine in rats (Arffman et al., 1981).

Organ toxicity The minimal effects observed in repeated exposure testing with biodiesel fuels suggest that biodiesel does not produce target organ effects.

Interactions Fatty acid metabolites from biodiesel may be utilized as nutrition. Essential fatty acids, such as linoleic acid (C18:2) and a-linolenic acid (C18:3), are those that the body cannot synthesize. Diets consisting solely of saturated fatty acids have been reported to lead to symptoms of fatty acid deficiency in rats, potentially through competition with essential fatty acids in metabolic processes

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(Smith and DeLuca, 1963). Such effects should be extremely unlikely through use or even accidental exposures. Interpretation of toxicological findings for studies that utilize prolonged high-dosage biodiesel FAME exposure should consider nutritional interactions as a possible confounding factor in test results.

Toxicogenomics A genome-wide transcriptomic comparative analysis of changes induced from the organic fractions of exhaust particles from rapeseed methyl ester found dysregulated genes in the bronchial epithelial cells of the assay. The altered genes are associated with antioxidant response, cell cycle regulation and p38 signaling (Selley et al., 2019).

Clinical management No clinical symptoms are predicted for acute exposure to biodiesel. Contaminated skin should be cleaned by thorough washing with soap and water, and contaminated clothing should be laundered. Exposed eyes should be flushed thoroughly with water or saline (ECHA REACH dossier, 2021a, b).

Environmental fate and behavior Biodiesel is predicted to partition primarily to sediment, approximately 86%, and to a lesser extent to water, approximately 11.5%, with minor amounts to soil and air (ECHA REACH dossier, 2021a, b). Shorter chain FAMEs are expected to be more mobile in sediment than longer chain-length FAMEs. Biodiesel is neither persistent nor bioaccumulative in the environment (OECD, 2021; ECHA REACH dossier, 2021a, b). Approximately 75–85% should degrade within 28 days. The faster biodegradation of biodiesel relative to petroleum diesel may result in less environmental migration than petroleum diesel; preferential biodegradation of biodiesel may result in delayed biodegradation of hydrocarbons from petroleum contamination. Biodiesel degradation by microbiota in sediment and soil produces free fatty acids, hydrocarbons, and methanol and can increase soil acidity; thus, biodegradability may not indicate an absence of environmental harm (Cruz et al., 2020). Biodiesel is lighter than water. If spilled onto waterways, biodiesel may produce an oily sheen on the water surface. The half-life of biodiesel in freshwater is approximately 6–7 days (ECHA REACH dossier, 2021a). Biodiesel is not predicted to bioaccumulate in tissues based upon modeling (ECHA REACH dossier, 2021a, b). It is metabolized and excreted if uptake occurs.

Ecotoxicity Biodiesel and individual FAMEs have demonstrated negligible toxicity to aquatic species (microbes, invertebrates, fish), with LC50 and EC50 values >1000 mg L−1 (ECHA REACH dossier, 2021a, b). FAMEs have low water solubility and should be tested for aquatic toxicity as a water-accommodated fraction (WAF); i.e., the biodiesel is blended into the aqueous medium to reach equilibrium, allowed to settle, and the aqueous phase is siphoned off for introduction into the test system. The liquid itself, as with petroleum diesel, may coat animals and plants and produce physical fouling. Biodiesel fuel tested in this way caused increasing immobility in water fleas over time, but was less toxic than petroleum diesel (Bamgbose and Anderson, 2018). Long-term exposure of the water-soluble fractions of FAMEs derived from castor oil, waste cooking oil, and palm oil were found to be toxic to marine micro algae and sea urchins; methanol, a metabolite, was the most prominent contaminant in samples stored for 120 days (Leite et al., 2010). Biodiesel may be toxic to plants at high contamination levels in the range of 1.76% (Bamgbose and Anderson, 2015). FAMEs derived from safflower and castor oils inhibited germination and survival of lettuce, alfalfa, wheatgrass, and radish, although biodiesel was less toxic than petroleum diesel fuel.

Exposure standards and guidelines The Michigan Department of Environmental Quality set an initial threshold screening level for methyl soyate of 16 mg m−3 (annual averaging time) (Michigan, 2014). Exposure standards for vegetable oil mist of 10 mg m−3 (total), 5 mg m−3 (respirable) (time weighted average) has been set by NIOSH and 15 mg m−3 (total), 5 mg m−3 (respirable) by US OSHA (US NIOSH, 2019).

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Other Biodiesel-soaked materials can spontaneously combust due to heat generated during degradation and should not be stored near combustible materials. Fuel containers can accumulate static charge if not properly grounded.

PubChem URLs CAS 68990-52-3: https://pubchem.ncbi.nlm.nih.gov/#query¼68990-52-3 CAS 85480-42-8: https://pubchem.ncbi.nlm.nih.gov/#query¼85480-42-8 CAS 67784-80-9: https://pubchem.ncbi.nlm.nih.gov/#query¼67784-80-9 CAS 68910-48-5: https://pubchem.ncbi.nlm.nih.gov/#query¼68910-48-5 CAS 73891-99-3: https://pubchem.ncbi.nlm.nih.gov/#query¼73891-99-3 CAS 61788-61-2: https://pubchem.ncbi.nlm.nih.gov/#query¼61788-61-2 CAS 91051-34-2: https://pubchem.ncbi.nlm.nih.gov/#query¼91051-34-2 CAS 67762-26-9: https://pubchem.ncbi.nlm.nih.gov/#query¼67762-26-9 CAS 67762-38-3: https://pubchem.ncbi.nlm.nih.gov/#query¼67762-38-3

Hydrotreated renewable fuels Synonyms For hydrotreated renewable fuels, synonyms may refer to materials with similar but not identical chemical composition due to differences in feedstock and synthesis processes. Hydroprocessed renewable jet fuel Bio-SPK (synthetic paraffin kerosene) Hydroprocessed esters and fatty acids (HEFA) Biomass-to-Liquid (BTL), Gas-to-Liquid (GTL) Naphtha (Fischer-Tropsch), light, C4-10-branched and linear Fischer-Tropsch kerosene (C8-16 branched and linear hydrocarbons (full range) - kerosine) Fischer-Tropsch Diesel (Distillates (Fischer-Tropsch), C8-26-branched and linear) Renewable Diesel: Alkanes, C10-20 -branched and linear Renewable hydrocarbons (diesel type fraction)

CAS numbers CAS numbers are provided as found and may be specific to a proprietary material. Renewable Diesel: alkanes, C10-20 -branched and linear: 928771-01-1 Naphtha (Fischer-Tropsch), light, C4-10-branched and linear: 848301-65-5 Fischer-Tropsch kerosene (C8-16 branched and linear hydrocarbons (full range) - kerosine): 848301-66-6 Fischer-Tropsch diesel (distillates (Fischer-Tropsch), C8-26-branched and linear): 848301-67-7 Renewable hydrocarbons (diesel type fraction): EC number 700-916-7

Molecular formula Variable, dependent upon the feedstock, manufacturing process, and final product carbon range. Renewable hydrocarbon fuels are saturated hydrocarbons composed primarily of linear alkanes of short carbon chain length in the naphtha range with increasing proportions of branched constituents as carbon length increases, containing low or no aromatic compounds.

Chemical structure Unspecified. Renewable fuels are substances of Unknown or Variable composition, Complex reaction products or Biological materials (UVCBs).

Background Hydrotreated renewable fuels are produced by processing biomass feedstocks such as animal fats, plant-based (e.g., vegetable, camelina) oils, or microbial (e.g., algae) oils with hydrogen and deoxygenation. In general, hydroprocessing produces hydrocarbons

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that are predominantly paraffinic alkanes, normal, branched or cyclic, with minimal aromatic content. Fuels in various carbon ranges, such as short hydrocarbons for blending with gasoline, longer hydrocarbons for blending with petroleum diesel, and carbons of intermediate length for blending with jet fuel or kerosene, can be separated by distillation, as boiling point increases with increasing carbon length. Biofuels, like conventional fuels, must meet strict performance specifications. Hydrotreated renewable fuels are compositionally similar to synthetic Fischer-Tropsch (F-T) fuels (Smagala et al., 2013). For biofuels synthesized through the F-T process, the carbon source, methane, is derived from the breakdown of biomass. The saturated hydrocarbons produced by this process have the formula CnH(2n+2), and are primarily linear alkanes in the naphtha range and increasingly branched (mostly methyl groups) with increasing carbon number. Because of the similarity of the composition of fuels produced by the F-T process whether the carbon source is biomass-derived methane or natural gas, some toxicity estimations below are based on F-T fuels data irrespective of the carbon source. Note that most synthetic fuels are made from either natural gas or coal, but they can be derived from any carbon source, including biomass (Pfleger, 2016). Thus, a fuel made by the F-T process from biomass would be both synthetic and renewable. The most common hydrotreated renewable fuel in the market today is renewable diesel (Smagala et al., 2013). Alcohol-to-jet (ATJ) fuels are produced through dehydration and oligomerization processes using ethanol or butanol feedstocks from sugars or lignocellulosic biomass (Sterner et al., 2016). The composition of ATJ fuels may be highly aliphatic or contain a percentage of aromatics approximately 5–20%, similar to that of petroleum jet fuel. The reliance upon renewable hydrocarbon fuels to meet performance specifications in combustion engines means that there is considerable overlap in composition with conventional petroleum fuels, with the exception that renewable hydrocarbon fuels contain very low to no aromatic constituents. Some toxicity effects in petroleum products have been attributed to the aromatic constituents, such as leukemia (benzene) and prenatal harm (polycyclic aromatic compounds). With significantly lower or negligible levels of aromatic constituents, renewable hydrocarbon fuels generally have less toxicity and fewer required classifications for hazardous effects.

Uses/occurrence Renewable fuels are predominantly used for transportation and may be blended with conventional petroleum fuels.

Exposure Exposure may occur in occupational settings through handling, quality control analysis, transportation, blending with petroleum fuel, or fueling operations. Light end, volatile hydrocarbon constituents within the gasoline and jet fuel carbon ranges present a potential for exposure by inhalation or through skin. The low volatility of renewable hydrocarbons in the diesel range indicate that inhalation should be a minimal exposure pathway unless oil mists are generated. Organic vapor respirators may be recommended or required to prevent potential inhalation of mists that might result in chemical pneumonitis. Consumer exposure may occur when vehicles are fueled or maintained. Exposure to exhaust emissions may occur to the public as currently occurs through the use of conventional petroleum fuels for transportation.

Toxicokinetics Absorption from the gastrointestinal tract is expected to be greater for the hydrocarbons of lower molecular weight than for larger hydrocarbons. For example, the absorption of a 14-carbon hydrocarbon is expected to be as much as 60%, while a hydrocarbon of twice this size would have very low absorption (Nordt, 2009). Volatile hydrocarbons are expected to be readily inhaled.

Mechanism of toxicity The skin irritation that may occur following repeated exposure to renewable hydrotreated fuels is likely due to defatting of the skin. The lipophilic nature of the hydrocarbons in these fuels endows them with the ability to dissolve dermal lipids. Conventional petroleum fuels pose a similar hazard to the skin. The low viscosity of renewable hydrocarbon fuels poses a risk of aspiration into the lungs if ingested, which can induce a potentially fatal chemical pneumonitis (Craan, 1996). This risk is also shared with petroleum fuels. Hydrotreated renewable fuels, as with a number of petroleum hydrocarbons, can produce kidney effects in male rats due to accumulation of a-2 m-globulin. Increased eosinophilic material in renal tubular epithelium was observed microscopically following 10 weeks of oral exposure. Additional microscopic renal findings noted for petroleum hydrocarbons include hyaline droplet accumulation, granular casts, corticotubular basophilia, and degeneration with regeneration of tubular epithelium. These lesions, characterized as hyaline droplet nephropathy, are attributed to accumulation of a-2m-globulin, a carrier protein produced in

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substantial quantities in male rat livers but in negligible amounts in female rats and humans. The a-2m-globulin protein, which transports endogenous pheromones into the kidneys of male rats, also has an affinity for hydrocarbons. The mechanism for hyaline droplet nephropathy is specific to male rats and is considered irrelevant to human health risk assessment (US EPA, 1991).

In vitro toxicity data In vitro test systems for eye (bovine corneal opacity and permeability) and skin (Episkin-SM®) irritancy, conducted with F-T fuels demonstrated concordance with in vivo test data, i.e., the in vitro assay results aligned with results from in vivo tests of skin irritancy for F-T fuels of C12 and shorter carbon lengths, and no classification for eye irritancy across the carbon range of C4-26 (Boogaard et al., 2017). Attempts to utilize an in vitro skin sensitization method, the direct peptide reactivity assay (DPRA) with F-T materials encountered technical challenges due to limited solubility in the assay (Boogaard et al., 2017). In vitro assays, bacterial reverse mutation and human lymphocyte chromosomal aberration, have been used to evaluate the genetic toxicity potential of F-T fuels (ECHA REACH dossier, 2021e, f, g); results were negative for mutagenicity and clastogenicity. Particulates from hydrotreated vegetable oil exhaust induced a more potent increase in TNF-a and MIP-2 secretion in mouse macrophage cells than the particulates from either petroleum diesel or rapeseed methyl ester biodiesel exhaust (Jalava et al., 2010), suggesting a stronger inflammatory response.

Acute and short-term toxicity Short-term exposure to renewable fuels is likely to have limited adverse effects consisting of defatting and possible irritation to the skin, central nervous system depression and respiratory irritation by inhalation of light hydrocarbons, and possible aspiration. Existing data indicate that the toxicity of renewable hydrocarbon fuels is less than or similar to conventional petroleum fuels. Inhalation of short-chain volatile hydrocarbons would be expected to cause central nervous system depression as can occur with conventional petroleum-derived fuels. Sensory irritation of the respiratory tract, measured as a depression in respiratory rate, indicated that F-T synthetic paraffinic kerosene was less irritating than conventional jet fuel (Mattie et al., 2018). Hydrocarbons in the range for diesel engine technology have low volatility; thus, inhalation effects are unlikely. Acute exposures by the oral and dermal routes are not expected to be harmful (NICNAS, 2012; Boogaard et al., 2017); studies conducted found no mortality or signs of effect. Inhalation data for F-T gas oils with a carbon range of C8-26 suggest that high aerosol exposures may cause local symptoms consistent with aspiration of the material into the lungs (Boogaard et al., 2017). Renewable diesel was slightly irritating to skin and eyes, but the effects were minor and transient, insufficient to classify as an irritant (NICNAS, 2012). Naphtha range, and to a lesser extent kerosene (jet) range, renewable fuels may produce skin irritation (Boogaard et al., 2017). The lipophilic nature of renewable hydrocarbon fuels may cause defatting of the skin. Longer or repeated exposure is likely to increase the severity of this effect. Skin sensitization testing of C8-12 F-T branched and linear hydrocarbons concluded that this material does not cause allergic skin reactions (ECHA REACH dossier, 2021g). The results of skin sensitization studies with renewable diesel, both a maximization test and a local lymph node assay, were equivocal (NICNAS, 2012). The saturated linear and branched hydrocarbons of renewable fuels have no structural alerts to suggest a potential to cause allergic skin reactions; regulatory agencies and manufacturers appear to concur that these paraffinic renewable fuels are not skin sensitizers. Few systemic effects from renewable diesel exposure were reported from a 10-week repeated-exposure satellite conducted as part of an oral-exposure two-generation rat reproduction study (NICNAS, 2012). Increased liver weights and hepatocellular hypertrophy were observed in both males and females; this effect was considered adaptive to the high dosage testing regimen rather than adverse. Male rats had increased amounts of eosinophilic material in the renal tubular epithelium in a dose-responsive manner. This was confirmed through Mallory Heidenhain staining as accumulation of a-2m-globulin, a carrier protein produced in substantial quantities in male rat livers but in negligible amounts in female rats and humans. The a-2 m-globulin protein, which transports endogenous pheromones into the kidneys of male rats, also has an affinity for hydrocarbons. This mechanism is specific to male rats and is considered irrelevant to human health risk assessment (US EPA, 1991). The no adverse effect level, NOAEL, for systemic effects in the study was concluded to be the highest dose tested, 1000 mg kg−1 d−1. Similar to conventional hydrocarbon fuels, low-viscosity renewable hydrocarbon fuels across the naphtha, jet, and diesel fuel ranges present a concern for aspiration if ingested, i.e., the low viscosity of the materials provides the potential for the fuel to enter the respiratory tract and may result in chemical pneumonitis, a potentially fatal condition (Craan, 1996).

Chronic toxicity No lifetime bioassay studies were located for renewable diesel.

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Immunotoxicity No studies specific to immune function were found for renewable hydrocarbon fuels. Renewable diesel was not considered to cause allergic skin reactions in either a guinea pig maximization test or the mouse local lymph node assay (NICNAS, 2012). Sensitization testing of F-T fuels across the naphtha to diesel range did not indicate a potential to produce allergic skin reactions (Boogaard et al., 2017). Exhaust particulates from hydrotreated vegetable oil had a larger inflammatory response than rapeseed methyl ester biodiesel in an in vitro mouse macrophage assay (Jalava et al., 2010).

Reproductive and developmental toxicity Toxicology studies conducted with F-T and other renewable hydrocarbon diesel materials suggest that these materials are unlikely to harm reproduction or prenatal development. Renewable diesel was evaluated in an oral exposure two-generation rat reproduction study (NICNAS, 2012; ECHA REACH dossier, 2021d). No adverse effects to reproduction or pups were observed in the study. There were no effects to estrous cyclicity. An increase in homogenization-resistant testicular spermatid counts was not considered adverse. The NOAEL for reproduction and development in the study was concluded to be the highest dose tested, 1000 mg kg−1 d−1. Similarly, an oral two-generation study performed with F-T gas oil, a C8-26 branched and linear diesel range material, found no defects to reproductive performance, litter parameters, anogenital distance, postnatal development during weaning, vaginal cytology, or most sperm measurements (Boogaard et al., 2017; ECHA REACH dossier, 2021g); small statistically significant increases in the age of F1 balanopreputial separation and abnormal sperm were within the normal historical control range. No microscopic effects were found in male or female reproductive organs. As with the F-T gas oil repeated exposure testing, interstitial alveolar inflammation was observed at the highest dose of 750 mg kg−1 d−1 in both male and female rats, and male kidneys had findings suggestive of hydrocarbon-induced a-2m-globulin. The prenatal toxicity potential of renewable and F-T diesel range fuels was evaluated in oral rat prenatal studies; there were no treatment-attributed effects upon maternal measurements, uterine parameters, or fetal development in either study; the no adverse effect levels were 1000 mg kg−1 d−1 and 750 mg kg−1 d−1 (ECHA REACH dossier, 2021d, g). The reproductive toxicity of linear and branched hydrocarbons in the naphtha range may be based upon assessment of reproductive organs in repeated-dose testing and read-across to petroleum hydrocarbons in this range (ECHA REACH dossier, 2021e). No macroscopic or histopathological adverse effects were observed in male or female rats in a 90-day repeated oral dosing test with F-T naphtha (Boogaard et al., 2017). Additionally, prenatal and reproductive testing conducted by inhalation exposure to gasoline vapor condensate, with a carbon range of C4-C11 and containing aromatic constituents, produced no adverse effects to fertility, reproductive performance, histopathological changes to reproductive organs, postnatal growth or adult reproductive performance (Gray et al., 2014). Despite the absence of adverse effects in these studies, naphtha-range renewable fuel may be classified for reproductive toxicity if n-hexane, a reproductive toxicant, is present in sufficient proportion.

Genotoxicity Renewable hydrocarbon fuels are unlikely to cause genetic toxicity. In vitro bacterial reverse mutation and human lymphocyte chromosomal aberration studies were negative for mutagenicity and clastogenicity for F-T naphtha, kerosine, and diesel range materials (Boogaard et al., 2017). F-T jet fuel did not induce mutations in bacteria with or without metabolic activation and did not cause chromosomal aberrations in human lymphocytes in vitro (Mattie et al., 2010). Additional mutagenicity testing conducted on other alternative jet biofuels using Salmonella and Escherichia Coli for the assays also found no evidence of mutagenic potential (Riccio et al., 2010). Renewable diesel was found to be non-genotoxic in in vitro bacterial reverse mutation and mouse lymphoma cell assays, as well as not clastogenic in an in vitro human lymphocyte chromosomal aberration assay (NICNAS, 2012).

Carcinogenicity Renewable hydrocarbon fuels have not been evaluated for carcinogenic potential. The absence of structural alerts for mutagenicity or carcinogenicity, and the lack of hyperplasia observed in repeated-exposure studies suggest that testing for carcinogenicity is unwarranted.

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Organ toxicity Effects to the central nervous system, including dizziness and CNS depression at high exposures, is associated with exposure to light, volatile hydrocarbons (McKee et al., 2015) and should be considered a potential effect of overexposure to renewable hydrocarbon fuels also. Microscopic findings observed in the kidneys of male rats after exposure to renewable diesel are consistent with an a-2 m-globulin mechanism for renal toxicity, considered irrelevant for human health risk assessment (US EPA, 1991).

Interactions F-T synthetic jet fuel is notable for an interaction that does not occur. JP-8, a petroleum fuel in the kerosene hydrocarbon range, has been shown to cause impaired hearing when co-exposed with noise (Fechter et al., 2012), although JP-8 alone exerted no significant effect upon auditory function. This hearing impairment has been associated with aromatic constituents, such as ethylbenzene, present in jet fuel. In contrast, no auditory impairment interaction occurred in rats due to exposure to F-T synthetic jet fuel alone or in combination with noise (Fechter et al., 2010).

Toxicogenomics Genome-wide assessments of renewable hydrocarbon fuels were not discovered. Genomic information for certain hydrocarbons within these renewable fuels may be found; for example, polymorphisms of CYP2E1, involved in the metabolism of n-hexane (CAS 110-54-3) to its neurotoxic metabolite 2,5-hexanedione, have been associated with risk of nerve damage in printing workers (CTD, accessed, 2022). Genome-wide transcriptomic comparative analysis of changes induced from the organic fractions of exhaust particles from hydrotreated renewable diesel derived from hydrotreated vegetable oil and waste animal fat were conducted. The analysis identified dysregulated genes in the bronchial epithelial cells of the assay related to antioxidant response, cell cycle regulation and proliferation (Selley et al., 2019).

Clinical management Individuals exposed to vapors or aerosols of renewable naphtha or kerosene fuels should be moved to fresh air; if breathing is impaired, medical attention should be sought. Management of effects from inhalation exposure to renewable diesel is unlikely due to its low vapor pressure, unless a heavy spray or mist has been generated. If this occurs, exposed individuals should be removed to fresh air. Contaminated skin should be washed with soap and water; contaminated clothing should be laundered. Eyes should be rinsed with large quantities of water. If ingested, vomiting should not be induced due to the risk of entry of fuel into the lungs which could cause chemical pneumonitis (Craan, 1996).

Environmental fate and behavior Linear and branched hydrocarbons in the ranges for transportation fuels, approximately C4 to C26, are expected to partition primarily to air and water in the environment, with little adsorption to soil and sediment. The majority of the lightest hydrocarbons, those within the naphtha range, are likely to partition primarily to air; as carbon length increases, partitioning will shift toward water. Low water solubility suggests that renewable diesel fuels will have limited distribution within bodies of water, although spills may produce an oily sheen on water surfaces. The linear and branched saturated hydrocarbons found in renewable hydrocarbon fuels are considered aerobically and anaerobically biodegradable (Rojo, 2009) and may meet the criteria for ready biodegradability. Linear constituents should be more rapidly metabolized than their branched counterparts, and shorter hydrocarbons are expected to undergo biodegradation more rapidly (ECHA REACH dossier, 2021e, g). This is supported by empirical biodegradation data for hydrotreated light naphtha from petroleum, 89% of which biodegraded in 28 days (as cited in ECHA REACH dossier, 2021e), and data for F-T diesel, 74–75% biodegraded in 28 days (ECHA REACH, 2021g). Similarly, renewable diesel is expected to biodegrade in the environment; 82% degraded in 29 days in laboratory testing (NICNAS, 2012).

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Biodegradation should minimize the potential for bioaccumulation by renewable hydrocarbon transportation fuels. For renewable diesel, the log Kow value, >6.5, suggests that renewable diesel may bioaccumulate, but rapid degradation should limit this likelihood (NICNAS, 2012).

Ecotoxicity Saturated hydrocarbons found in renewable hydrocarbon fuels have limited water solubility and therefore are best tested for aquatic toxicity as water accommodated fractions (WAFs) to provide information on the toxicity of the dissolved constituents. Ecotoxicity testing of naphtha, kerosine, and diesel range hydrocarbons produced by the F-T process and of renewable diesel indicate that saturated renewable hydrocarbons are less toxic than their conventional petroleum fuel counterparts and do not meet classification criteria. Short term exposure to renewable diesel is unlikely to harm aquatic ecosystems. The acute exposure EL50 values for freshwater fish (96-h EL50 > 1000 mg L−1 WAF), invertebrates (48-h EL50 > 100 mg L−1), and algae (72-h EL50 > 100 mg L−1) indicate a low concern for acute toxicity (NICNAS, 2012). Chronic exposure could potentially be harmful; testing in water fleas for 21 days found no effects upon adult immobilization or reproduction at 100 mg L−1, but fewer live young per female; the NOAEL in the study was 1 mg L−1. Comparable results were found for F-T fuels, and invertebrates were again the most sensitive species in the aquatic toxicity testing of F-T materials. Acute exposure of F-T naphtha to freshwater fish was not harmful, and exposure for 28-days post-hatching did not impair survival or growth of freshwater fish larvae; in both tests, EL50 > 100 mg L−1 WAF (ECHA, 2021e). Acute exposure to freshwater algae at the highest concentration of 100 mg L−1 WAF was also without effect. Immobilization of invertebrates was the most sensitive endpoint in acute exposure tests; the acute EL50 was 18–32 mg L−1, although 21-day exposure had no effect upon immobilization and reproduction at the highest concentration tested, 22 mg L−1 WAF (Whale et al., 2018). F-T kerosine, composed of C8-C16 branched and linear hydrocarbons, produced no effects in acute exposure to freshwater fish, invertebrates or algae and had no effect upon fish early life stage testing and limited effect in invertebrate reproduction studies; all EL50 values for these studies were greater than 100 mg L−1 WAF (Whale et al., 2018). For the larger, primarily branched hydrocarbons in the F-T diesel fuel range, acute testing in freshwater fish, invertebrates and algae produced no effects at the highest concentration tested, 1000 mg L−1 WAF, and similarly no effects to growth and survival to freshwater fish larvae, EL50 > 100 mg L−1 WAF, but produced some effect to invertebrate mobility and reproduction (NOEC ¼ 32 mg L−1, EL50 > 100 mg L−1 WAF) (Whale et al., 2018). Additional environmental testing with F-T diesel fuel to assess potential effects to sediment species (midges, earthworms) and plant toxicity did not discover toxicity (Whale et al., 2018).

Exposure standards and guidelines No regulatory exposure standards were found for any of the renewable hydrocarbon fuels. Individual hydrocarbons contained within the composition of renewable fuels may have exposure limits that would be applicable in occupational settings. One example of a regulated constituent is n-hexane, found in naphtha-range fuels, which has a recommended exposure limit of 20 ppm (70 mg m−3) TWA in South Africa and in the European Union (ECHA, 2022), and a recommended exposure limit of 50 ppm by ACGIH and the US NIOSH (OSHA, 2021). Renewable diesel is not classified under the International Maritime Dangerous Goods Code. Occupational exposure limits of 200 mg m−3 (vapor) and 5 mg m−3 (aerosol), matching the ACGIH 8-h TWA for kerosene/jet fuel, have been proposed for alcohol-to-jet and F-T synthetic paraffin kerosene fuels (Sterner et al., 2016; Mattie et al., 2018, 2020), based upon similar or lesser toxicity than conventional petroleum-derived jet fuel.

Other Renewable fuels are flammable or combustible materials. If stored in containers that are heated, pressure inside the container will build and may cause the container to explode. Containers can accumulate static charge if not properly grounded.

PubChem URLs CAS 928771-01-1: https://pubchem.ncbi.nlm.nih.gov/#query¼928771-01-1 CAS 848301-65-5: https://pubchem.ncbi.nlm.nih.gov/#query¼848301-65-5 CAS 848301-66-6: https://pubchem.ncbi.nlm.nih.gov/#query¼848301-66-6 CAS 848301-67-7: https://pubchem.ncbi.nlm.nih.gov/#query¼848301-67-7

Conclusion The term “biofuels” refers to fuels derived from biomass and applies to a wide range of renewable products synthesized to run in engines designed for gasoline, jet, and diesel fuels. Most biofuels have variable compositions. Current commercial usage is greatest

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for ethanol, biodiesel, and renewable hydrocarbon fuels synthesized for compatibility with naphtha, kerosene, or gas oil (diesel) fuels. The primary potential human health hazards for biodiesel and renewable hydrocarbon fuels are aspiration due to low viscosity, and skin irritation or defatting; light renewable hydrocarbons, as with light petroleum hydrocarbons, may also impair the central nervous system. Biodiesel and renewable fuels should degrade in the environment and are likely to be less toxic to aquatic ecosystems than petroleum fuels. Combustion will produce exhaust emissions that may have similar properties to petroleum-derived exhaust emissions. Current information suggests that biofuel exhaust emissions may have mutagenic or inflammatory effects.

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California Poison Control System, Spring. 2009 https://calpoison.org/news/hydrocarbon-toxicity-abuse. Accessed 29 November 2021. NTP (2021a) US National Toxicology Program Ames Conclusions (updated February 4, 2021), Methyl soyate. https://doi.org/10.22427/NTP-DATA-022-00002-0001-000-7. Accessed 18 November 2021. NTP (2021b) US National Toxicology Program In Vivo Micronucleus (updated February 4, 2021), Methyl soyate. https://doi.org/10.22427/NTP-DATA-022-00002-0011-000-8. Accessed 18 November 2021. OECD (2021) Categorization results from the Canadian Domestic Substances List. CAS 68990-52-3. https://canadachemicals.oecd.org/ChemicalDetails.aspx? ChemicalID¼BFFF3010-C4EE-41BA-A305-0882826F8C48. Accessed 18 November 2021. Pfleger BF (2016) Commentary: Microbes paired for biological gas-to-liquids (Bio-GTL) process. PNAS 113: 3717–3719. https://www.pnas.org/content/pnas/113/14/3717.full.pdf. Poon R, Valli VE, Rijden M, Rideout G, and Pelletier G (2009) Short-term oral toxicity of three biodiesels and an ultra-low sulfur diesel in male rats. Food and Chemical Toxicology 47: 1416–1424. https://doi.org/10.1016/j.fct.2009.03.022. Renner HW (1986) The anticlastogenic potential of fatty acid methyl esters. Mutation Research 172: 265–269. https://doi.org/10.1016/0165-1218(86)90064-9. Riccio ES, Green CE, and Mattie DR (2010) Evaluation of five jet fuels in the Salmonella-Escherichia coli / microsome plate incorporation assay. In: US Air Force Research Laboratory Report AFRL-RH-WP-TR-2010-0138. https://apps.dtic.mil/sti/pdfs/ADA536581.pdf. Accessed 29 November 2021. Rojo F (2009) Degradation of alkanes by bacteria. Environmental Microbiology 11: 2477–2490. https://doi.org/10.1111/j.1462-2920.2009.01948.x. Ross JA, Nelson GB, Mutlu E, Warren SH, Gilmour MI, and DeMarini DM (2015) DNA adducts induced by in vitro activation of extracts of diesel and biodiesel exhaust particles. Inhalation Toxicology 27: 576–584. https://doi.org/10.3109/08958378.2015.1068892. Sager TM and Castranova V (2009) Surface area of particle administered versus mass in determining the pulmonary toxicity of ultra fine and fine carbon black: Comparison to ultrafine titanium dioxide. Particle and Fibre Toxicology 6: 15. https://doi.org/10.1186/1743-8977-6-15. Selley L, Phillips DH, and Mudway I (2019) The potential of comics approaches to elucidate mechanisms of biodiesel-induced pulmonary toxicity. Particle and Fibre Toxicology 16(1): 4. https://doi.org/10.1186/s12989-018-0284-y. Shvedova AA, Yanamala N, Murray AR, Kisin ER, Khaliullin T, Hatfield MK, Tkach AV, Krantz QT, Nash D, King C, Gilmour MI, and Gavett SH (2013) Oxidative stress, inflammatory biomarkers, and toxicity in mouse lung and liver after inhalation exposure to 100% biodiesel or petroleum diesel emissions. Journal of Toxicology and Environmental Health Part A 76: 907–921. https://doi.org/10.1080/15287394.2013.825217. Siriarchavatana P, Banchonglikitkul C, Thanmongkol Y, Sematong T, Khayungarnnawee A, Laovitthayanggoon S, and Arunpairojana V (2010) The subacute toxicity test of biodiesel from Jatropha seed. Thai Journal of Toxicology 25: 47–56. https://li01.tci-thaijo.org/index.php/ThaiJToxicol/article/view/243919. Smagala TG, Christensen E, Christison KM, Mohler RE, Gjersing E, and McCormick RL (2013) Hydrocarbon renewable and synthetic diesel fuel blend stocks: composition and properties. Energy & Fuels 27: 237–246. https://doi.org/10.1021/ef3012849. Smith J and DeLuca HF (1963) Essential fatty acid deficiency and rat liver homogenate oxidations. Journal of Nutrition 79: 416–422. https://doi.org/10.1093/jn/79.4.416. Sterner TR, Wong BA, Mumy KL, and Mattie DR (2016) Human health assessment of alcohol-to-jet (ATJ) synthetic kerosenes. In: US Air Force Research Laboratory Report AFRL-RH-WP-TR-2017-0007, July 2016. https://apps.dtic.mil/sti/pdfs/AD1028990.pdf. Accessed 29 November 2021. Stoeger T, Reinhard C, Takenaka S, Schroeppel A, Kary E, Ritter B, Heyder J, and Schulz H (2006) Instillation of six different ultrafine carbon particles indicates a surface area threshold dose for acute lung inflammation in mice. Environmental Health Perspectives 114: 328–333. https://doi.org/10.1289/ehp.8266. Swanson KJ, Kado NY, Funk WE, Pleil JD, Madden MC, and Ghio AJ (2009) Release of the pro-inflammatory markers by BEAS-2B cells following in vitro exposure to biodiesel extracts. The Open Toxicology Journal 3: 8–15. https://doi.org/10.2174/1874340400903010008. Swern D, Wieder R, McDonough M, Merans DR, and Shimkin MB (1970) Investigation of fatty acids and derivatives for carcinogenic activity. Cancer Research 30: 1037–1046. Sylvester PW, Russell M, Ip MM, and Ip C (1986) Comparative effects of different animal and vegetable fats fed before and during carcinogen administration on mammary tumorigenesis, sexual maturation, and endocrine function in rats. 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Further reading Arguelles-Arguelles A, Amezcua-Allieri MA, and Ramirez-Verduzco LF (2021) Life cycle assessment of green diesel production by hydrodeoxygenation of palm oil. Frontiers in Energy Research 9: 690725. https://doi.org/10.3389/fenrg.2021.690725. Burnett D, Baustian J, Hoffman G, Parker R, and O’Callighan J (2016) Inhalation health effects testing of isobutanol gasoline blend: A promising new biofuel. Toxicologist 150(1): 345. https://www.cdc.gov/niosh/nioshtic-2/20047693.html. Bushnell PJ, Beasley TE, Evansky PA, Martin SA, McDaniel KL, Moser VC, Luebke RW, Norwood J Jr., Copeland CB, Kleindienst TE, Lonneman WA, and Rogers JM (2015) Toxicological assessments of rats exposed prenatally to inhaled vapors of gasoline and gasoline-ethanol blends. Neurotoxicology and Teratology 49: 19–30. https://doi.org/ 10.1016/j.ntt.2015.02.004. DeMarini DM, Mutlu E, Warren SH, King C, Gilmour MI, and Linak WP (2019) Mutagenicity emission factors of canola oil and waste vegetable oil biodiesel: Comparison to soy biodiesel. Mutation Research, Genetic Toxicology and Environmental Mutagenesis 846: 403057. https://doi.org/10.1016/j.mrgentox.2019.05.013. Douki T, Corbière C, Preterre D, Martin PJ, Lecureur V, André V, Landkocz Y, Pottier I, Keravec V, Fardel O, Moreira-Rebelo S, Pottier D, Vendeville C, Dionnet F, Gosset P, Billet S, Monteil C, and Sichel F (2018) Comparative study of diesel and biodiesel exhausts on lung oxidative stress and genotoxicity in rats. Environmental Pollution 235: 514–524. https:// doi.org/10.1016/j.envpol.2017.12.077. ECHA Registration Dossier (2021) Renewable Hydrocarbons (Diesel Type Fraction). https://echa.europa.eu/registration-dossier/-/registered-dossier/10505/7/3/2. Edwards JT, Shafer LM, and Klein JK (2012) U.S. Air Force hydro processed renewable jet (HRJ) fuel research. US Air Force Research Laboratory Report AFRL-RQ-WP-TR-2013-0108, July 2012. https://apps.dtic.mil/sti/pdfs/ADA579552.pdf. Accessed 29 November 2021. Hawrat-Paw M, Koniuszy A, Zaja˛ c G, and Szyszlak-Bargłowicz J (2020) Ecotoxicity of soil contaminated with diesel fuel and biodiesel. Scientific Reports 10: 16436. https://doi.org/ 10.1038/s41598-020-73469-3. Hoekman SK, Gertler A, Broch A, and Robbins C (2009) Investigation of biodistillates as potential blend stocks for transportation fuels. In: Coordinating Research Council (CRC) Project No. AVFL-17 Final Report, Desert Research Institute, Reno NV 89512, https://citeseerx.ist.psu.edu/viewdoc/download?doi¼10.1.1.626.8428&rep¼rep1&type¼pdf. O’Malley J and Searle S (2021) Air quality impacts of biodiesel in the United States. International Council on Clean Transportation. https://theicct.org/sites/default/files/publications/USbiodiesel-impacts-mar2021.pdf. Smith A, Lincoln R, Simones T, and Johnson A (2017) Review of scientific literature regarding the human health effects of emissions produced by the combustion of ethanol containing gasoline and the effect of increasing ethanol blends on emissions. In: Report to the Maince Center for Disease Control and Prevention and the Maine Department of Environmental Protection, January 1, 2017. https://www.maine.gov/governor/mills/sites/maine.gov.governor.mills/files/inline-files/Ethanol%20in%20Gasoline%20Report%20FINAL%201.01. 17.pdf.

Relevant websites https://211bresearchgroup.org/peer-review-publications :American Petroleum Institute, Compilation of Peer-Reviewed Publications Stemming from the US EPA Clean Air Act 211(b) Test Program on Gasoline, Including Links to Each Paper (open access). https://ww2.arb.ca.gov/resources/documents/fuels-multimedia-evaluation-biodiesel :California Air Resources Board, Fuels Multimedia Evaluation of Biodiesel: A portal to the Full Multimedia Tiered Evaluation Reports for Biodiesel, Including Aquatic Toxicity and Biodegradation Information. https://ebb-eu.org/wordpress/wp-content/uploads/2021/05/REACH-Consortium-Substances-covered-list.pdf :EBB (2009). EBB Biodiesel REACH Consortium; Substances covered by the EBB Biodiesel REACH Consortium Agreement; 23/06/2009. https://afdc.energy.gov/fuels/emerging_hydrocarbon.html :US Department of Energy Alternative Fuels Data Center. Renewable Hydrocarbon Biofuels; Overview of Types of Renewable Hydrocarbon Biofuels. https://www.osha.gov/green-jobs/biofuels :US Department of Labor Green Job Hazards: Biofuels; A Brief Overview of Types of Biofuels and Links to Informative Government Websites on Physical Hazards. https://www.eia.gov/energyexplained/biofuels/biodiesel-and-the-environment.php :US Energy Information Administration, Biofuels Explained: A general Overview of Biodiesel Information. https://www.epa.gov/renewable-fuel-standard-program/approved-pathways-renewable-fuel :US [email protected] Protection Agency Renewable Fuel Standard Program: Approved Pathways for Renewable Fuels: A Listing of Fuel Types, Feedstocks and Production Process Requirements.

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Biological products in medicine* Marzieh Danialia,b, Taraneh Mousavia,b, and Mohammad Abdollahia,b, aDepartment of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran; bToxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran © 2024 Elsevier Inc. All rights reserved.

Introduction Vaccines Inactivated vaccines Live attenuated vaccines Subunit vaccines Toxoid vaccines Conjugate vaccines Recombinant vaccines Blood products Hormone extracts Insulin Glucagon Growth hormone Gonadotropins Gene/cell therapy Monoclonal antibodies Conclusion Acknowledgment References Further reading

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Abstract Biotherapeutics, possessing 32.3% of all pharmaceutical sales in the United States, have attracted tremendous attention in human diagnostic, preventive, and treatment applications. Vaccines, blood products, hormone extracts, gene/cell-based therapies, tissue-engineered products, and monoclonal antibodies are considered cutting-edge in the management of cancer, autoimmune and immune-based diseases, transplantation, heredity disorders, and, more recently, Coronavirus disease of 2019 (COVID-19). Their large size, complexity, parenteral administration, dysregulation of the immune system balance, toxicity issues from different aspects of carcinogenicity, genotoxicity, reproductive toxicity, ecotoxicity, and clinical safety are key issues that must be taken into account. This chapter focuses on the preclinical and clinical toxicity aspects of different groups of biologics.

Keywords Biological products; Biologics; Biotherapeutics; Blood products; Cell therapy; Gene therapy; Hormone; Monoclonal antibody; Vaccines

Introduction As one of the major sectors of the pharmaceutical industry, biological products or biologics, or biotherapeutics has been garnering substantial interest over the recent years, with processing almost half of new medicine approvals (Johnson, 2018). Market analysis research has revealed that the biologics market values USD 268.51 bn in 2021 and is expected to reach USD 420.55 bn in 2025, with a compound annual growth rate (CAGR) of 12% (Markets, 2021). According to the United States Food and Drug Administration (USFDA) definition (USFDA, 2021d), biologics are those derived from living sources, i.e., humans, microorganisms, animals, or plants (Johnson, 2018; Klaassen, 2013; Peters and Hennessey, 2020). They are classified into vaccines, blood products, hormone extracts, gene/cell-based therapies, tissue-engineered products, and monoclonal antibodies (Johnson, 2018, Klaassen, 2013, Peters and Hennessey, 2020). As they target specific sites responsible for the pathogenesis of different diseases, biologics can cover a broad

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The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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spectrum of indications from diagnosis and prevention to treatment of medical conditions, including but not limited to cancer, autoimmune and immune-based diseases, transplantation, respiratory diseases, migraine, and more recently, Coronavirus disease of 2019 (COVID-19), particularly in severe refractory cases (Matucci et al., 2016; Ingrasciotta et al., 2018). Biologics, especially blood products, are also among vital therapies or even the only available treatments in life-threatening conditions, envenomation, or heredity diseases, e.g., hemophilia. Hence, considering toxicity issues from different aspects of carcinogenicity, genotoxicity, reproductive toxicity, environmental toxicity, and clinical safety matters is essential in the ever-increasing use of these products. Such toxicity concerns mainly arise from the large size, complexity, and parenteral route of administration of biologics which directly impacts immune system balance given either immunosuppression or activation (Peters and Hennessey, 2020; Klaassen, 2013). Resultantly, hypersensitivity reactions, including infusion/injection-related reactions and increased risk of infections, should be carefully monitored and managed upon the clinical use of biotherapeutics (Klaassen, 2013; Matucci et al., 2016; Peters and Hennessey, 2020). Each class of biotherapeutics is comprehensively discussed in this chapter, focusing on their preclinical and clinical toxicity aspects.

Vaccines Vaccines are biological products that activate the human immune system toward an infectious disease and resemble microorganisms (CDC, 2021). According to the world health organization (WHO), vaccination protects humans from harmful and life-threatening diseases by constructing resistance toward the organisms (WHO, 2020). Vaccines are among the most critical and bright discoveries in the history of biological science. With the improvements in understanding the pathogenesis of infectious diseases in the 17th century, the first vaccine, the smallpox vaccine, was developed in 1798. Besides, increases in the mortality rate of contagious diseases encouraged scientists to focus on this life-saving product (Plotkin, 2014; Plotkin, 2005). Fig. 1 summarizes the history of vaccine development. Vaccination is an outstanding achievement associated with saving billions of people from being infected worldwide; however, some people consider it unsafe, which can threaten their lives (Dubé et al., 2013). Several types of vaccines are also investigated in Fig. 1. Inactivated, live attenuated, subunit, toxoid, conjugate, and recombinant vaccines will be aptly discussed in the following (HHS, 2021).

Inactivated vaccines Inactivated or killed vaccine is the first type of vaccine introduced to the market. This type of vaccine contains a microorganism cultured under in-vitro conditions and then killed to minimize its infectivity. Heat, chemicals (formaldehyde), radiation, and physical methods destroy the microorganism. Inactivated vaccines can be classified based on the inactivating way (Nunnally et al., 2015; CDC, 2019).

Live attenuated vaccines This type of vaccine is a weakened living microorganism; thus, the risk of infectious diseases decreases significantly. The immune system’s response toward live-attenuated vaccines is associated with better antibody production. The negative aspect of this vaccine type is the probability of genetic mutation, which is possible in live microorganisms (Nunnally et al., 2015, CDC, 2019).

Subunit vaccines Subunit vaccines contain antigens of microorganisms to provoke the immune system. However, some subunit vaccines contain the epitope, the target site of antigen. Due to the lack of unnecessary sections in this type of vaccine, the risk of adverse effects lowers significantly (Nunnally et al., 2015, CDC, 2019).

Toxoid vaccines This type of vaccine is accessible in microorganisms releasing toxins associated with the most essential and life-threatening disease symptoms. The toxin of tetanus and diphtheria is responsible for disease symptoms; therefore, the protein-based toxin known as toxoid is used as the antigen stimulating the immune system (WHO, 2021d). Formalin, formaldehyde, and sterilized water treatment are ways which detoxify and inactivate this toxin (Nunnally et al., 2015). Moreover, aluminum and calcium are used as adjuvant salts to increase the immune system’s response. Although the risk of infection with toxoid vaccines is shallow, and these vaccines are less sensitive to environmental changes, some anaphylaxis cases are reported with tetanus toxoid (WHO, 2021d).

Conjugate vaccines A bacterium with an outer part (capsule) of polysaccharides is subjected to develop conjugate vaccines. This protective outer layer plays the role of the antigen and can stimulate the human immune system easily (Goldblatt, 2000). The binding of the polysaccharide capsule with a protein to carry the antigen-like section is responsible for activating the T-cell-dependent activation

Biological products in medicine

Fig. 1 Summary of human vaccine development.

119

120

Biological products in medicine

of the immune system. Hemophilus influenza type b (Hib) was the first conjugate vaccine introduced to the market. The protein used in this vaccine as the carrier is the antigen of the microorganism (Crowe Jr, 2001). Recent studies on the development of Escherichia coli vaccines reported that the protective polysaccharide layer of this bacteria is already attached to the protein carrier which is also called natural bioconjugates (Rappuoli et al., 2019).

Recombinant vaccines Recombinant vaccines are usually produced by benefiting from bacteria, yeast, mammalian, and insect cells. This type of vaccine requires the insertion and transference of the DNA section responsible for encoding the antigen. Among the mentioned cells, bacterial expression is the most frequently utilized type that does not need modifications associated with mammalians’ and insects’ cells (Simpson et al., 2020). Most of the recombinant vaccines developed in the recent decays are classified as recombinant protein vaccines. The downside of recombinant protein vaccines is their high cost and limited accessibility. However, the safety profile of this type is significantly better than others (Nascimento and Leite, 2012). In some live recombinant vaccines, an attenuated virus or bacterium is used as the carrier vector. These carriers make the immune system’s response very similar to that of natural infectious microorganisms. The microorganism’s DNA attaches to the vector and stimulates the human immune system (Nunnally et al., 2015; CDC, 2019). Another type of recombinant vaccine is the DNA vaccine, also known as a genetic vaccine. The naked DNA plasmid is injected intramuscularly or through the “Gene Gun” delivery system. This vaccine can trigger T-cells significantly, and the cost of purification of this vaccine is lower than other recombinant vaccines (Wolff et al., 1990). Messenger RNA (mRNA) vaccines include the mRNA as an intermediate between the DNA and the protein. Although mRNA vaccines have high potency, their use has been restricted in some countries because of their instability in animal studies (Pardi et al., 2018). Currently, mRNA vaccines are in the first step of their enhancement, and further studies are required to establish the role of mRNA vaccines in both cancer and infectious diseases (Weide et al., 2008).

Blood products Blood transfusion and plasma-derived medicinal products have become an integral and surviving component in global clinical settings since the first successful blood transfusion between dogs in 1665. This was followed by transfusions from sheep to humans in 1667 and the first human blood transfusion to treat postpartum hemorrhage in 1818 (The American National Red Cross, 2021; Grindon, 2009). After that, as of 1940, plasma-derived medicinal products, including fibrinogen, albumin, gamma globulin, have become available for clinical use through developing cold ethanol fractionation. Almost 40 years later (1985), lyophilized factors VIII and IX were produced, leading to the introduction of a recombinant type of these factors in the 1990s (Grindon, 2009; Silva et al., 2020). According to the World Health Organization (WHO), “A blood product is any therapeutic substance derived from human blood, including whole blood and other blood components for transfusion, and plasma-derived medicinal products” (WHO, 2021a). Fractionated plasma products include albumin, alpha-1 proteinase inhibitors, antihemophilic factors, antithrombins, thrombin, anti-inhibitor coagulant complexes, C1 esterase inhibitors, coagulation factors, fibrin, fibrinogen, and immune globulins. Besides, blood grouping and phenotyping reagents and infectious disease tests are among other USFDA licensed products in this regard (USFDA, 2021b); the latter are used for detecting hepatitis B/C, human immunodeficiency virus (HIV) type 1/2, human T-lymphotropic type 1/2, Trigonoscuta cruzi, West Nile virus, Zika virus, and Babesia species in whole blood specimens. As summarized in Table 1, the use of fractionated plasma products ranges from addressing heredity diseases, child mortality, anemia, immune thrombocytopenia, transplantation, maternal health and immune-related syndromes to bleeding management in acquired diseases, critical care, and surgeries (Harris and Crookston, 2021; Burnouf, 2019; WHO, 2021b; WHO, 2021c). More to the point is the broad application of immune globulins in postexposure prophylaxis of botulism, anthrax, rabies, hepatitis B, vaccinia, varicella-zoster, along with being the only pharmacological treatments for scorpion, crotalid, and rattlesnake envenomation (Shannon and Haddad, 2007; Nelson et al., 2019). Due to the increasing population age and the prevalence of acquired life-threatening diseases, there has been a considerable surge in demand. Hence, the market size of these products, is projected to hit USD 10,253 million by 2027, at a CAGR of 4.5% (Allied Market Research, 2020). Based on the WHO report, about 120 million units of blood are donated each year (WHO, 2021c), most of which are transfused to children under 5 and patients over 60 years of age in low- and high-income countries, respectively (WHO, 2021b). Resultantly, the availability and safety of these products are an essential concern. To this end, global organizations have been established to monitor the safety of blood products. Among, WHO and The Center for Biologics Evaluation and Research (CBER) have released standards and guidelines on monitoring the so-called “hemovigilance” or manufacturing, storage, supply, and post-marketing safety issues with these products (USFDA, 2021a; WHO, 2021c). Sterile equipment, qualified personnel, meeting blood donor requirements, and safe preparation playing a major role in this regard, are comprehensively discussed elsewhere (Harris and Crookston, 2021). Previous research was limited to acute toxicity and repeated dose toxicity assessments only for some products for preclinical toxicity assessments. Indeed, there is a lack of data on carcinogenicity, genotoxicity, reproductive toxicity, and ecotoxicity aspects of blood products. Reasons indicated in labels of products include the development of antibodies and further harm to animals over chronic testing, and the impossibility of interference with DNA or other chromosomal materials, especially for human plasma-derived products. Moreover, according to the guidance on the environmental risk assessment of medicinal products for human use provided by the European Medicines Agency (EMA), ecological risk assessment for amino acids/proteins is not required since they do not seem to exert significant risks to the

Table 1

The clinical safety profile of USFDA approved biologic products.

Product Approval year Fractionated plasma products Albumin solution KedbuminW (2011) AlbuminexW (2018) Human-derived alpha-1 proteinase inhibitor ZemairaW (2003) Prolastin-CW (2009) GlassiaW (2010) Anti-inhibitor coagulant complex FEBIAW (1986) Recombinant antithrombin CinryzeW (2008) ATrynW (2009) Human-derived C1 esterase inhibitor BerinertW (2009) HaegardaW (2017) Recombinant C1 esterase inhibitor RuconestW (2014)

Contraindications

Box warning

ARDS (25% solution), adjunct treatment of cirrhotic ascites, erythrocyte resuspension, hypovolemia, neonatal hemolytic disease, acute nephrosis, OHSS treatment (25% solution) Severe heredity deficiency of alpha-1 antitrypsin (long term maintenance and augmentation therapy)

Fever, rash, chills, N/V, tachycardia, and hypotension

None

Respiratory tract infection, COPD exacerbation, cough, headache, injection site reaction, urinary tract infection, myalgia, and nausea

Hypersensitivity to any of the formulation’s components or albumin; risk of volume overload (i.e., renal insufficiency, stabilized chronic anemia, HF, severe anemia), dilution with sterile water for injection Hypersensitivity to any of the formulation’s components and alpha-1 proteinase inhibitor; IgA deficient patients with IgA antibodies

N/V, diarrhea, anemia, hemarthrosis, hepatitis B surface antibody positive, hypersensitivity, and thromboembolic events (stroke, PE, DVT)

Hypersensitivity to any of the formulation’s components such as kinin generating system factors; DIC; thrombosis or embolism

Thromboembolic events

Hemorrhage and infusion site reaction

Hypersensitivity to goat or goat-milk proteins

None

Injection site reaction, skin rash, nasopharyngitis, and headache

Hypersensitivity to any of the formulation’s components; anaphylaxis to C1 esterase inhibitors

None

Antibody development, headache, diarrhea, nausea, vertigo, erythema, burning sensation, " CRP and fibrinogen, lipoma, back pain, angioedema, and sneezing Pruritus, skin rash, " factor VIII inhibitors, headache, catheter infection, cough, arthralgia, fever, and nasopharyngitis

Hypersensitivity to any of the formulation’s components; anaphylaxis to C1 esterase inhibitors or allergy to rabbit or rabbit-derived formulations Hypersensitivity to any of the formulation’s components and mouse/hamster/bovine protein

None

Arthralgia, myalgia, malaise, rash, and headache

Hypersensitivity to any of the formulation’s components or antihemophilic factor

None

Antibody development

Hypersensitivity to any of the formulation’s components or antihemophilic factor

None

Hemophilia A and B with inhibitors (prevention and control of bleeding; management of perioperative bleeding; routine prophylaxis to # or prevent bleeding) Heredity antithrombin deficiency (prevention of perioperative and peripartum thromboembolism) Heredity angioedema berinert: acute abdominal, laryngeal, facial attacks treatment in adults and pediatrics; cinryze: prevention of attacks in pediatrics 6 YO, adolescents, and adults; hegarda: prevention of attacks in adolescents and adults Heredity angioedema (acute attacks treatment in pediatrics and adults) Hemophilia A (prevention and control of bleeding in pediatrics and adults; management of perioperative bleeding; routine prophylaxis to # or prevent bleeding)

Hemophilia A (prevention and control of bleeding in pediatrics and adults; management of perioperative bleeding; routine prophylaxis to # or prevent bleeding) Acquired hemophilia A

None

None

121

Porcine recombinant coagulation factor VIII ObizurW (2014)

Adverse events

Biological products in medicine

Human recombinant coagulation factor VIII RecombinateW (1992) KogentateW (1993) AdvateW (2003) XynthaW (2008) NovoeightW (2013) NuwiqW (2015) AfstylaW (2016) KovaltryW (2016) JiviW (2018) Human recombinant coagulation factor VIII with Fc fusion EloctateW (2014)

USFDA approved indication (s)

(Continued )

(Continued)

122

Table 1

USFDA approved indication (s)

Adverse events

Contraindications

Box warning

Recombinant coagulation factor IX with albumin fusion IdelvionW (2016)

Hemophilia B (prevention and control of bleeding in pediatrics and adults; management of perioperative bleeding; routine prophylaxis to # or prevent bleeding) Hemophilia B (prevention and control of bleeding in pediatrics and adults; management of perioperative bleeding; routine prophylaxis to # or prevent bleeding) Hemophilia B (prevention and control of bleeding in pediatrics and adults; management of perioperative bleeding; routine prophylaxis to # or prevent bleeding) Hemophilia B (prevention and control of bleeding in pediatrics and adults; management of perioperative bleeding) Bleeding episodes of perioperative management

Headache and dizziness

Life-threatening hypersensitivity to factor IX or any of the formulation’s components

None

Antibody development and headache

Life-threatening hypersensitivity to factor IX, hamster protein, or any of the formulation’s components; DIC and signs of fibrinolysis (rixubis) Life-threatening hypersensitivity to factor IX or any of the formulation’s components

None

Life-threatening hypersensitivity to factor IX, hamster protein, or any of the formulation’s components None

None

Recombinant coagulation factor IX BeneFIXW (1997) RixubisW (2013) IxinityW (2015) Recombinant coagulation factor IX with Fc fusion AlprolixW (2014) Recombinant glycopegylated coagulation factor IX RebinynW (2017) Recombinant coagulation factor VII activated NovoSevenRTW (1999) Human-derived coagulation factor X CoagadexW (2015) Recombinant coagulation factor XIII A-subunit TrettenW Human-derived coagulation factor XIII human concentrate CorifactW (2011) Human fibrin sealant TisseelW (1998) EvicelW (2003) ArtissW (2008) TachoSilW (2010) EvarrestW (2012) RaplixaW (2015)

Bleeding episodes and perioperative management of bleeding Factor XIII A-subunit deficiency Adults and pediatrics with congenital factor XIII deficiency (prevention of bleeding and controlling perioperative surgical bleeding) Colonic anastomosis sealing and skin graft adhesion (Tisseel); facial rhytidectomy (Artiss); adjuvant hemostasis

Headache, obstructive uropathy, and oral paranesthesia Injection site reaction, pruritus, hypersensitivity reaction Fever, hemarthrosis, headache, injection site discomfort, dizziness, antibody development, # fibrinogen, HTN, and thrombosis Fatigue, back pain, and infusion-site pain/ erythema Headache, " fibrinolysis, antibody development, and injection site pain Fever, hypersensitivity reactions, hematoma, arthritis, chills, arthralgia, headache, pruritus, " LDH, erythema, and skin rash AF, HTN, N/V, " transaminases, antibody development, and pleural effusion

Hypersensitivity to any of the formulation’s components or coagulation factor X Hypersensitivity to any of the formulation’s components or factor X Hypersensitivity to any of the formulation’s components; known anaphylactic reactions to human plasma-derived products Artiss: hypersensitivity aprotinin or any of the formulation’s components; direct injection into the circulatory system or highly vascularized tissue Evarrest: severe hypersensitivity to human blood products, IV application, bleeding from large defects in visible arteries/veins treatment Evicel: hypersensitivity to any of the formulation’s components, severe arterial bleeding, direct IV injection Raplixa: hypersensitivity to any of the formulation’s components, severe arterial bleeding, direct IV injection TachoSil: hypersensitivity to any of the formulation’s components, human blood products or horse protein, IV application Tisseel: hypersensitivity aprotinin or any of the formulation’s components, direct IV injection, severe arterial bleeding

None

Thrombosis None None None

Biological products in medicine

Product Approval year

Human-derived fibrinogen concentrate RiaSTAPW (2009) FibrygaW (2017) Coagulation factor IX complex Profilnine SDW

Congenital fibrinogen deficiency, i.e., hypofibrinogenemia and afibrinogenemia (controlling acute bleeding in pediatrics and adults) Factor IX deficiency or hemophilia B or Christmas disease (prevention/control of bleeding)

Human-derived prothrombin complex concentrate (PCC) KcentraW (2013)

Treatment and prevention of bleeding (rapid reversal of vitamin K antagonists’ effect in patients with acute bleeding or candidates of acute/invasive procedure

Protein C concentrate from human plasma CeprotinW (2007)

Severe congenital protein C deficiency (prevention/treatment of thrombosis and purpura fulminans in neonates, pediatrics and adults) Hemostasis aid when standard surgical techniques are ineffective or impractical, and there is oozing blood and minor bleeding from capillaries and small venules

Thrombin EvithromW (human) (2007) RecothromW (recombinant) (2008)

von Willebrand disease

Human-derived von Willebrand factor complex AlphanateW (1978) WilateW (2009) Rabies immune globulin KedrabW (2017) Horse-derived antithymocyte globulin AtgamW (1981) Rabbit-derived antithymocyte globulin ThymoglobulinW (1998) Hepatitis B immune globulin HepaGam BW (2006) Nabi-HBW (1999)

Hemophilia A (prevention and treatment of in adults and pediatrics); von Willebrand disease (surgical and/or invasive procedures in pediatric and adults) Postexposure prophylaxis for suspected rabies exposure Aplastic anemia (treatment of moderate-severe cases for whom bone marrow transplantation is not suitable) Prevention/treatment of renal transplant rejection Postexposure prophylaxis; hepatitis B virus recurrence prevention following liver transplantation

Severe hypersensitivity reactions to any of the formulation’s components, fibrinogen or other human plasma-derived products

None

Thrombosis, flushing, skin rash, chills, urticaria, headache, N/V, paresthesia, drowsiness, lethargy, DIC, anaphylactic shock, dyspnea, fever, and antibody development #" BP, AF, tachycardia, PE, pulmonary edema, CVA, chest pain, DVT, venous thrombosis, ICH, headache, insomnia, mental changes, hypervolemia, N/V, hypokalemia, constipation, diarrhea, anemia, " AST/ALT, arthralgia, prolonged bleeding time, pleural effusion, rales, and respiratory distress Rash, itching, and lightheadedness

None

None

Hypersensitivity to PCC or any of the formulation’s components, including human albumin, factors II, VII, IX, X, protein C and S, antithrombin III; DIC; known HIT

Complications of arterial and venous thromboembolism

None

None

Hypersensitivity to any formulation’s components, known anaphylactic/severe systemic reactions to blood products (evithrom) or hamster protein (recothrom), direct injection into the circulatory system (recothrom), severe/brisk arterial bleeding treatment (evithrom and recothrom) Life-threatening hypersensitivity reactions to hamster/mouse proteins, von Willebrand factor or any components of the formulation

Severe bleeding and thrombosis complications

Hypersensitivity reactions to any formulation’s components, human-derived products or antihemophilic/von Willebrand factor

None

Headache and injection site pain

None

None

Fever, chills, arthralgia, headache, leukopenia, thrombocytopenia, dermatological reaction

Previous anaphylactic reaction to the administration of antithymocyte globulin or other horse-derived gamma globulin preparations Hypersensitivity to any of the formulation’s components or rabbit proteins; active acute/ chronic infection HempaGam B: anaphylaxis to human globulin preparations; IgA deficiency; postexposure prophylaxis in patients with severe thrombocytopenia/coagulation disorders Nabi-HB: anaphylaxis to human globulin preparations

Anaphylaxis

Thromboembolism, pruritus, antibody development, N/V, " INR, PT and PTT, " neutrophils, # lymphocyte, and hypersensitivity reaction Tremor, paranesthesia, antibody development, hot flash, chest discomfort, DVT, HTN, tachycardia, pruritus, vertigo, dizziness, vomiting, and dysgeusia Nausea and postoperative pain, and hemorrhage

UTI, Abd pain, HTN, N/V, headache, anxiety, fever, chills, shortness of breath, hyperkalemia, and # platelets and WBC Headache and erythema

None

Should only be used by an experienced physician None

Biological products in medicine

von Willebrand factor (recombinant; without factor VIII) VonvendiW (2015)

Erythema, vomiting, weakness, fever, pruritus, and headache

123 (Continued )

(Continued)

124

Table 1

USFDA approved indication (s)

Adverse events

Contraindications

Box warning

Anti-D immune globulin WinRho SDFW (1995) RhophylacW (2004)

Immune thrombocytopenia; pregnancy and other obstetric conditions; transfusion

Fever, asthenia, " serum bilirubin, chills, and headache

Intravascular hemolysis

Immune globulin GamaSTANW, GammagardW (1994) GamaplexW (2003) FlebogammaW (2006) PrivigenW (2007) HizentraW (2010) BivigamW (2012) HyQviaW (2014) OctagamW (2014) CuvitruW (2016) CutaquigW (2018) PanzygaW (2018) AscenivW (2019) XenbifyW (2019) Crotalidae polyvalent ovine immune Fab CroFabW (2000) Crotalidae equine-derived immune F(ab´)2 antivenom AnavipW (2015) Cytomegalovirus (CMV) immune globulin CytoGamW

Chronic inflammatory demyelinating polyneuropathy (Hizentra, Privigen); chronic lymphocytic leukemia (Gammagard SD), chronic ITP (Flebogamma 10%, Gammagard SD, Gammaplex, Octagam, Panzyga, Privigen); immunodeficiency syndrome (except GamaSTAN); Kawasaki syndrome (Gammagard SD); multifocal motor neuropathy (Gammagard); passive immunity (GamaSTAN)

Common adverse effects are cardiovascular, CNS, dermatologic, GI, hematologic and oncologic, hepatic, immunologic, local, renal, respiratory, musculoskeletal reactions, as well as fever

Previous anaphylactic reaction to the administration of human immune globulin; IgA deficiency with antibodies to IgA and previous hypersensitivity; administration to neonate whose mother received the drug postpartum (Rhophylac); autoimmune hemolytic anemia with preexisting/high risk of hemolysis (WinRho SDF); suppression of RhD isoimmunization in infants (WinRho) Previous anaphylactic reaction to the administration of immune globulin or any of the formulations’ components; IgA deficiency with antibodies to IgA and previous hypersensitivity (except Gammagard SD); Hizentra and Privigen: hyperprolinemia GamaSTAN SD: severe thrombocytopenia/ coagulation disorders Octagam 5%: hypersensitivity to corn Gamaplex 5%: fructose heredity intolerance or neonate/infants with unknown fructose heredity intolerance

Crotalid envenomation in pediatrics and adults

HTN, chills, skin rash, nausea, pruritus, anorexia, urticaria, and hypersensitivity reaction

Hypersensitivity to any of the formulation’s components

None

Rattlesnake envenomation in pediatrics and adults

Pruritus, skin rash, arthralgia, peripheral edema, headache, N/V, limb pain, and erythema

None

None

Prevention of CMV due to kidney/lung/liver/ pancreas/heart transplant as monotherapy or in combination with ganciclovir

Flushing, chills, N/V, wheezing, back pain, and arthralgia

None

Scorpion antivenom AnascorpW (2011) Anthrax immunoglobulin AnthrasilW (2015)

Scorpion envenomation

N/V, rash, fever, and pruritus

Hypersensitivity to any of the formulation’s components or CMV immune globulin/other immune globulin preparations; selective IgA deficiency None

Inhalation exposure to anthrax (treatment in pediatrics and adults)

Headache, back pain, nausea, swelling, injection site pain

Interaction with glucose monitoring systems, thrombosis

Botulism immune globulin BabyBIGW (2003)

Infant botulism treatment due to toxin type A or B

Mild and transient erythematous rash, "# BP, edema, contact dermatitis, dysphagia, irritability, otitis media, nasal congestion, fever, atelectasis, # SaO2, and vomiting

A previous severe systemic reaction to anthrax/ other human immunoglobulin preparations or any of the formulation’s components; IgA deficiency with antibodies to IgA and previous hypersensitivity A previous severe systemic reaction to human immunoglobulin preparations or any of the formulation’s components; selective IgA deficiency with antibodies to IgA and previous hypersensitivity

Thrombosis, renal dysfunction, and ARF (except Cutaquig, Cuvitru, Hizentra, HyQvia, GamaSTAN SD, Xembify)

None

None

Biological products in medicine

Product Approval year

Digoxin immune Fab DigiFabW (2001) Varicella zoster human immune globulin VarizigW (2012)

Digoxin toxicity

Human vaccina immune globulin VIGIVW (2005)

Eczema vaccinatum; severe generalized or progressive vaccinia; vaccinia infection in patients with a skin condition; aberrant infections

Headache, dizziness, nausea, and rigors

T1D and adults’ T2D

Hypoglycemia, allergic reactions, lipodystrophy, upper respiratory tract infection Hypoglycemia, allergic reactions, cough, and sore throat Fluid retention-weight gain Low potassium Limp feeling Numbness or tingling Skin allergic reactions, lipodystrophy

Postexposure prevention of varicella-zoster in high-risk groups

Hypokalemia, hypersensitivity reaction, orthostatic hypotension, phlebitis Injection site pain, headache, fatigue, chills, nausea, and skin rash

None

None

A previous severe systemic reaction to human immunoglobulin preparations; IgA deficiency with antibodies to IgA and previous hypersensitivity Isolated vaccinia keratitis; previous severe systemic reaction to human immunoglobulin preparations; IgA deficiency with antibodies to IgA and previous hypersensitivity

None

Do not use during episodes of hypoglycemia

None

Do not use during episodes of hypoglycemia, chronic lung disease Do not use during episodes of hypoglycemia and in hypersensitive patients

Chronic lung diseases

Hypersensitivity to insulin aspart

None

Fluid retention-weight gain, hypoglycemia

Hypokalemia, liver problems

Severe hypoglycemic risk None None None

Interaction with glucose monitoring systems

Hormones Insulins Admelog insulinW (2017) Afrezza insulinW (2014)

Adults with T1D and T2D

Apidra insulinW (2004)

Adults and children with diabetes mellitus

Fiasp insulinW (2017) Humalog insulinW (2015)

Children more than 2 YO and adults with diabetes mellitus Adults and children with diabetes mellitus

Novolog insulinW (2000) Humulin R insulinW (1982) Novolin R insulinW (1991) Humulin N insulinW (1982) Novolin N insulinW (1991) Basaglar Kwikpen insulinW (2015)

Adults and children with diabetes mellitus Adults and children with diabetes mellitus Adults and children with diabetes mellitus, allergic reactions Adults and children with diabetes mellitus Adults and children with diabetes mellitus Adults and children with diabetes mellitus

Hyperglycemia, allergic reactions Hyperglycemia, allergic reactions Hyperglycemia, allergic reactions

Hypoglycemia, hypokalemia, liver problems Hypoglycemia, hypokalemia, liver problems Hypoglycemia, hypokalemia, liver problems

Hyperglycemia, allergic reactions Hyperglycemia, allergic reactions Hyperglycemia, allergic reactions

Lantus insulinW (2000)

Adults and children with diabetes mellitus

Hyperglycemia, allergic reactions

Levemir insulinW (2005)

Adults and children with diabetes mellitus

Hyperglycemia, allergic reactions

Toujeo insulinW (2015)

Children more than 6 YO and adults with diabetes mellitus Adults with diabetes mellitus

Hyperglycemia, allergic reactions

Hypoglycemia, hypokalemia, liver problems Hypoglycemia, hypokalemia, liver problems Do not use in the treatment of diabetic ketoacidosis, hypokalemia Do not use in the treatment of diabetic ketoacidosis Do not use in the treatment of diabetic ketoacidosis Hypersensitivity to glargine Hypokalemia Do not use in the treatment of diabetic ketoacidosis, hypoglycemia, hypokalemia, liver problems Hypersensitivity to glucagon

None

None None None None

Severe hypoglycemia in T1D Inhibit motility of GIT for radiologic tests

Allergic reactions

Chronic obesity

Hypoglycemia, constipation

Hypoglycemia, suicide history, gallbladder inflammation

Risk of thyroid c-cell carcinoma

T2D adjunct therapy in children more than 10 YO and adults

Loss of appetite, abdominal discomfort, constipation

Pancreatitis, bile flow blockage, thyroid hyperplasia

Risk of thyroid c-cell and pancreatic carcinoma

T2D adjunct therapy for reducing the risk of cardiovascular events

Abdominal discomfort

Diabetes retinopathy, hypoglycemia, thyroid cancer, or history of medullary thyroid carcinoma

Risk of thyroid c-cell carcinoma (Continued )

125

Glucagon GlucaGenW (glucagon) (1998) Saxenda W (Liraglutide, GLP1 receptor agonist) (2014) VictozaW (Liraglutide, GLP1 receptor agonist) (2019) OzempicW (Semaglutide, GLP1 receptor agonist) (2020)

Hyperglycemia, allergic reactions

None None None

Biological products in medicine

Tresiba FlexTouch insulinW (2015)

None

Table 1

(Continued)

Product Approval year

USFDA approved indication (s)

Adverse events

Contraindications

Box warning

TrulicityW (Dulaglutide, GLP1 receptor agonist) (2014) RevestiveW (Teduglutide, GLP2 receptor agonist) (2012) BaqsimiW (2019)

T2D adjunct therapy for reducing the risk of cardiovascular events

Frequent bowel movements, abdominal discomfort

Risk of thyroid c-cell carcinoma

126

Short bowel syndrome

Abdominal pain, respiratory tract infections

Diabetes retinopathy, hypoglycemia, thyroid cancer, or history of medullary thyroid carcinoma Hypersensitivity to Revestive

None

Children more than 4 and adults for the management of severe hypoglycemia Idiopathic short stature, Turner syndrome

Nasal congestion, nasal and eye itching, redness of eyes Headache, abdominal pain

Pheochromocytoma, insulinoma

None

Acute critical illness after open-heart, abdominal surgery

None

GH deficiency

Headache, abdominal pain

Intracranial tumor

None

Turner syndrome, Prader-Willi syndrome, GH deficiency

Headache, abdominal pain, Joint pain

None

Biological products in medicine

Nutropin AQW (1995)

GH deficiency-induced shortage, Turner syndrome, CKD

Headache, abdominal pain, muscle pain

ZorbtiveW (2003) SerostimW (1996) TransconW (2021) Gonadotropin Hormones Gonal FW (FSH) (2004) OvaleapW (FSH) (2013) BemfolaW (FSH) (2014) PuregonW (FSH) (2004) FollistimW (FSH) (2004) Rekovelle W (FSH) (2004) ElonvaW (FSH, long-acting) (2014) LuverisW (LH) (2004) PergoverisW (FSH/LH) (2004) OvitrelleW (hCG) (2000) Gene and cellular therapy ABECMAW (idecabtagene vicleucel) (2021)

Short bowel syndrome

Headache, abdominal pain,

Personal or family history of cancer, diabetic retinopathy, reduced function of adrenal glands, Carpal-tunnel syndrome Personal or family history of cancer, diabetic retinopathy, reduced function of adrenal glands, Carpal-tunnel syndrome Active malignancy

None

Cachexia in HIV patients

Headache, abdominal pain, muscle pain

Hypersensitivity to Serostim

None

Regulation of calcium and phosphate in bone and blood, GH deficiency Women: ovulation promotion help fertility Men: Spermatogenesis

Headache

None

None

Headache, abdominal pain, breast tenderness

Uncontrolled thyroid or adrenal diseases

None

Women: ovulation promotion help fertility Men: Spermatogenesis Women: ovulation promotion help fertility Men: Spermatogenesis Women: ovulation promotion help fertility Men: Spermatogenesis Women: ovulation promotion help fertility Men: Spermatogenesis Use in the IVF process

Injection site reactions

Pregnancy and lactation, high levels of FSH

None

Injection site reactions, headache

Pregnancy and lactation, high levels of FSH

None

Abdominal pain

None

Headache, pelvic discomfort

Pregnancy and lactation, high levels of FSH, thyroid dysfunction, adrenal dysfunction Pregnancy and lactation, high levels of FSH, thyroid dysfunction, adrenal dysfunction Pituitary gland tumors, ovarian cysts

None

Use in the IVF process

Headache, pelvic discomfort

Ovarian, breast, and pituitary tumors

None

Ovulation stimulating in women with a deficiency in LH and FSH Ovulation stimulating in women with a deficiency in LH and FSH Trigger ovulation, corpus luteum developing, use in IVF process, oligo-ovulatory Relapsed or refractory multiple myeloma, AntiCD-38 monoclonal antibody

Tiredness, headache

Pituitary gland tumors, thyroid dysfunction, ovarian cysts Pituitary gland tumors, enlargement of ovarian, ovarian gynecological hemorrhages None

None

Growth Hormone HumatropeW (1986) NorditropinW (2000) GenotropinW (2001)

Abdominal pain, headache, pelvic discomfort

Ovarian cysts, injection site reactions Headache, abdominal pain, ovarian hyperstimulation symptoms Fatigue, fever, shivering, headache, dizziness, and lightheadedness

CMV infection, lower platelets in the blood, neutrophilia, pregnancy

None

None

None None None

ALLOCORDW (HPC Cord Blood) (2019)

The procedure of unrelated donor for hematopoietic progenitor cell transplantation

Allergic reactions, infusion reactions

Allergy to DMSO

BREYANZIW (lisocabtagene maraleucel) (2021) CLEVECORDW (HPC Cord Blood) (2016)

Relapsed or refractory large B cell lymphoma

None

The procedure of unrelated donor for hematopoietic progenitor cell transplantation

Fatigue, fever, shivering, headache, dizziness, and lightheadedness HTN, bradycardia, fever, fatigue, flushing

DUCORDW (HPC Cord Blood) (2019)

The procedure of unrelated donor for hematopoietic progenitor cell transplantation

Blurred vision, chest discomfort, lightheadedness, shortness of breath

Hypersensitivity to DMSO

GINTUITW (Allogeneic Cultured Keratinocytes and Fibroblasts in Bovine Collagen) (2012) HEMACORDW (HPC, cord blood) (2011)

Allogeneic cellularized scaffold product for the management of vascular wound induced by surgery, adult’s mucogingival conditions The procedure of unrelated donor for hematopoietic progenitor cell transplantation

Oral pain, aphthous stomatitis, facial hypoesthesia

Pregnancy and lactation

HTN, vomiting, bradycardia, fever

Hypersensitivity to DMSO

IMLYGICW (talimogene laherparepvec) (2015) KYMRIAH W (tisagenlecleucel) (2017) LAVIVW (Azficel-T) (2011) MACI W (Autologous Cultured Chondrocytes on a Porcine Collagen Membrane) (2016) PROVENGEW (sipuleucel-T) (2010)

local treatment of unresectable lesions in recurrent melanoma ALL in children more than 3 YO, refractory large B cell lymphoma in adults Nasolabial fold wrinkles in adults. Repair symptomatic cartilage defects of the knee

Abdominal discomfort, constipation, or diarrhea

Immunocompromised patients and presence of active infection None

Refractory large B cell lymphoma in adults Treatment of Spinal Muscular Atrophy

None Hypersensitivity to gentamycin and aminoglycosides

Fatal infusion reactions, engraftment syndrome, and failure of the graft None Cytokine Release Syndrome None None

Fever, shaking chills, severe pain in bones, muscle, and joints, hypotension Abdominal pain, constipation, dry mount, fatigue

None

None

Hypersensitivity to plasminogen

None

Fever, low white and red blood cells, tachycardia, slurred speech Cytokine release syndrome, hypotension, encephalopathy, headache Vomiting, elevated amounts of aminotransferases

None

Seizure, decreased consciousness Allergic reactions

Hypersensitivity to DMSO High levels of bilirubin, abnormal liver function, decreased blood platelets

None

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Abd: Abdominal; AF: Arterial fibrillation; ALT: Alanine transaminase; ALL: Acute lymphoblastic leukemia; ARDS: Acute respiratory distress syndrome; ARF: Acute renal failure; AST: Aspartate transaminase; BP: Blood pressure; CMV: Cytomegalovirus; CKD: Chronic kidney disease; CNS: Central nervous system; COPD: Chronic obstructive pulmonary disease; CVA: Cerebrovascular accident; DIC: Disseminated intravascular coagulation; DMSO: Dimethyl sulfoxide; DVT: Deep vein thrombosis; FSH: Follicle stimulating hormone; GI: Gastrointestinal; GIT: Gastrointestinal tract; GLP1: Glucagon like peptide 1; GLP2: Glucagon like peptide 2; HF: Heart failure; HIT: Heparin-induced thrombocytopenia; HIV: Human immunodeficiency virus; HTN: Hypertension; ICH: Intracerebral hemorrhage; INR: International normalized ratio; IV: Intravenous; IVF: In vitro fertilization; LDH: Lactate dehydrogenase; LH: Luteinizing hormone; MCL: Mantle cell lymphoma; N/V: Nausea and vomiting; OHS: Ovarian hyperstimulation syndrome; PCC: Prothrombin complex concentrate; PE: Pulmonary embolism; PT: Prothrombin time; PTT: Partial thromboplastin time; SaO2: Arterial hemoglobin oxygen saturation; T1D: Type 1 diabetes; T2D: Type 2 diabetes; USFDA: United States Food and Drug Administration; UTI: Urinary tract infection; WBC: White blood cell; YO: Years old; ": Increase; #: Decrease.

Biological products in medicine

RYPLAZIM W (plasminogen, human-tvmh) (2021) TECARTUSW (brexucabtagene autoleucel) (2020) YESCARTA W (axicabtagene ciloleucel) (2017) ZOLGENSMAW (onasemnogene abeparvovec-xioi) (2019)

Metastatic castrate-resistant prostate cancer every Plasminogen deficiency type 1 (hypoplasminogenemia) Relapsed and refractory MCL

Fever, shaking chills, severe pain in bones, muscle, and joints, hypotension Injection site reactions, redness, swellings, edema Pain in bones, muscle, and joints, arthralgia, tendonitis

None

Fatal infusion reactions, engraftment syndrome, and failure of the graft Cytokine Release Syndrome Fatal infusion reactions, engraftment syndrome, and failure of the graft Fatal infusion reactions, engraftment syndrome, and failure of the graft None

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environment (EMA, 2006). Nevertheless, preclinical toxicity assessment of biotherapeutics is still in its infancy and needs the development of methods to overcome the existed limitations. At the clinical level, strict regulations should be globally implemented to detect infectious diseases before donation/transfusion (Harris and Crookston, 2021). WHO recommends mandatory testing for HIV, hepatitis B/C, and syphilis (WHO, 2021b), while USFDA suggests further screening for T. cruzi, West Nile virus, Zika virus, human T-lymphotropic type 1/2, and cytomegalovirus (Frazier et al., 2017). As well, blood compatibility testing must be done before transfusion (WHO, 2021b). To avoid mismatching, new technologies have been implemented to remove antigens A and B and produce engineered red blood cells (RBCs); however, they have not been successful enough in the clinical place (Grindon, 2009). Since available infectious disease tests cannot fully determine all pathogens, listing the name of unsuitable donors as well as asking for a clear medical history of blood donors to find out any risk factors related to infectious diseases are other regulations provided by the USFDA (USFDA, 2021a; Harris and Crookston, 2021). Even with thoroughly following all regulations, clinicians should be prepared for managing possible blood donation and/or transfusion-related reactions. Blood donation might be associated with nausea, faint, vasovagal reaction, hyperventilation, delayed syncope (Sultan et al., 2016), arterial puncture, and nerve damage (Harris and Crookston, 2021), as well as local reactions, including hematoma, inflammation, bruising and hemorrhage. However, donation-related side effects vary across countries and depend on donor characteristics (Taheri Soodejani et al., 2020). More concerning than blood donation side effects are transfusion-related reactions, which arise in 1 out of 100 transfusions (Harris and Crookston, 2021). They occur either immediately (acute) or days after the procedure (delayed). They include a range of clinical presentations from mild flu-like symptoms and infection transmission (Ramirez-Arcos et al., 2016) to post-transfusion purpura (PTP), transfusion-related acute lung injury (TRALI), hypotension, dyspnea, febrile non-hemolytic transfusion reaction (FNHTR), acute/delayed hemolytic transfusion reaction (AHTR/DHTR), delayed serologic transfusion reaction (DSTR), and transfusion-associated circulatory overload (TACO) (Harris and Crookston, 2021; Suddock and Crookston, 2021; Frazier et al., 2017). Blood transfusion should be abrupted upon each of the mentioned conditions, and supportive treatments such as antihistamines/antipyretics can be administered. Noteworthy, vital signs should be closely monitored, an intravenous line should be remained open, and samples from blood and possibly equipment (bag and tubing) should be taken for further examinations. Leukoreduction and irradiation to gamma UV prior to transfusion might be valuable in this regard (Suddock and Crookston, 2021, Frazier et al., 2017). Contraindications to these products include a history of hypersensitivity to the formulation components, or other blood-derived products in most cases (Table 1) (Harris and Crookston, 2021). Volume overload should also be ruled out prior to administering blood products, as it is linked to transfusion-related reactions (Harris and Crookston, 2021). Regarding all these, enforcing national regulations on safety, quality, and rational use of blood products/transfusion should be globalized. This is a crucial concern in low-income countries with a higher incidence of transfusion-related reactions and a lack of enough legislative coverage (39%) compared to middle-income (63%) and high-income (79%) countries (WHO, 2021b).

Hormone extracts Hormones, as biological products, have therapeutic roles in humans and other living organisms. These products were initially extracted from animals and humans; after that, due to the improvements in biological technologies, hormones are produced through new methods such as recombinant DNA technology. Insulin and analogs, glucagon and analogs, growth hormone, and gonadotropins are the most frequently produced and used hormones (Silva et al., 2020).

Insulin Insulin is a hormone consisting of 51 amino acids released from pancreatic islets in the body. Insulin regulates glucose levels in the blood, provoking glycogen and fatty acid production in the liver and muscles, and adipocytes, respectively (Rhodes and White, 2002). Insulin is beneficial in various types of diabetes and was first extracted from the pancreas of bovines and porcine. With the improvements in technology and side effects associated with insulin derived from natural resources such as contamination and allergic reactions, insulin was produced through recombinant DNA (Herring and Russell-Jones, 2018). Humulin® was the first recombinant insulin used in humans with recombinant DNA techniques in Escherichia coli. However, Saccharomyces cerevisiae and Pichia pastors were also used to produce insulin. Also, insulin analogs were developed by changing some consequences of amino acids from natural insulin (Johnson, 1983). The most recent form of insulin is pulmonary insulin which was introduced to the market in Brazil under the brand name Afrezza®. The development of Afrezza® is based on non-invasive methods of administration (Muheem et al., 2016). Also, IN-105® insulin, which is currently under phases 2 and 3 of clinical trials, is an oral insulin analog (NIH, 2018).

Glucagon Glucagon is another hormone that is released naturally from pancreatic islet cells. This 29-amino acid hormone is produced in cells. Unlike insulin, glucagon is released to increase blood glucose levels and stimulate glycogenolysis and gluconeogenesis in the liver (Street et al., 2003). Moreover, glucagon analogs, including glucagon-like peptide 1 (GLP1) and glucagon-like peptide 2 (GLP2), are released from intestinal endocrine L cells. GLP1’s activity is to promote pancreatic islets to release insulin; however, GLP2 aims to decrease the motility of the proximal bowel (TOLBORG, 2018).

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First, glucagon hormones in the universal market were purified from animals like bovine and porcine. After that, GlucaGen®, as recombinant glucagon synthesized in Saccharomyces cerevisiae, was introduced to the market in 1998 (Hövelmann et al., 2018). Nasal powder glucagon, Baqsimi®, was recently approved by USFDA as a non-invasive alternative (Lilly, 2021).

Growth hormone Growth hormone (GH), commonly known as somatotropin, has a significant role in regulating the growth of the body and metabolisms of human cells. This 109-amino acid hormone is naturally released from the hypothalamus gland, from the anterior section. The first presence of GH in the universal market was in 1950, which was natural GH purified from human resources. In 1980, with the improvements in biological technology, GH was synthesized in Escherichia coli (USFDA, 2021c). Transcon® is a recently approved form of GH attached to the methoxy polyethylene glycol (mPEG) to increase its efficacy and tolerability in patients (Sprogøe et al., 2017).

Gonadotropins The anterior section of the pituitary is also responsible for synthesizing gonadotropin hormones, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) (Aubuchon et al., 2012). FSH and LH have different sexual roles in males and females. These hormones contain a and b subunits. Additional gonadotropin hormones have been used in humans for reproductive issues. Like GH, first gonadotropin hormones were obtained from human resources such as menopausal urine and pregnant women for FSH and LH and hCG, respectively. Afterward, mammalian cell lines were utilized for clinical fertility management. However, recombinant forms of gonadotropin hormones are proper alternatives that are frequently benefited nowadays (Bernard et al., 2019; Crommelin, 2008) (32)

Gene/cell therapy Improvements in the knowledge of genetics encouraged scientists to think more about local modifications in the cellular genome to manage specific treatments (Tebas et al., 2014). Gene therapy contains treatments that alter the genes for a particular and genome-relating disease’s management. The main alteration of gene therapy is whether to add a gene or replace the problematic gene. Although gene therapy is among the recent treatment approaches, various studies demonstrated the efficacy of gene therapy in cancer, cystic fibrosis, diabetes, cardiovascular diseases, hemophilia, and AIDS. However, the application of gene therapy in a laboratory environment is now available (Linden, 2010). The main aim of gene therapy is to cure the disease and eliminate the leading cause of symptoms rather than symptom therapy or improving the immune system. To cure disease through gene therapy, different manners are performed:

• • •

Correction of the mutated genes Changing the mutated genes with healthy ones Making the cells more obvious and evident to the body’s immune system

Although gene therapy is associated with beneficial impacts in managing diseases, different side effects, including life-threatening adverse effects, are observed from gene therapy treatment. As the genes are transformed through vectors, and microorganisms like viruses and bacteria are the most commonly used vectors, allergic reactions are easily observed. Also, there is a possibility of infectious while these vectors might infect other cells, resulting in cancers and tumors, as a life-threatening adverse effect (Clinic, 2017). The first step of the gene therapy process is to add the normal and healthy gene into the genome and then enter the modified genome in the stem cell through vectors. DNA microinjection, cationic polymers and liposomes, and bombard of particles are ways to release the gene into the target stem cell (Yang et al., 1994). This vector should have specific properties, such as not being detected by the immune system, easy purification in large quantities, the ability to transfer and express the desired genes (Gardlík et al., 2005). Viral vectors are the most commonly used ones which are reported to have some limitations. Acute immune response and allergic reactions are associated with the use of viruses as vectors. Due to the everyday use of viral vectors, genetic modifications are based on two different approaches, virus-mediated and physical techniques. Retrovirus, Lentivirus, Herpes virus, and Adenovirus are frequently used as viral vectors (Gonçalves and Paiva, 2017). Cellular therapy (CT) is the process of human cells transplantation to replace the damaged cell or tissue. Hematopoietic stem cells, skeletal muscle stem cells, mesenchymal stem cells, pancreatic islet cells, lymphocytes, and dendritic cells might be used in CT. Among stem cells, hematopoietic cells are ideal cells for the process of gene therapy and gene transfer. Also, hematopoietic stem cell transplantation is the most commonly performed CT beneficial for various blood cancers (AABB). Induced pluripotent stem cells (iPS) are a great example of gene therapy for hematopoietic stem cells. Patients who require liver transplants can receive iPS instead of liver transplantation (Kay, 2011). Another example of gene therapy is chimeric antigen recipient T (CAR-T). T cells are manipulated and reprogrammed. The main aim of CAR-T is to enhance the detection of the tumor by influencing the tumor-specific epitope (Chambers and Allison, 1997). CT products such as cellular immunotherapies, vaccines for cancers, autologous and allogeneic cells are used to manage many life-threatening disorders. They are approved for being used in the clinical area. The list of CT products is available in Table 1.

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Monoclonal antibodies With allocating USD 146,642 million in 2020 and reaching USD 390,582 million by 2030 (Allied Market Research, 2021), monoclonal antibodies (mAbs or moAbs) have been attracting tremendous attention due to their broad use in multiple aspects. These include, but are not limited to, immune-based and autoimmune diseases, cancer, viral/bacterial infections, migraine, asthma, dermatitis, osteoporosis, hypercholesterinemia, and COVID-19 (2012). The introduction of the hybridoma technique in 1975 (Cooper and Negrusz, 2013) revolutionized the pharmaceutical industry, with around 100 mAbs having gotten USFDA approval so far (Zahavi and Weiner, 2020). By applying this technique, mAbs are processed by infusion murine/other species lymphocytes with myeloma cells. This is followed by separating and cloning the produced hybridomas, which secret monoclonal antibodies particular to an epitope/antigen (National Institute of Diabetes and Digestive and Kidney Diseases, 2012; Cooper and Negrusz, 2013). Considering the murine source of the first USFDA approved mAb, muromonab (anti-CD3), a range of adverse effects (mainly immunogenicity) have been reported in clinical trials (Pintea et al., 2021; Niebecker and Kloft, 2010). Accordingly, the design and development process of mAbs has resulted in the emergence of chimeric mouse-human and entirely human mAbs, with better safety profiles (National Institute of Diabetes and Digestive and Kidney Diseases, 2012; Niebecker and Kloft, 2010). Nonetheless, there are still serious side effects to be considered prior to mAbs administration, e.g., hypersensitivity and infusion-related reactions, immunosuppression and malignancies, hematological toxicity, cardiovascular diseases, and hepatic disorders. According to Pichler’s updated classification, immune-mediated hypersensitivity reactions, whether acute or delayed, are classified as beta-type reactions that are mostly IgE mediated. Some cases are related to IgG and the complement system (Pintea et al., 2021, Niebecker and Kloft, 2010). Among them, injection-related, or mostly, infusion-related reactions are commonly seen with different mAbs, more commonly at the first dose of administration (Matucci et al., 2016). These side effects seem to be also due to alpha-type reactions (cytokine overproduction) rather than just being beta-type (Pintea et al., 2021), which have been more reported with muromonab, cetuximab, natalizumab, rituximab, and omalizumab (Hansel et al., 2010). Acute reactions mainly appear in the shape of local itching, erythema, flushing, urticaria, fever, flu-like symptoms, or, in more severe cases, chest pain, tachypnea, hypotensive shock, anaphylaxis, anaphylactoid reactions, and death, within 1–2 h post-administration (Hansel et al., 2010; Matucci et al., 2016). Delayed systemic infusion reactions usually manifest as serum sickness, patchy lung infiltrates, psoriasiform plaques, cytokine release syndrome, tumor lysis syndrome, generalized maculopapular exanthema, erythema multiforme, and skin necrotizing vasculitis (Matucci et al., 2016; Hansel et al., 2010). Hypersensitivity to mAbs may also happen given anti-antibody production, even with the fully humanized products. These are classified into human anti-murine antibodies, human anti-chimeric antibodies, human anti-fusion antibodies, and human anti-human antibodies (Niebecker and Kloft, 2010). Due to the extensive reports of hypersensitivity reactions occurrence with mAbs, it is vital to determine true hypersensitivity reactions. If confirmed, mAbs administration must be stopped, and suitable symptomatic therapy with corticosteroids, bronchodilators, analgesics, antihistamines, oxygen, and normal saline should be conducted. Further trials in patients with a history of hypersensitivity to biological agents, recombinant humanized/murine proteins, and Chinese hamster ovary-derived products should be avoided. In case of robust rationality to continue taking the medication, proper pre −/post-treatment approaches ought to be implicated. That is desensitization, gradually increasing infusion rate, premedication with corticosteroids/antihistamines, and monitoring for cytokine release syndrome (Niebecker and Kloft, 2010; Hansel et al., 2010). Another significant issue with mAbs, is gamma-type reactions in the shape of immunosuppression and hyperactivation of the immune system; thereby, increasing the incidence of systemic or organ-specific autoimmune disorders (Pintea et al., 2021; Johnson, 2018; National Institute of Diabetes and Digestive and Kidney Diseases, 2012). Immune suppression may lead to the emergence of opportunistic bacterial or viral infections like hepatitis B, cytomegalovirus (CMV), JC virus, tuberculosis, pneumonia, and Varicella zoster which has been widely reported with anti-TNF-a therapies, natalizumab, tocilizumab, alemtuzumab, and rituximab (Matucci et al., 2016) (Pintea et al., 2021; National Institute of Diabetes and Digestive and Kidney Diseases, 2012). Accordingly, a complete screening for the mentioned infections and progressive multifocal leukoencephalopathy (PML) should be done before imitating treatment. Appropriate treatments for latent tuberculosis have to be done as well. Nevertheless, mAbs in patients with active and severe infections and highly immunocompromised cases are contraindicated. Candidates for mAbs therapy are also suggested not to receive live-attenuated vaccines during the treatment course. All patients should be monitored considering the development of infections, particularly those at high risk, i.e., anti-TNF-a receivers, cases of surgery, active skin lesions, symptomatic PML, hepatitis B, and CMV. Upon confirmed diagnosis, recurrence, and severe infections, mAbs’ discontinuation is preferred (Niebecker and Kloft, 2010). Epsilon-type reactions are non-immunological side effects caused by some members of mAbs, for which the reason is not entirely known in some cases (Pintea et al., 2021). Examples are anti-TNF-a therapies/bevacizumab-induced heart failure, cardiotoxicity (trastuzumab and alemtuzumab), bleeding (abciximab), dermatologic toxicity (cetuximab and panitumumab), gastrointestinal disorders (bevacizumab), thyroid toxicity (alemtuzumab), cytopenia (anti-TNF-a agents, abciximab, alemtuzumab, and rituximab) (Niebecker and Kloft, 2010; Matucci et al., 2016), hepatotoxicity, depression, exacerbation of asthma, and pulmonary fibrosis. Adverse effects, contraindications, and clinical management of mAbs-induced issues are comprehensively discussed elsewhere (Hansel et al., 2010; Johnson, 2018; Niebecker and Kloft, 2010). Although in the majority of cases, managing the expected side effects of mAbs is feasible, life-threatening conditions should not be overlooked. One should keep in mind that the intensity, severity, and duration of side effects vary among subjects, based on the indication of treatment, medications history, and underlying diseases. Hence, personalized approaches should be implemented to maintain patients’ safety (Johnson, 2018).

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Conclusion Considering the ever-increasing use of biologics in different fields of diagnosis, prevention, and treatment of a wide range of diseases, thoroughly following aseptic principles in each step of the manufacturing process is crucial. Due to the production of neutralizing antibodies and possible harm to animals, preclinical toxicity assessment of biotherapeutics is still in its infancy, mainly because of carcinogenicity, mutagenicity, reproductive toxicity, and ecotoxicity. Hence, alternative preclinical procedures should be designed to make chronic toxicity studies possible. Since biological products are commonly associated with hypersensitivity and/or immunosuppression, proper monitoring and management of side effects must be implemented in clinical settings. As with other pharmaceutical compounds, robust global regulations should be in place for preclinical and post-marketing safety assessments of these products.

Acknowledgment This chapter is the outcome of an in-house, financially non-supported study. Authors appreciate their institute for providing digital library and electronic access to literature. Iran National Science Foundation (INSF) is acknowledged for the Seat Award directed to the corresponding author.

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Oman Medical Journal 31: 124–128. Taheri Soodejani M, Haghdoost AA, Okhovati M, Zolala F, Baneshi MR, Sedaghat A, and Tabatabaei SM (2020) Incidence of adverse reaction in blood donation: A systematic review. American Journal of Blood Research 10: 145–150. Tebas P, Stein D, Tang WW, Frank I, Wang SQ, Lee G, Spratt SK, Surosky RT, Giedlin MA, Nichol G, Holmes MC, Gregory PD, Ando DG, Kalos M, Collman RG, Binder-Scholl G, Plesa G, Hwang W-T, Levine BL, and June CH (2014) Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. New England Journal of Medicine 370: 901–910. The American National Red Cross (2021) History of Blood Transfusion. Washington, United States: The American National Red Cross. Available: https://www.redcrossblood.org/ donate-blood/blood-donation-process/what-happens-to-donated-blood/blood-transfusions/history-blood-transfusion.html (Accessed 10 September, 2021). TOLBORG (2018) Glucagon analogues. USFDA (2021a) Blood & Blood Products. Maryland, United States: U.S. Food and Drug Administration. Available: https://www.fda.gov/vaccines-blood-biologics/blood-blood-products (Accessed 03 September 2021). USFDA (2021b) Fractionated Plasma Products. Maryland, United States: U.S. Food and Drug Administration. Available: https://www.fda.gov/vaccines-blood-biologics/approved-bloodproducts/fractionated-plasma-products (Accessed 02 September, 2021). USFDA (2021c) Somatropin Information. Maryland, United States: U.S. Food and Drug Administration. Available: https://www.fda.gov/drugs/postmarket-drug-safety-informationpatients-and-providers/somatropin-information (Accessed 08 September 2021). USFDA (2021d) What Are “Biologics” Questions and Answers. Maryland, United States: U.S. Food and Drug Administration. Available: https://www.fda.gov/about-fda/center-biologicsevaluation-and-research-cber/what-are-biologics-questions-and-answers (Accessed 02 September, 2021). Weide B, Carralot JP, Reese A, Scheel B, Eigentler TK, Hoerr I, Rammensee HG, Garbe C, and Pascolo S (2008) Results of the first phase I/II clinical vaccination trial with direct injection of mRNA. Journal of Immunotherapy 31: 180–188. WHO (2020) Vaccines and Immunization: What is Vaccination? Geneva, Switzerland: WHO. Available: https://www.who.int/news-room/q-a-detail/vaccines-and-immunization-whatis-vaccination (Accessed 8 September, 2021). WHO (2021a) Blood Products. Geneva, Switzerland: World Health Organization (WHO). Available: https://www.who.int/health-topics/blood-products (Accessed 08 September, 2021). WHO (2021b) Blood Safety and Availability. Geneva, Switzerland: World Health Organization (WHO). Available: https://www.who.int/en/news-room/fact-sheets/detail/blood-safety-andavailability (Accessed 10 September, 2021). WHO (2021c) Blood Transfusion Safety. Geneva, Switzerland: World Health Organization (WHO). Available: https://www.who.int/health-topics/blood-transfusion-safety (Accessed 10 September 2021). WHO (2021d) Toxoid Vaccines. Geneva, Switzerland: World Health Organization. Available: https://vaccine-safety-training.org/toxoid-vaccines.html (Accessed 08 September, 2021). Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, and Felgner PL (1990) Direct gene transfer into mouse muscle in vivo. Science 247: 1465–1468. Yang Y, Nunes FA, Berencsi K, Furth EE, Gönczöl E, and Wilson JM (1994) Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proceedings of the National Academy of Sciences of the United States of America 91: 4407–4411. Zahavi D and Weiner L (2020) Monoclonal antibodies in cancer therapy. Antibodies (Basel, Switzerland) 9: 34.

Further reading AABB (2021) Facts About Cellular Therapies. Zürich, Switzerland: AABB. Available: https://www.aabb.org/news-resources/resources/cellular-therapies/facts-about-cellular-therapies (Accessed 08 September, 2021).

Relevant websites https://www.fda.gov/vaccines-blood-biologics :Food and Drug Administration. https://www.who.int/health-topics/biologicals :World Health Organization.

Biomarkers, human health Solange Costaa,b and Filipa Estevesa,b, aEnvironmental Health Department, National Institute of Health, Porto, Portugal; bEPIUnit-Instituto de Saúde Publica da Universidade do Porto, Porto, Portugal © 2024 Elsevier Inc. All rights reserved. This is an update of PCS Coelho, JP Teixeira, Biomarkers, Human Health, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 479–482, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00365-1.

Introduction Role of biomarkers in the development of medicine Biomarkers as tools in human health risk assessment Conclusion Acknowledgments References Further reading

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Abstract The term “biomarker” comes from the Greek term “biο-“, meaning life, in combination with “mark”, Old English of “mearcere”, meaning “writer, notary”, or literally “one who marks”. In scientific literature the word first emerged in the 1970s, but the concept has been part of biomedical research for decades. Various definitions were established over the years, the simplest describes it as a “biological endpoint (or change of ) measured in the organism” used as an indicator in clinical practice, drug development and risk assessment research. As a result, several subtypes have been defined according to the field/subject of the study in question.

Keywords Biomarkers; Classification; Clinical application; Disease; Drug development; Drug discovery; Human biomonitoring; Toxicology

Key points

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Biomarker definition ad scope. Role of biomarkers in medicine progress. Biomarkers as tools in human health risk assessment.

Introduction What is a biomarker and why do we need it? A reasonable starting point for any explanation of what is a “biomarker” and its context in human health would be with the general definition of the term. A comprehensive definition may be a parameter (or change of ) measured in a biological system as an indicator of exposure, effect, susceptibility, or clinical disease. The definition of biomarkers has evolved in recent decades, particularly considering the increased interest of different research, and applied sciences in developing and applying markers for the prediction or occurrence of specific adverse health endpoints (Fig. 1). In late 1990s anticipating the growth in the use of biomarkers in different areas of biomedical research, particularly in clinical trials, an expert group was convened by the U.S. National Institutes of Health (U.S. NIHs) to reach a consensus on biomarker terminology and definitions (Strimbu and Tavel, 2010). A biological marker or biomarker was described as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” (Biomarkers Definitions Working Group, 2001). The World Health Organization (WHO) defined biomarker in 2001 as “any substance, structure or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease” (World Health Organization and International Programme on Chemical Safety, 2001). More recently, the U.S. NIHs in a joint initiative with the Food and Drug Administration Agency (FDA-NIH), have published BEST (Biomarkers, EndpointS, and other Tools) Resource, an open-online biomarker glossary (FDA-NIH Biomarker Working Group, 2016). The FDA-NIH Biomarker Working Group “opens” the definition and describes it as “a defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes, or biological responses to an exposure or intervention, including therapeutic interventions. Biomarkers may include molecular, histologic, radiographic, or physiologic characteristics. A biomarker is not a measure of how an individual feels, functions,

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Fig. 1 Number of publications per year within Biomarkers, Human Health; 170,369 articles were retrieved from PubMed database using the subject heading “biomarker human health” filtered with “humans” to identify articles using human populations/samples/cells.

or survives”(FDA-NIH Biomarker Working Group, 2016). All the definitions proposed in the last decades are broad by necessity, since the term crosses several biological systems, biochemical and clinical fields and can incorporate any of the modern technologies applied on biology, pharmacology, or clinical research. Historically, the concept of measuring a variation of a biological endpoint is not new, in practice biomarkers have been an integral component of biomedical research for decades. But the formalization of the concept in the different related areas, such as, toxicology, clinical biochemistry, and pharmaceutical sciences with the consequent development of unique terminology and conceptual models is as described above relatively recent. One can say that the growing interest in biomarkers has mainly stemmed from the emerging needs of two areas: clinical/ pharmaceutical development of new bioactive compounds (e.g., for commercial interest) and hazard identification/risk assessment. In clinical practice and pharmaceutical industry, biomarkers are used for the prognosis/diagnosis/treatment of disease and drug discovery and development, respectively. In the field of hazard identification and risk assessment, the focus is prevention of disease, surveillance, and identification of risk factors, either from the environment itself (e.g., water, outdoor and indoor air) or behavior and lifestyle (e.g., diet, smoking habits). Within this context, biomarkers are seen as indicators of the continuum of biological events that can occur between the exposure to an external agent (or xenobiotic) and its effect and/or disease. The starting point for any biological endpoint to be considered a biomarker is the cause-effect of the association with an outcome, either disease or exposure. In 1965, Sir Austin Bradford Hill proposed nine “aspects of association” to evaluate whether the epidemiologic associations observed—between occupational and environmental exposures and disease outcomes-were causal (Hill, 1965). These guidelines, referred as the Bradford Hill Criteria might be useful when looking for information that can be used to establish a causal association between a biomarker and an exposure or clinical outcome (Aronson, 2005). However, developments in molecular biology, toxicology, -omics, exposure science, and statistics have increased our analytical capacity to explore potential causal associations and a better understanding of the complexity of the onset and progression of human disease (Fedak et al., 2015). But the concepts that underlie the Bradford Hill Criteria still can be interpreted and applied to a variety of methodologies to help answer some questions about causation (Fedak et al., 2015). Fig. 2 depicts the 9 Bradford Hill Criteria. Depending on the study (e.g., clinical trial, therapeutical intervention, cross-sectional) biomarkers may provide data on disease status, therapeutic uses, toxicity profiles, mechanisms of action, toxicokinetic processes, toxicodynamic processes, dose-response relationships, target dose and tissue, among others. All critical information used in biomedical research, either for medical practice or risk evaluation. The sections below summarize the role of biomarkers in these pivotal fields of health research.

Role of biomarkers in the development of medicine We all know biomarkers without realizing it. Actually, biomarkers are used regularly in clinical practice, for example serum cholesterol (levels) used to monitor the risk of cardiovascular disease. In fact, throughout history the research on biomarkers has been focus on the identification of endpoints that could be used to detect the early stages, the status or the disease itself. Some early examples, includes descriptive markers or semi-quantitative

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Fig. 2 Bradford Hill Criteria. Based on Aronson JK (2005) Biomarkers and surrogate endpoints. British Journal of Clinical Pharmacology 59(5): 491–494. doi:10.1111/j.1365-2125.2005.02435.x; Fedak KM, Bernal A, Capshaw ZA and Gross S (2015) Applying the Bradford Hill criteria in the 21st century: How data integration has changed causal inference in molecular epidemiology. Emerging Themes in Epidemiology 12: 14. https://doi.org/10.1186/s12982-015-0037-4.

measures, such as blood pressure to assess the risk of cardiovascular disorders at the end of the 19th century (Fuchs and Whelton, 2020). Over the second half of the 20th century with the rapid progress of bioanalytical chemistry and physiological measurements, more specific, quantifiable, and mechanistically relevant types of markers were characterized. Some examples still commonly used today are glucose and hemoglobin A1c levels to monitor prediabetes/diabetes or C-reactive protein and cardiac troponin levels for cardiovascular disease. Other breakthrough occurred in 1965, when Dr. Phil Gold & Dr. Samuel O. Freedman found a substance in the blood of patients with colon cancer that was only found in fetal tissues, the carcinoembryonic antigen (CEA). This biomarker became the first test used by clinicians to identify tumors revolutionizing the diagnosis and management of cancer (Hall et al., 2019). By the end of the 1970s, other potential serum tests/markers were developed for a variety of cancers (Turriziani et al., 2012; Kankanala and Mukkamalla, 2022). Besides being used for the diagnosis or disease control (Boccardi et al., 2021), biomarkers are used for prognosis (Wurzba et al., 2022) or to test the efficacy of a therapeutic intervention (Ho et al., 2020). Biomarkers have played an increasingly prominent role in drug research. In this context, these tools are used across all stages of drug development, ranging from enrichment, stratification, and patient selection to safety, efficacy, and performance assessment. In recent years, advances in −omics technologies and bioinformatics have driven biomarker research expanding the biological knowledge of the disease process and identifying drug candidates to perform at a molecular level. Indeed, the increased sophistication of drug discovery processes in the beginning of the 21st century has led to the development of drugs targeting specific molecular pathways and more effective and efficient treatment of disease. In short, to a more personalized and precision medicine. Based on their putative applications in the different areas of biomedical research the FDA-NIH Biomarker Working Group has categorized biomarkers in six main classes (FDA-NIH Biomarker Working Group, 2016): diagnostic, monitoring, pharmacodynamic/response, predictive, prognostic, safety, and susceptibility/risk biomarkers (Fig. 3). Important to note is that one biomarker may meet multiple criteria for different uses, but it must develop evidence for each definition. So, even though some definitions may overlap, they also have clear distinguishing characteristics that identify specific uses. For example, some biomarkers can be both prognostic and predictive. Prognostic biomarkers are mainly identified from observational data and are routinely used to identify patients more likely to have a specific outcome (FDA-NIH Biomarker Working Group, 2016). A predictive biomarker is generally identified comparing a treatment to a control in patients with and without the

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Fig. 3 Classification of biomarkers for clinical and pharmacology applications according to FDA-NIH Biomarker Working Group (2016).

biomarker (FDA-NIH Biomarker Working Group, 2016). A biomarker that is both prognostic and predictive, will show for example a higher survival rate among patients positive for the biomarker and receiving a specific treatment compared to others not receiving it (or receiving other treatment); and shorter but similar survival rate among patients negative for the biomarker receiving treatment or not (or receiving other treatment) (Mukohara, 2015; FDA-NIH Biomarker Working Group, 2016). Effectively identifying and validating biomarkers is essential for accurate therapeutic development. Validation is an important step to guarantee that a test, tool, or instrument is appropriate for its intended use. In general, it involves gathering and analyzing data about the analytical and clinical performance of the test, tool, or instrument (FDA-NIH Biomarker Working Group, 2016). Hence, data from exploratory studies are not sufficient for biomarker validation. The biomarker validation process consists of assessing its performance characteristics, and identifying the range of conditions under which the biomarker will give reproducible, consistent, and accurate data; as well as their ability to tell us something important about our health or disease (Hunter et al., 2010). According to the FDA-NIH Biomarker Working Group validation is “critical to establish that the test measures what it was intended to measure (i.e., analytical validation) and that the biomarker (through its test) can predict or measure the relevant clinical concept (i.e., clinical validation). By establishing whether biomarkers (and the tests used to assess them) are fitfor-purpose, validation informs essentially any potential use of a biomarker in all the biomarker categories” (FDA-NIH Biomarker Working Group, 2016). Essential to any validation process is the purpose, the condition, for which the test, tool, or instrument will be used and knowing the potential benefits and risks associated with that use. A valid biomarker is defined as being “measured in an analytical test system with well-established performance characteristics and for which there is an established scientific framework or body of evidence that elucidates the physiologic, toxicologic, pharmacologic, or clinical significance of the test result” (Hunter et al., 2010).

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Biomarkers as tools in human health risk assessment Humans are daily exposed to a variety of potentially harmful agents in the air they breathe, the liquids they drink, the food they eat and products they use. The aim of hazardous/risk assessment research is the prevention or reduction of exposures to environmental agents that contribute, either directly or indirectly, to increased rates of premature death, disease, discomfort, or disability. Human biomonitoring (HBM) is a frequently used approach to provide information on early warning signals of excessive exposure to harmful substances and for prediction of health risks. In general, exposure is estimated by measuring concentrations of the agent in air, dust, food, or other media that a population or individual is in contact with. However, this external exposure evaluation only provides information on the external dose that an individual or population is exposed, and at best an estimation of the internal exposure (agent dose or its metabolite at the critical target in the organism). HBM research (e.g., exploratory, longitudinal, interventional, or cross-sectional) aims to assess the internal exposure and to investigate the relationship between exposure and the biological effects induced by the exposure. Since it reflects the overall systemic exposure (internal dose) and effect (biological effective) of the organism regardless of the source (e.g., water, air, soil) or pathway (e.g., inhalation, dermal, ingestion) the data provided is used for risk assessment. The tools used in HBM are biomarkers, i.e., chemicals, metabolic products or by-products, their complexes with cellular components, or other cellular parameters, measured in human samples (e.g., blood, urine, saliva, sweat, breast milk). Selection of the optimal biomarker for analysis for any exposure situation is based on the type of exposure and duration, the exposed population characteristics, endpoint mechanism, and the expected target tissue. It should be added that, in this context, the detection of a biomarker in a human sample (tissue or fluid) is not indicative of illness or toxic process, but only exposure of the organism to a substance (Kamrin, 2003). Historically, biomarkers have been used to identify and regulate hazardous human exposures occurring in the environment (e.g., mercury poisoning Minamata) or/and in the workplace (e.g., exposure to carcinogens such as benzene, formaldehyde). One of the most significant examples of the impact of biomarkers on health and public policies occurred in 1943, with the first published data linking late mental development to lead poisoning (Byers and Lord, 1943). Studies that followed confirmed this association establishing a causal effect between early-life exposure to lead (e.g., through toys, paints, or urban pollution) and neurocognitive impairment (Rocha and Trujillo, 2019). The biomarker used to demonstrate this link was blood lead levels, still used today to monitor lead exposure (McFarland et al., 2022). From the mid-1970s, driven by the increasing public concern, regulatory changes and public education programs were implemented to control lead concentrations in the environment. Some of the mitigation measures were reduction or elimination of leaded gasoline, food packaging, house paint, solder used in plumbing, water pipes/plumbing and soft drink cans (Pohl et al., 2017). These initiatives resulted in a substantial decline of lead levels, confirmed by human biomonitoring campaigns carried during the following years (Rocha and Trujillo, 2019; McFarland et al., 2022). Accumulated evidence shows that 80% of all cancers are caused by environmental factors (e.g., diet, lifestyle, work) (Anand et al., 2008). Estimations also show that at least one-third of all cancer cases of environmental and occupational origin can be prevented based on current knowledge (Espina et al., 2013). Epidemiologic studies of occupational groups have been central to the identification of human carcinogens. The use of a biomarkers in occupational studies has expanded the possibilities for identifying human carcinogens and for understanding the disease process. Often, workers are in contact with hazardous agents at higher levels than the general population, resulting in accumulation of exposure effects over lifetime. Many occupational diseases, including work-related cancers, are characterized by long latency periods and therefore are difficult to recognize until the clinical manifestation of their symptoms. Early detection of a professional risk situation can significantly decrease the occurrence of deleterious effects on worker’s health. Thus, preventing individual exposure to carcinogens may prevent the disease. Biomarkers are helpful in reinforcing the causality between exposure and health effect or to serve as a warning signal to identify carcinogenic potential when epidemiological and toxicological data concerning the exposure-effect (or disease) relationship is inconclusive or unavailable (Shaham and Ribak, 1996). To facilitate the use of these tools in the assessment of health risk biomarkers were divided into categories representing the continuum of biological events that can occur between exposure to an external agent and the disease (Fig. 4). Each of these biomarkers provides different information and consequently has its own set of advantages and disadvantages relating to its specificity, relevance to the toxic pathway and difficulty of analysis. The three main classes are: biomarkers of exposure, biomarkers of effect and biomarkers of susceptibility. In short, different biomarkers reflecting various stages of the exposure continuum can be used to provide information on the etiology of disease, to monitor exposed populations and to identify susceptible subgroups of the population. A biomarker of exposure is the xenobiotic chemical itself, its metabolites, or the product of an interaction between a chemical and its target molecule or cell that is measured in a compartment within an organism (World Health Organization and International Programme on Chemical Safety, 2001). Generally, their presence only indicates that an exposure has occurred. A biomarker of exposure can be defined as a biomarker of internal dose, when the amount of a chemical or its metabolites is quantified in a biologic tissue or fluid (e.g., lead in blood, volatile organic compounds in breath) or as a biomarker of biologically effective dose (e.g., DNA-adducts or protein-adducts) (Coelho and Teixeira, 2012). Biomarker of effect is defined as a measurable biochemical, physiological, behavioral, functional, or other alteration within an organism that might be elicited by the exposure. It may represent an event that can be correlated with, and possibly predictive of, a deleterious health effect (World Health Organization and International Programme on Chemical Safety, 2001). Cytogenetic

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Fig. 4 A simplified diagram of the cascade of events from exposure to clinical disease showing the relationships among exposure, effect, and susceptibility biomarkers. Adapted from Manini P, De Palma G and Mutti A (2007) Exposure assessment at the workplace: Implications of biological variability. Toxicology Letters 168(3): 210–218.

alterations, such as chromosomal aberrations and micronucleus test are the most used endpoints. The measurements of biomarkers of effect generally reflect events that take place at the latter end of the continuum between exposure and disease manifestation. Although they are less specific, they can be more predictive of the ultimate health effect (Coelho and Teixeira, 2012). Biomarker of susceptibility- is an indicator of an inherent or acquired limitation of an organism to respond to the challenge of exposure to a specific chemical agent(s) (World Health Organization and International Programme on Chemical Safety, 2001). Generally, it is used to identify whether a person or a group are susceptible to the effect caused by the exposure to certain xenobiotic. Usually includes (epi)genetic (e.g., polymorphic genes, enzyme function, co-factors) or non-genetic factors (e.g., lifestyle, age). These factors are independent of exposure, but they identify those individuals in a population who may be more susceptible or resistant to the effects of a hazardous exposure. Thus, it may help explain why individuals exposed to the same environmental stressor produce markedly different levels of biomarkers of exposure and/or biomarkers of effect, which in turn may correlate with different severities of clinical symptoms and illness (Coelho and Teixeira, 2012). The information produced is also used to set environmental limit exposure values and to support regulatory policies aimed at minimizing the likelihood of significant health risks. Exposure limit values are established concentrations of a specific contaminant in the air which if not exceeded, will not generally cause adverse effects to the health of the subject(s) exposed. Based on available evidence the ACGIH (American Conference of Governmental Industrial Hygienists) has establish Biological Exposure Indices (BEIs). These are guidance values used especially in occupational settings to assess human exposure. BEIs are biomarkers of exposure (chemical itself, their metabolites, or a biochemical change) sampled in urine, blood, or exhaled air (ACGIH (American Conference of Governmental Industrial Hygienists), 2021). And like the environmental limit exposure values, it generally indicates a concentration below which nearly all individuals should not experience adverse health effects. BEIs can help the occupational health professional to assess body burden, monitor work practices or test the efficacy of personal protective equipment or other mitigation measures. It serves as a complement to exposure assessment by environmental sampling and medical surveillance.

Conclusion Biomarker definition has evolved over the years and will certainly continuing to change along with scientific development and technical advances. Biomarkers can expand our knowledge about the mechanisms underlying the disease and consequently, the identification of potential new therapeutic key targets. Furthermore, they are also valuable tools used in hazard identification and risk assessment providing data on the different stages of the continuum between exposure and disease. Biomarkers can be measured via a wide range of techniques, from direct biological and morphological observations to quantitative biochemical and genomic assays among others. Particularly important is biomarker validation either to develop the best targeted therapeutical interventions or to prevent diseases induced by environmental exposures. The continuous advances on systems biology coupled with our capacity to store and compute massive amounts of information will change our understanding of both biology and clinical outcomes. One

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promising field are minimally invasive and non-invasive biomarker sensors. These sensors have a tremendous potential for medical and risk assessment fields since biomarker evaluation is done using minimal invasive biological matrices such as sweat or breath, and the information is given on real-time.

Acknowledgments Filipa Esteves work is supported by Fundação para a Ciência e Tecnologia Filipa Esteves and by the European Social Fund (ESF), grant number UI/BD/150783/2020.

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Current Drug Targets 11(5): 536–545. https://doi.org/10.2174/138945010791011947. Kamrin M (2003) Traces of Environmental Chemicals in the Human Body. New York: American Council of Science and Health. [Internet: http://www.acsh.org]. Kankanala VL and Mukkamalla SKR (2022) Carcinoembryonic antigen. In: StatPearls. StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK578172/. McFarland MJ, Hauer ME, and Reuben A (2022) Half of US population exposed to adverse lead levels in early childhood. Proceedings of the National Academy of Sciences of the United States of America 119(11), e2118631119. https://doi.org/10.1073/pnas.2118631119. Mukohara T (2015) PI3K mutations in breast cancer: Prognostic and therapeutic implications. Breast Cancer (Dove Medical Press) 7: 111–123. https://doi.org/10.2147/BCTT. S60696. Pohl H, Ingber S, and Abadin H (2017) Chapter 13: Historical view on Lead: Guidelines and regulations. In: Sigel A, Sigel H, and Sigel R (eds.) Lead: Its Effects on Environment and Health, pp. 435–470. Berlin, Boston: De Gruyter. https://doi.org/10.1515/9783110434330-013. Rocha A and Trujillo KA (2019) Neurotoxicity of low-level lead exposure: History, mechanisms of action, and behavioral effects in humans and preclinical models. Neurotoxicology 73: 58–80. https://doi.org/10.1016/j.neuro.2019.02.021. Shaham J and Ribak J (1996) The role of biomarkers in detecting early changes relating to exposure to occupational carcinogens. Journal of Occupational Health 38: 170–178. https://doi.org/10.1539/joh.38.170. Strimbu K and Tavel JA (2010) What are biomarkers? Current Opinion in HIV and AIDS 5(6): 463–466. https://doi.org/10.1097/COH.0b013e32833ed177. Turriziani M, Fantini M, Benvenuto M, Izzi V, Masuelli L, Sacchetti P, Modesti A, and Bei R (2012) Carcinoembryonic antigen (CEA)-based cancer vaccines: Recent patents and antitumor effects from experimental models to clinical trials. Recent Patents on Anti-Cancer Drug Discovery 7(3): 265–296. https://doi.org/10.2174/157489212801820020. World Health Organization & International Programme on Chemical Safety (2001) Biomarkers in Risk Assessment: Validity and Validation. World Health Organization. https://apps.who. int/iris/handle/10665/42363. Wurzba S, Salo TA, and Coletta RD (2022) Editorial: Prognostic biomarkers for oral cancer. Frontiers in Oral Health 3: 994387. https://doi.org/10.3389/froh.2022.994387.

Further reading Manini P, De Palma G, and Mutti A (2007) Exposure assessment at the workplace: Implications of biological variability. Toxicology Letters 168(3): 210–218.

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Relevant websites https://www.hbm4eu.eu/ :European Human Biomonitoring Initiative (HBM4EU). https://www.ncbi.nlm.nih.gov/books/NBK326791/ :FDA-NIH Biomarker Working Group. https://www.eu-parc.eu/ :European Partnership for the Assessment of Risks from Chemicals (PARC). https://prevention.cancer.gov/research-groups/cancer-biomarkers/about-cancer-biomarkers :U.S. National Cancer Institute, Division of Cancer Prevention, Cancer Biomarkers Research Group. https://www.phri.ca/about/ :Population Health Research Institute (PHRI). https://www.iarc.who.int/cards_page/iarc-publications/ :International Agency for Research on Cancer (IARC).

Biomonitoring Carla Costaa,b,c and João Paulo Teixeiraa,b,c, aEnvironmental Health Department, National Institute of Health, Porto, Portugal; bEPIUnit, Institute of Public Health, University of Porto, Porto, Portugal; cLaboratory for Integrative and Translational Research in Population Health (ITR), Porto, Portugal © 2024 Elsevier Inc. All rights reserved. This is an update of C. Costa, J.P. Teixeira, Biomonitoring, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 483–484, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01000-9.

Introduction Biomonitoring—A matter of environmental and human health Environmental biomonitoring Human biomonitoring Conclusion References

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Abstract Biomonitoring refers to the systematic measurement of compounds and/or detection of cell or cell molecules alterations in living organisms with the purpose of identifying or assessing potential hazardous exposure and effects to chemicals or other hazardous agents. The goal of this chapter is to provide an overview on biomonitoring, both from the environmental and human health perspectives. Therefore, the main concepts of environmental and human biomonitoring are here described, alongside a few considerations on its application, limitations and challenges.

Keywords Bioindicators; Biomarkers; Biomonitors; Biosensors; Environmental biomonitoring; Hazardous agents; Human biomonitoring; Human biomonitoring assessment values; Pollution; Risk

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Many of the chemicals used nowadays are hazardous to the environment and human health; Biomonitoring may refer both to measurement of chemicals and their effects in the environment (environmental biomonitoring) and in human health (human biomonitoring); Environmental biomonitoring includes different tools such as bioindicators, biosensors, and biotests; Human biomonitoring relies on different biomarkers (dose, effect, and susceptibility) to associate exposure to human health outcomes.

Introduction At the same time that chemicals are considered essential to the development of new energy- and resource-efficient technologies, materials and products, they can also be hazardous to both environment and human health (EC, 2020). The increasing production, diversity and use of chemicals around the world (UNEP, 2019), alongside their release in large quantities, are key contributors to environmental pollution, making the sound management of chemicals and waste a critical issue for a sustainable development (UNEP, 2016). A toxic-free environment is now the main goal of different international policies (van Dijk et al., 2021) to be achieved with the refrainment in usage of harmful and non-essential chemicals, and a safe and sustainable use of the remaining (EC, 2020). But this is not yet the global scenario given the uneven progress on environmental concerns across countries and regions due to lack of awareness, capacity and financial resources, among other factors (UNEP, 2020). According to the latest report of WHO (WHO, 2021), 2 million lives and 53 million disability-adjusted life-years were lost in 2019 due to exposures to selected chemicals, an estimate that surpasses the ones disclosed in 2016 (WHO, 2018) and 2012 (WHO, 2016). The impact in human health, environmental degradation and loss of biodiversity due to chemical contamination are now expected to accelerate in combination with the increasing number of extreme weather events driven by climate change (Mal et al., 2017). For all the above-mentioned, it is clear that despite the political and societal efforts toward environment protection, both the environment and human health are still significantly threatened by pollution and climate change. To prevent and manage these risks it is necessary to make use of available tools that may contribute to the risk management process and pollution control (de Zwart, 1995),

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such as environmental monitoring and biomonitoring (also designated biological monitoring). Environmental monitoring includes analysis of air, soil, water, and sediments, and may refer to physical or chemical monitoring (Lam and Gray, 2003). Biomonitoring, on the other hand, relies on monitoring of biological communities, or of individual organisms to identify and characterize risk (Bartram and Ballance, 1996). These strategies should not be considered alternatives to each other, but complementary, as physical or chemical monitoring of environmental matrices are often needed for a proper interpretation of results (Bartram and Ballance, 1996). One of the main advantages of biomonitoring is that, depending on the chosen indicator, it can: (i) integrate total uptake of chemicals by different routes of absorption (Angerer et al., 2007) with individual variability in absorption, metabolism and excretion (Ladeira and Viegas, 2016); (ii) integrate bioavailability of the compounds with their concentration, and intrinsic toxicity (de Zwart, 1995); (iii) integrate biological responses to different contaminants of multiple sources (Albertini et al., 2006), and therefore provide information about the overall effect in the organism, even if in mixtures (Lam and Gray, 2003); (iv) offer information on the result of prolonged exposure to adverse stimuli (Boogaard, 2009); (v) indicate the ability of an organism to tolerate and respond a chemical insult (Holt and Miller, 2010); (vi) in some cases, provide cheaper, more precise, and more sensitive information than chemical analysis (in the case of environmental biomonitoring; Lam and Gray, 2003).

Biomonitoring—A matter of environmental and human health The definition of biomonitoring is controversial; some researchers associate this concept to the measurement of chemicals and/or metabolites, or their effects in the human biological samples only, while others include also measurements in other species, communities and ecosystems; the current chapter builds on both concepts, as it will consider both environmental and human health but in two different sections (environmental biomonitoring -EBM and human biomonitoring—HBM). The biomonitoring concept refers to repeated, standardized measurements, observations, evaluations and reporting of the environment or human health in order to define status and trends (de Zwart, 1995; Ladeira and Viegas, 2016). Biomonitoring, both EBM and HBM, must be carried out under strict quality assurance in order to guarantee the production of reliable data for further use in risk assessment.

Environmental biomonitoring There are different possibilities to catalog tools and approaches used in EBM (de Zwart, 1995). Ecological methods, for example, mainly refer to the structural aspects of ecosystems, such as species abundance and distribution, community structure (species composition), and trophic structure (food web complexity, and niche occupation) (de Zwart, 1995). On the other hand, responses to stress or adverse stimuli by individual organisms, namely behavioral, physiological or morphological changes can also be studied (Bartram and Ballance, 1996). In this context, EBM includes both the observation of so called bioindicators, species that usually present a moderate tolerance to environmental variability (Holt and Miller, 2010), and analytical studies of biological samples that act as passive samplers accumulating pollutants (Kuczy nska et al., 2005). EBM may also build on the use of bioanalytics that employ biologically active substances acting as receptors of certain pollutants, namely biosensors and biotests (Kuczy nska et al., 2005). Bioindicators are usually defined as organisms or communities of organisms, whose content of certain pollutants and/or whose morphological, histological or cellular structure, metabolic-biochemical processes, behavior or population structure(s) provide information on qualitative alterations of the environment (Markert et al., 2003). Their utility depends of their sensitivity, specificity, and prevalence of a response, that together describe their predictive capacity (Gerhardt, 2002). Furthermore, their methodological (e.g., easy to use) and societal relevance (e.g., cost-effectiveness) must not be overlooked (Burger, 2006). A great number of species or tissues can be used as bioindicators, such as algae, plants (Gadzała-Kopciuch et al., 2004), invertebrates, fish, birds, etc. (Burger, 2006). If these bioindicators provide also information on the quantitative alterations of the environment, they can be designated of biomonitors (Markert et al., 2003). Biomonitors can be catalogued as sensitive or accumulative (Conti, 2008). The first one can be used as early warning systems of environmental stress, while the latter present the ability to store pollutants and thereby, to provide an integrated measure of these contaminants in the environment (Conti, 2008). Lichens constitute an example of biomonitors (Augusto et al., 2016). Biosensors are devices that integrate biological constituents, such as enzymes or antibodies, with an electronic component to generate a measurable signal (Naresh and Lee, 2021). On the other hand, in biotests, a biological sample is used to assess the presence of pollutants in the environment or to assess their toxicity on a live organism (in comparison with a control sample) (Kuczy nska et al., 2005). Among the current limitations of EBM, high cost, and limited coverage are probably the most relevant, increasing the need to find better methodologies, able to respond to the widespread and rapid environmental alterations; in this context, Next-Generation Biomonitoring (NGB) approaches are expected to deliver better and more accurate information, at a cheaper price, in the next decades (Bohan et al., 2018). Furthermore, EHM is expected to benefit from advances in the field of molecular ecology, remote sensing, network science and ecoinformatics (Bohan et al., 2018).

Human biomonitoring Human biomonitoring (HBM) can be seen as an intermediate area falling between environmental monitoring and public health surveillance (Kyle, 2019). In fact, it is generally accepted that HBM investigates the continuum that links chemical exposure, internal

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dose and health impairment (Louro et al., 2019; Tan et al., 2012), thereby contributing to advances in exposure science and epidemiology (Louro et al., 2019). HBM was been widely used in many epidemiological studies, both in the environmental and occupational settings, to associate exposures to health outcomes (Ladeira and Viegas, 2016). HBM helps environment- and occupational health scientists to understand who is exposed, which are the exposure trends and which chemicals need to be prioritized (Tan et al., 2012). When combined with environmental measurements, HBM can also offer data on exposure sources and routes for exposure assessment; and clarify exposure and dose levels for risk assessment (Tan et al., 2012). HBM comprises not only the determination of hazardous substances in body fluids (biomarkers of dose), but also of early, reversible effects observable in cells and tissues (biomarkers of effects) and the existence of individual susceptibility factors (biomarkers of susceptibility) (Angerer et al., 2007; Au, 2007). Several human biomonitoring programs have been established in the last decades, in different countries for a longitudinal surveillance of population’s exposure, usually to chemicals of concern. These include the National Health and Nutrition Examination Study (NHANES), in the United States of America (Sobus et al., 2015); the Canadian Health Measures Survey, in Canada (Tremblay et al., 2007); the German Environmental Surveys (GerES), in Germany (Kolossa-Gehring et al., 2011); and the Arctic Monitoring and Assessment Program, in Denmark (Hansen, 1998), and HBM4EU (Gilles et al., 2021), among others (Ganzleben et al., 2017; Kolossa-Gehring, 2011). Nonetheless, in order to obtain reliable data, a number of requirements must be strictly met, namely the access to a suitable biological matrix (Angerer et al., 2007), in the necessary amount (Dennis et al., 2017), within the acceptable timing for collection (Clewell et al., 2008), and with the possibility to adequately store it (Dennis et al., 2017). Furthermore, it is necessary that suitable biomarkers, able to reflect exposure or effects, have been previously identified (Angerer et al., 2007) and guarantee access to fit-for-purpose methods (Dennis et al., 2017). This usually demands extensive knowledge on toxicokinetics, individual variability (temporal, spatial and genetic variability) (Tan et al., 2012), and technical expertise, what often limits the use and/or implementation of HBM programs. A detailed and reliable example of an evaluation process for biomarker, human matrices and analytical methods selection in an HBM program can be found in Vorkamp et al., 2021. Evaluation and interpretation of results is a key step in HBM, that in the least complex approach (a descriptive one) involves a simple comparison with pre-established HBM assessment values (NAP, 2006). One possibility is to use reference values (RV95) that constitute a statistical description of the range of concentrations usually detected in a reference population; but this reference value does not provide any information of the possible health effects caused by that exposure (Angerer et al., 2011). On the other hand, HBM assessment values may derive from exposure-effect relationships or from tolerable daily intake values; that is the case of biological exposure indices (BEIs; used in occupational health), and HBM I, HBM II, and biomonitoring equivalents (used in the general population; Angerer et al., 2011). Even though extremely useful, these values are unfortunately unavailable for most substances available on the market (Kolossa-Gehring, 2011). In opposition to environmental monitoring, HBM raises important ethical issues; individuals must be informed and agree to what will be measured, how, why and what will be done with results (Jones, 2020). This point can be particularly problematic in the case of susceptible populations, such as pregnant women (Arbuckle, 2010) or children (Pedersen et al., 2007). Other limitations of HBM include the use of surrogate matrices, such as blood and urine that imply that measurements are in fact only proxies of chemical burden in accessible biological matrices rather than the real concentration at (all) target organs (Louro et al., 2019). Additionally, HBM does not allow the identification of exposure sources, nor provide information on the risk posed by the chemical to human health (Kolossa-Gehring, 2011). Simultaneously, HBM has been dealing with some significant challenges. The need to harmonize procedures for suspect screening and non-targeted analyses (Pourchet et al., 2020), to develop new biomarkers for non-invasive biomonitoring (Esteban and Castaño, 2009), and to establish effective and timely communication of HBM results (Exley et al., 2015) are just a few of examples of topics that need to be further explored in the next years for HBM advancement.

Conclusion Put together, biomonitoring information contributes to knowledge on the effects that certain chemicals (or other agents) may have on the environment and human health and lead to the discussion of risk reduction strategies. Data provided by environmental and human biomonitoring may provide support to political decision-making processes, urgently needed to balance environmental and human health with current challenges associated to chemical usage and climate change extreme events.

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Au WW (2007) Usefulness of biomarkers in population studies: From exposure to susceptibility and to prediction of cancer. International Journal of Hygiene and Environmental Health 210(3–4): 239–246. Augusto S, Shukla V, Upreti DK, Paoli L, Vannini A, Loppi S, Nerín C, Domeño C, and Schuhmacher M (2016) Biomonitoring of Airborne Persistent Organic Pollutants Using Lichens. Nova Science Publishers, Inc. Bartram J and Ballance R (eds.) (1996) Water Quality Monitoring: A Practical Guide to the Design and Implementation of Freshwater Quality Studies and Monitoring Programmes. CRC Press. Bohan D, Dumbrell A, Woodward G, and Jackson M (2018) Next Generation Biomonitoring: Part 1. Academic Press. Boogaard PJ (2009) Biomonitoring of the workplace and environment. In: General, Applied and Systems Toxicology, 3rd edn, pp. 2559–2589. Hoboken: Wiley. Burger J (2006) Bioindicators: Types, development, and use in ecological assessment and research. Environmental Bioindicators 1(1): 22–39. 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International Journal of Hygiene and Environmental Health 210(3–4): 479–482. Pourchet M, Debrauwer L, Klanova J, Price EJ, Covaci A, Caballero-Casero N, Oberacher H, Lamoree M, Damont A, Fenaille F, and Vlaanderen J (2020) Suspect and non-targeted screening of chemicals of emerging concern for human biomonitoring, environmental health studies and support to risk assessment: From promises to challenges and harmonisation issues. Environment International 139: 105545. Sobus JR, DeWoskin RS, Tan YM, Pleil JD, Phillips MB, George BJ, Christensen K, Schreinemachers DM, Williams MA, Hubal EAC, and Edwards SW (2015) Uses of NHANES biomarker data for chemical risk assessment: Trends, challenges, and opportunities. Environmental Health Perspectives 123(10): 919–927. Tan YM, Dary CC, Chang EM, Ulrich JM, Van Emon JM, Xue J, Pleil JD, Kenneke JF, Sobus J, Sheldon LS, and Morgan MK (2012) Biomonitoring–An Exposure Science Tool for Exposure and Risk Assessment. 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Bioremediation Eric A Seagren, Department of Civil, Environmental, and Geospatial Engineering, Michigan Technological University, Houghton, MI, United States © 2024 Elsevier Inc. All rights reserved. This is an update of M. Megharaj, K. Venkateswarlu, R. Naidu, Bioremediation, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 485-489, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01001-0.

Introduction Biodegradation mechanisms The spectrum of biodegradation Energy metabolism versus cometabolism Factors limiting biodegradation potential in the field Genetic potential Environmental conditions Primary substrates Other nutrients Temperature pH Moisture Toxicity Contaminant availability Bioremediation strategies and applications The spectrum of bioremediation approaches Site characterization Intrinsic in situ bioremediation and monitored natural attenuation Engineered in situ bioremediation Water circulation systems Air sparging and biosparging Bioventing and bioslurping In situ bioreactive barriers Phytoremediation Ex situ bioremediation Land treatment Composting and biopiles Slurry-phase treatment Summary and conclusion Acknowledgment References

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Abstract Bioremediation uses biodegradation processes to either eliminate environmental contaminants or reduce their risks. Biodegradation processes transform organic contaminants, sometimes resulting in complete conversion to inorganic products (mineralization), or alter the transport of inorganic contaminants. In some cases, naturally-occurring intrinsic bioremediation is sufficient for risk reduction; however, numerous factors can limit biodegradation in the environment. Engineered bioremediation strategies focus on promoting biodegradation and overcoming limitations. In situ bioremediation approaches treat the contamination in place, and are classified by the technique for adding stimulating amendments. Ex situ bioremediation technologies involve excavation of the contaminated materials and treatment in different types of bioreactors.

Keywords Bioaugmentation; Biodegradation; Bioremediation; Biostimulation; Biotransformation; Cometabolism; Engineered bioremediation; Ex situ bioremediation; In situ bioremediation; Intrinsic bioremediation; Mineralization; Monitored Natural Attenuation; Phytoremediation

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Bioremediation uses biodegradation processes to eliminate environmental contaminants. A major advantage of bioremediation is that biodegradation processes possess the potential to convert contaminants to harmless products. Biodegradation processes for organic contaminants range from mineralization, i.e., the complete conversion to inorganic products and elimination of the contaminant’s hazard, to biotransformation reactions, which do not result in significant changes to the parent compound’s structure, and may or may not be reduce risk. Most inorganic contaminants cannot be degraded, and bioremediation is focused on altering their transport properties. A number of biological and physical/chemical factors can impact whether contaminant biodegradation occurs and the rate of biodegradation, including: genetic potential, environmental conditions, toxicity, and bioavailability. Intrinsic bioremediation, i.e., contaminant biodegradation using the naturally occurring capabilities of the native microorganisms, is sometimes sufficient for risk reduction. Engineered bioremediation strategies focus on promoting and enhancing biodegradation by modifying the site to overcome any limiting factors. These strategies may be performed in situ or ex situ, and vary in their aggressiveness. In situ bioremediation approaches use different methods for adding amendments to the subsurface, including water circulation, air injection, bio-barriers, and agronomic techniques. Ex situ bioremediation involves excavation of the contaminated materials followed by treatment in a bioreactor, e.g., land treatment, composting/biopiles, and slurry phase treatment.

Introduction Bioremediation has been defined as “the intentional use of biodegradation processes to eliminate environmental pollutants from sites where they have been intentionally or inadvertently released” (Madsen, 1997). Contaminants that are commonly treated via bioremediation include oil and refined petroleum products, solvents, and pesticides (USEPA, 2012). Biodegradation processes are typically mediated by microorganisms (e.g., bacteria, fungi) and/or plants (i.e., phytoremediation). Bioremediation applications at contaminated sites may require the control, manipulation, and monitoring of the biological process(es) of interest in the soil or subsurface (i.e., in situ (in place) technologies), or in surface reactors (i.e., ex situ (above ground) technologies). Bioremediation has a number of advantages. Perhaps most importantly, biodegradation holds the potential to convert contaminants to harmless products, as opposed to transferring contaminants from one environmental medium to another (e.g., from water to air), as some physical-chemical treatment methods do. It is also often considered to be a “natural” process because it typically uses existing native microorganisms in the soil and groundwater that transform contaminants, and can be accomplished at a contaminated site (USEPA, 2012). The latter is advantageous because it eliminates the need to excavate or pump and transport contaminated waste off site and avoids the associated risks to human and environmental health. For example, in situ bioremediation can be carried out with minimal site disruptions, fugitive emissions, and contact risks to residents. Finally, bioremediation may not require as much equipment, labor, or energy as some other remedial approaches, making it a lower cost alternative (USEPA, 2012). However, although bioremediation may have lower capital costs than many methods, it also may result in higher overall operation and maintenance costs if the remediation must occur over a longer period than more aggressive methods. Despite its advantages, there are a number of challenges associated with the implementation of bioremediation, as summarized by Becker and Seagren (2010). For example, as with all remediation approaches, there are the difficulties associated with the inaccessibility and heterogeneity of many contaminated environments. There are also challenges specific to bioremediation because of the need to understand how contaminants are biodegraded. For example, difficulties may be encountered in achieving cleanup goals with bioremediation if the contaminants are non- or partially-biodegradable, or if the contaminant levels required cannot be achieved microbially. In some cases, toxic byproducts may be formed during biotransformation and, as a result, the process must be carefully monitored to ensure effectiveness of degradation. In addition, it is necessary to understand how microbial activities are impacted by environmental conditions and interactions with other microbial populations, and how the beneficial microbial processes can be enhanced through engineering activities. This description of bioremediation is organized around the broad themes of possibilities, limitations, and applications. First, the potential mechanisms by which microorganisms can possibly destroy or transform contaminants are reviewed. This mechanistic summary is followed by a review of the factors that can limit or influence biodegradation effectiveness and efficiencies in the field, and a summary of the various bioremediation strategies and applications that are implemented in the field to overcome those limitations.

Biodegradation mechanisms The spectrum of biodegradation Bioremediation’s potential lies in the wide range of chemical transformations that are possible via biodegradation processes (Gao et al., 2010). The term biodegradation refers to the biological transformation of a substance to another form without regard to the

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extent, desirability, or whether it supports microbial growth (ASCE, 2004). The biodegradation of organic contaminants, which have been the focus of most bioremediation applications, can be divided into two broad classes of transformations: mineralization, and biotransformation. Mineralization refers to the complete conversion of organic chemicals to inorganic products (e.g., CO2, H2O, Cl−, NH+4). An example of mineralization is the aerobic oxidation of normal alkanes (e.g., hexadecane) found in petroleum products to CO2 and H2O: CH3 ðCH2 Þ14 CH3 + 24:5 O2 ➔16 CO2 + 17 H2 O

(1)

Because the products of mineralization are generally harmless at the concentrations resulting from microbial metabolism, mineralization will usually eliminate the toxic hazard associated with the chemical of concern. In comparison, biotransformation refers to the conversion of an organic contaminant by biochemical reactions that do not result in the complete conversion to inorganic products. An example of a biotransformation reaction is the reductive dehalogenation of tetrachloroethene (PCE) to trichloroethene (TCE): Cl2 C ¼ CCl2 + H2 ➔ClCH ¼ CCl2 + H+ + Cl −

(2)

The extent to which biotransformation reactions reduce the hazard associated with the contaminated media depends on the relative toxicity of the substrates and products. Accordingly, some biotransformation reactions reduce risk, some increase it, and others are risk neutral. For example, the sequential anaerobic conversion of PCE to TCE, and then dichloroethene (DCE) and vinyl chloride (VC) transforms a likely carcinogen into a known carcinogen, whereas complete dehalogenation to ethene results in an acceptable product (Freedman and Gossett, 1989). Bioremediation can also be applied to inorganic contaminants such as metals (e.g., chromium), radionuclides (e.g., uranium), and anions (e.g., nitrates, and cyanides); however, most inorganic contaminants cannot be degraded (Bolton Jr. and Gorby, 1995). Rather, the focus of bioremediation is on altering their transport properties by immobilizing (i.e., bioimmobilization), mobilizing (i.e., biomobilization), or biotransforming the inorganic contaminants. For example, microbial reduction of oxidized uranium, U(VI), to reduced uranium, U(IV), shown here coupled to oxidation of acetate, is a possible mechanism for bioremediation of uranium-contaminated groundwater (Lovley et al., 1991): CH3 COO − + 4UðVIÞ + 4H2 O➔4UðIV Þ + 2HCO3  + 9H+

(3)

U(VI) is soluble and readily transported in groundwater, whereas U(IV) is insoluble and precipitates out of solution, thereby immobilizing and concentrating the uranium and decreasing the aqueous phase concentration and associated hazard (Barkay and Schaefer, 2001).

Energy metabolism versus cometabolism Biodegradable substrates can also be divided into primary and secondary substrates based on their ability to support microbial growth. Primary substrates are those that support microbial growth by being able to serve as sources of carbon and/or energy. These substrates can be further subdivided into two categories: (1) substrates that are used in energy-generating pathways, such as electron-donor substrates that are oxidized during metabolism (e.g., hexadecane in Eq. (1), H2 in Eq. (2), and acetate in Eq. (3)) and electron-acceptor substrates that are reduced during metabolism (e.g., O2 in Eq. (1), PCE in Eq. (2), and U(VI) in Eq. 3); and (2) precursors for cell synthesis, including carbon sources (e.g., organic compounds or CO2), macronutrients (e.g., N, P, S, K, Na, Ca, Mg), and micronutrients (e.g., Fe, Co, Se, Cu, Mn, etc.) (ASCE, 2004). The mineralization reactions discussed above are often the result of metabolic processes and linked to energy conservation and biomass synthesis (Alexander, 1981), with the contaminant serving as either an electron donor or an electron acceptor. Often, the electron donor is also the carbon source for the microbe. Metabolic biodegradation processes facilitate bioremediation because the contaminants that serve as growth substrates naturally select for microbial populations that can carry out those reactions, and as the microbial population grows on the contaminant, the size of the population increases, thereby increasing the biodegradation rates. This has important practical implications because the potential for successful implementation of bioremediation is the greatest when the biodegradation process leads to mineralization and complete contaminant detoxification (Becker and Seagren, 2010). In fact, many organic contaminants are highly reduced and can serve as metabolic electron donors and/or carbon source during biodegradation reactions (e.g., Eq. 1). In comparison, chlorinated organic compounds are highly oxidized and frequently contribute to growth by serving as the terminal electron acceptor in dehalorespiration processes (Fetzner, 1998) (e.g., Eq. 2). If the complementary electron acceptor or donor required to sustain the biodegradation process is not available in sufficient quantities, then the limiting growth substrate must be provided via one of the various bioremediation strategies. Secondary substrates do not support microbial growth, meaning that the degrading organisms must also be supplied a primary substrate. Secondary substrates are unable to support microbial growth for one of two reasons (ASCE, 2004). One, the compound is present at too low of concentrations to supply sufficient energy to meet the microorganism’s maintenance energy requirements and support biosynthesis. This differs from primary utilization only with respect to the bioavailable substrate concentration. Two, the compound is biodegradable, but the transformation does not yield free energy or carbon that can be used by the microorganisms mediating the reactions. The latter are referred to as cometabolic processes (Horvath, 1972). Dalton and Stirling (1982) defined cometabolism as “the transformation of a non-growth substrate in the obligate presence of a growth substrate or another transformable compound.” However, the presence of a growth substrate or other transformable compound is not always required,

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provided that the enzymes responsible for the cometabolic reaction are present and the cells have been grown under conditions in which the cell has endogenous reserves that are able to generate the necessary cofactors for the transformation, thereby allowing the non-growth substrate to be biotransformed fortuitously (Rittmann et al., 1994). Cometabolism usually results from the non-specificity of an enzyme that is involved in the normal catabolic pathways of the cometabolizing organism. As a result, the target contaminant can bind to the enzyme’s active site and be transformed by a reaction analogous to that normally catalyzed by the enzyme. For example, the toluene dioxygenase enzyme from Pseudomonas putida F1 has been shown to act on over 100 substrates of varying structural characteristics (Gao et al., 2010). Often, the regular substrate for the enzyme must be available to the cells to induce the required enzyme synthesis. Thus, in some cases, the growth substrate may compete with the cometabolic substrate for the enzyme. Cometabolism may also be detrimental to cells because it diverts energy from growth and/or generates highly reactive products that are toxic to cells. For example, aerobic cometabolic biotransformation of TCE by a dioxygenase enzyme generates short-lived products, which can inactivate enzymes and other cellular components, resulting in biodegradation and growth rates decreasing over time (Wackett and Householder, 1989). Finally, successful bioremediation may sometimes require coupling cometabolic reactions that only slightly modify contaminant structures with additional biodegradation processes. For example, the toluene dioxygenase of P. putida F1 cometabolically converts TCE to glyoxylate and formate (Li and Wackett, 1992), which may ultimately may be mineralized by other populations in mixed microbial cultures.

Factors limiting biodegradation potential in the field Despite the broad potential for microbially mediated biodegradation of environmental contaminants, a number of factors can cause contaminant compounds to resist biodegradation in the environment (Rittmann et al., 1994). Key biological and physical/ chemical factors impacting whether contaminant biodegradation occurs and the rate of biodegradation include: genetic potential, environmental conditions, toxicity, and bioavailability.

Genetic potential A common strategy for bioremediation is to stimulate the growth and biodegradation potential of the indigenous microorganisms, e.g., via addition of a key limiting substrate such as an electron donor or acceptor, i.e., biostimulation. This requires that the appropriate genes encoding for enzymes that degrade the contaminants of interest must be present and expressed by the indigenous microbial community (Rittmann et al., 1994). Thus, the necessary genetic potential or capability must exist. All remediation projects begin with site characterization, and for bioremediation projects, this provides an opportunity to assess the genetic capability of the site (Becker and Seagren, 2010). Preliminary laboratory treatability studies (e.g., slurry microcosms, soil column tests) may be conducted if there is any uncertainty about the suitability of the contaminant or site characteristics for biodegradation (Rittmann et al., 1994). Such assessments are typically not necessary for proven bioremediation scenarios that do not depend on unique organisms or conditions. However, treatability assays are needed for biodegradation of contaminants that are highly site-specific. In addition, conventional treatability studies are increasingly being complemented by molecular-biology based environmental molecular diagnostics (EMDs), which can be used during site characterization to directly evaluate what microbes are present and their relative abundance, and what metabolic capabilities are present and being expressed (ITRC, 2011a). One of the more commonly applied molecular-biology based EMDs has been quantitative polymerase chain reaction (qPCR), which quantifies a target gene, and can be used to quantify abundance and expression of specific functional genes, specific microorganisms, or groups of related microorganisms (ITRC, 2011b). For example, indigenous microbial populations with the capacity to respire PCE and TCE and reductively dechlorinate them to DCE are generally assumed to be present at PCE and TCE contaminated sites, but the subsequent reductive dichlorination of DCE and VC is not observed at many sites and appears to require the presence of specific Dehalococcoides strains that can dehalorespire these lesser chlorinated ethenes using hydrogen as the electron donor (Ritalahti et al., 2006). Therefore, qPCR-based methods can be used to detect and quantify the VC reductive dehalogenation genes in Dehalococcoides and assess sites for the potential for complete detoxification of PCE and TCE. In addition, reverse transcriptase qPCR (RT-qPCR) can also be used to detect the enzyme products of specific genes when they are expressed (ITRC, 2011b). For example, RT-qPCR assays that quantify the expression of dehalogenase genes in Dehalococcoides strains can be applied at these sites, thereby offering even greater potential for describing the activity of these organisms, and the likelihood for successful bioremediation under different conditions (Cupples, 2008). If it is necessary to increase the genetic capability, the approach used depends on the degradation mechanism (Rittmann et al., 1994). If the contaminant is a growth substrate, present at sufficient concentrations to support substantial growth, accumulation of sufficient biomass is relatively simple. Specifically, bacteria capable of utilizing the contaminant (after specific genes are expressed and enzymes produced) are likely to gain a competitive advantage, and accumulate to be a significant portion of the biomass (i.e., selective enrichment occurs). It may be necessary to promote the selective enrichment by biostimulation. However, if the required contaminant-degrading population is not native to the site, it may be possible to add a laboratory-grown culture with large numbers of the required organisms through an engineered bioremediation process known as bioaugmentation. Biodegradation processes that are highly site-specific may be good candidates for bioaugmentation, if indicated by treatability studies or molecular-biology based EMDs. For example, bioaugmentation of sites with DCE- and VC-respiring Dehalococcoides strains may be necessary for complete detoxification of PCE and TCE if key dehalogenases are lacking at a site or not being expressed (Hazen, 2010).

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Environmental conditions Environmental conditions potentially affecting microbial growth and biotransformation, include primary substrate and nutrient levels, temperature, pH, and moisture levels (Rittmann et al., 1994; Alexander, 1999). These conditions are sometimes modified as part of bioremediation strategies, as described below, although it is not always possible or economical to modify or control some of these factors.

Primary substrates If the appropriate microorganisms are present, the next key requirement for chemotrophic microbes (i.e., microbes that use chemical energy sources) is an appropriate electron-donor and -acceptor (Cookson, 1995). Many organic contaminants can serve as the sole carbon and energy source or as an electron acceptor. However, bioremediation can be limited by very low concentrations of the contaminants serving in these roles. If the target organic contaminant concentration is too low to support growth, or cannot support growth for some other reason, it must be degraded as a secondary substrate, with another primary substrate supporting microbial growth (Cookson, 1995). On the other hand, if readily utilizable organic contaminants are introduced into the environment at sufficiently high levels, one or more of the previously non-limiting substrates or nutrients may become limiting (Alexander, 1999). For example, high concentrations of organic contaminants like hydrocarbons that can serve as an electron donor may result in low electron-acceptor concentrations. This is important because the redox condition will limit the species of microorganisms active in a particular region and the potential for the biotransformation of contaminants (McNabb and Dunlap, 1975). For hydrocarbons, the preferred electron acceptor is oxygen, and biodegradation rates are more rapid under aerobic conditions than anaerobic (Alexander, 1999). Sites where significant hydrocarbon contamination is present will often be anaerobic (Alexander, 1999). As a result, many bioremediation technologies are focused on the addition of oxygen, as discussed further below. In addition, electron acceptors other than oxygen are frequently present in the subsurface environment (e.g., sulfate, carbon dioxide, nitrate, manganese oxides and iron oxyhydroxides) (McNabb and Dunlap, 1975; Matthess, 1982; Ghiorse and Wilson, 1988), and numerous organic compounds can be biodegraded anaerobically. Nevertheless, high levels of contamination could quickly result in the depletion of these electron acceptors as well, with natural rates of replenishment likely to be very slow (Ghiorse and Wilson, 1988). Given the limited natural capacity for electron acceptor replenishment, the coupling of contaminant transport via groundwater flow and biodegradation is likely to result in spatial gradients of electron acceptor concentrations (Bouwer, 1992), with a progression in redox conditions expected to occur along the groundwater flow path moving away from a contaminated zone. Heterotrophic microorganisms are predicted to sequentially use the available electron acceptors in the following order, according to the free energy released during respiration: oxygen, nitrate, Mn(IV) and Fe(III), sulfate, and carbon dioxide.

Other nutrients Chemotrophic microorganisms also require nutrients for the synthesis of cellular materials. These nutrients are often classified as the: (1) “major” elements C, H, O, N; (2) “minor” elements P, K, S, Mg; (3) growth factors (e.g., vitamins, amino acids); and (4) trace elements (Pirt, 1975). If one or more of these substances are lacking, further microbial growth or metabolic activity is effectively limited. The carbon and energy sources discussed above are often also sources of oxygen and hydrogen (Pirt, 1975), and the supply of K, S, Mg, Ca, Fe, and micronutrient elements is almost always greater than the demand (Alexander, 1999). Thus, the nutrients usually in short supply are N and/or P. The addition of N and P has been shown to stimulate biodegradation in soils (Efroymson and Alexander, 1994) and groundwater samples (Jamison et al., 1975). Accordingly, bioremediation applications have often incorporated inorganic nutrient additions (e.g., see in situ bioremediation examples in Lee et al. (1988) and Staps (1990)). Nevertheless, the conclusions drawn from laboratory and field data regarding the impact of added nutrients on bioremediation have been mixed, with many laboratory studies indicating nutrient additions increase degradation rates, while field observations have often suggested there is little effect (Cookson, 1995).

Temperature Soil and groundwater temperatures will fluctuate seasonally from the surface down to a depth of approximately 10 m, below which the groundwater temperature is determined largely by the region’s mean annual temperature (Kuznetsov et al., 1963). This is important for bioremediation because temperature is a key environmental factor impacting microbial growth and biotransformation rates (Alexander, 1999). For example, temperature affects hydrocarbon biodegradation via complex interactions between effects on: (1) the physical nature and chemical composition of the hydrocarbons, (2) the rate of hydrocarbon metabolism by microorganisms, and (3) the microbial community composition (Atlas, 1981; Leahy and Colwell, 1990). In general, biodegradation rates decrease with decreasing temperature, although seasonal changes may also select for a shift in the composition of the microbial community to contaminant-degrading microorganisms adapted to the ambient temperature (Atlas and Bartha, 1973). Nevertheless, it is uncommon to intentionally alter temperatures for in situ bioremediation because of energy costs.

pH The pH of groundwater typically is in the range 6–9 (Freeze and Cherry, 1979; Matthess, 1982), but the pH of soil can range from acidic, in areas where sufficient rainfall occurs to leach bases from the soil, to alkaline, primarily in arid and semi-arid regions (Hartel, 1998). Bacterial biodegradation rates tend to be fastest at near neutral pH (Alexander, 1999), although fungi prefer acidic

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conditions (Maier, 2000). For acidic soils and subsoil materials, or in soil and groundwater with low alkalinity that have become acidic due to microbial activities (e.g., production of organic acids or HCl from reductive dehalogenation (Eq. 2), it is common to add lime to neutralize the pH (Cookson, 1995). Alternatively, oxidation of reduced sulfur compounds by sulfur bacteria results in the production of protons and can be used to lower the pH of alkaline soils or groundwater (Madigan et al., 1997).

Moisture Microbes require moisture to sustain growth and activity; thus, moisture levels that are insufficient can severely restrict biodegradation in surface soils, which are subject to drying to suboptimal levels (Alexander, 1999). The optimum moisture level is a function of the soil properties, specific compound, and if the desired biotransformation is aerobic or anaerobic (Alexander, 1999). For example, Dibble and Bartha (1979) observed that the optimum moisture level for biodegradation of oily sludges was 30–90% of the soil’s water-holding capacity. When moisture levels are high, there is less air-filled pore space and the soil soon becomes anaerobic due to the slow rate of oxygen diffusion through water. However, if the moisture levels become too low, degradation rates also decrease (Hinchee and Arthur, 1991)

Toxicity Many of the common groundwater contaminants (e.g., hydrocarbons, and halogenated hydrocarbons), or the products of their metabolism, may limit biodegradation potential by exerting an inhibitory effect on cellular metabolism (i.e., microbial growth and/or substrate consumption), thereby preventing the development of the contaminant degrading population (Rittmann et al., 1994). Like biodegradability, toxicity depends on the physiology of the microorganisms and the contaminant chemical characteristics. Inhibition of microbial growth and substrate utilization by toxic compounds can result from two classes of mechanisms: (1) interference with a specific metabolic pathway, or (2) more general, non-specific interactions with microbial cells. Inhibition by the first mechanism occurs if the toxicant inhibits a single enzyme in a key metabolic pathway, whereas inhibition by the second mechanism occurs with compounds that interfere with the normal functions of the cell membrane, e.g., when the cell membrane becomes leaky and concentration gradients across the membrane cannot be maintained. There are also toxicants that inhibit microbial growth in a way that is neither completely specific or nonspecific, e.g., by inhibiting enzymes from participating in the reactions that are their normal function.

Contaminant availability Although the environmental conditions clearly impact microbial activity, modifying them (e.g., nutrient additions) as part of bioremediation strategies may not have a positive impact in the field (Cookson, 1995). This could be a result of inadequate delivery and distribution of nutrients in the field, adequate N and P levels in the soil or present in the organic pollutants, or the existence of some other, more rate-limiting factor, such as contaminant availability (Cookson, 1995; Alexander, 1999). Indeed, field and laboratory studies suggest that a large fraction of the contaminants present in environmental systems is unavailable for microbial degradation (Alexander, 1995; Beck et al., 1995; Bosma et al., 1997). Such observations indicate that the overall biotransformation rate observed depends not only on the microbially mediated biodegradation rates and the environmental conditions that affect them, but also on physicochemical constraints that control contaminant bioavailability, i.e., the fraction of contaminant molecules that are dissolved and available to the microorganisms (Ramaswami and Luthy, 1997; Sturman et al., 1995). Two key phenomena potentially limiting contaminant bioavailability at many sites are their presence in the form of a nonaqueous phase liquid (NAPL) and sorbed to solid surfaces. Many of the organic contaminants of concern (e.g., hydrocarbons, chlorinated solvents) are present as NAPLs, which are transported or used as separate-phase liquids and have relatively low water solubility. Spills, leaks, and improper disposal of NAPLs can migrate through the subsurface as a separate liquid phase, ultimately becoming trapped in the subsurface in various configurations ranging from blobs to pools (AGU, 2005). Entrapped NAPLs can serve as long-term sources of contamination as they slowly dissolve into the groundwater and volatilize into the soil gas. These NAPL contaminant sources can also limit the bioavailability of contaminants as microbial substrates due to interphase mass-transfer limitations and partitioning into a NAPL, which can reduce the aqueous phase concentrations, and decrease biodegradation rates (Fu and Alexander, 1995; Ghoshal et al., 1996; Labare and Alexander, 1995; Yang et al., 1995). When biodegradation rates are limited by the NAPL dissolution rate, possible techniques for enhancing the overall biotransformation rate during bioremediation include cosolvent or surfactant additions (Zhang et al., 1998). Given the large surface area present in subsurface porous media, sorption processes may determine the physical/chemical conditions by sorption of microbial cells and alteration of aqueous contaminant concentrations (Ghiorse and Wilson, 1988; Madsen and Ghiorse, 1993; van Loosdrecht et al., 1990). If the sorption sink is significant, it can limit the bioavailability of contaminants due to interphase mass-transfer limitations and by reducing the aqueous concentration, thereby reducing the bioavailable substrate (Mihelcic and Luthy, 1991; Miller and Alexander, 1991; Scow and Alexander, 1992; van Loosdrecht et al., 1990). However, if solute concentrations are toxic to the microorganisms, sorption may also positively influence biodegradation rates by sufficiently reducing the solute concentration to allow biodegradation (Apajalahti and Salkinoja-Salonen, 1984; Ehrhardt and Rehm, 1985; Morsen and Rehm, 1987). If the biodegradation rates are limited by the sorption/desorption rate, some means of

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improving the desorption kinetics is required. Methods used to accomplish this in the field include application of surfactants (e.g., Franzetti et al., 2010, and thermal approaches (e.g., Kosegi et al., 2000). Mass transport via advection and dispersion can also significantly impact contaminant distribution and the availability of substrate, nutrients, and electron acceptors to microorganisms capable of contaminant biodegradation (Sturman et al., 1995). For example, flushing of water through the system can potentially accelerate NAPL dissolution and desorption by concomitantly increasing the mass-transfer coefficient and the concentration gradient (e.g., Miller et al., 1990; Powers et al., 1991; Seagren et al., 1999; Seagren and Moore, 2003). However, if the rate of advection of substrate is too fast compared to the biodegradation rate, there is not sufficient time for biodegradation (Seagren et al., 1993). Only if increased flushing lowers solute concentrations below toxic levels, is the biodegradation sink increased (Seagren et al., 2002).

Bioremediation strategies and applications Up to this point, the focus has been on biodegradation mechanisms and the factors that limit that biodegradation potential in the field. The bioremediation technologies covered in the remainder of this review use the physiological potential of microorganisms and/or plants to eliminate pollutants at field sites. The selected bioremediation strategy must reduce the pollutant concentration(s) to levels that are acceptable to the site owners and regulatory agencies involved.

The spectrum of bioremediation approaches The strategies for using bioremediation to reduce or eliminate contaminant concentrations can be categorized based on the location where the contaminants are metabolized, and the aggressiveness of the remediation (Madsen, 1997). With respect to location, bioremediation technologies are categorized as being in situ or ex situ. In situ bioremediation is performed with the contaminated material left in its natural or original position. In comparison, ex situ bioremediation involves removing the contaminated material from its original position, and treating it in some type of bioreactor system. Applications of in situ and ex situ technologies include solid-, slurry-, and vapor-phase systems. In terms of aggressiveness, in situ and ex situ bioremediation approaches can be divided into two broad classes (NRC, 1993; Madsen, 1997). At the passive end of the spectrum is what is sometimes referred to as intrinsic bioremediation, also known as “natural”, “passive”, and “spontaneous” bioremediation and “bioattenuation.” This approach is based solely on using the innate capabilities of the naturally-occurring microorganisms to degrade the contaminants. Because this occurs with the native microbes and contaminated material in their original location, it is by definition an in situ approach. At the aggressive end of the scale, bioremediation applications incorporate actions to modify the site or contaminated material to overcome any factor(s) limiting biodegradation, with a goal of promoting and enhancing the desired biodegradative activities, e.g., via biostimulation or bioaugmentation. Generally, these technologies are referred to as “engineered” or “enhanced” bioremediation approaches.

Site characterization Site characterization and treatability studies are performed as part of bioremediation projects to understand if it is necessary to engineer the system and, if so, how (e.g., what limitations need to be addressed). Evaluating the potential for applying bioremediation first requires development of a conceptual site model (Hazen, 2010), which is used to initiate development of a general understanding of the site, evaluate potential risks to human and environmental health, and set priorities for site activities (USEPA, 1989). For most contaminated sites, the initial site assessment will have established the nature and the extent of contamination, and provided some general site geology and groundwater hydrology data (e.g., flow direction, hydraulic gradients, general nature of the aquifer) (Cookson, 1995). However, these data are insufficient for evaluating the appropriateness of bioremediation, or designing a bioremediation system. Site characterizations for bioremediation must also define the potential biological systems, and evaluate site characteristics that impact biodegradation and process control. The first step in any bioremediation project is to evaluate the contaminant’s potential for biodegradation, delineate the microbial processes capable of providing degradation, and verify the potential for a successful bioremediation (Cookson, 1995). This requires appropriate treatability studies, and assessment of microbial community structure and function, as discussed above. Next, the site characterization activities need to: (1) delineate any environmental modifications necessary for optimizing biodegradation, and (2) define the site-specific characteristics important to bioremediation (Cookson, 1995). Information required for optimizing the biodegradation processes includes: (1) potential carbon and energy sources for the microbes, (2) electron acceptor availability and the redox condition, (3) existing microbial activity and potential toxicity, (4) availability of nutrients (especially nitrogen and phosphorus), and (5) the status of the site’s environmental parameters significant to microbial activity (e.g., temperature, pH, moisture). Key site-specific characteristics important to bioremediation are those necessary for manipulating the microbial processes in the subsurface via engineering process control and operation. One particularly important site characteristic is the capacity for the porous medium to transmit water (i.e., the hydraulic conductivity, K), or fluids in general (i.e., the intrinsic permeability, k), because the subsurface conditions are often controlled via injection and/or extraction of either water or air. For engineered bioremediation systems using groundwater circulation, K should be at least on the order of 10−4 cm s−1; for systems using air circulation, the intrinsic permeability should be >10−9 cm2 (NRC, 1993). In the case of intrinsic bioremediation, it

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is important that the velocity and direction of groundwater flow be relatively consistent. Other key hydrogeological site characteristics include: natural groundwater gradients, flow direction and velocity; soil grain size distribution and classification; porosity; bulk density; and heterogeneities in an aquifer. The heterogeneous and complex nature of the subsurface and its effect on fluid flow can cause poor distribution of amendments, and generally make completely reliable predictions of contaminant fate and transport impossible (NRC, 1993; Cookson, 1995). Based on the type and extent of contamination, the microbially mediated removal processes effective for those contaminants, the nature of the site, and the goals for managing the site (e.g., simple containment, meeting specific regulatory standards, etc.), a bioremediation strategy for encouraging sufficient growth of the appropriate microbes can be developed (NRC, 1993).

Intrinsic in situ bioremediation and monitored natural attenuation Intrinsic in situ bioremediation exploits the naturally occurring capabilities of the native microorganisms to biodegrade the contaminants of concern, thereby containing the contaminants and preventing their migration away from the source (NRC, 1993; Rittmann and McCarty, 2001). This occurs without attempting to engineer the site to enhance the process. However, this is not a “do-nothing” alternative, because it requires thorough documentation of the role played by the native microbes in removing the contaminants. This documentation can take the form of tests performed in the field or using site-derived samples. In addition, the site must be monitored to demonstrate the effectiveness of the intrinsic bioremediation for preventing contaminant migration. Intrinsic bioremediation also requires a careful site characterization. Important site characteristics include consistent groundwater speed and direction, as well as the presence of minerals such as carbonates to buffer pH changes, and high ambient concentrations of electron acceptors (or donors) and elemental nutrients (especially N and P). Intrinsic bioremediation can be used in combination with other remedial technologies, such as engineered bioremediation. In addition, intrinsic bioremediation is a component of the broader remedial approach known as monitored natural attenuation, which refers to contaminant reduction via all types of naturally occurring biological, physical, and chemical processes, including biodegradation, dispersion, dilution, sorption, volatilization, and chemical reactions.

Engineered in situ bioremediation Engineered in situ bioremediation strategies are designed to enhance the intrinsic biodegradation of contaminants. This is typically accomplished via the input of amendments, i.e., stimulatory materials, into the contaminated region (Rittmann et al., 1994). By supplying limiting substrates in such a way that no contaminant escapes biodegradation, a biologically active zone (BAZ) can be established. Therefore, the success of a bioremediation approach depends upon the effectiveness of the system used to input the substrates designed to overcome the limiting factor(s). Correspondingly, the engineered in situ bioremediation techniques can be broadly classified based on how the stimulating amendments are added to the subsurface.

Water circulation systems When the contaminants are present in the saturated zone, then a water circulation system can be used to apply the stimulatory materials (NRC, 1993; Rittmann et al., 1994; Rittmann and McCarty, 2001). Typically, a water circulation system couples the injection of dissolved limiting substrates, with hydraulic control of the plume migration to force the flow of the contaminated water through the BAZ (Fig. 1). Input of limiting substrates is commonly accomplished using: (1) vertical or horizontal wells and (2) infiltration galleries. Wells are the most direct method, but they are more expensive and more prone to clogging. In comparison, infiltration galleries are less expensive and less prone to clogging and can also be used to remediate the unsaturated zone; however, they are not effective as wells if the contaminated zone is deep below the water table. Techniques for avoiding clogging include the pulsing of the input of stimulatory materials, so that less intense microbial growth is promoted at some distance from the injection point. The groundwater capture system commonly includes wells, or in some cases trenches, which typically extract more water than is injected. Extraction is usually followed by treatment (e.g., using air stripping, biological treatment, activated carbon, etc.) and return to the groundwater via the injection system. Often a portion of the extracted water must be treated and exported off site. A fundamental problem with water circulation systems is how to supply sufficient oxygen to the subsurface to support aerobic biodegradation (NRC, 1993; Norris, 1994; Reinhard, 1994; Bouwer, 1992). This is due to the limited amount of oxygen (8–10 mg L−1) that can be delivered from the air into water, coupled with the high oxygen demand (Eq. 1), which results in the need to pump large quantities of water. The use of pure oxygen instead of air can reduce the water required significantly, but it also increases the cost. Hydrogen peroxide, which disproportionates to produce O2 (H2O2 + H2O2 ! O2 + 2H2O), can theoretically be used to reduce the volume of water required due to the greater water solubility of H2O2 compared to O2. However, hydrogen peroxide can be consumed by competing abiotic processes, can be toxic to microorganisms and, if catalysts in the soil cause disproportionation to occur too quickly, can result in formation of oxygen gas bubbles, which reduces O2 bioavailability and lowers aquifer permeability (Bouwer, 1992; Leeson and Hinchee, 1997). Alternatively, other electron acceptors can be used, e.g., nitrate, which may be present as a co-contaminant (Bouwer, 1992). If the contaminant is used as an electron acceptor (e.g., chlorinated ethenes), the challenge then becomes adequate supply of dissolved electron donor.

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Fig. 1 Conceptual diagram of a water circulation system for bioremediation of contaminated groundwater. From FRTR (2022) Federal Remediation Technologies Roundtable [Online]. U.S.A.: United States Government. Available: https://frtr.gov [Accessed March 31 2022].

Fig. 2 Schematic of an air sparging or biosparging system. From FRTR (2022) Federal Remediation Technologies Roundtable [Online]. U.S.A.: United States Government. Available: https://frtr.gov [Accessed March 31 2022].

Air sparging and biosparging Air sparging and bioventing, which is described below, were developed based on the use of air to supply oxygen for aerobic biodegradation processes, because air is a more efficient carrier for delivering oxygen to the BAZ than water (NRC, 1993; Brown, 1994; Rittmann and McCarty, 2001). In air sparging systems, air is injected under pressure directly into the saturated zone below the water table via vertical or horizontal sparge wells, thereby displacing water and creating a transient air-filled space (Fig. 2). This enhances aerobic biodegradation by increasing oxygen transfer, in addition to also promoting contaminant removal by volatilization (Gierke et al., 1998). Thus, low volatility contaminants are removed primarily via biodegradation, and high volatility contaminants primarily via volatilization. When biodegradation is the primary contaminant removal mechanism instead of volatilization, the technology is sometimes referred to as biosparging (FRTR, 2022). The resulting contaminant-laden air arriving at the water table is collected using conventional soil vapor extraction technology, and off-site migration of dissolved contaminants is prevented by using a groundwater capture system. In the case of biosparging, the volatilization rate may be reduced to a level

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where the vapor extraction system is not required. Gaseous stimulatory amendments (e.g., methane) may also be added via the air injection, or dissolved amendments may be added via a water circulation system, as described above. This technology is most effective in unconfined aquifers with homogeneous, high-permeability soils, that are contaminated by aerobically-biodegradable volatile organic compounds, with little or no NAPL.

Bioventing and bioslurping Bioventing is the process of supplying air or oxygen to soil to stimulate the aerobic biodegradation of contaminants in the BAZ, with some volatilization (NRC, 1993; Leeson and Hinchee, 1997; Rittmann and McCarty, 2001). It is one of the most cost effective and efficient technologies for hydrocarbon remediation in the unsaturated zone (also known as the vadose zone) above the water table. The goal is to provide air flow rates and configurations to ensure adequate oxygenation for aerobic biodegradation, while minimizing production of hydrocarbon off-gases (Fig. 3). Thus, systems are typically set up to operate in an air injection mode, rather than using a soil gas extraction system. Air injection is generally performed using blowers, in what is known as active bioventing, but at some sites passive bioventing is performed by relying on barometric pressure changes or tidal fluctuations (FRTR, 2022). Nutrient addition may be desirable, either in dissolved form, or as gases (see “Air sparging and biosparging” section), but field research to date indicates it is not necessary. Bioslurping is an approach that combines bioventing with light NAPL (LNAPL) recovery by using vacuum-enhanced pumping to recover free-phase LNAPLs, while contributing to vapor extraction and bioventing in the unsaturated zone when free product is not present (Domenico and Schwartz, 1998; Miller, 1996).

In situ bioreactive barriers In situ bioreactive barriers, also known as biobarrier or biowall systems, are a bioremediation approach used to prevent further transport of a contaminant plume in the saturated zone (Rittmann and McCarty, 2001; FRTR, 2022). Specifically, a biobarrier is a type of permeable reactive barrier in which a BAZ is created perpendicular to the plume’s path via the input of stimulatory materials, and possibly microorganisms (Fig. 4). The stimulatory materials used to create the biobarrier can be added to the saturated zone either in dissolved form via aqueous injection wells, infiltration galleries, or recirculating wells, or by placing slow-release amendments (e.g., proprietary chemical sources of electron donors or acceptors, mulch, wood chips, iron filings, etc.) in wells or trenches. In some cases, hydraulic or physical controls (e.g., a funnel-and-gate system) may also be applied to ensure that the plume passes through the BAZ.

Phytoremediation Phytoremediation is a bioremediation technology that uses various plants and their associated microbiota, coupled with soil amendments and agronomic techniques, to degrade, extract, contain, or immobilize contaminants from shallow soil and

Fig. 3 Schematic of an active bioventing installation. From FRTR (2022) Federal Remediation Technologies Roundtable [Online]. U.S.A.: United States Government. Available: https://frtr.gov [Accessed March 31 2022].

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Fig. 4 Illustration of an in situ bioreactive barrier. From FRTR (2022) Federal Remediation Technologies Roundtable [Online]. U.S.A.: United States Government. Available: https://frtr.gov [Accessed March 31 2022].

groundwater (FRTR, 2022). This technology can offer an innovative, cost-effective alternative to the more established treatment methods used at hazardous waste sites. To implement phytoremediation, a specific plant or community of plants are established that have been selected to provide one or more of the necessary phytoremediation mechanisms applicable to contaminated media (FRTR, 2022; USEPA, 2000) (Fig. 5). One mechanism by which phytoremediation can work is via physical stabilization/containment effects. For example, by taking up contaminated groundwater, plants, especially trees, with deep root systems that are capable of high groundwater uptake rates, can exert hydraulic control on the dissolved contaminant plume (phytohydraulics), and transpire volatile contaminants into the atmosphere (phytovolatilization). Plants can also contribute to remediation via removal/degradation mechanisms, such as phytodegradation in which metabolism within the plant results in production of degradative enzymes (e.g., dehalogenases and oxygenases), which help to catalyze degradation of contaminants taken up by the plant. Plants can also function as accumulators (i.e., phytoaccumulation or phytoextraction) by taking up contaminants such as dissolved metals through transpiration mechanisms and accumulating them in their roots, with later translocation into shoots and leaves. Finally, plants can contribute to enhanced rhizosphere biodegradation (rhizodegradation) via the release of exudates (e.g., sugars, acids, alcohols) and O2, which stimulate the growth of contaminant degrading microorganisms in the soil. Although phytoremediation is typically applied as an in situ remedial approach as described here, it can be implemented as an ex situ technology whereby contaminated groundwater is extracted and treated via use of constructed wetlands or hydroponics (FRTR, 2022).

Ex situ bioremediation Ex situ bioremediation refers to remedial strategies that require the excavation of the contaminated materials followed by their biological treatment. Thus, by its nature, ex situ bioremediation is an engineered approach and primarily applicable to sites with small, highly contaminated sources, or when a rapid cleanup is required (Rittmann and McCarty, 2001). The three main approaches used–land treatment, composting/biopiles, and slurry phase treatment—are summarized below. These technologies vary primarily in how the contaminated material is manipulated, especially how it is aerated, and the degree of water saturation.

Land treatment In land treatment, after excavation, the contaminated materials are spread out in relatively thin layers, typically in a specially constructed, above-grade treatment system, or land treatment unit (LTU) (Fig. 6) (Baker and Herson, 1994; Eweis et al., 1998; Cookson, 1995). In general, LTUs incorporate controls for containing the contaminated materials, such as a liner and leachate collection system, and possibly a cover system for containing volatile emissions. After emplacement in the LTU, the contaminated material is tilled to aerate the soil and mix it, thereby reducing mass-transfer limitations. Biodegradation is further enhanced by monitoring, and adjusting as necessary, the soil nutrient levels, moisture content, and pH. Other soil amendments may include a

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Fig. 5 Illustration of various phytoremediation mechanisms. From FRTR (2022) Federal Remediation Technologies Roundtable [Online]. U.S.A.: United States Government. Available: https://frtr.gov [Accessed March 31 2022].

Fig. 6 Illustration of land treatment. From FRTR (2022) Federal Remediation Technologies Roundtable [Online]. U.S.A.: United States Government. Available: https://frtr.gov [Accessed March 31 2022].

bulking agent (e.g., wood chips, shredded bark, saw dust, etc.) or organic material to improve porosity and permeability. Contaminant removal is primarily via aerobic biodegradation, although photochemical oxidation may play a role and volatilization may be a concern.

Composting and biopiles In composting, the contaminated material is placed in piles rather than spreading it out in thin layers, which affects the degree of heat entrapment that occurs, and the mode of aeration (Cookson, 1995; Eweis et al., 1998). The contaminated material to be composted is typically mixed with an organic bulking agent (see “Land treatment” section) to improve airflow in the pile by increasing the porosity, and possibly a thermal source whose degradation results in an increase in pile temperature. The bulking agent and the thermal source may be the same material (e.g., manure). Water is also added as necessary to adjust the moisture

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Fig. 7 Schematic of a biopile system. From FRTR (2022) Federal Remediation Technologies Roundtable [Online]. U.S.A.: United States Government. Available: https://frtr.gov [Accessed March 31 2022].

content. Typically, one of three basic composting process designs are used: (1) windrows on an impervious surface, which are aerated by mechanical mixing at regular intervals; (2) static piles on an impervious surface, which are either aerated passively, or via a forced air system; or (3) in-vessel reactors, which are mechanically mixed, possibly in combination with forced aeration. In low-temperature composting, also known as biopiles or soil heaping (Fig. 7), the contaminated soil is possibly mixed with a poorly-degraded bulking agent and then placed in a static pile, which is aerated passively or by forced air, and irrigated to control moisture, pH, and nutrients (Cookson, 1995; Fahnestock et al., 1998).

Slurry-phase treatment Unlike land treatment and composting/biopiles, bioremediation via slurry-phase treatment is conducted using water-saturated conditions (Cookson, 1995, Eweis et al., 1998). Treatment is performed in a reactor that is typically operated in a batch or semi-batch mode, and may be open or closed. The contaminated material is pretreated as necessary and mixed with sufficient water to allow continuous mixing. Formation, mixing, and aeration of the slurry improves mass-transfer and oxygen availability, as well as reaction rates. Amendments for improving biodegradation (e.g., nutrients, neutralizing agents, supplemental bacteria, surfactants, dispersants, etc.) are added as required. In some cases, sludges resulting from the long-term storage of contaminated material may be treated in situ using existing lagoons or impoundments; however, an impermeable layer should be present under the system to prevent contaminant migration. Post-treatment requires dewatering to separate the treated water and soil, which are then either treated further, reused, or disposed of, as appropriate.

Summary and conclusion Application of bioremediation for cleanup of contaminated environments has several advantages compared to other remedial technologies. In particular, biodegradation processes possess the potential to convert contaminants to harmless products. Nonetheless, there are a number of challenges associated with bioremediation including the need to understand how microorganisms degrade contaminants, how those microbial activities are impacted by environmental conditions and interactions with other microbial populations, and how the beneficial microbial processes and their rates can be enhanced through engineering activities. The spectrum of biodegradation processes applied to organic contaminants range from mineralization to biotransformation reactions. During mineralization reactions, organic chemicals are converted completely to inorganic products, thereby eliminating the toxic hazard associated with the chemical of concern. Mineralization is often the result of metabolic processes, with the contaminant serving as a primary growth substrate. In comparison, the conversion of the contaminant substrate during biotransformation reactions does not result in significant changes in the structure of the parent compound, and may or may not be reduce risk. Biotransformations often fall under the category of secondary substrate utilization, or cometabolic transformations, in which case the contaminants are biodegradable, but incapable of supporting microbial growth. In comparison, most inorganic contaminants cannot be degraded, and bioremediation instead is focused on altering their transport properties.

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Despite the great potential for biodegradation of contaminants via one of these mechanisms, a number of factors can cause contaminant compounds to resist biodegradation in the environment. These biological and physical/chemical factors impacting whether contaminant biodegradation occurs and the rate of biodegradation include: genetic potential, environmental conditions, toxicity, and bioavailability. In some cases, the naturally occurring capabilities of the native microorganisms to biodegrade the contaminants of concern are sufficient to contain the contaminants and prevent their migration away from the source via what is known as intrinsic bioremediation. Intrinsic bioremediation is often a component of the broader remedial approach known as monitored natural attenuation. Engineered bioremediation strategies intentionally enhance biodegradation by overcoming any factor(s) limiting biodegradation in the field. These strategies may be performed in situ, or ex situ. Bioremediation approaches also vary in their aggressiveness, ranging from intrinsic bioremediation at the passive end of the scale, to engineered or enhanced bioremediation applications that incorporate actions like biostimulation and bioaugmentation to modify the site and promote and enhance the biodegradative activities of microorganisms. Engineered in situ bioremediation approaches differ with respect to the technique used for adding amendments to the subsurface, including water circulation, air injection, bio-barriers, and agronomic techniques. In comparison, ex situ bioremediation involves the excavation of the contaminated materials followed by treatment in a bioreactor, e.g., land treatment, composting/ biopiles, and slurry phase treatment.

Acknowledgment The author thanks Prof. John S. Gierke of the Department of Geological and Mining Engineering and Sciences at Michigan Technological University for his helpful comments on an earlier version of this document.

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Biotransformation/metabolism Natalia Guevara, Marta Vázquez, and Pietro Fagiolino, Facultad de Química, Universidad de la República. Montevideo, Uruguay © 2024 Elsevier Inc. All rights reserved. This is an update of J.L. Rourke, C.J. Sinal, Biotransformation/Metabolism, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 490–502, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00007-5.

Enzymatic reactions Oxidation reactions Cytochrome P450 monooxygenase system Flavin-containing monooxygenases Alcohol and aldehyde dehydrogenases Monoamine oxidases H2O2-dependent peroxidases Reduction reactions Cytochrome P450-dependent reactions Flavoprotein-dependent reactions Carbonyl reductases Hydrolysis reactions Epoxide hydrolase Carboxylesterases/amidases Conjugation reactions UDP-glucuronosyltransferases Sulfotransferases Glutathione S-transferases Mercapturic acid biosynthesis Cysteine conjugate b-lyase/thiomethylation Acyl-coenzyme A: Amino acid N-acyltransferases N-acetyltransferases N- and O-methyltransferases ABC transporters Regulation of biotransformation Variability Genetics Sex Age Epigenetics Phenytoin: An example to summarize the complexity of drug metabolism and its consequences References

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Abstract Lipophilic xenobiotics and endobiotics are converted in the body by enzymatic reactions to hydrophilic products that are readily excreted in urine and/or bile. For this purpose, sequential enzymatic reactions take place, and several enzymes are involved. In addition, efflux transporters also have an important role in metabolism. Owing to the importance of biotransformation in reducing xenobiotics concentration and blocking functional groups, the process is subject to transcriptional regulation. Nevertheless, there are multiple factors that account for intra and interindividual variability in biotransformation.

Keywords Age; Biotransformation; Efflux transporters; Enzymatic reactions; Epigenetics; Genetics; Phases I–II enzymes; Sex; Transcriptional regulation; Xenobiotic and drug metabolism

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Enzymes and efflux transporters cooperate in xenobiotic and drug biotransformations. The mechanisms by which biotransformation is regulated is basically transcriptional. The expression of both enzymes and transporters follows circadian rhythms and could be regulated by xenobiotic cell concentrations. Genetics of individuals, their sex and age are some of the sources of variability in biotransformation apart from epigenetics causes.

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Enzymatic reactions Biotransformation refers to the process by which lipophilic (fat-soluble), xenobiotic (foreign) or endobiotic (endogenous) chemicals are converted in the body by enzymatic reactions to products that are more hydrophilic (water-soluble). In this context, metabolism, metabolic transformation and biotransformation are synonyms. A xenobiotic is a relatively small (molecular weight 5 joints per week) were found to have lower verbal and memory scores at age 2 years. A few studies have demonstrated a possible increased risk of non-lymphoblastic leukemia, rhabdomyosarcoma, and astrocytoma in children whose mothers reported using cannabis during their pregnancies (Bonomo et al., 2019). As of current, Epidiolex is unassigned a pregnancy category stating there is no adequate data on the developmental risks but cautiously states that CBD administration has demonstrated developmental toxicity in animal studies (FDA, 2021).

Genotoxicity In a recent study performed on wistar rats, the rats exposed to a daily dose of 7 mg/kg of THC demonstrated effects of low-level DNA damage in white blood cells and brain cells, and greater susceptibility to DNA breakage in brain cells (Kopjar et al., 2019). Furthermore, another recently conducted study demonstrated that low concentrations of CBD and CBDV (cannabidivarin) caused single and double strand breaks in DNA, oxidation of DNA bases, and chromosomal aberrations in human-derived cell lines (Russo et al., 2018).

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Carcinogenicity There is mixed evidence on the effects of cannabis on cancer. Cannabis has carcinogenic effects when smoked. A study examining the long-term effects of marijuana smoking on men aged 18–20 years old over a course of 40 years demonstrated that heavy cannabis smoking was associated with over a twofold risk of developing lung cancer (Callaghan et al., 2013). A plethora of studies have demonstrated anti-cancer effects of cannabinoids through inhibiting cancer cell invasion, inducing apoptosis of cancer cells, and regulating/inhibiting the proliferation of cancer cells in pheochromocytoma, thyroid epithelioma, skin carcinoma, prostate cancer, leukemia, mantle cell lymphoma, pancreatic cancer, breast cancer, rhabdomyosarcoma, cervical cancer, cholangiocarcinoma, colon cancer, gastric cancer, neuroblastoma, non-small cell lung cancer, hepatocarcinoma, head and neck squamous cell carcinoma, bladder carcinoma and multiple myeloma. Cannabinoids have also demonstrated reduced chemoresistance and enhanced sensitivity to chemotherapy when used concomitantly with antineoplastic drugs proving its benefit as an adjuvant and/or synergist in cancer treatment (Ramer et al., 2017).

Pulmonary toxicity Transient bronchodilation is the most prominent effect after an acute exposure. Heavy smokers or chronic users experience increased cough, sputum production, and wheezing. These complaints are augmented by concurrent tobacco use. A gradual decline in respiratory function is greater among marijuana smokers compared to tobacco smokers. Marijuana cigarettes contain the same components as tobacco smoke, including bronchial irritants, tumor initiators (mutagens), and tumor promoters, in addition to nicotine. Estimates based on smoking had revealed that the amount of tar in a marijuana cigarette is three times higher compared to a tobacco cigarette, with one-third greater deposition in the respiratory tract. Bronchitis, squamous metaplasia of the tracheobronchial epithelium, and emphysema are the commonest features among chronic users. Smoking marijuana demonstrated a greater than 2-fold risk of developing lung cancer even after statistical adjustment for tobacco use and respiratory conditions (Callaghan et al., 2013). Studies have also indicated an association between inhalational marijuana and spontaneous pneumothorax, bullous emphysema, or COPD (Martinasek et al., 2016). Most illegally obtained marijuana is believed to be contaminated with a biological carcinogen aflatoxin (produced by the fungus Aspergillus species) which can cause invasive pulmonary aspergillosis in immunocompromised users.

Neurotoxicity Cannabinoids have mixed evidence on its effect on neural health revealing both neurotoxic and neuroprotective results. In one study, CBD at low concentrations produced neuroprotective effects in primary hippocampal neurons against hydrogen peroxide (Kim et al., 2021). Neurotoxic effects from cannabis are dependent on dose, age of use, and duration of use. One study showed a loss of IQ points evident in adulthood in subjects using persistently and frequently since adolescence (Meier et al., 2012). Another study analyzed MRI scans revealing that heavy marijuana users have lower levels of grey matter volumes in comparison to non-users (Filbey et al., 2014). A strong correlation exists between cannabis use and mental health. Adolescent abusers commonly suffer one or more comorbid health or behavioral problems. Several studies have demonstrated marijuana abuse to coexist with attention deficit hyperactivity disorder, other learning disabilities, depression, and anxiety. Cohort and well-designed cross-sectional studies suggest a modest association between early, regular, or heavy cannabis use and depression. An association exists between cannabis use and schizophrenia. Several well-controlled studies with well-defined samples looked at cannabis use and psychosis and concluded an overall two-fold increase in the relative risk for developing schizophrenia, although it was not determined whether cannabis use is necessary or sufficient to cause schizophrenia. Cannabis use is believed to worsen schizophrenic psychotic symptoms (Broyd et al., 2016).

Cardiovascular toxicity A sudden 20–100% rise in heart rate is very common with naïve user, lasting up to 2–3 h. Peripheral vasodilatation causes postural hypotension, which may lead to dizziness or syncope. The methods by which this occurs is through increased sympathetic nervous system activity, inhibition of parasympathetic innervation to the heart, and the effects of vasodilation causing reflex tachycardia (Prakash et al., 1975) Cardiac output increases by as much as 30%. In addition, the cardiac oxygen demand is also increased. Tolerance to these effects can develop within a few days of use. Naive users can experience angina. In addition, users with pre-existing coronary artery disease or cerebrovascular disease may experience myocardial infarctions, congestive heart failure, and strokes. Reports of myocardial infarction in otherwise healthy young patients with no prior CVD risk have been reported. CB1 and CB2 receptors can be found on platelet membranes and when cannabinoids are ingested in high concentrations, activation can induce irreversible platelet aggregation (Levy et al., 1976). Chronic users may experience a combination of all these effects prior to

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onset of persistent cardiac anomalies. One study analyzing the differences between users and non-users in hypertension, marijuana users showed an increased risk of hypertension-associated mortality compared to non-users (Yankey et.al.,2017). Cannabis use has demonstrated adverse effects on the cardiovascular system in an array of studies. Occurrences of arterial vasospasm, thrombus formation, atherosclerosis, and arteritis have been reported in otherwise healthy cannabis users (Subramaniam et al., 2019). Cannabis has shown to have anti-atherogenic effects at the CB2 receptor, but also atherogenic effects at the CB1 receptor. CB1 receptor agonist activates MAPK, which increases AT1 receptor expression- yielding formation of Reactive Oxygen Species (ROS) promoting endothelial injury. In addition to this, oxidized LDL increases CB1 and CB2 receptor expression and endogenous cannabinoid production, causing overall lipid accumulation in macrophages through elicitation of an inflammatory cascade (Singla et al., 2012; Jiang et al.,2009).

Clinical management Despite marijuana’s lower risk of abuse potential and promising therapeutic effects, it still has poses a risk for adverse effects with its misuse. Regardless of its legalization and availability of recreational purchase in stores, users- especially naïve, may be unaware of quantifying the dose necessary to achieve therapeutic effects and may suffer acute toxicity. Depressive, hallucinatory, or psychotic reactions should be treated by placing the patient in a quiet area, providing reassurance that no permanent effects will occur. Benzodiazepines are preferred drugs for treatment of extreme agitation and marijuana-withdrawal induced insomnia. When psychotic phenomena predominate, haloperidol 5 mg i.m. is recommended. The patient should be kept well hydrated. As of currently, the FDA has not approved any medications for managing marijuana-use disorder, however forms of behavioral and motivational therapy have been shown effective (National Institute on Drug Abuse, 2021).

Ecotoxicology There is limited data relating to the ecotoxicology of cannabinoids. Due to its recent and expanding legalized use in many countries and cities, cannabis use and demand has increased exponentially waging increased cultivating of cannabis which may have negative ecological effects. For example, research shows that cultivating cannabis plants produces volatile organic compounds that act as ozone-degrading aerosols. The hazards of cannabis presence in waste waters is less known and there are currently no regulations circling cannabis metabolites in wastewater (Wang et al., 2019).

Exposure standards and guidelines The average THC potency of cannabis has increased due to cultivation of improved biotechnology-based plant breeding in the last two decades. Marijuana cigarettes in the past contained approximately 10 mg of THC, but today a commercially-available joint may contain approximately 60–150 mg. Because the effects of THC are dose-dependent, modern cannabis users may experience greater morbidity than their predecessors. Cannabis is available in the following forms: Marijuana is a combination of the C. sativa flowering tops and leaves. The THC content is 0.5–5%. Several preparations are as follows: (1) Bhang – dried leaves and tops; (2) Ganja – leaves and tops with a higher resin content, which results in greater potency; (3) Hashish is dried resin collected from the flowering tops. The THC concentration is 2–20% in hash oil, a liquid extract that contains up to 15% THC. Sinsemilla is unpollinated flowering tops derived from the female cannabis plant in which THC content is as high as 20%, and in Dutch hemp (netherweed) has a THC concentration as high as 20%. Commercially available cannabinoids such as Dronabinol (synthetic) and Nabilone (synthetic) and Epidiolex are approved drugs used in anorexia associated with weight loss patients with AIDs, nausea and vomiting with cancer chemotherapy, and seizures, respectively. Epidiolex is commercially available as an oral solution of 100 mg/mL and the recommended starting dose is 2.5 mg/kg by mouth twice daily (FDA, 2018). Although cannabis is currently legal for either recreational or medical use in 36 states, federally it is still considered a Schedule I drug and is illegal (Bonomo et al., 2019).

Conclusion Regarded as a multi ¼ tasking herbal medicine and recreational substance for thousands of years, cannabis is one of the most widely consumed substances in the World. Globally, approximately 180 million people use cannabis for nonmedical, or recreational, purposes and that
13 million people are dependent on cannabis (Bonomo et al., 2019). Its reputation as “non-addictive” and “safe” remains contentious. The therapeutic potential and their relevance to public health of cannabinoids continues to go through intense debates and risk vs. the benefit ratio remains undetermined. Use of unknown concentrations of cannabinoids by the public for therapeutic purposes continues to be a challenge to the medical community. Rigorous clinical trials are needed to inform the healthcare community regarding its safe and beneficial use. Further research and awareness regarding its toxicological profile should also be meaningful. Its primary route of exposure (via smoking) has been proven to be highly carcinogenic, and data investigating the biochemical mechanisms of marijuana in humans demonstrate its potential organotoxic effects. Consistent with the principles of evidence-based medicine, it is about time to invest resources to generate high quality clinical data for proper medical use of cannabinoids in the light of disease prevention.

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Centers for Disease Control and Prevention. https://www.cdc.gov/marijuana/factsheets/pregnancy. htm#10.  Cerne K (2020) Toxicological properties of D9-tetrahydrocannabinol and cannabidiol. Archives of Industrial Hygiene and Toxicology 71(1): 1–11. https://doi.org/10.2478/aiht-202071-3301. Chilakapati J and Fariss FF (2014) Cannabinoids. In: Encyclopedia of Toxicology, 3rd edn, 649–654. https://www.sciencedirect.com/science/article/pii/B9780123864543002670. Dunne EM, et al. (2019) Problem solving reduces sexual risk associated with sensation seeking, substance use, and depressive symptoms among African-American adolescents. Journal of Child & Adolescent Substance Abuse 28(2): 113–118. https://doi.org/10.1080/1067828x.2019.1610679. FDA (2018) EPIDIOLEXW (cannabidiol) Oral Solution. FDA Access Data FDA. https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/210365lbl.pdf. FDA (2021) FDA Regulation of Cannabis and Cannabis-Derived Products: Q&A. U.S. Food and Drug Administration. https://www.fda.gov/news-events/public-health-focus/fdaregulation-cannabis-and-cannabis-derived-products-including-cannabidiol-cbd. Filbey FM, et al. (2014) Long-term effects of marijuana use on the brain. Proceedings of the National Academy of Sciences 111(47): 16913–16918. https://doi.org/10.1073/ pnas.1415297111. Golombek P, et al. (2020) Conversion of cannabidiol (CBD) into psychotropic cannabinoids including tetrahydrocannabinol (THC): A controversy in the scientific literature. Toxics 8(2): 41. https://doi.org/10.3390/toxics8020041. Gonçalves J, et al. (2019) Cannabis and its secondary metabolites: Their use as therapeutic drugs, toxicological aspects, and analytical determination. Medicine 6(1): 31. https://doi. org/10.3390/medicines6010031. History.com Editors (2017) Marijuana. History.com A&E Television Networks. https://www.history.com/topics/crime/history-of-marijuana. Huestis MA (2007) Human cannabinoid pharmacokinetics. ChemInform 38(47). https://doi.org/10.1002/chin.200747256. Huestis MA, et al. (2019) Cannabidiol adverse effects and toxicity. Current Neuropharmacology 17(10): 974–989. https://doi.org/10.2174/1570159X17666190603171901. Jiang LS, et al. (2009) Role of activated endocannabinoid system in regulation of cellular cholesterol metabolism in macrophages. Cardiovascular Research 81: 805–813. https://doi. org/10.1093/cvr/cvn344. Kim J, et al. (2021) Neuroprotective effect of cannabidiol against hydrogen peroxide in hippocampal neuron culture. Cannabis and Cannabinoid Research 6(1): 40–47. https://doi.org/ 10.1089/can.2019.0102. Kopjar N, et al. (2019) DNA damaging effects, oxidative stress responses and cholinesterase activity in blood and brain of wistar rats exposed to D9-Tetrahydrocannabinol. Molecules 24(8): 1560. https://doi.org/10.3390/molecules24081560. Levy R, Schurr A, Nathan I, Dvilanski A, and Livne A (1976) Impairment of ADP-induced platelet aggregation by hashish components. Thrombosis and Haemostasis 36: 634–640. PMID: 1037158. Martinasek M, et al. (2016) A systematic review of the respiratory effects of inhalational marijuana. Respiratory Care 61(11): 1543–1551. https://doi.org/10.4187/respcare.04846. Meier MH, et al. (2012) Persistent cannabis users show neuropsychological decline from childhood to midlife. Proceedings of the National Academy of Sciences 109(40): E2657–E2664. https://doi.org/10.1073/pnas.1206820109. Morales P, et al. (2017) Molecular targets of the phytocannabinoids: A complex picture. Progress in the Chemistry of Organic Natural Products 103: 103–131. https://doi.org/ 10.1007/978-3-319-45541-9_4. National Institute on Drug Abuse (2021) Available Treatments for Marijuana Use Disorders. National Institute on Drug Abuse. https://www.drugabuse.gov/publications/researchreports/marijuana/available-treatments-marijuana-use-disorders. NIDA (2021) What are marijuana’s effects on lung health? https://nida.nih.gov/publications/research-reports/marijuana/what-are-marijuanas-effects-lung-health. Prakash R, et al. (1975) Effects of marihuana and placebo marihuana smoking on hemodynamics in coronary disease. Clinical Pharmacology and Therapeutics 18: 90–95. https://doi. org/10.1002/cpt197518190. Ramer R, et al. (2017) Cannabinoids as anticancer drugs. Cannabinoid Pharmacology 397–436. https://doi.org/10.1016/bs.apha.2017.04.002. Rieder S, et al. (2010) Cannabinoid-induced apoptosis in immune cells as a pathway to immunosuppression. Immunobiology 215(8): 598–605. https://doi.org/10.1016/j. imbio.2009.04.001. Rudd J (2020) CBD vs THC – What are the main differences? Analytical Cannabis. https://www.analyticalcannabis.com/articles/cbd-vs-thc-what-are-the-main-differences-297486. Russo C, et al. (2018) Low doses of widely consumed cannabinoids (cannabidiol and cannabidivarin) cause DNA damage and chromosomal aberrations in human-derived cells. Archives of Toxicology 93(1): 179–188. https://doi.org/10.1007/s00204-018-2322-9. Secades-Villa R, et al. (2015) Probability and predictors of the cannabis gateway effect: a national study. The International Journal on Drug Policy 26(2): 135–142. https://doi.org/ 10.1016/j.drugpo.2014.07.011. Sharma P, et al. (2012) Chemistry, metabolism, and toxicology of cannabis: clinical implications. Iranian Journal of Psychiatry 7(4): 149–156. Singla S, et al. (2012) Cannabinoids and atherosclerotic coronary heart disease. Clinical Cardiology 6: 329–335. https://doi.org/10.1002/clc.21962. Subramaniam V, et al. (2019) The cardiovascular effects of marijuana: Are the potential adverse effects worth the high? Missouri Medicine 116(2): 146–153. Thompson GR, et al. (1973) Comparison of acute oral toxicity of cannabinoids in rats, dogs and monkeys. Toxicology and Applied Pharmacology 25: 363–372. https://doi.org/ 10.1016/0041-008x(73)90310-4. Turner AR, et al. (2019) Marijuana toxicity. In: StatPearls. Treasure Island (FL): StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK430823/. USDOJ: DEA-Diversion Control Division (2001) Toxic Effects of marijuana and THC. https://www.deadiversion.usdoj.gov/fed_regs/notices/2001/fr0418/fr0418h.htm. Wang CT, et al. (2019) Leaf enclosure measurements for determining volatile organic compound emission capacity from Cannabis spp. Atmospheric Environment 199: 80–87. https:// doi.org/10.1016/j.atmosenv.2018.10.049. Watanabe K, et al. (2007) Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes. Life Sciences 80(15): 1415–1419. https://doi.org/10.1016/j.lfs.2006.12.032. Wiese BM, et al. (2020) Cannabinoid-2 agonism with AM2301 mitigates morphine-induced respiratory depression. Cannabis Cannabinoid. https://doi.org/10.1089/can.2020.0076 Epub ahead of print PMID: 33998869. Yankey BA, et al. (2017) Effect of marijuana use on cardiovascular and cerebrovascular mortality: A study using the National Health and Nutrition Examination Survey linked mortality file. European Journal of Preventive Cardiology 17: 1833–1840. https://doi.org/10.1177/2047487317723212. Zou S, et al. (2018) Cannabinoid receptors and the endocannabinoid system: Signaling and function in the central nervous system. International Journal of Molecular Sciences 19(3): 833. https://doi.org/10.3390/ijms19030833.

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Further reading Caitlin D (2019) The effects of cannabis on female and male reproduction. BCMJ 61(7): 282–285. https://bcmj.org/articles/effects-cannabis-female-and-male-reproduction. NCCIH (2019) Cannabis (Marijuana) and Cannabinoids: What You Need To Know. In: National Center for Complementary and Integrative Health. U.S. Department of Health and Human Service. https://www.nccih.nih.gov/health/cannabis-marijuana-and-cannabinoids-what-you-need-to-know. PubChem. Cannabidiol National Center for Biotechnology Information. PubChem Compound Database. US-NLM: https://pubchem.ncbi.nlm.nih.gov/compound/Cannabidiol. PubChem. delta9-Tetrahydrocannabinol. National Center for Biotechnology Information. PubChem Compound Database. U.S. National Library of Medicine. https://pubchem.ncbi.nlm. nih.gov/compound/delta9-Tetrahydrocannabinol. Zavala CA, et al. (2020) Cannabinoid CB2 receptor activation attenuates fentanyl-induced respiratory depression. Cannabis Cannabinoid Res. https://doi.org/10.1089/ can.2020.0059.

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Captafol Iva Srdanovic, Azhar Hussain, and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of P. Raman, Captafol, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 655–658, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00268-2.

Chemical profile Introduction Uses Environmental fate and behavior Exposure routes and pathways Toxicokinetics Mechanism of toxicity Acute and short-term toxicity (or exposure) Animal Human Chronic toxicity (or exposure) Animal Human Genotoxicity Carcinogenicity Clinical management Exposure standards and guidelines Conclusion References Further reading

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Abstract Captafol is a chloroalkylthio fungicide which was used for the control of fungal diseases of fruits, vegetables, ornamental plants and turf grasses, including controlling certain seed and soil-borne organisms. It was banned in countries worldwide since 2010 due to its toxicity to the skin, eyes and respiratory tract of humans. Persons with a skin rash following exposure were found to have dermal as well as systemic disorders. In vitro and in vivo short-term genotoxicity studies support mutagenicity as a mechanism of carcinogenesis. Animal studies in rodents show captafol to be genotoxic with DNA strand breaks, micronucleus formation, and dominant lethal mutations, as well as carcinogenic in both mice and rats with the vascular, gastrointestinal systems and the liver being the sites of tumor formation. The formulated product of captafol may pose an environmental risk if released into the aquatic environment. Fungicides are normally used to limit the growth of molds on food crops. Captafol (Folcid; Arborseal), folpet, dithiocarbamates, pentachlorophenol, and the mercurial are well-known in the industry because of their fungicidal activity. Foodborne fungicides do not pose significant risks of health hazards because their exposure levels are very low, and their accumulation pattern in the environment is not alarming.

Keywords Captafol degradation; Captafol volatilization; Carcinogenicity; Chloroalkylthio fungicide; Conjunctivitis; Dermatitis; Genotoxicity; Respiratory sanitization; Sulfanilamide; Tetrahydrophthalimide

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Captafol’s use has been discontinued since 2010 in most countries worldwide Main routes of exposure are: dermal, ocular, inhalation and through direct contact for workers in the farm industry. Captafol has been known to cause allergic reactions and/or contact dermatitis, eye irritation/conjunctivitis and irritation of the respiratory tract. Captafol is genotoxic and carcinogenic in animal in vitro and in vivo models. Captafol is classified as a probable human carcinogen of either Group 2A or Group 2B by International Agency for Research on Cancer and US EPA, respectively; but it has been recognized as being non-carcinogenic in humans by the American Conference of Governmental Industrial Hygienists (ACGIH)

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In vitro and in vivo short-term genotoxicity studies support mutagenicity as a mechanism of carcinogenesis in bacterial lines, non-mammalian systems, animal models and humans

Chemical profile

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Name: Captafol Chemical Abstracts Service Registry Number: 2425-06-1 Synonyms: 2-(1,1,2,2-tetrachloroethylsulfanyl)-3a,4,7,7a-tetrahydroisoindole-1,3-dione; Folcid; Difolatan Molecular Formula: CJOH9Cl4NO2S Chemical Structure:

Introduction Captafol is a chloroalkylthio fungicide, which is colorless to pale yellow in color with a distinct odor. It has low water solubility of 1.4 mg 1−1 at 20
C and a melting point in the range of 160–162
C. It is mostly soluble in organic solvents. Captafol is found in trade name products, such as, Crisfolatan, Difolatan, Difosan, Folcid, Haipen, Kenofol, Pillar-tan, and Sanspor. Captafol is available as dusts, wettable powders and flowable formulations. The formulated product of captafol may pose an environmental risk if released into the aquatic environment. Captafol is a non-systemic antifungal chemical widely used to control foliage and fruit diseases of tomatoes, berry disease of coffee, potato blight, and tapping panel disease of rubber trees. It is also used in the lumber and timber industries to prevent growth of wood rot fungi in logs and a variety of wood products. Animal studies showed captafol to be hepatotoxic and to induce potentially pre- neoplastic glutathione S-transferase placental form positive (GST-P+) foci in the liver of male F344 rats in both the initiation and promotion phases of studies of tumor development. In addition to these findings, promotion with captafol increased the incidences of hyperplasia of the forestomach and adenoma of the small intestine, thyroid follicular-ce.11adenoma, and the expression of a marker of cell proliferation (proliferating-cell nuclear antigen) in the kidneys in F344 rats. In addition to direct genotoxic activity, several epigenetic mechanisms depleted cellular thiol groups (non-protein and protein), inhibited DNA replication enzymes (DNA topoisomerases and polymerases), DNA synthesis, RNA synthesis, and induced CYPP450 monooxygenases, suggesting underlying causes of tumor formation pathogenesis (Tamano et al., 1990, 1991, 1993; Pubchem, n.d.; NTP, 2011).

Uses Captafol is a widely used broad-spectrum contact chloroalkylthio fungicide belonging to the class of sulfanilamides. It is used to control fungal diseases of fruits, vegetables, ornamental plants and grasses, and as a seed treatment (IARC, 1991; IPCS, 1990). It is widely used outside the United States to control foliage and fruit diseases on apples, citrus, tomato, cranberry, sweet corn, barley, wheat, and several other plants. Additionally, it is used for the purpose of reducing the losses from wood rot fungi in logs and wood products. Mixed formulations of this compound include (captafol +) triadimefon; ethirimol; folpet; halacrinate; propiconazole; and pyrazophos. Captafol is compatible with most plant-protection products, with the exception of alkaline preparations and formulating materials (Zang et al., 2008). Captafol was used as a fungicide in the United States until 1987, when all registrants of captafol products requested voluntary cancellation of their registrations. Even though legal use of existing stocks was allowed after 1987, the USEPA further restricted its use in 1999, and all captafol tolerances were revoked except those for onions, potatoes, and tomatoes. These were also revoked in

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2006. Captafol was still used until mid-2000s in several countries that exported agricultural products to the United States, including Mexico and Brazil even though many countries had banned its use. However, as of 2010, no countries were banned from using captafol on food crops (IPCS, 1990; IARC, 1991; NTP, 2008).

Environmental fate and behavior Captafol is not persistent in the environment. Captafol is stable under ordinary environmental conditions and rapidly degrades in soil, the rate of degradation being a function of soil type and pesticide concentration. It does not leach from basic soils and is unlikely to contaminate groundwater. Captafol sprayed on most crops has a half-life of less than 5 days. Captafol and/or its metabolites and degradation products are readily absorbed by roots and shoots of plants. If released to air, an extrapolated vapor pressure of 8.27  10–9 mmHg at 25
C indicates captafol will exist solely in the particulate phase in the ambient atmosphere. Particulate-phase captafol will be removed from the atmosphere by wet and dry deposition. If released to soil, captafol is expected to have slight mobility based on Koc values of 2073 and 2120. Volatilization from moist soil surfaces is not expected to be an important fate process based on a Henry’s Law constant of 2.7  10–9 atm-cu m mol−1. In a laboratory setting, the biodegradation half-life of captafol in three soils was found in the range of 23–55 days. The overall half-life of captafol in soil is about 11 days, independent of soil type or initial concentration. If released into water, captafol is expected to adsorb to suspended solids and sediment based on the Koc. Volatilization from water surfaces is not expected to be an important fate process based on this compound’s estimated Henry’s Law constant. An estimated bio-concentration factor of 170 suggests the potential for bio-concentration in aquatic organisms is high, provided the compound is not altered physically or chemically after being released to the environment. The half-lives for the hydrolysis of Difolatan at pH 3.0, 7.0, and 8.0 were 77.8, 6.54, and 0.72 h, respectively. Hydrolysis is likely to be the predominant pathway of degradation in the aquatic environment (IARC, 1991; NTP, 2011; Pubchem, n.d.; Shinde et al., 2019).

Exposure routes and pathways Exposure routes to captafol include inhalation, skin absorption, ingestion, skin and/or eye contact. Dermal and ocular exposures are the most common routes of exposure. Contact dermatitis has been reported after exposure to captafol. Occupational exposure to captafol has been reported to cause severe irritation of the respiratory tract, eye damage and other systemic effects. No systemic poisonings have been reported in humans (IARC, 1991; Pubchem, n.d.).

Toxicokinetics Captafol is poorly absorbed from the gastrointestinal tract. The primary sites of captafol metabolism are the liver and the gastrointestinal tract. Captafol is eliminated via urine, feces, and exhaled air. The major single metabolite, tetrahydrophthalimide (THPI), was detected in blood, urine, and feces, but most of the activity in the blood and urine was in the form of more water-soluble metabolites. Following oral administration in animals, captafol is hydrolyzed to THPI and dichloroacetic acid, where upon THP is degraded to tetrahydrophthalimidic acid and further to phthalic acid and ammonia (Whyatt et al., 2003; IARC, 1997; Pubchem, n.d.; Raman, 2014).

Mechanism of toxicity The primary toxicity following captafol exposure probably occurs through a hypersensitivity mechanism. Most experiments suggest captafol to be DNA active (NTP, 2011; Pubchem, n.d.).

Acute and short-term toxicity (or exposure) Animal The oral LD50 for captafol in rats is 2500–6200 mg/kg bw, while the dermal LD50 in rabbits is 15,400 mg/kg bw (Ben-Dyke et al., 1970). In rats, single intraperitoneal doses of captafol at 5 mg/kg bw increased plasma transaminase levels and decreased liver monoxygenase content and activity and (Dalvi and Mutinga, 1990). Another test for captafol-induced eye irritation in rabbit showed corneal opacity and iris and conjunctival irritation, all symptoms being present for 21 days. It is also mildly irritating to skin in rabbits (Pubchem, n.d.). Most animal feeding studies have shown that most of the captafol is excreted unchanged. When rats, dogs, and monkeys were fed 14 (C) captafol, almost 80% was excreted within 36 h, mainly in the urine, and none via expired carbon dioxide. In mammals, major captafol metabolite in blood, urine and feces is tetrahydrophthalimide. Captafol is moderately toxic to birds, honeybees, earthworms, and most aquatic organisms (Pubchem, n.d.).

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Human The primary symptoms of captafol exposure reported in humans include contact dermatitis and conjunctivitis. The reaction may be severe and may include stomatitis and painful bronchitis. Persons with a skin rash following exposure to captafol were found to have systemic as well as dermal disorders. Vomiting and diarrhea may occur following ingestion of large amount of captafol. Allergic dermatitis, contact dermatitis, respiratory sensitization and conjunctivitis are also known to occur. Respiratory sensitization manifests as wheezing due to bronchospasms. Following captafol exposures, systemic disorders including hypertension, hepatic and renal disturbances, usually paralleling the degree of dermatitis, have been reported. Wheezing due to bronchospasm, contact dermatitis and vomiting and diarrhea are the features of exposure to captafol by inhalation, skin contact and ingestion respectively (NTP, 2011).

Chronic toxicity (or exposure) Animal Rats exposed to captafol at dietary levels of 1500 and 5000 ppm demonstrated growth depression, some liver and kidney changes, and an increased mortality. Following exposure to 300 or I 00 mg/kg of captafol, dogs suffered frequent vomiting and diarrhea during the first 4 weeks and were observed to be slightly anemic and deficient in growth during a 2 year study. Dogs at dosages of 30 mg/kg or greater developed both absolute and relative increases in the weights of the liver and kidney. Oral administration in mice produced a high incidence of adenocarcinomas of the small intestine, vascular tumors of the heart, and spleen and hepatocellular carcinomas. In a 2-year rat-feeding study, a dose-related increased incidence of neoplastic nodules in the liver of females was reported. The USEPA reported a NOEL for nononcogenic effects at 56 ppm, based on a chronic toxicity study in rats. There is sufficient evidence in experimental animals for carcinogenicity of captafol. Several teratology studies have been conducted in many mammalian species, including nonhuman primates, which concluded low to no teratogenic potential after administering captafol throughout organogenesis (Raman, 2014).

Human Captafol is also known to be a skin sensitizer, causing dermatitis upon repeated and prolonged exposure. Repeated or prolonged inhalation exposure to captafol may cause asthma (IPCS, CED, 1994; HSDB-Hazmap, 2023). Breakdown products may contribute to the skin irritation and sensitization associated with captafol (NTP, 2011). Chronic exposure can cause hypertension, depression of liver function, dermatitis, conjunctivitis and anemia. Classified as Group B, probable human carcinogen based on mammary-gland and liver tumors in female Sprague-Dawley rats, kidney tumors in both male and female rats, and lymphosarcoma and hemangiosarcoma in both male and female CD-1 mice, with Harderian-gland tumors in male mice (NTP, 2008). A recent study by Bhat et al. (2020) shows induction of intestinal tumors in mice by agents very similar to captafol (captan and folpet).

Genotoxicity Captafol is an alkylating agent causing genotoxicity. In vitro and in vivo short-term genotoxicity studies support mutagenicity as a mechanism of carcinogenesis. Captafol caused mutations in Salmonella typhimurium (base-pair mutations) and Escherichia coli and in non-mammalian in vivo systems (Aspergillus nidulans and Drosophila melanogaster) (Raman, 2014; Nazir et al., 2003; Rahden-Staron, 2002). Captafol has been shown to cause DNA single-strand breaks, micronucleus formation, sister chromatid exchange, chromosomal aberrations, polyploidy (in one of two studies), mitotic spindle disturbances, and cell transformation in vitro in cell lines from rodents and other mammals (NTP, 2011). In human cells in vitro, it caused DNA single-strand breaks, sister chromatid exchange, micronucleus formation, and chromosomal aberrations (Robbiano et al., 2004). In rodents exposed in vivo, captafol caused DNA strand breaks, micronucleus formation, and dominant lethal mutations in rats (NTP, 2008); however, it did not cause mutations in the host-mediated assay in rats or dominant lethal mutations in albino mice (Kennedy et al., 1975).

Carcinogenicity Experimental evidence shows captafol to be carcinogenic in mice and rats. In mice, the vascular system, gastrointestinal system, and liver were subject to tumor formation, and included the following: (1) cancer of the lymphoid tissue (lymphosarcoma) in CD-1 mice, (2) blood vessel cancer (hemangiosarcoma) in B6C3F1 and CD-1 mice, (3) benign tumors of blood vessels of the spleen (splenic hemangioma) in B6C3F1 mice, (4) benign and malignant tumors of the small intestine in B6C3F1 mice, and (5) liver cancer (hepatocellular carcinoma) in B6C3F1 mice (NTP, 2011). Benign Harderian-gland tumors (adenoma) also were observed in CD-1 males (Ito et al., 1984; Quest et al., 1993). In rats, tumor sites were predominantly the liver and the kidney (NTP, 2011). However, the task force for evaluation found captafol to be active in a wide range of tests for genetic and related effects, including the generally insensitive in vivo assay for dominant lethal mutation. Based on the above observation, captafol was labeled as probable

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human carcinogen (Group 2A) by the International Agency for Research on Cancer, whereas USEPA categorized this compound as Group 2B: probable human carcinogen, and the American Conference of Governmental Industrial Hygienists (ACGIH) recognized it as not classifiable as a human carcinogen (Ito et al., 1996; Whyatt et al., 2003; NTP, 2008, 2011; Shinde et al., 2019).

Clinical management Exposed eyes and skin should be flushed with water in case of exposure. Patients exposed through the respiratory route to captafol should be monitored for respiratory distress, so that artificial ventilation may be provided and symptomatic treatment may be administered as necessary.

Exposure standards and guidelines Captafol is a pesticide no longer sold or used in the United States. The US Occupational Safety and Health Administration threshold limit value for captafol is reported to be 0.1 mg m−3. Captafol is a restricted use fungicide and as of 2010, the use of captafol on food crops has been banned. ACGIH Threshold Limit Value: 0.1 mg m−3 time-weighted average (1WA) (skin); Appendix A4 (Not Classifiable as a Human Carcinogen). National Institute for Occupational Safety and Health Recommended Exposure Limit: 0.1 mg m−3 lWA (skin) potential carcinogen (Pubchem, n.d.).

Conclusion Captafol is no longer used as a pesticide in many countries world-wide. It has been banned in the United States since 2010. Captafol exposure gives rise to various acute and chronic toxicities in humans, including those of the skin, the eye and respiratory tract. It is particularly carcinogenic in animals. Chemical fungicides and pesticides are used for effective control of phytopathogens. However, the deleterious effects of these chemicals on human health and environment strongly demand the search for eco-friendly approaches for pathogen control. In this regard, research is in progress with Chitosan, a biodegradable, biocompatible, and nontoxic cationic polymer, which is being investigated to develop micro- and/or nano-formulations. Because of its ability to chelate with many organic/inorganic compounds, it is being explored for its bioavailability, stability, and biocidal activity of a variety of fungicides or pesticides or misc. other agricultural products. Recent literature shows that advanced analytical methods for the multiresidue analysis of captan (plus its metabolite, tetrahydrophthalimide), folpet (plus its metabolite, phthalimide), captafol, and iprodione in cereals using liquid chromatography tandem mass spectrometry (LC-MS/MS) are available (Shinde et al., 2019).

References Ben-Dyke R, Sanderson DM, and Noakes DN (1970) Acute toxicity data for pesticides. World Review of Pest Control 1970(9): 119–127. Bhat VS, et al. (2020) An adverse outcome pathway for small intestinal tumors in mice involving chronic cytotoxicity and regenerative hyperplasia: A case study with hexavalent chromium, captan, and folpet. Critical Reviews in Toxicology 50(8): 685–706. https://doi.org/10.1080/10408444.2020.1823934. Dalvi RR and Mutinga ML (1990) Comparative studies of the effects on liver and liver microsomal drug-metabolizing enzyme system by the fungicides captan, captafol and folpet in rats. Pharmacology & Toxicology 66(3): 231–233. HSDB-Captafol (2023) https://haz-map.com/Agents/340. IARC (1991) Occupational exposure to insecticide application and some pesticides. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans. vol. 53, pp. 353–369. Lyon, France: International Agency for Research on Cancer. https://pubmed.ncbi.nlm.nih.gov/1842583/. IARC (1997) Captafol (IPCS-INCHEM). https://inchem.org/documents/iarc/vol53/09-captafol.html. IPCS (1990) Captafol. Health and Safety Guide No. 49. International Program on Chemical Safety. http://www.inchem.org/documents/hsg/hsg/hsg049.htm. IPCS, CEC (1994) International Chemical Safety Card on Captafol (April 1994). https://www.inchem.org/pages/icsc.html. Ito N, et al. (1996) Effects of pesticide mixtures at the acceptable daily intake levels on rat carcinogenesis. Food and Chemical Toxicology 34(11−12): 1091–1096. Ito N, Ogiso T, Fukushima S, Shibata M, and Hagiwara A (1984) Carcinogenicity of captafol in B6C3F1 mice. Gann 75(10): 853–865. https://ntp.niehs.nih.gov/ntp/roc/twelfth/2007/ expertpanelmtgs/captafol_report_parta_508.pdf. Kennedy GL Jr., Arnold DW, and Keplinger ML (1975) Mutagenicity studies with captan, captofol, folpet and thalidomide. Food and Cosmetics Toxicology 13(1): 55–61. Nazir A, Saxena DK, and Kar Chowdhuri D (2003) Induction of hsp70 in transgenic Drosophila biomarker of exposure against phthalimide group of chemicals. Biochimica et Biophysica Acta 1621(2): 218–225. https://europepmc.org/article/med/12726998. NTP: National Toxicology Program (2008) Final Report on Carcinogens Background Document for Captafol. Rep. Carcinog. Backgr. Doc. 8-5974 [1-xvi] 1–118. https://ntp.niehs.nih. gov/ntp/roc/twelfth/2010/finalbds/captafol_final_508.pdf. NTP-National Toxicology Program (2011) http://ntp.niehs.nih.gov/ntp/roc/twelfth/profiles/Captafol.pdf. Pubchem: Captafol. n.d. https://pubchem.ncbi.nlm.nih.gov/compound/17038#section¼Adverse-Effects Quest JA, et al. (1993) Evaluation of the carcinogenic potential of pesticides. 4. Chloroalkylthiodicarboximide compounds with fungicidal activity. Regulatory Toxicology and Pharmacology 17(1): 19–34. https://pubmed.ncbi.nlm.nih.gov/8441825/. Rahden-Staron I (2002) Toe inhibitory effect of the fungicides captan and captafol on eukaryotic topoisomerases in vitro and lack of recombinagenic activity in the wing spot test of Drosophila melanogaster. Mutation Research 518(2): 205–213. https://pubmed.ncbi.nlm.nih.gov/12113771/. Raman P (2014) Captafol. In: Encyclopedia of Toxicology, 3rd edn, 655–658. https://www.sciencedirect.com/science/article/pii/B9780123864543002682.

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Robbiano L, et al. (2004) DNA damage and micronuclei induced in rat and human kidney cells by six chemicals carcinogenic to the rat kidney. Toxicology 204(2–3): 187–195. Shinde R, et al. (2019) Multi-residue analysis of captan, captafol, folpet, and iprodione in cereals using liquid chromatography with tandem mass spectrometry. Food Additives & Contaminants. Part A, Chemistry, Analysis, Control, Exposure & Risk Assessment 36(11): 1688–1695. https://doi.org/10.1080/19440049.2019.1662953. Tamano S, Kurata Y, Kawabe M, et al. (1990) Carcinogenicity of captafol inF344/DuCrj rats. Japanese Journal of Cancer Research 81(12): 1222–1231. Tamano S, Kurata Y, Shibata M, Tanaka H, Ogiso T, and Ito N (1991) 13-Week oral toxicity study of captafol inF344/DuCrj rats. Fundamental and Applied Toxicology 17(2): 39G–398. Tamano S, Kawabe M, Sano M, Masui T, and Ito N (1993) Subchronic oral toxicity study of captafol in B6C3F1 mice. Journal of Toxicology and Environmental Health 38(1): 69–75. Whyatt RM, et al. (2003) Contemporary-use pesticides in personal air samples during pregnancy and blood samples at delivery among urban minority mothers and newborns. Environmental Health Perspectives 111(5): 749–756. Zang X, Wang J, Wang O, et al. (2008) Analysis of captan, folpet, and captafol in apples by dispersive liquid-liquid microextraction combined with gas chromatography. Analytical and Bioanalytical Chemistry 392(4): 749–754. https://pubmed.ncbi.nlm.nih.gov/18665351/.

Further reading Hayes WJ Jr. (1982) Pesticides Studied in Man. Baltimore/London: Williams and Wilkins. p. 672. https://books.google.com/books/about/Pesticides_Studied_in_Man.html? id¼mWDL9vCkBDYC. Thompson CM, et al. (2017) Comparison of toxicity and recovery in the duodenum of B6C3F1 mice following treatment with intestinal carcinogens captan, folpet, and hexavalent chromium. Toxicologic Pathology 45(8): 1091–1101. https://doi.org/10.1177/0192623317742324.

Relevant websites http://pmep.cce.comell.edu/profiles/extoxnet/24d-captan/Captafol-ext.html :Cornell University. http://ull.chemistry.uakron.edu/erd :The Chemical Database - The Department of Chemistry at the University of Akron. https://www.epa.gov/pesticide-science-and-assessing-pesticide-risks/evaluating-pesticides-carcinogenic-potential :EPA criteria Classification of Pesticides.

Captan Ida Adeli, Hosna MohammadSadeghi, and Behnaz Bameri, Toxicology and Disease Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of T Song, Captan, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 575–577, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00105-6.

Chemical profile Background Uses/occurrence Environmental fate and behavior Toxicokinetics Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Chronic toxicity Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines PubChem URL CompTox URL References

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Abstract Captan is a broad-spectrum fungicide that has been used in agriculture since 1951. Captan exerts its fungicidal effect by inhibiting metabolism and the respiratory pathway. It is also used in industries other than agriculture. Exposure to captan may happen through ingestion, inhalation, skin or eye contact, and it immediately gets absorbed through the oral route. Captan is rapidly distributed, metabolized, and excreted; and does not accumulate in the body. It is genotoxic, carcinogenic, and immunotoxic and causes a wide range of harmful effects on the body. Furthermore, captan has deleterious impacts on both the reproductive system and the fetus.

Keywords Agriculture; Captan; Dicarboxamide; Fungicide; Tetrahydrophthalimide; Thiazolidine-2-thione-4-carboxylic acid

Chemical profile

• • • • •

Chemical Profile: Dicarboxamide. Name: 2-(trichloromethylsulfanyl)-3a,4,7,7a-tetrahydroisoindole-1,3-dione. Synonyms: N Trichloromethylthio 4 cyclohexane 1,2 dicarboximide, N-Trichloromethylthio-4-cyclohexane-1,2-dicarboximide, Vancide 89. CAS Number: 133-06-2. Molecular Formula: C9H8Cl3NO2S H

O

Cl

Cl Cl

•

N

Chemical Structure:

H Encyclopedia of Toxicology 4th Edition

S

O

https://doi.org/10.1016/B978-0-12-824315-2.00331-6

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Captan

Background Captan is an odorless compound that can exist in the forms of white to cream powder, colorless crystals, or white crystalline powder (PubChem, 2022). It was first applied in 1951 in agriculture due to its fungicidal activity (Fernandez-Vidal et al., 2019). Captan reacts with endogenous substances in fungus and inhibits its respiration and metabolism (Saha et al., 2022).

Uses/occurrence Captan is a broad-spectrum fungicide used to control diseases of vegetables, fruits, crops, and ornamental plants (Scariot et al., 2017). It is also used in food crop packaging boxes. Other applications include a preservative for cosmetic products, fungicide and bacteriostatic agent in soaps, fungicide in fabrics, plastics, paints, and leather (PubChem, 2022).

Environmental fate and behavior The half-life of captan is 1–10 days in a soil environment, and hours to days in water depending on acidities and temperatures. Captan is not mobile in soil but can significantly evaporate from the soil surface. It is quickly degraded in neutral water (PubChem, 2022). Exposure and Exposure Monitoring: Exposure to captan may occur through inhalation, ingestion, skin contact, or eye contact (PubChem, 2022).

Toxicokinetics Captan promptly gets absorbed through the gastrointestinal tract. As well, it is rapidly metabolized and eliminated from the body. Although captan is widely distributed, it doesn’t accumulate in tissues due to fast excretion (PubChem, 2022; Anastassiadou et al., 2020). After exposure via the oral route, the captan gets metabolized and forms two metabolites: tetrahydrophthalimide (THPI) and thiazolidine-2-thione-4-carboxylic acid (TTCA). Degradation in the gut seems to be an important step during captan metabolism. During experiments on rats, THPI metabolite was detected in both urine and feces, while TTCA metabolite was detected in urine. 92% and 96% of THPI were excreted within 48 and 96 h (85% in the urine and 12% in the feces) (PubChem, 2022). Besides, captan is metabolized by CYP450 enzymes, such as CYP3A, CYP1A1, CYP1A2, CYP2A1, and CYP2B1 in the liver (Bossou et al., 2020).

Mechanism of toxicity Captan increases intracellular Ca2+ and Zn2+ respectively by increasing the membrane permeability to Ca2+ and release of Zn2+ in the cell. Both calcium and zinc are intracellular messengers, and an increase in their amount causes intracellular signaling disruption. Furthermore, an increase in Ca2+ and Zn2+, a captan-induced decrease in non-protein thiols, and an increase in superoxide ion (O2%–) lead to risen oxidative stress (Inoue et al., 2018). Additionally, repeated exposure to relatively high doses of captan may impact liver enzymes. During in vitro studies, captan caused mitochondria swelling in rat liver and loss of intracellular potassium in human erythrocytes. Captan inhibits the function of mitochondria and is assumed to cause its inner membrane breakdown (Wexler, 2014).

In vitro toxicity data In isolated rat hepatocytes, the captan increased the oxidative damage parameters (Inoue et al., 2018). In an in vitro study, captan reduced the dehydroepiandrosterone-mediated estrogen level in JEG-3 cells at concentrations higher than 100 nmol/L. As well, it inhibited the activity of cytochrome P19A1 over 50% at 100 nmol/L concentration (Ge et al., 2018). Moreover, captan showed genotoxic effects within in vitro studies (PubChem, 2022; Anastassiadou et al., 2020).

Acute and short-term toxicity During a study, a single oral administration of captan to male rats with doses up to 15,000 mg/kg bw/day, resulted in suppressed body weight. While within two other studies in which the maximum dose was 14,000 and 13,786 mg/kg b.w./day, a single oral administration exerted different results. Haematuria in male rats, reduced motor activity in female rats, and diarrhea in both genders was observed in the first study. The result of the second study was rhinorrhea, lacrimation, salivation, and loose watery feces in both

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male and female rats. A single-dose administration of captan via the oral route (the maximum dose was 3000 mg/kg b.w./day) to male mice brought about reduced motor activity and loose watery feces. A developmental toxicity study done by administering an oral single dose of captan to male rabbits showed death. In another study, captan caused increased embryo resorption and post-implantation loss. In the same study with a higher maximum dose, increased post-implantation loss rate, increased dead embryos number, and alterations in external, skeletal, and soft tissue were observed. In the developmental toxicity study in which a single administration of captan via oral route was performed, embryo resorption, decrease in the number of viable fetuses, deformation of the tail, whole-body edema, and complex abnormality emerged (Food Safety, 2017). Evidence shows that captan is generally toxic to embryo and adult zebrafish. Captan inhibited the heartbeat of embryos of zebrafishes at a concentration of 0.76 mg/L. it also inhibited the hatching and decreased the body length of hatched larvae (Zhou et al., 2019). In an acute toxicity study on Folsomia candida, captan was lethal to this animal at 1000 mg/kg dry weight (d.w.) (Alves et al., 2014). Within an acute toxicity study on mice, two of them exposed to 16,000 ppm captan died in the first few days. Four mice exposed to 12,000 ppm showed dehydration and hunched posture and one mouse exposed to 6000 ppm became thin. Also, the food intake by mice exposed to 12,000 ppm dramatically decreased. Furthermore, crypts epithelial hyperplasia, villi enterocytes hypertrophy, increased villi enterocytes, an increase in mononuclear cell infiltrates in villi, and rarefaction of the lamina propria were observed (Thompson et al., 2017). Moreover, humans’ acute dermal exposure to captan caused dermatitis and conjunctivitis (PubChem, 2022).

Chronic toxicity Captan caused suppression in rats’ body weight within two chronic toxicity studies (Food Safety, 2017). Clarias batrachus fishes undergoing chronic exposure to sub-lethal doses showed a lower value of hematocrit, hemoglobin, and mean corpuscular hemoglobin (MCH), serum protein, k-factor, and lower specific growth rate. Besides, evidence revealed that captan exposure may impose stress-specific effects at the biochemical and physiological levels, which affects the whole health condition and lifespan of such animals negatively (Saha et al., 2022). Furthermore, prolonged or repeated exposure to captan may result in skin sensitization or dermatitis (PubChem, 2022).

Immunotoxicity The reduction in red blood cell antibodies and stimulation of splenic lymphoblasts by phytohemagglutinin (PHA) and lipopolysaccharide (LPS) was observed in animals fed 0.3% (w/w) for 42 days (Aroonvilairat et al., 2018).

Reproductive and developmental toxicity Developmental toxicity studies on rats showed that captan causes offspring, embryos, and fetuses to have lower body weight. Within two developmental studies on rabbits, in which the maximum dose of captan was 60 mg/kg b.w./day, captan caused suppression in maternal, fetus, and embryos body weight and non-teratogenic skeletal variations in fetuses and embryos. On the other hand, captan showed suppression in maternal body weight and teratogenic skeletal variations in fetuses and embryos during the developmental toxicity study in which the maximum dose was 100 mg/kg b.w./day. Moreover, in the developmental toxicity study on hamsters, an increased mortality rate in mothers and teratogenic suppressed body weight in embryos and fetuses were observed. Although captan was not recognized as teratogenic during a developmental toxicity study on monkeys, it led to miscarriage and death of mothers, embryos, and fetuses (Food Safety, 2017). In a study designed to evaluate the captan effects on mice reproductive system, lower body and ovary weight, disrupted follicle growth, a decrease in pre-ovulatory follicle numbers, an increase in the ratio of atretic follicles, and decreased levels of the follicle development-related molecules estrogen receptor beta (ER-b), luteinizing hormone receptor (LHR), and anti-Müllerian hormone (AMH) was observed as a result. Other outcomes include oxidative stress, inflammation, autophagy, apoptosis, decreased oocyte growth potential, and mitochondrial damage (Saha et al., 2022). Captan was observed to have teratogenic impacts on zebrafishes embryos as it caused pericardial edema, yolk sac edema, tail bending, spine curvature, and deformities (Zhou et al., 2019).

Genotoxicity Captan can directly interact with DNA and/or proteins related to chromatin like histones, polymerases, or topoisomerase II. In addition, it can induce the break of DNA in mammalian cells (Fernandez-Vidal et al., 2019). In humans, captan may cause pair mutation, gene conversion, and recombination (Scariot et al., 2017). The genotoxic effects of captan on mammalian cells were perceived in the study performed by Fernandez-Vidal et al. (Fernandez-Vidal et al., 2019).

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Carcinogenicity The US EPA classified Captan as B2, a probable human carcinogen. Captan is considered by the US National Institute for Occupational Safety and Health (NIOSH) to be a potential occupational carcinogen (PubChem, 2022).Three carcinogenicity studies (26 months, 22 months, and 80 weeks) revealed that captan causes duodenal mucosal hyperplasia, increased duodenal adenoma, and adenocarcinoma lymphoid infiltration in the duodenum, and lower average body weight (Food Safety, 2017). Within a study on the relationship between agricultural exposure to pesticides and breast cancer among Latina in a specific region in California, the captan was detected to correlate with breast cancer (Mills et al., 2019). Exposure to captan for 7 years is related to a three-fold increase in multiple myeloma risk (Presutti et al., 2016). In vivo studies indicated that captan leads to the development of gastric and duodenal tumors following the mucosal damage it brought about (Saha et al., 2022).

Organ toxicity A study on private pesticide applicators in the United States demonstrated that captan causes olfactory impairment (Shrestha et al., 2020). In a prospective cohort study in pesticide applicators, captan was distinguished to increase the risk of age-related macular degeneration (AMD) (Montgomery et al., 2017). Captan exposure via skin causes dermatitis and skin sensitization. Persistent erythema, itching, and desquamation were observed on the face and back of the hand in men occupationally exposed to captan. Other symptoms following the overexposure to captan include blurred vision, dyspnea, diarrhea, vomiting, and conjunctivitis (PubChem, 2022). Agricultural workers’ disease, asthma, contact dermatitis, fatty liver, gastrointestinal neoplasms, glucose intolerance, hyperglycemia, insulin resistance, kidney neoplasm, non-Hodgkin lymphoma, rhinitis, uterine neoplasm, and weight gain are diseases and disorders mentioned to be related to captan (PubChem, 2022).

Clinical management Intoxication after acute captan exposure is unlikely. Treatment is needed if symptoms occur. Immediate first aid: stop the patient from being contacted to material and decontaminate them (remove the clothes if needed and wash the body with water and soap). Start artificial breathing if the patient is not breathing. If eyes are exposed to captan, remove contact lenses and wash eyes with water for at least 15 min (PubChem, 2022). Basic treatment: make sure that the patient is breathing and use ventilation if necessary. Irrigate each eye with 0.9% normal saline. If exposure was via the oral route, do not make the patient vomit. Rinse mouth with water and administer 5 ml/kg up to 200 ml of water to dilute captan. Use activated charcoal. Be careful about the seizure occurrence (PubChem, 2022).

Environmental fate and behavior Even though captan is supposed to be high to moderately mobile, studies on six different sites showed that it is relatively immobile to slightly mobile. Based on its vapor pressure, 9.0  10−8 mmHg, captan is not expected to volatilize from the moist or dry soil surface (PubChem, 2022). Captan is expected to adsorb to suspended solids. Based on Henry’s Law constant of 7.0  10−9 atm-cu m/mol, its vapor pressure, and its solubility in water, which is 5.1 mg/L, volatilization from the water surface is not an important fate for captan. Captan quickly becomes hydrolyzed in 5–9 pH (PubChem, 2022). Captan is expected to exist as vapor and particles in the atmosphere. Its vapor degrades due to its reaction with photochemically-produced hydroxyl radicals and ozone. Captan does not undergo photolysis by sunlight as it does not have chromophores absorbing at wavelengths >290 nm (PubChem, 2022).

Ecotoxicology In a study that evaluated residues of different compounds, captan residue was detected in soil, plant leaves, flowers, pollen, honeybee bodies, honeybee broods, wax, honey samples, and crops (Piechowicz et al., 2021). Additionally, captan was found to decrease the microbial biomass in soils, thereby affecting mycetophagous nematodes (Alves et al., 2014).

Exposure standards and guidelines Based upon the studies carried out on rats, the acceptable daily intake (ADI), the acute reference dose (ARfD), the acceptable operator exposure level (AOEL), and the acute acceptable operator exposure level (AAOEL) for captan are respectively 0.25, 0.9, 0.25, and 0.9 mg/kg b.w. per day (Food Safety, 2017). Captan is an environmentally hazardous compound in solid and liquid phases, so it should not be transported with food. Besides, captan is a dangerous air pollutant and causes serious health problems, therefore it should be prevented from emission (PubChem, 2022).

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PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/8606

CompTox URL https://comptox.epa.gov/dashboard/chemical/details/DTXSID9020243

References Alves PR, et al. (2014) Seed dressing pesticides on springtails in two ecotoxicological laboratory tests. Ecotoxicology and Environmental Safety 105: 65–71. Anastassiadou M, et al. (2020) Peer review of the pesticide risk assessment of the active substance captan. EFSA Journal 18(9): e06230. Aroonvilairat S, et al. (2018) Effects of topical exposure to a mixture of chlorpyrifos, cypermethrin and captan on the hematological and immunological systems in male Wistar rats. Environmental Toxicology and Pharmacology 59: 53–60. Bossou YM, et al. (2020) Impact of pesticide coexposure: An experimental study with binary mixtures of lambda-cyhalothrin (LCT) and captan and its impact on the toxicokinetics of LCT biomarkers of exposure. Archives of Toxicology 94(9): 3045–3058. Fernandez-Vidal A, et al. (2019) Exposure to the fungicide captan induces DNA base alterations and replicative stress in mammalian cells. Environmental and Molecular Mutagenesis 60(3): 286–297. Captan (Pesticides). Food Safety 5(2): 61–66. Ge H, et al. (2018) Effects of folpet, captan, and captafol on human aromatase in JEG-3 cells. Pharmacology 102(1–2): 81–87. Inoue T, et al. (2018) Captan-induced increase in the concentrations of intracellular Ca(2+) and Zn(2+) and its correlation with oxidative stress in rat thymic lymphocytes. Environmental Toxicology and Pharmacology 63: 78–83. Mills PK, et al. (2019) Agricultural exposures and breast cancer among Latina in the San Joaquin Valley of California. Journal of Occupational and Environmental Medicine 61(7): 552–558. Montgomery MP, et al. (2017) Pesticide use and age-related macular degeneration in the agricultural health study. Environmental Health Perspectives 125(7): 077013. Piechowicz B, et al. (2021) Assessment of risk to honey bees and honey consumers resulting from the insect exposure to captan, thiacloprid, penthiopyrad, and l-cyhalothrin used in a commercial apple orchard. Environmental Monitoring and Assessment 193(3): 129. Presutti R, et al. (2016) Pesticide exposures and the risk of multiple myeloma in men: An analysis of the North American Pooled Project. International Journal of Cancer 139(8): 1703–1714. PubChem (2022) PubChem Compound Summary for CID 8606, Captan. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Captan (03/18/2022) Saha S, et al. (2022) Physiological (haematological, growth and endocrine) and biochemical biomarker responses in air-breathing catfish, Clarias batrachus under long-term CaptanW pesticide exposures. Environmental Toxicology and Pharmacology 90: 103815. Scariot FJ, et al. (2017) Necrotic and apoptotic cell death induced by Captan on Saccharomyces cerevisiae. World Journal of Microbiology and Biotechnology 33(8): 159. Shrestha S, et al. (2020) Occupational pesticide use and self-reported olfactory impairment in US farmers. Occupational and Environmental Medicine. https://doi.org/10.1136/oemed2020-106818. Thompson CM, et al. (2017) Comparison of toxicity and recovery in the duodenum of B6C3F1 mice following treatment with intestinal carcinogens captan, folpet, and hexavalent chromium. Toxicologic Pathology 45(8): 1091–1101. Wexler P (2014) Encyclopedia of Toxicology, 3rd edn. Elsevier, pp. 1–999. Zhou Y, et al. (2019) Toxicity effects of captan on different life stages of zebrafish (Danio rerio). Environmental Toxicology and Pharmacology 69: 80–85.

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Carbamate pesticides Horacio Heinzen and María Verónica Cesio, Grupo de Análisis de Contaminantes Traza (GACT), Facultad de Química, Universidad de la República, Montevideo, Uruguay © 2024 Elsevier Inc. All rights reserved. This is an update of R.C. Gupta, Carbamate Pesticides, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 661–664, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00106-8.

Chemical structure and uses Historical background Exposure routes Mechanism of toxicity Acute toxicity Intermediate syndrome Chronic toxicity Interaction with other anticholinesterase pesticides Biomarkers and biomonitoring Human risk Clinical management Ecotoxicology Environmental fate Future directions References Further reading

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Abstract The carbamic acid substitution rules the pesticide activity. The N-methyl esters of carbamic acid are insecticides. Other derivatives of carbamic, thiocarbamic, and dithiocarbamic acids are herbicides and fungicides. Carbamate insecticides were broadly applied, but nowadays their use is declining all over the world. Nevertheless, benzimidazole carbamates are still widely employed. N-methylcarbamate insecticides are toxic through acetylcholinesterase inhibition. Mammals overexposure to them results in poisonings, causing hypercholinergic activity toxic symptoms. Carbamate-induced excitotoxicity also involves hyperactivation of N-methyl-D-aspartate receptors. Treatment of N-methylcarbamates poisonings rests with atropine sulfate. Carbamate herbicides and fungicides show low mammalian toxicity, but chronic effects are of concern.

Keywords Aldicarb; Carbamate fungicides; Carbamate herbicides; Carbamate insecticides; Carbaryl; Carbofuran; Cholinesterase inhibitors; Dithiocarbamates; Immunotoxicity; Methomyl; Molinate; Thiocarbamates

Key points

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The pesticide bioactivity of carbamates and thiocarbamates is reviewed. Carbamates can show insecticidal, fungicidal and herbicidal action. The toxicological profile of each carbamate motif is reviewed. Human toxicity of carbamates (insecticides, fungicides and herbicides) is updated. Attention should be paid to chronic exposure to carbamates.

Chemical structure and uses The carbamate compounds are subdivided into three main groups: carbamates, thiocarbamates, and dithiocarbamates (see basic structures in Fig. 1). The relationship between carbamate structure and bioactivity is shown in Fig. 2. N-methylcarbamates are usually used as insecticides, such as bendiocarb, carbaryl, carbofuran, methomyl, oxamyl, propoxur, and many others. Currently, carbaryl and propoxur are the only two carbamates that are also recommended for the control of ectoparasites on animals. Some of the carbamates, such as aldicarb, carbofuran, and propoxur, are commonly encountered in malicious poisonings in dogs and

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A

B

C

D

E

Fig. 1 General formulas of carbamate function according to the pesticide activity: A&B insecticides, C & D herbicides, E, fungicides.

Fig. 2 General formulas for carbamates.

mammalian and avian wildlife. Derivatives of carbamic acid (asulam, barban, chloropropham, chlorbupham, karbutilate, and phenmedipham), thiocarbamic acid (butylate, cycloate, diallate, EPTC, molinate, and triallate), and dithiocarbamic acid (metham sodium) are used as herbicides. The N-benzimidazole carbamates are largely employed fungicides. They can be also classified as benzimidazole fungicides. Other Fungicides of this group include, mancozeb, maneb, and thiram. Carbamate insecticides and herbicides were widely used in the XX century to protect crops as well as human and animal health from insect-vector-mediated diseases but, as evidence accumulate on their toxicity and negative ecotoxicological consequences, either acute or chronic, most of them have been banned or their use strictly restricted. Since 2011, the major agencies (US-EPA, EU-EFSA) have issued restrictions and/or prohibitions to the use of these compounds (aldicarb, asulam, molinate, benomyl, among others). . .

Historical background During the mid-nineteenth century, the first carbamate compound physostigmine (eserine alkaloid) was extracted from the Calabar beans (ordeal poison) of a perennial plant Physostigma venenosum commonly found in tropical West Africa. It was not until the 1960s and 1970s that dozens of carbamates (esters of carbamic acid) were synthesized for pesticidal use. Carbaryl was the first carbamate to be used as an insecticide. The knowledge of autonomic pharmacology, especially the cholinergic system, enabled the synthesis of more potent carbamates and to understand their mechanism of toxicity. Aldicarb was synthesized to mimic the chemical structure of acetylcholine (ACh). Currently, aldicarb has the maximal potential for mammalian toxicity and it was commonly marketed globally under the trade name Temik®. The high leachability of aldicarb caused serious damages in the aquatic fauna and threatened human health, as it reached freshwater sources. Although thousands of carbamates have been synthesized, not more than two dozen compounds were used practically as insecticides and ectoparasiticides. Despite very little residue persists in the environment and mammalian system compared with organochlorines and organophosphates, the negative effects on health and the environment, the advent of new and less toxic compounds, has limited enormously their use. Since the 1940s, derivatives of carbamic acid, including ethylene bisdithiocarbamates (EBDCs), have been widely used as fungicides throughout the world. Common examples of this group of fungicides include mancozeb, maneb, metiram, nabam, and zineb. In general, thiocarbamates and dithiocarbamates are of low mammalian toxicity because they do not inhibit acetylcholinesterase (AChE) activity, and therefore pose less risk when compared with N-methylcarbamate insecticides. Carbamate herbicides, as they have different modes of action, can affect life in different ways. Some of them are banned (molinate) for being an endocrine disruptor by inhibiting testosterone biosynthesis in rats, or their use is restricted as their presence in potable water cannot be precluded (asulam).

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Exposure routes Most carbamates are easily absorbed following oral, respiratory, and parenteral exposure. Carbamates can also be absorbed through the skin, although the absorption rate appears to be relatively slow. It is established that following absorption; these pesticides are well distributed in tissues throughout the body. Being lipophilic, maximum levels of these compounds are usually found in the adipose tissue and the brain. Carbamates are usually metabolized in the liver to less toxic or nontoxic metabolites. However, some of the metabolites of carbamates are quite toxic. For example, the two major metabolites of carbofuran (3-hydroxycarbofuran and 3-ketocarbofuran) have a significant impact on the overall toxicity of carbofuran. This is partly because these metabolites are toxic and they are trapped in enterohepatic circulation. Because of the extensive metabolism of carbamates in the body, rarely are parental carbamates detected in the urine. Also, only a few metabolites are detected in the urine that can be used as biomarkers of carbamate insecticides exposure. In essence, N-methylcarbamate insecticides are readily absorbed, widely distributed, and extensively metabolized before being excreted in the urine and/or bile. Residues of some carbamates and their metabolites can be detected in the milk. In the case of herbicides such as asulam, the presence of glucose, acetyl and malonyl conjugates as well as sulfanilamide and their derivatives can be used as markers of the exposure to the herbicide (Arena et al., 2018). Carbamic acid derivative fungicides, such as EBDCs, are readily absorbed, rapidly metabolized, and excreted within 24 h through urine and feces, with no evidence of long-term bioaccumulation.

Mechanism of toxicity Acute clinical signs of N-methylcarbamate insecticides toxicity are primarily associated with the inhibition of acetylcholinesterase (AChE) at synapses in the brain and neuromuscular junctions in skeletal muscles. Of course, these insecticides can also bind to many other enzymes, receptors, and proteins. Carbamates inhibit AChE activity by carbamylation, and as a result, acetylcholine (ACh) accumulates at the nerve endings of all cholinergic nerves and causes an overstimulation of electrical activity. Inhibition of AChE >70% leads to a toxic-level accumulation of ACh at cholinergic junctions (e.g., central nervous system, neuromuscular junction, autonomic preganglionic and parasympathetic postganglionic synapses, and the sympathetic innervation of the adrenal and sweat glands). Carbamates interact with AChE in the same manner as the natural substrate ACh, except the rates of hydrolysis and reactivation of AChE (decarbamylation) appear to be drastically slower than for the hydrolysis of the acetylated enzyme. The turnover time for ACh is of the order of 150 ms, whereas the carbamylated enzyme t½ for hydrolysis is substantially slower (15–30 min). Accumulated ACh overstimulates muscarinic receptors (mAChRs) and nicotinic receptors (nAChRs), and consequently the symptoms of hyper cholinergic preponderance are seen. Evidence also suggests that some carbamates, such as aldicarb, bendiocarb, physostigmine, and propoxur, directly interact with ACh receptors. Some carbamate insecticides induce a variety of toxic effects through noncholinergic mechanisms. Evidence of noncholinergic mechanisms was presented by the involvement of glutamate release, causing activation of N-methyl-D-aspartate (NMDA) receptors. In addition, the adenosinergic, gamma-aminobutyric acid (GABAergic), and monoaminergic systems may also be involved in the seizures and lethality associated with carbamates. Carbamate-induced neuronal cell death is a consequence of a series of extracellular and intracellular events leading to the intracellular accumulation of Ca2+ ions and the generation of free radicals. Excessive free radical production causes oxidative/nitrosative stress, to which the brain is especially vulnerable. These events result in mitochondrial damage and dysfunction, neuronal energetic dysfunction, and neurodegeneration and neuronal death. Toxicity of N-methylcarbamates has been related to a drop in the immune response, that it was correlated with cancer incidence. The mechanisms of carbamates insecticides to attack the immune system is depicted in Fig. 3. N-methylcarbamates negatively influence immune responses via parallel mechanisms such as the inhibition of serine hydrolases in immune cells, oxidative damage in the spleen and the thyroid and modulation of signal transduction pathways in women (Corsini et al., 2013) as well as in children (Jones et al., 2014). The over expression T regs cells (CD4+CD25+ FoxP3+ regulatory T cells) which is one of the ways tumors elude the immune system, is mediated by carbamate insecticides in the surroundings cancerous cells. This fact causes a complication in cancer immunotherapy (Dhouib et al., 2015). In addition, N-methylcarbamates enhance the apoptosis of T cells, down regulating the immune response of the organism (Li et al., 2015). Herbicides of the carbamate family show different modes of action, either inhibiting folic acid biosynthesis (asulam) or mitosis, disrupting the organization of microtubules (molinate). EFSA reasoned opinion (Arena et al., 2018) on the herbicide asulam stressed that “further data are needed to conclude on the human relevance of the thyroid toxicity observed in test species exposed to asulam; the outcome of which may require further consideration in terms of endocrine disrupting potential” and also “data gaps are identified concerning the available genotoxicity studies, the developmental thyroid toxicity studies and dermal toxicity studies.” Thiocarbamates and dithiocarbamates are of low mammalian toxicity and have received less attention, and consequently their mechanisms of action are less understood. Among thiocarbamate herbicides, molinate and diallate are of concern because these two compounds are relatively more toxic. Molinate inhibit lipid synthesis and decreases aldehyde dehydrogenase, an enzyme important in the catabolism of many neurotransmitters, which may account for its central and peripheral neurotoxicity in several species. Metabolites of molinate can also interfere with testicular esterases, inhibiting testosterone production and leading to reproductive toxicity in laboratory animals. It is classified as endocrine disrupting chemical.

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Fig. 3 Pathways of direct immunotoxicity of carbamates. From Dhouib I, et al. (2015) Carbamates pesticides induced immunotoxicity and carcinogenicity in human: A review. Journal of Applied Biomedicine, https://doi.org/10.1016/j.jab.2016.01.001.

Fungicides such as mancozeb, maneb, and metiram produce toxicity in the thyroid. These compounds inhibit the synthesis of thyroid hormones thyroxine (T4) and triiodothyronine (T3), leading to elevated levels of thyroid-stimulating hormone (TSH) via feedback stimulation of the hypothalamus and pituitary. Continuous and prolonged elevation of TSH levels results in hypertrophy and hyperplasia of the thyroid follicular cells, leading to development of follicular nodular hyperplasia, adenoma, and/or carcinoma.

Acute toxicity Depending on the dose, frequency, and length of exposure, carbamate insecticides can produce minor health effects, such as mild discomfort or chest pain, or effects as serious as convulsions, seizures, coma, and death. By employing in vivo and in vitro models, AChE inhibiting carbamates are known to produce a variety of toxicologic effects on the central nervous system, peripheral nervous system, musculoskeletal, cardiovascular, ocular, immunologic, reproductive, and other body systems, in addition to oxidative stress, apoptosis, endocrine disruption, and carcinogenesis. Based on acute toxicity, some of the carbamate compounds, such as aldicarb, carbofuran, oxamyl, methomyl, and many others are extremely toxic to mammalian and avian species. In general, onset of clinical signs appears within less than an hour. Symptoms of acute poisoning with carbamate insecticides result from overstimulation of both muscarinic and nicotinic ACh receptors because of accumulation of ACh resulting from AChE inactivation. The muscarinic symptoms include hypersalivation, excessive tracheobronchial secretions, gastrointestinal cramps, lacrimation, dacryorrhea, nausea, excessive sweating, urinary incontinence, diarrhea, miosis, and bradycardia. The nicotinic receptor-associated effects include muscle fasciculations, tremors, muscle weakness, flaccid paralysis, blurred vision, vomiting, and paralysis of respiratory muscles. Exposure to high doses of a carbamate can lead to symptoms of CNS origin, including restlessness, tremors, convulsions, partial or generalized seizures, mental disturbance, incoordination, cyanosis, and coma. Finally, death ensues within a few hours because of cardiac and respiratory failure. Clinical signs of acute poisoning usually resolve within a few hours of exposure, but some symptoms of a neuropsychological nature appear to persist for a longer period. The surviving patient may exhibit symptoms such as schizoid reactions, paranoid delusions, poor sleep because of hallucinations and nightmares, and deficits in memory and attention. Signs and symptoms of thiocarbamate toxicity include anorexia, squinting, hypersalivation, lacrimation, piloerection, dyspnea, ataxia, hypothermia, incoordination, depression, paresis, muscular fibrillation, convulsions, and death. Thiobencarb has been

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shown to cause toxic neuropathies in neonatal and adult rats. Diallate poisoning is reported in many species with signs and symptoms of anorexia, ataxia, muscular contractions, exhaustion, and prostration. The cat appears to be the most sensitive species.

Intermediate syndrome In the late 1980s, intermediate syndrome (IMS) was reported for the first time in human patients who were poisoned with large quantities of OP insecticides. Recently, a carbamate insecticide carbofuran was demonstrated to cause IMS in patients accidentally or intentionally exposed to large doses of this insecticide. Clinically, IMS is characterized by acute paralysis and weakness in several cranial motor nerves, neck flexors and facial, extraocular, palatal, nuchal, proximal limb, and respiratory muscles 24–96 h after poisoning. Generalized weakness, depressed deep tendon reflexes, ptosis, and diplopia are also evident. From mechanisms and treatment viewpoints, IMS is better characterized for OPs than carbamates, because recovery is much faster with carbamates. IMS with OPs involves depressed AChE, expressed nAChR mRNA, and increased oxidative stress. But the central mechanism seems to be the defect(s) at the neuromuscular endplate and postsynaptic level involving nAChRs. It can be hypothesized that carbamate-induced IMS may involve similar mechanisms as described for OPs.

Chronic toxicity Interestingly, carbamates do not cause peripheral neuropathy, as do some organophosphates (OPs). This is because carbamates may inhibit neurotoxic esterase activity (the “first step” in the precipitation of the neuropathy), but do not “age” (the definitive step in precipitation of the neuropathic response). Also, tolerance development has been known for OPs following chronic exposure for almost half a century, but to date no tolerance development is reported for any carbamate insecticides. In addition to inhibition of AChE activity, carbamates have been reported to cause skin and eye irritation, hemopoietic alterations, degeneration of the liver, kidneys, and testes, as well as functional and histopathologic changes in the nervous system after long-term, high-dose exposures. Moreover, some carbamates are known to produce reproductive and teratogenic effects. Fetuses of mothers dosed with a carbamate have been reported to exhibit increased mortality and decreased weight gain. Men chronically exposed to carbofuran have semen of low quality, as the spermatozoa and spermatids are found to be multinucleated. Carbamates are also considered embryotoxic, fetotoxic, teratogenic, mutagenic, and carcinogenic. In a long study on the exposure to carbofuran of pregnant women and children in China, a delayed physical development at the age of seven as well as reduced weight of the newborn correlated negatively with carbamate exposure (Zhang et al., 2022). Chronic studies with thiophanate (a methyl-benzimidazole carbamate) fungicide caused an increase in liver weight in rats, and increased thyroid weights in rats and dogs. The major concern with EBDC pesticides (including maneb and metiram) is that their major metabolite, ethylene thiourea (ETU), is goitrogenic. ETU is known to interfere with thyroid peroxidase. In a 2-year chronic study in rats, ETU produced thyroid follicular hyperplasia at 50 ppm and malignant thyroid neoplasia at 250 ppm. ETU is also known to cause hepatocellular adenomas, anterior pituitary adenomas, and reproductive and developmental abnormalities. Mancozeb can produce small effects on thyroid morphology and a depression of iodine uptake.

Interaction with other anticholinesterase pesticides Carbamate and OP insecticides are often used in combination, with the objective of achieving synergistic interaction and controlling a wide range of insects, including those that are resistant. Therefore, exposure of the environment as well as humans and animals to multiple pesticides is inevitable. Under such circumstances, exposure to a single AChE-inhibiting insecticide is at the subtoxic level, but simultaneous exposure to more than one can sometimes lead to devastating health effects because of an additive or potentiating interaction. Studies based on laboratory animals have revealed potentiating toxicity following simultaneous exposure to OPs (P]S type) and N-methylcarbamate insecticides. Thus, avoiding such interactions in nontarget species are the challenges that we face today, although there are better options at hand.

Biomarkers and biomonitoring Biomonitoring data are useful for a variety of applications, from exposure assessment to risk assessment. Because carbamates are unstable compounds, their metabolites are also considered to be determined in serum/plasma, blood, and urine to estimate the exposure levels of carbamates. Carbamates that are commonly encountered in poisonings include aldicarb, bendiocarb, benomyl, carbaryl, carbofuran, carbosulfan, methomyl, pirimicarb, and propoxur. Major metabolites of carbaryl (1-naphthol, 2-naphthol, and/or 4-hydroxycarbarylglucuronide), carbofuran (3-hydroxycarbofuran and 3-ketocarbofuran), and propoxur (2-isopropoxyphenol) are also analyzed in addition to the parent compounds in case of carbamate poisoning, as well as aldicarb sulfoxide and sulfone. Carbamates and their metabolites are also measured in saliva for pharmacokinetics and dosimetry. Recently, liquid chromatography–mass spectrometry has been employed to identify novel biomarkers of AChE-inhibiting pesticide exposure

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by detecting their adducts on serine of butyrylcholinesterase and tyrosine of albumin. Both scientific and regulatory communities have recognized that erythrocytes-AChE inhibition is a sensitive biomarker of exposure to carbamates and OPs, because it serves as a sensitive surrogate endpoint for the inhibition of brain-AChE. However, AChE inhibition measurement cannot ascribe to a specific pesticide exposure event.

Human risk Risks to humans are significant from overexposure to N-methylcarbamate insecticides, whereas risks are minimal from thiocarbamate and dithiocarbamate herbicides and fungicides.. The occurrence of sulfanilamine, a metabolite of asulam is of concern for risk assessment studies, as it showed to be the major degradation product of house preparations of asulam containing vegetables (Arena et al., 2018).

Clinical management Humans and animals acutely poisoned with carbamate insecticides usually show the signs and symptoms of hypercholinergic preponderance, such as salivation, lacrimation, diarrhea, nausea, tremors, miosis, bradycardia, headache, confusion, and sometimes death. Dogs exposed to large doses of a carbamate insecticide in a malicious intent usually die. Both in humans and dogs, signs and symptoms are reported within minutes of exposure and can last for hours. However, because of reversibility of the inhibition of AChE, recovery is usually apparent within 24 h, depending on the dose of the carbamate and severity of poisoning. Metabolites of carbamates in the urine and inhibition of erythrocytes-AChE activity can be used for biological monitoring. In acute poisoning cases, atropine sulfate is recommended with a full dose. It should be repeated at half a dose on hourly intervals, until all hypersecretory signs completely subside. Oximes are typically contraindicated. Supportive therapy is highly recommended.

Ecotoxicology When applied directly to the soil, many carbamates can cause significant reduction in microflora and worms. Bees are especially sensitive to some carbamate pesticides. Some of the carbamate insecticides, such as aldicarb, carbofuran, and propoxur are deadly toxic to both mammalian and avian wildlife. Morbidity and mortality have been noted in wildlife even when some carbamates were used at the recommended levels. Deaths in many nontarget species have been reported as a result of malicious intent or secondary poisoning.

Environmental fate In general, carbamates are degraded into metabolites of lesser toxicity, and they have very little impact in terms of environmental persistence, but their impact in the local fauna or flora in acute when spills and similar incidents occur, is noteworthy. Carbamates can be degraded by microorganisms, soil, water, light, and animals. These compounds do not bioaccumulate in the food chain or environment. Of course, groundwater can have carbamate residue for an extended period of time. There are serious concerns about EBDC-based fungicides that produce a toxic metabolite ethylene thiourea (ETU) in the environment.

Future directions In every aspect, carbamates insecticides have received less attention compared with organophosphates, because carbamates produce toxicity by a similar mechanism and the toxic effects are reversible. Evidently, one area that needs attention is the clinical management of carbamates’ poisoning, because atropine is inadequate to cover the entire spectrum of toxicity. Evidence is being accumulated on toxic side effects of carbamates to which no attention has been paid in the past, such as the negative modulation of immune system and their relation with cancer development.

See also: Aldicarb; Benomyl; Carbaryl; Carbofuran; Cholinesterase inhibition; Dithiocarbamates; Methomyl; Molinate; Propoxur

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References Arena M, et al. (2018) Peer review of the pesticide risk assessment of the active substance asulam (variant evaluated asulam-sodium). EFSA Journal. https://doi.org/10.2903/j. efsa.2018.5251. Corsini E, et al. (2013) Pesticide induced immunotoxicity in humans: A comprehensive review of the existing evidence. Toxicology 307: 123–135. Dhouib I, et al. (2015) Carbamates pesticides induced immunotoxicity and carcinogenicity in human: A review. Journal of Applied Biomedicine. https://doi.org/10.1016/j. jab.2016.01.001. Jones K, Everard M, and Harding A-H (2014) Investigation of gastrointestinal effects of organophosphate and carbamate pesticide residues on young children. International Journal of Hygiene and Environmental Health 217(2–3): 392–398. Li Q, Kobayashi M, and Kawada T (2015) Carbamate pesticide induced apoptosis in human T lymphocytes. International Journal of Environmental Research and Public Health 12(4): 3633–3645. Zhang J, et al. (2022) Carbamate pesticides exposure and delayed physical development at the age of seven: Evidence from the SMBCS study. Environment International 01604120160(2022): 107076. https://doi.org/10.1016/j.envint.2022.107076.

Further reading Gupta RC (2004) Brain regional heterogeneity and toxicological mechanisms of organophosphates and carbamates. Toxicology Mechanisms and Methods 14: 103–143. Gupta RC (2006) In: Gupta RC (ed.) Toxicology of Organophosphate and Carbamate Compounds, pp. 1–763. Amsterdam: Academic Press/Elsevier. Gupta RC and Crissman JW (2012) Agricultural chemicals. In: Haschek-Hock WM, Rousseaux CG, and Wallig MA (eds.) Handbook of Toxicologic Pathology, 3rd edn. Amsterdam: Elsevier. Gupta RC and Milatovic D (2012a) Organophosphates and carbamates. In: Gupta RC (ed.) Veterinary Toxicology: Basic and Clinical Principles, pp. 573–585. Academic Press/Elsevier. Gupta RC and Milatovic D (2012b) Toxicity of organophosphates and carbamates. In: Marrs TC (ed.) Mammalian Toxicology of Insecticides, pp. 104–126. Cambridge: RSC Publications. Jokanovic M (2010) Medical treatment of poisoning by organophosphates and carbamates. In: Satoh T and Gupta RC (eds.) Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology, pp. 583–597. Hoboken: John Wiley and Sons. Satoh T and Gupta RC (2010) In: Satoh T and Gupta RC (eds.) Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology, pp. 1–625. Hoboken: John Wiley and Sons.

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Carbamazepine Anca Oana Doceaa, Valentina Patricia Predoib, Daniela Calinac, and Andreea Letitia Arsened, aDepartment of Toxicology, University of Medicine and Pharmacy of Craiova, Craiova, Romania; bFaculty of Pharmacy, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania; cDepartment of Clinical Pharmacy, University of Medicine and Pharmacy of Craiova, Craiova, Romania; dDepartment of Microbiology, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania © 2024 Elsevier Inc. All rights reserved. This is an update of J.C.Y. Lo, Carbamazepine, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 665–667, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00705-3.

Chemical profile Background Uses Environmental fate and behavior Exposure routes and pathways Toxicokinetics Mechanism of toxicity Acute and short-term toxicity (or exposure) Animal Human Chronic toxicity (or exposure) Animal Human Immunotoxicity Reproductive Toxicity Animal Human Genotoxicity Animal Human Carcinogenicity Clinical management Ecotoxicology Other hazards References

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Abstract Carbamazepine is a synthetic iminostilbene derivative used as a prescription anticonvulsant with antimuscarinic activities. It is slowly and incompletely absorbed during therapeutical use, and delayed absorption in overdose due to its ability to decrease gut motility and formation of pharmacobenzoars. Carbamazepine toxicity results from its multiple pharmacological effects, including antimuscarinic, anti-N-methyl-D-aspartate, and sodium channel actions. Acute toxicities include ataxia, nystagmus, coma, seizure, hypotension, and tachycardia. Chronic toxicities include hepatotoxicity, hyponatremia, hypersensitivity syndrome, and teratogenic effects. It is an inducer of CYP 3A4 that can lead to a wide variety of interactions with food or drugs metabolized by the same enzymatic system.

Keywords Anticonvulsant; Antimuscarinics; Ataxia; Carbamazepine; Coma; Hyponatremia; Multidose activated charcoal; Nystagmus; Pharmacobezoars; Seizure; Whole bowel irrigation

Key points

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Carbamazepine is used as a prescription anticonvulsant with antimuscarinic activities Carbamazepine toxicity results from its multiple pharmacological effects, including antimuscarinic, anti-N-methyl-D-aspartate, and sodium channel actions. Acute toxicities include ataxia, nystagmus, coma, seizure, hypotension, and tachycardia. Chronic toxicities include hepatotoxicity, hyponatremia, hypersensitivity syndrome, and teratogenic effects

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Chemical profile

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Name: Carbamazepine Chemical Abstracts Service Registry Number: 298-46-4 Synonyms: CBZ; 5H-dibenz(b,f )-azepine-5-carboxamide; Tegretol Molecular Formula: C15H12N2O Chemical Structure: NCONH2

Background Carbamazepine is a synthetic iminostilbene derivative structurally similar to imipramine, a tricyclic antidepressant. While unrelated structurally, carbamazepine shares a similar therapeutic action with phenytoin. Carbamazepine was first discovered in 1953 by Swiss chemist Walter Schindler. Throughout the 1960s, antimuscarinic was used and marketed for trigeminal neuralgia and as an anticonvulsant. By the 1970s, it was being used as a mood stabilizer for patients with bipolar disorder (Grzesiak et al., 2003).

Uses Carbamazepine is used in the treatment of epilepsy, mixed seizures, partial seizures, generalized tonic-clonic seizures and trigeminal neuralgia. Well-designed trials suggested that off-label use of carbamazepine has beneficial effects for refractory schizophrenia, improving both positive and negative symptoms, for the treatment of restless leg syndrome, for diminishing the agitation and aggression associated with dementia, and for the treatment of fibromyalgia and neuropathic pain. Unlabeled uses include treatment of postherpetic pain syndrome, neurogenic diabetes insipidus, alcohol withdrawal, and cocaine dependence. Carbamazepine can also be used in the treatment of bipolar I disorder with manic episodes and mixed manic-depressive episodes. In myoclonic seizures and petit mal seizures, carbamazepine is not usually effective.

Environmental fate and behavior Environmental exposure occurs via direct release into water or via vaporization into the air. It is susceptible to photolysis and is thought to have a half-life of roughly 63 days in lake water in vitro. However, when dissolved and exposed to direct photolysis, it has a half-life of approximately 1 day (Donner et al., 2013).

Exposure routes and pathways The exposure pathway for carbamazepine is exclusively oral (ingestion of tablets, extended-release tablets, suspension and solutions).

Toxicokinetics Carbamazepine is slowly and incompletely absorbed during therapeutic use. With large ingestions, absorption may be delayed and unpredictable, producing peak levels from 4 to 72 h after the overdose. The absorption phase in an overdose is highly variable because of carbamazepine’s poor solubility, ability to significantly decrease gut motility, and to form pharmacobezoars (Mohamed et al., 2017; Tolou-Ghamari et al., 2013). One of the primary metabolites of carbamazepine is carbamazepine-10,11-epoxide (CBZE), which also has anticonvulsant activity. A minor pathway results in iminostilbene formation. Further hydrolysis and conjugation produce six other known metabolites including 10,11-dihydroxycarbamazepine. Protein binding is 75% for carbamazepine and 50% for CBZE. However, the percentage of protein binding may decrease in massive overdose due to saturable binding sites. The volume of distribution is 0.8–1.9 L kg−1. The hydrolyzed and conjugated metabolites are eliminated through the kidneys, with only 1.2% free carbamazepine being found in the urine and 28% is eliminated unchanged in the feces. Carbamazepine induces drug-metabolizing enzymes so that the drug half-life is reduced in chronic use. The half-life in healthy adults ranges from 18 to 65 h in a single dose to 8–17 h during chronic administration. In newborns and children, the half-life is below 9 h (Puranik et al., 2013).

Mechanism of toxicity Carbamazepine is both an important anticonvulsant in therapeutic doses and a powerful proconvulsant in overdose. The therapeutic anticonvulsant mechanism is primarily related to blockade of presynaptic voltage-gated sodium channels. Blockade of the sodium channels is believed to inhibit the release of synaptic glutamate and possibly other neurotransmitters.

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Carbamazepine is also a powerful inhibitor of the muscarinic and nicotinic acetylcholine receptors, N-methyl-D-aspartate (NMDA) receptors, and the central nervous system (CNS) adenosine receptors. In addition, carbamazepine is structurally related to the cyclic antidepressant imipramine and in massive overdose, it may affect cardiac sodium channels.

Acute and short-term toxicity (or exposure) Animal Carbamazepine is not commonly used in animals. Limited information on toxicity exits. Tachyarrhythmias, hypotension, and seizures have been seen.

Human Doses of carbamazepine above 24 g have been correlated with fatal outcomes. The primary and common toxic event involves the CNS. Cardiac conduction delays and ventricular arrhythmias can be seen but are infrequent. Sinus tachycardia and hypotension are more commonly seen. In the few deaths directly attributable to carbamazepine toxicity, ventricular dysrhythmias have been the terminal event (Kasarskis et al., 1992). Coma, seizures, and respiratory depression are commonly seen in adults at levels greater than 40 mg mL−1 (170 mmol L−1). Status epilepticus has been reported. The incidence of serious toxicity is similar in adults and children. However, serum levels are less predictive in children (Bridge et al., 1994). Therefore, coma, seizures, and apnea may be seen at lower serum levels than in adults. Other manifestations of neurological toxicity are nystagmus, ataxia, choreoathetoid movements, encephalopathy, absence of corneal reflexes, decreased deep tendon reflexes, urinary retention, and dystonias. A cyclic clinical course can be seen, with a waxing and waning of symptoms. This may be due to the presence of a pharmacobezoar in the gut or more commonly due to a decrease in gastrointestinal motility produced by the prominent anticholinergic effects of carbamazepine.

Chronic toxicity (or exposure) Animal Male albino rats given injections of carbamazepine over 3 months demonstrated decreased prostate weight and decreased sperm motility. These changes did not affect fertility (Cohn et al., 1982).

Human Idiopathic hepatotoxicity has been reported as a rare manifestation of chronic therapy and is not dose-related (Dertinger et al., 1998). Hyponatremia is a common adverse effect associated with carbamazepine exposure, but mild and reversible. Increased antidiuretic hormone secretion and increased aquaporin 2 expression are proposed mechanisms. Hypersensitivity syndrome, or drug rash with eosinophilia and systemic symptoms, and Stevens–Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) have been described with carbamazepine administration (Ganeva et al., 2008; Roma
nska-Gocka et al., 2010). In vitro toxicity studies of carbamazepine on rat cerebellar granule cells have shown inhibition of NMDA-stimulated calcium entry in a rapid and reversible manner. These findings occurred in therapeutic concentrations of carbamazepine, which may help explain the antiseizure activity of carbamazepine. It is believed that the toxic cerebellar effects of carbamazepine may be due to this mechanism (Savidge and Bristow, 1997). The anticholinergic effects determine the increase of delirium risks in the elderly population, along with other anticholinergic effects such as constipation, urinary retention and an increase of intraocular pressure (Mizuno et al., 2000). Carbamazepine can lead to the development of homocysteinemia that can exacerbate the heart failure or be the cause of cardiac dysfunctions in healthy patients (Kasarskis et al., 1992).

Immunotoxicity Isolated cases of transient hypogammaglobulinemia have been reported with carbamazepine administration. IgG, IgA, and IgM were not found to be significantly reduced in patients treated with carbamazepine in comparison to control subjects who did not receive carbamazepine. An increase in cytotoxic activity of natural killer cells was noted in patients treated with carbamazepine. Carbamazepine has been associated with a potentially fatal idiosyncratic reaction known at antiepileptic hypersensitivity syndrome. This is characterized by eosinophilia, fever, rash, coagualopathy, and hepatotoxicity. Pancytopenia has also been reported with carbamazepine including neutropenia and agranulocytosis (Shao et al., 2013).

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Reproductive Toxicity Animal Rats born to mothers chronically fed carbamazepine during gestation demonstrated challenges with maintaining balance and had more difficulty lifting their hind legs than controls. Mice born to mothers who received intraperitoneal administration of carbamazepine demonstrated eye malformation, spina bifida, anencephaly and oligodactyly as well as vertebral and calvarial deformities, brachydactyly and short tail (Elshama et al., 2015). The exposure to carbamazepine caused a reduction in fetal weight and external congenital malformations (Jose et al., 2017).

Human Carbamazepine is highly teratogenic. Neural tube defects are associated with carbamazepine exposure during pregnancy and can lead to spina bifida. Carbamazepine is pregnancy category D (Nie et al., 2016).

Genotoxicity Animal High-dose carbamazepine resulted in an increased number of mutations per Drosophila wing (Sarikaya and Yüksel, 2008).

Human Associations have been shown for carbamazepine and human leukocyte antigen (HLA)-B∗1502 and HLA-A∗3101-induced cutaneous adverse drug reactions. Genetic studies showed that in patients of HAN Chinese ancestry, SJS/TEN is strongly associated with the HLA-B 1502 gene, while no risk is observed in Iranian patients having the same gene (Zhang et al., 2011). Another allele associated with dermatological severe reactions such as SJS/TEN, and DRESS syndrome (drug reaction with eosinophilia and systemic symptoms and toxic epidermic necrolysis is HLA 3101 found in European, Korean and Japanese ancestry (McCormack et al., 2011).

Carcinogenicity High-dose (25 mg kg−1 day−1) carbamazepine administration for more than 2 years caused hepatocellular tumors in female rats and benign interstitial tumors of the testes in male rats.

Clinical management Basic and advanced life-support measures should be utilized as necessary. Gastrointestinal decontamination procedures should be used as appropriate. Activated charcoal effectively binds carbamazepine. Multiple-dose activated charcoal (0.5 g kg−1 every 4 h) has been shown to decrease the half-life of carbamazepine. Generally, supportive measures are all that is required in carbamazepine overdose. Seizures initially should be managed with diazepam or lorazepam. However, persistent seizures may require advancement to phenobarbital or pentobarbital. Ventricular arrhythmias should be managed with lidocaine. Patients that present with widening of the QRS complex can be given sodium bicarbonate in 50 meq boluses. The presence of persistently high serum levels or fluctuating elevated serum levels may suggest the presence of a pharmacobezoar in the gut. Removal should be attempted, in the presence of an active bowel, with whole bowel irrigation using a polyethylene glycol–electrolyte solution. Hemodialysis and charcoal hemoperfusion have been used in carbamazepine overdose (Spiller, 2001)). There is no antidote available. Charcoal hemoperfusion can be difficult and the procedure entails risks (e.g., thrombocytopenia, hypothermia, hypocalcemia, hypophosphatemia, and hypoglycemia). Patients managed with early charcoal hemoperfusion experienced lower peak carbamaz epine concentrations, fewer cases of respiratory depression, seizures, and shorter hospitalizations compared with patients not treated with extracorporeal elimination.

Ecotoxicology Carbamazepine is not expected to produce acute ecotoxicological effects (van den Brandhof and Montforts, 2010).

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Other hazards Carbamazepine is an inducer of CYP 3A4. Coadministration of carbamazepine and contraceptive drugs may result in decreased level of contraceptive drugs and permit ovulation. Patients receiving carbamazepine should avoid grapefruit juice due to fluctuations in plasma drug levels and CYP3A4 inhibition is the proposed mechanism. Monoamine oxidase inhibitors (Selegiline, Tranylcypromine, Phenelzine, etc.) should not be associated with carbamazepine because significant side reactions (nausea, vomiting, tremor, rigidity, diaphoresis, hypertensive crises, seizures, coma) may occur. Coadministration of carbamazepine and verapamil may lead to increased plasma levels of carbamazepine. The administration in patients with abnormal ECG should be avoided due to the risk for the second or third-degree AV heart block. The withdrawal of carbamazepine should be done gradually due to the risk of status epilepticus that can appear in immediate withdrawal (Fuhr et al., 2021).

See also: Lidocaine; Polyethylene glycol

References Bridge TA, Norton RL, and Robertson WO (1994) Pediatric carbamazepine overdoses. Pediatric Emergency Care 10: 260–263. Cohn DF, Homonnai ZT, and Paz GF (1982) The effect of anticonvulsant drugs on the development of male rats and their fertility. Journal of Neurology, Neurosurgery, and Psychiatry 45(9): 844–846. Dertinger S, Dirschmid K, Vogel W, and Drexel H (1998) Immunosuppressive therapy for carbamazepine-induced hypersensitivity syndrome and hepatitis. Journal of Hepatology 28(2): 356–357. Donner E, Kosjek T, Qualmann S, Kusk KO, Heath E, Revitt DM, Ledin A, and Andersen HR (2013) Ecotoxicity of carbamazepine and its UV photolysis transformation products. Science of the Total Environment 443: 870–876. Elshama SS, Osman HE, and Ael-M E-K (2015) Teratogenic effect of carbamazepine use during pregnancy in the mice. Pakistan Journal of Pharmaceutical Sciences 28: 201–212. Fuhr LM, Marok FZ, Hanke N, Selzer D, and Lehr T (2021) Pharmacokinetics of the CYP3A4 and CYP2B6 inducer carbamazepine and its drug-drug interaction potential: A physiologically based pharmacokinetic modeling approach. Pharmaceutics 13(2): 270. Ganeva M, Gancheva T, Lazarova R, Troeva J, Baldaranov I, Vassilev I, Hristakieva E, and Tzaneva V (2008) Carbamazepine-induced drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome: Report of four cases and brief review. International Journal of Dermatology 47(8): 853–860. Grzesiak AL, Lang M, Kim K, and Matzger AJ (2003) Comparison of the four anhydrous polymorphs of carbamazepine and the crystal structure of form I. Journal of Pharmaceutical Sciences 92(11): 2260–2271. Jose M, Sreelatha HV, James MV, Arumughan S, and Thomas SV (2017) Teratogenic effects of carbamazepine in mice. Annals of Indian Academy of Neurology 20(2): 132–137. Kasarskis EJ, Kuo CS, and Berger R (1992) Carbamazepine-induced cardiac dysfunction: Characterization of two distinct clinical syndromes. Archives of Internal Medicine 152: 186–191. McCormack M, Alfirevic A, Bourgeois S, Farrell JJ, Kasperaviciute_ D, Carrington M, Sills GJ, Marson T, Jia X, de Bakker PI, Chinthapalli K, Molokhia M, Johnson MR, O’Connor GD, Chaila E, Alhusaini S, Shianna KV, Radtke RA, Heinzen EL, Walley N, Pandolfo M, Pichler W, Park BK, Depondt C, Sisodiya SM, Goldstein DB, Deloukas P, Delanty N, Cavalleri GL, and Pirmohamed M (2011) HLA-A 3101 and carbamazepine-induced hypersensitivity reactions in Europeans. The New England Journal of Medicine 364(12): 1134–1143. Mizuno K, Okada M, Murakami T, Kamata A, Zhu G, Kawata Y, Wada K, and Kaneko S (2000) Effects of carbamazepine on acetylcholine release and metabolism. Epilepsy Research 40(2–3): 187–195. Mohamed A, Adnan A, Seifert S, Smolinske S, Castresana D, Parasher G, et al. (2017) An endoscopic end to coma. Toxicology Communications 1: 6–9. Nie Q, Su B, and Wei J (2016) Neurological teratogenic effects of antiepileptic drugs during pregnancy (review). Experimental and Therapeutic Medicine 12(4): 2400–2404. Puranik YG, Birnbaum AK, Marino SE, Ahmed G, Cloyd JC, Remmel RP, Leppik IE, and Lamba JK (2013) Association of carbamazepine major metabolism and transport pathway gene polymorphisms and pharmacokinetics in patients with epilepsy. Pharmacogenomics 14(1): 35–45. Roma
nska-Gocka K, Gocki J, Placek W, Zegarska B, and Krause P (2010) Stevens Johnson syndrome after carbamazepine and SJS/TEN overlap syndrome after amoxicillin: Case reports and a review. Archives of Medical Science 6(1): 130–134. Sarikaya R and Yüksel M (2008) Genotoxic assessment of oxcarbazepine and carbamazepine in drosophila wing spot test. Food and Chemical Toxicology 46(9): 3159–3162. Savidge JR and Bristow DR (1997) Routes of NMDA- and K(+)-stimulated calcium entry in rat cerebellar granule cells. Neuroscience Letters 229(2): 109–112. Shao J, Katika MR, Schmeits PCJ, Hendriksen PJM, van Loveren H, Peijnenburg AACM, et al. (2013) Toxicogenomics-based identification of mechanisms for direct immunotoxicity. Toxicological Sciences 135(2): 328–346. Spiller HA (2001) Management of carbamazepine overdose. Pediatric Emergency Care 17: 452–456. Tolou-Ghamari Z, Zare M, Habibabadi JM, and Najafi MR (2013) A quick review of carbamazepine pharmacokinetics in epilepsy from 1953 to 2012. Journal of Research in Medical Sciences: The Official Journal of Isfahan University of Medical Sciences 18(Suppl 1): S81–S85. PMID: 23961295. PMC3743329. van den Brandhof EJ and Montforts M (2010) Fish embryo toxicity of carbamazepine, diclofenac and metoprolol. Ecotoxicology and Environmental Safety 73(8): 1862–1866. Zhang Y, Wang J, Zhao LM, Peng W, Shen GQ, Xue L, Zheng XX, He XJ, Gong CY, and Miao LY (2011) Strong association between HLA-B 1502 and carbamazepine-induced Stevens-Johnson syndrome and toxic epidermal necrolysis in mainland Han Chinese patients. European Journal of Clinical Pharmacology 67(9): 885–887.

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Carbaryl Leona D Scanlana and Svetlana E Koshlukovab, aOffice of Environmental Health Hazard Assessment, California Environmental Protection Agency, Sacramento, CA, United States; bDepartment of Pesticide Regulation, California Environmental Protection Agency, Sacramento, CA, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.E. Koshlukova, N.R. Reed, Carbaryl, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 668–672, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00107-X.

Chemical profile Background Regulatory history Illness reports US EPA summary of illness report data California department of pesticide regulation pesticide illness surveillance program Uses/occurrence Exposure and exposure monitoring Toxicokinetics (ADME) Mechanism of toxicity New approach methodologies Toxicity forecaster (ToxCast) Endocrine disruption screening program for the 21st century (EDSP21) Physiologically based pharmacokinetic-pharmacodynamic (PBPK-PD) modeling Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Animal Human Developmental neurotoxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Other hazards Toxicogenomics Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines PubChem and CompTox Acknowledgment References Further reading

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Abstract Carbaryl (CAS 63-25-2) is a broad-spectrum N-methyl carbamate insecticide applied worldwide for control of agricultural and structural pests and as a molluscicide. It causes toxicity by inhibiting the enzyme acetylcholinesterase in the nervous system, which leads to accumulation of acetylcholine and cholinergic hyper-stimulation. Immature animals are more sensitive to cholinesterase (ChE) inhibition than adults. Carbaryl causes reproductive and developmental toxicity, including neurodevelopmental perturbations, and may alter the immune response in mammals. It exhibits high toxicity to non-target organisms including fish, aquatic invertebrates and honeybees. Human exposure occurs through residues in food, skin contact and air dispersion. It is not expected to bioaccumulate in aquatic or terrestrial food chains. Concerns regarding

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human and ecological risks have resulted in increased regulatory restrictions and decreased use in most developed countries. However, carbaryl is still widely applied around the world. The United States Environmental Protection Agency classified carbaryl as “likely to be carcinogenic in humans”. In California, carbaryl is listed as a developmental and reproductive toxin and a carcinogen and is designated as a restricted material.

Keywords 1-Naphthol; Acetylcholinesterase; Atropine; Biomonitoring; Carbamate; Carcinogen; Cholinergic effects; Developmental toxicity; Neurotoxicity; N-methyl carbamate; Organophosphate; Pesticide

Glossary

CYP Cytochrome P450 monooxygenase LC50 Lethal concentration resulting in death of 50% of test animals LD50 Lethal dose resulting in death of 50% of test animals PEL OSHA enforceable 8-h time weighted permissible occupational exposure limit Skin Occupation exposure limit designated as the potential for dermal absorption

Key points

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Carbaryl is a broad-spectrum N-methyl carbamate insecticide. It primarily causes toxicity through inhibition of the enzyme acetylcholinesterase in the nervous system. Carbaryl causes reproductive, developmental, and neurodevelopmental toxicity in mammals. Carbaryl is highly toxic to fish, aquatic invertebrates and honey bees. It is classified by the United States Environmental Protection Agency as “likely to be carcinogenic in humans”. Concerns regarding human and ecological risks have resulted in increased regulatory restrictions and decreased use in most developed countries. However, carbaryl is still widely applied around the world. Cases of human poisoning with carbaryl are twice as likely to show life-threatening effects as poisoning by other pesticides; the frequency and severity of carbaryl cases have declined with decreased use.

Abbreviations

AChE CMG NMC PBPK-PD PISP SMAV

Acetylcholine esterase Common mechanism group N-methyl carbamate Physiological Based Pharmacokinetic-Pharmacodynamic model Pesticide Illness Surveillance Program Species mean acute values

Chemical profile

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Name: Naphthalen-1-yl methylcarbamate Synonyms: Carbaril, Sevin, 1-naphthyl N-methylcarbamate, 1-naphthyl methylcarbamate, 1-naphthalenyl methylcarbamate, Caprolin, Carbatox, Carbavur, Carpolin CAS Number: CAS 63-25-2 Chemical class: Carbamate; Insecticide, Acaricide, Molluscicide Molecular Formula: C12H11NO2 Molecular weight: 201.22 g mole−1 Density: 1.23 g cm−3 at 25
C

Carbaryl

• •

Vapor pressure: 0.00000136 mmHg at 25
C Chemical structure:

O

O N

• • • • • • •

501

H

Boiling Point: Decomposes Decomposes Melting Point: 145
C Flash Point: 193–202
C Conversion Factor (ppm to mg m−3): 1 ppm ¼ 0.82 mg m−3 (IPCS, 1997). Appearance: White or gray crystalline solid Odor: Odorless

Background Carbaryl is a N-methyl carbamate manufactured as an insecticide and molluscicide. Its most prominent toxicity to insects and mammals is inhibition of the enzyme acetylcholinesterase (AChE) (DPR, 2014). First sold in 1959, carbaryl is used worldwide to control agricultural and structural pests and mosquitos (US EPA, 2017a). Formulations include dusts, liquids, emulsifiable concentrates, granules and baits. Uses include fruit and nut trees, fruit, vegetable and grain crops, and ornamental plants (US EPA, 2017a).

Regulatory history Originally manufactured by Union Carbide, carbaryl was first registered in the United States in 1959 for use on cotton (DPR, 2014). In the late 1990s, it was one of the top selling pesticides for agriculture, turf management, residential pet products, and lawn treatments. Over the last several decades, concerns regarding human and ecological risks have limited its use. Carbaryl was phased out of use in the United Kingdom by 1998, and in the European Union by 2007 under the Council Directives 79/117/EEC and 91/414/EEC (Pan Europe, 2006). Use limitations were imposed in Australia in 2007 (APVMA, 2007). In the United States, about 80% of products were canceled, and many agricultural and residential uses were restricted by 2005. All registered indoor residential uses for carbaryl were canceled by 2017 in the United States (US EPA, 2017b). This included all carbaryl products used on pets (pet collars, dips, kennel treatments, bedding treatments and flea powders). In California, carbaryl is listed as a developmental and reproductive toxin, and as a carcinogen under Proposition 65, the Safe Drinking Water and Toxic Enforcement Act (OEHHA, 2019). In 2020, California’s Department of Pesticide Regulation (DPR) designated most agricultural pesticide products containing carbaryl as restricted materials, thereby limiting their purchase and use to certified applicators only. This action was taken to reduce residential and bystander exposure after applications in and around residences, scenarios identified in the DPR’s, 2014 risk characterization document as posing acute health risks via dermal and inhalation exposure pathways (DPR, 2014; DPR, 2019).

Illness reports Data on illnesses or injuries associated with exposure to carbaryl are available from numerous sources. US EPA reported that cases of carbaryl poisoning were twice as likely to show life-threatening effects, cause significant disability, require hospitalization, or

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involve critical care as poisoning by other pesticides (US EPA, 2007a). This pattern was not seen with occupational workers, suggesting handling by non-professionals is associated with an increased health risk (US EPA, 2007a).

US EPA summary of illness report data In 2017, US EPA published a review of human incidents and epidemiology based on data from several sources, including the Incident Data System database, the National Pesticide Information Center (NPIC), NIOSH SENSOR-Pesticides, DPR’s Pesticide Incident Surveillance Program (PISP), and the Agricultural Health Study (AHS) under the auspices of the National Institutes of Health and the National Cancer Institute (US EPA, 2017b). The report concluded that the frequency and severity of carbaryl cases since 2010 have declined or have occurred at low frequencies, data which track with increasingly stringent use requirements and limitations. Clinical signs of exposure were similar in each database, and included neurological, respiratory, gastrointestinal, dermal, and cardiovascular effects. Public and worker exposures occur through the oral (ingestion), dermal and inhalation routes. The Incident Data System database contained 356 cases involving carbaryl, 34 of which included one or more additional active ingredients. The NIOSH SENSOR database contained 287 cases of carbaryl exposure from 1998 to 2013, 71% of which were from residential exposure. These cases were also reviewed by the US Department of Agriculture (USDA) (USDA, 2019).

California department of pesticide regulation pesticide illness surveillance program PISP maintains a database of pesticide-related illnesses and injuries reported in California. Case reports are received from physicians and from workers’ compensation records. To expand on the information above, and include the latest data available, there were 130 reports linked to carbaryl from 1992 to 2017 (DPR, 2021). The health effects attributed to exposure to carbaryl alone, or in combination with other pesticides, were rated as definite (23 cases), probable (38 cases) or possible (69 cases). Three cases of definite carbaryl exposure involved self-harm or purposeful ingestion (suicide attempts).

Uses/occurrence In the United States, about 3.9 million pounds were sold for over 400 uses during 1992–2001. Half of this amount was used outside of agriculture. Since 2003, more than two-thirds of all registered carbaryl products were canceled. Nevertheless, carbaryl is still among the most widely applied pesticides in the United States for agriculture, in professional ornamental plant production and turf management, for public health programs, for forestry applications and in use on residential lawns and gardens (US EPA, 2021a). From 2013 to 2017 over 700,000 pounds of carbaryl were applied to 650,000 acres of agricultural crops annually in 39 states (US EPA, 2020). Almost half of all carbaryl agricultural use (in pounds) was made to two crops, apples and soybeans, while half of the total acres treated was applied to apples, pecans, and soybeans. An estimated 1.3 million pounds of carbaryl is applied annually outside of domestic dwellings. Almost 300,000 pounds are applied annually by lawn care professionals and landscape contractors, at institutional turf facilities, on golf courses and at sod farms (US EPA, 2020).

Exposure and exposure monitoring Exposure to carbaryl occurs through ingesting residues in food and water, inhaling vapors, and skin contact. Prior to 2004, residential exposure was widespread. Carbaryl was the most frequently detected pesticide in indoor and outdoor air in the United States and Canada (Yearly and Leonard, 1993). It was found in house dust in association with its agricultural use (Davis et al., 2021). Today, there are fewer potential residential exposure scenarios because of the numerous product cancelations and use restrictions. Exposure to the general public occurs mainly via the diet, ambient air, and as a result of outdoor uses in residential settings or as a result of spray drift from agricultural applications (US EPA, 2017a). Workers’ exposures are mainly dermal and inhalation from handling, applying or re-entering treated fields (US EPA, 2017a). Carbaryl “tolerances” or Maximum Residue Levels (MRLs) are established for more than 70 food commodities in the United States (Code of Federal Regulations, 2021). Tolerances are the highest levels allowable in or on these commodities. Carbaryl can be applied directly to growing crops, which may result in surface and ground water contamination (US EPA, 2017a). At a given exposure concentration, children generally have higher body burden due to their higher intake (inhalation volume, amount of food and water intake) or contact on a per body weight basis.

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The Comparative Toxicogenomics Database (accessed 12 August 21) lists 22 exposure monitoring studies, with thousands of study subjects, which show carbaryl or its metabolite 1-naphthol detected in house dust, carpet, hand-wipes, urine and air (Davis et al., 2021).

Toxicokinetics (ADME) The oral absorption of carbaryl is 74–100% in rats, mice and humans (ACGIH, 2008; Gunasekara et al., 2008). In rats, peak blood concentrations of carbaryl and its metabolites occur within 15–30 min after dosing. Dermal absorption is estimated to be 34–74% in rats and humans; dermal absorption in rabbits was nearly complete (ACGIH, 2008; Baron, 1991; Gunasekara et al., 2008). Inhalation absorption is indicated by the inhibition of cholinesterase (ChE) activities (Baron, 1991). Carbaryl is metabolized by the liver cytochrome P450 enzymes (CYPs) (ACGIH, 2008). The main routes of metabolism are similar in humans, rats, mice, guinea pigs, monkeys and sheep (Baron, 1991). Hydrolysis results in 1-naphthol, CO2 and methylamine, while alkyl oxidation forms N-hydroxymethylcarbaryl. Hydroxylation produces 5-hydroxycarbaryl, 4-hydroxycarbaryl, 5-6-dihydro-5-6-dihydroxycarbaryl and 3,4-dihydro-3,4-dihydroxycarbatyl, via epoxide intermediates (Gunasekara et al., 2008). In animals, the highest levels of carbaryl and its major metabolite 1-naphthol are found in the kidney, blood, liver and brain (Baron, 1991, Gunasekara et al., 2008). In a case of self-poisoning, carbaryl was found in the gastrointestinal tract, blood, liver, kidneys, and urine of a human adult (ACGIH, 2008). Carbaryl binds to plasma proteins, e.g., albumin. In rats and mice, transplacental transfer is evidenced by fetal brain ChE inhibition and the presence of carbaryl in the fetal eye, liver and brain (Cambon et al., 1980; Declume and Benard, 1977). Urine is the main route of elimination for carbaryl, where 68–74% of a single oral dose is found within 24 h (Baron, 1991; Blacker et al., 2010; Gunasekara et al., 2008). About 2–11% is excreted in the bile/feces. Enterohepatic circulation is significant. The major metabolites in excreta are 1-naphthol, 5-hydroxycarbaryl, glucuronide and sulfate conjugates. Urinary 1-naphthol is commonly used in human biomonitoring studies. A shift in the urinary metabolite pattern, with increases in the hydroxylated compounds derived from epoxide intermediates, is observed in rats and mice at high doses of carbaryl that caused severe toxicity.

Mechanism of toxicity Like other N-methyl carbamates (NMC), the mechanism of carbaryl toxicity is related to its binding to and inhibition of serine hydrolase AChE. AChE hydrolyzes the neurotransmitter acetylcholine, thereby terminating its synaptic action (Baron, 1991). Carbaryl inhibits AChE activity by carbamylating the serine hydroxyl group in the active site of the enzyme. This causes a persistent increase of acetylcholine at the synapse, and leads to cholinergic overstimulation, autonomic and neuromuscular dysfunction, and at higher levels, coma and death (Knaak et al., 2008; Zhang et al., 2010). AChE is also a target of organophosphorus insecticides. Organophosphates and NMC differ in their interaction with the ChE enzyme. Inhibition of AChE via carbamylation is a reversible binding process that allows for rapid reactivation of the enzyme (US EPA, 2017a). The time to peak inhibition for NMCs is typically between 15 and 45 min, while complete recovery is within minutes to hours (US EPA, 2017a). Repeated daily exposure to NMCs, including carbaryl, therefore does not result in steadily increasing inhibition of AChE because enzyme recovery is complete before the next acute exposure. In addition, carbaryl shows significant metabolic clearance within 24 h (Baron, 1991; Gunasekara et al., 2008; Blacker et al., 2010). Therefore, only acute exposure or a series of acute exposures may be of concern for neurotoxic effects (US EPA, 2017a). An adverse outcome pathway (AOP) was developed for AChE inhibitors (carbamates and organophosphates, see Fig. 1) (US EPA, 2016a). The initiating event for both classes of pesticides is inhibition of AChE, which leads to accumulation of acetylcholine and ultimately to neurotoxicity (US EPA, 2016a). Carbaryl also inhibits other esterases, including butyrylcholinesterase and neuropathy target esterase (Baron, 1991). Butyrylcholinesterase may function as a molecular scavenger for anticholinesterase compounds in the blood or substitute for AChE where it is low. Neuropathy target esterase may be involved in the delayed neurotoxicity syndrome (Baron, 1991). Other non-ChE actions may influence the overall toxicity of carbaryl. The potential targets include macromolecule synthesis, chromatin protein, mitotic spindle formation, neurotransmitter receptors, the aryl hydrocarbon receptor (AhR), serotonin and catecholamine metabolism, and protein levels in developing brain (Baron, 1991; Boronat et al., 2007; Lee et al., 2015; Oziolor et al., 2017). Carbaryl also induces hepatic CYPs (Delescluse et al., 2001). Carbaryl’s major metabolite, 1-naphtol, is unlikely to be an effective cholinesterase inhibitor (DPR, 2014). Both carbaryl and 1-naphthol induce mitotic abnormalities (Gunasekara et al., 2008; Söderpalm-Berndes and Önfelt, 1988). Hydroxycarbaryl and

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Inhibition of AChE at active site

Accumulation of ACh

Neurotoxicity

Fig. 1 Adverse outcome pathway for acetylcholinesterase (AChE) inhibition by organophosphates and carbamates such as carbaryl.

dihydro-dihydroxycarbaryl are implicated in tumor induction in mice and rats based on their possible formation via epoxide intermediates (IPCS, 2001).

New approach methodologies New Approach Methodologies (NAMs) utilizing in vitro and alternative methods to replace conventional mammalian testing are increasingly explored for informing toxicity and chemical hazard in risk assessment. For carbaryl, a large body of published studies conducted in primary and established cell lines exists. Carbaryl has also been screened for changes in biological activity in vitro using automated screening technologies. Finally, models have been developed to predict carbaryl concentrations that cause AChE inhibition in human blood or brain. Different applications of in vitro/in silico technologies for carbaryl are described below.

Toxicity forecaster (ToxCast) Carbaryl was included in the ToxCast program by the US EPA, which aims to reduce the number of animals used in toxicity testing by validating high-throughput screening technologies and in vitro toxicity data (US EPA, 2016b). The results from ToxCast assays are used to prioritize chemicals for subsequent animal toxicity testing and to inform on chemical hazards. Carbaryl was active in 104 out of tested 918 assays (accessed 29 July 2021) (US EPA, 2021c). However, the active assays were either flagged for poor quality (e.g., confounded by overfitting, or had borderline activity) or the AC50 values were higher than 10 mM, which may indicate false positives (US EPA, 2021b; Judson et al., 2016).

Endocrine disruption screening program for the 21st century (EDSP21) Part of the ToxCast assay suite, EDSP assays are specific for interactions at the thyroid, androgen and estrogen receptors and for steroidogenesis processes. Carbaryl was tested in 5/21 assays for estrogen, 2/16 assays for androgen, 1/11 assays for thyroid and 6/27 assays for steroidogenesis (accessed 29 July 21) (US EPA, 2021c). All of the assays had precautionary flags or had borderline activity. The US EPA in 2015 concluded that there was no convincing evidence for interaction of carbaryl with estrogen, thyroid or androgen receptors and, based on weight of evidence analysis, did not recommend carbaryl for Tier 2 testing (US EPA, 2015).

Physiologically based pharmacokinetic-pharmacodynamic (PBPK-PD) modeling A human life-stage PBPK-PD model was developed for carbaryl to predict its distribution in the body and AChE inhibition in RBC and in brain after oral, inhalation, and dermal exposure. For this model, tissue partitioning, plasma protein binding, and hepatic metabolism parameters for carbaryl in humans were experimentally determined using in vitro methods (Knaak et al., 2008; US EPA, 2021a). In vitro methods were also used to determine PD parameters such as bimolecular inhibition rate constants to describe carbaryl’s reversible inhibition of AChE (US EPA, 2021a). Biologically appropriate scaling was done with in vitro to in vivo extrapolation (IVIVE) (Yoon et al., 2012). In addition, the model included age-specific physiological parameters and metabolic clearance of carbaryl (Yoon et al., 2012). The carbaryl PBPK-PD model underwent an external scientific review under the auspices of the FIFRA Scientific Advisory Committee. The panel evaluated the model coding and determined the model applicable for use in human health risk assessment (Versar, 2018; US EPA, 2021a). Subsequently, US EPA used the model in their 2021 draft risk assessment to estimate human points of departure and to reduce the default interspecies uncertainty factor from 10 to 1 (US EPA, 2021a).

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Acute and short-term toxicity Animal Carbaryl is classified by US EPA as a moderate acute oral toxicant (Toxicity Category II) with low acute toxicity by the dermal and inhalation routes (Toxicity Categories III and IV, respectively) (US EPA, 2021a). The acute oral LD50 values range from 108 to 1500 mg kg−1 for rats, mice, rabbits, guinea pigs, dogs, cats, swine, monkey and deer (DPR, 2014). The dermal LD50 in rats and rabbits is >2000 mg kg−1 day−1. The 4-h inhalation LC50 in rats is >0.9 mg L−1. Carbaryl is neither a dermal or eye irritant nor a dermal sensitizer in animal studies, but incidents of dermal irritation allergic response have been reported in humans (US EPA, 2021a). The main target of short-term oral toxicity is the nervous system. Cholinergic syndromes from overstimulation of muscarinic and nicotinic acetylcholine receptors include hypersalivation, respiratory distress, miosis, muscular twitches, tremors, ataxia, diarrhea and vomiting. Other non-lethal effects include hematological and liver enzyme changes, alterations in brain enzymes and neurotransmitter levels, changes in catecholamine metabolism, renal effects, hypothermia, and body weight decreases (DPR, 2014). No delayed neuropathy was observed in hens receiving a 300–560 ng kg−1 intraperitoneal (ip) dose of carbaryl (Ehrich et al., 1995). Young animals are twofold more sensitive to ChE inhibition than adults. Applying Benchmark Dose (BMD) analysis, US EPA established a BMDL (lower bound of BMD) at 1.1 mg kg−1 day−1 for 10% brain AChE inhibition in postnatal day (PND) 11 pups after a single oral exposure (US EPA, 2017a). DPR set a critical acute oral point of departure (POD) of 1 mg kg−1 based on cholinergic signs and decreased bodyweight gain in adult rats (DPR, 2014).

Human Human deaths have occurred from intentional ingestion of carbaryl. Pulmonary edema was reported in one case involving an approximate exposure (ingestion) of 5700 mg kg−1 (Baron, 1991). Initial signs included tremors, disturbed vision and severely inhibited RBC ChE. Carbaryl was detected post-mortem in the gastrointestinal tract, blood, liver, kidney and urine. Non-lethal effects include CNS, cardiovascular and respiratory systems. Five possible cases of suicide by carbaryl poisoning were reported in the DPR PISP database (DPR, 2021). Symptoms in the suicide cases included nausea, vomiting, diarrhea, shaking, sweating, hypotension, muscle twitching, salivation, and respiratory failure. Treatment included charcoal or atropine administration and intubation. Common clinical signs of cholinergic toxicity in humans are lacrimation, salivation, tremors, nausea, miosis and muscle incoordination. Abdominal pain, profuse sweating, lassitude and vomiting occurred after a single oral dose of 2.8–5.5 mg kg−1 (Baron, 1991). Rash, burning, skin irritation and depressed plasma ChE levels are reported in workers following dermal contact or after spraying carbaryl (Baron, 1991; DPR, 2021). Signs of CNS toxicity include dizziness, anxiety, mental confusion, convulsion and coma, and depression of respiration (IPCS, 1997). Unlike in animals, there are limited human data to determine the irritant properties of carbaryl (Baron, 1991). In studies of male adults, the acute LOEL for plasma and RBC ChE inhibition is 2 mg kg−1 (Cranmer, 1986). Children are more sensitive to carbaryl poisoning and are more likely to present with CNS symptoms (coma, seizures, hypertension cardiorespiratory depression) (Roberts and Reigart, 2013). In 2021, the US EPA revised their carbaryl risk assessment and included the PBPK-PD model to derive human equivalent doses and concentrations for several human populations (infants, children and adults) over acute (24 h) and longer-term (180 days or 6 months) exposure durations (US EPA, 2021a). PODs were based on 10% AChE inhibition in red blood cells (RBC), as the data showed that the RBC AChE inhibition was more sensitive than brain AChE inhibition (US EPA, 2021a). PODs were derived for each exposure scenario (for example, from food, drinking water, residential and occupational settings) (US EPA, 2021a). The acute dietary POD of 0.42 mg kg−1 was based on 10% RBC AChE inhibition in children 3–5 years old, while the acute PODs for drinking water exposure was calculated for seven different age groups (infants to adults) and ranging from 17.55 mg L−1 to 68 mg L−1 (US EPA, 2021a). Based on the PBPK-PD modeling, the US EPA also reduced the interspecies uncertainty factor from 10 to 1 and reduced the FQPA safety factor to 1 for all exposure scenarios (US EPA, 2021a).

Chronic toxicity Animal Non-lethal LOELs have been estimated in the following ranges: 10–17 mg kg−1 day−1 for thyroid follicular and liver hepatocellular hypertrophy in rats and globular deposits in the bladder epithelium in mice; 10–31 mg kg−1 day−1 for decreased pupil size, reduced rearing activity, tremors, and salivation in rats and dogs; 24 mg kg−1 day−1 for increased liver weight in rats; 30–79 mg kg−1 day−1 for decreases in body weight, body weight gains and food consumption in dogs and rats; and, 145–350 mg kg−1 day−1 for progressive nephropathy and opaque eyes in mice, and bladder epithelial hyperplasia, pelvic urothelial hyperplasia, cataracts, degeneration of

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sciatic nerve and muscle, chromodacryorrhea and alopecia in rats (Blacker et al., 2010; DPR, 2014; US EPA, 2007a; IPCS, 1997; IPCS, 2001).

Human Human volunteers who ingested carbaryl for 6 weeks reported abdominal changes and difficulty in sleeping at the LOEL of 0.13 mg kg−1 day−1 (ACGIH, 2008). Case reports suggested possible effects of chronic neurological or psychological problems (Branch and Jacqz, 1986). Effects on sperm morphology and increased risk for multiple tumors are discussed below under Reproductive Toxicity and Carcinogenicity sections.

Immunotoxicity Carbaryl given as a single or repeated dose at 0.8–500 mg kg−1 day−1 impaired the humoral immune response in mice and chickens, decreased the phagocytic activity of leukocytes and antibody formation, and disrupted the ability of the immune system to combat bacterial infections from Erysipelothrix rhusiopathiae and Staphylococcus in rats and rabbits (Baron, 1991; Cranmer, 1986; Km and Jb, 2010). Carbaryl enhanced the replication of varicella zoster virus in human lung cells (Baron, 1991). There were no significant immunotoxic effects in guideline studies submitted to US EPA for pesticide registration (US EPA, 2017a). Carbaryl reduced thymus and spleen weights, suppressed lymphocyte proliferation in rats and decreased markers of impaired tadpole innate antiviral immune responses at both 0.1 and 1.0 ppb (De Jesús Andino et al., 2017; Jorsaraei et al., 2014).

Reproductive and developmental toxicity Animal The developmental and reproductive toxicity of carbaryl has been studied in more than 10 mammalian species including rats, mice, guinea pigs, hamsters, gerbils, dogs, sheep and rhesus monkey (Baron, 1991; IPCS, 2001; Iyer and Makris, 2010; Coppock and Dziwenka, 2011; Mathur and Bhatnagar, 1991). Fetal growth retardation and malformations were observed in the presence of maternal toxicity when pregnant rats received up to 30 mg kg−1 day−1 carbaryl orally on gestation days (GD) 6–20 or when pregnant rabbits received up to 150 mg kg−1 day−1 on GD 6–29. Carbaryl caused severe malformations in offspring of pregnant dogs and pigs at 4–50 mg kg−1 day−1. In 2-generation studies, rats fed 4.7–111 mg kg−1 day−1 carbaryl mated normally and exhibited normal pregnancy. Parental body weights and food consumption were impacted only at the high dose. However, decreased pup survival occurred with a LOEL of 24 mg kg−1 day−1 (IPCS, 2001). Decreased sperm motility and count occurred after 90 days of oral dosing at as low as 5 mg kg−1 day−1 (Baron, 1991).

Human Collective results from several epidemiological studies suggest toxicity to the human reproductive system. A study with farm families in Canada indicated an increased risk of miscarriages following carbaryl usage by males (Savitz et al., 1997). In the United States and China, carbaryl-exposed factory workers showed decreased sperm count, sperm abnormality and sperm chromosomal aberrations (Tan et al., 2005; Xia et al., 2005). An association with various indicators of sperm toxicity (decreased motility and concentrations, DNA damage) were reported when urinary 1-naphthol was used as a biomarker of exposure (DPR, 2014; Meeker et al., 2004).

Developmental neurotoxicity In a developmental neurotoxicity study in rats, gestational and early post-natal exposure to 10 mg kg−1 day−1 carbaryl produced changes in brain structures (decreased cerebellar length in pups and thickened cerebral cortex) of the offspring later in life (US EPA, 2017a). Maternal toxicity at this dose included decreased body weight gain, pinpoint pupils, tremors, gait abnormalities and plasma, RBC and brain ChE inhibition (US EPA, 2017a). In a developmental neurotoxicity study in mice, pups received a single gavage dose of carbaryl (0.5–20 mg kg−1) on postnatal day 10, which corresponds to the peak of the murine perinatal brain growth spurt (Lee et al., 2015). Carbaryl caused alternations in motor activity and levels of proteins in the hippocampus and cortex lasting up to 4 months at the LOEL of 0.5 mg kg−1 day−1, and in

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the absence of pup brain ChE inhibition. The shift in brain levels of the proteins CaMKII, GAP-43, synaptophysin and tau was suggested to play a role in the developmental impairments by affecting neuronal structure or transmitter systems.

Genotoxicity Carbaryl was negative in reverse mutation tests in multiple strains of Salmonella and in the Chinese hamster ovary cell/ hypoxanthine-guanine phosphoribosyl-transferase (CHO/HGPRT) forward mutation assay (DPR, 2014). In Chinese hamster fibroblasts (CHF), carbaryl caused gene mutation in vitro in a ouabain resistance study, but did not cause gene mutation in a thioguanine resistance study (DPR, 2014). Chromosomal aberrations were observed in in vitro assays with CHO cells in the presence of metabolic activation and in CHF without metabolic activation, but carbaryl was negative in in vivo micronucleus assays in rats and mice. Unscheduled DNA synthesis was not found in a rat hepatocyte assay, but was detected in transformed human cells (DPR, 2014). Mitotic spindle abnormalities and sister chromatid exchange were detected in vitro in CHF. Protein and DNA binding in the liver were not detected in vivo in mice. The Joint FAO/WHO Meeting on Pesticide Residues (JMPR) concluded in 1996 that carbaryl is not genotoxic (IPCS, 1996) In 2021, US EPA concluded that the overall findings indicate that there is a concern for mutagenicity in vitro, but the concern is lessened because no genotoxic effects have been observed in vivo (US EPA, 2021a).

Carcinogenicity Carbaryl carcinogenicity is evident in 2-year dietary inclusion studies with rats and mice (DPR, 2014). Neoplastic lesions in Sprague-Dawley rats at the highest tested level of 7500 ppm included urinary bladder transitional cell papilloma and carcinoma in the males and females, kidney transitional cell carcinoma and thyroid adenoma and carcinoma in the males, and liver adenoma in the females. Neoplastic lesions in CD-1 mice included vascular hemangioma and hemangiosarcoma in males at all dose groups (100–8000 ppm) and in females at 8000 ppm. Additional lesions at 8000 ppm included kidney tubular cell adenoma and carcinoma in males, and hepatocellular adenoma, carcinoma, hepatoblastoma in females. In both studies, excess toxicity occurred at the highest tested dose. US EPA classified carbaryl as a Group C carcinogen—“Likely to be carcinogenic in humans” based on increased hemangiosarcomas in male mice (US EPA, 2002). Regulatory agencies have calculated different cancer potency slope factors for carbaryl using the same study in mice (NRC, 2015). US EPA established a human equivalent upper bound potency slope (Q ) of 8.75  10−4 mg kg−1 day−1 based on the incidence of hemangiosarcomas in mice. The DPR Q value of 9.72  10−3 mg kg−1 day−1 was based on combining the incidence of hemangiosarcomas and hemangiomas, and excluded the high-dose data based on the substantial toxicity observed at that dose. Health Canada calculated a Q value of 1.08  10−3 mg kg−1 day−1 after combining tumor types and excluding the high dose. The International Programme on Chemical Safety (IPCS), European Food Safety Authority (EFSA) and Australian Pesticides and Veterinary Medicines Authority (APVMA) used a threshold approach that assumed carbaryl is not genotoxic and established an acceptable daily intake (ADI) of 0.008 mg kg−1 day−1 (based on a safety factor of 2000). Several epidemiological investigations in Canada and the United States during the past three decades showed associations between carbaryl exposure and non-Hodgkin’s lymphoma, cutaneous melanoma, and prostate cancer. The North American Pooled Project pooled these case-control study data and used regression analysis to estimate odds ratios for association between chemical use and incidences of non-Hodgkin’s lymphoma (Koutros et al., 2019). For carbaryl, an odds ratio of 1.62 (confidence intervals: 1.20 and 2.18) characterized the risk of NHL among people who had ever used carbaryl compared to those who had never used carbaryl. In this study, 86–89% of the participants were men.

Organ toxicity Carbaryl affects the nervous system, respiratory system, cardiovascular system, reproductive system, immune system, skin, blood, endothelium and thyroid (NIOSH, 2019; Baron, 1991).

Interactions Concomitant exposure to other N-methyl carbamates with similar mechanism of action may result in cumulative toxicity (US EPA, 2007b). For example, exposure to other AChE inhibitors (OPs) can result in additive and/or synergistic effects (Laetz et al., 2009).

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Other hazards Age is a modifying factor for the acute toxicity of carbaryl due to low level of carboxylesterases in the young for detoxification (Moser et al., 2010). Nutritional factors can also influence the toxicity of carbaryl. Rats fed high protein diets showed higher toxicity than those on an ordinary diet (LD50 of 67 and 575 mg kg−1 day−1, respectively), possibly due to decreased metabolism (Tanaka et al., 1981; Tanaka et al., 1980; Boyd and Boulanger, 1968). Carbaryl metabolism is inhibited by other environmental chemicals (e.g., fipronil and chlorpyrifos) that are substrates for common CYPs (CYP3A4, CYP2B6) and share biotransformation pathways (Tang et al., 2002; Hodgson and Rose, 2007). In vitro, toxicity is increased by agents that inhibit liver metabolic enzymes. In contrast, acute toxicity in mice is decreased by CYP inducers (e.g., phenobarbital) or compounds that accelerate carbaryl urinary excretion (e.g., chlordane) (Baron, 1991; Lucier et al., 1972).

Toxicogenomics Curated carbaryl data were retrieved from the Comparative Toxicogenomics Database (Davis et al., 2021) (accessed 29 July 21). Carbaryl’s most commonly identified interaction was with the AChE (57 interactions). Carbaryl also interacted with the AH receptor (9 interactions) and with proteins involved in metabolism and transport (CYP1A1, albumin, and CYP1A2, with 8, 5 and 4 interactions, respectively). Carbaryl exposure has moderate evidence of association with 21 different diseases, including seizures, liver neoplasms, asthma and melanoma.

Clinical management The muscarinic signs of carbaryl poisoning are antagonized by atropine, which blocks acetylcholine at muscarinic receptors (Roberts and Reigart, 2013). Atropine can be given intramuscularly or intravenously. Induction of vomiting is not recommended due to risk of sudden seizures, coma, or respiratory depression (IPCS, 1997). Instead, activated charcoal can be administrated. Intubation may be required for severe respiratory distress. Oximes, widely used to treat nicotinic effects of organophosphates, are ineffective in carbaryl poisoning (Roberts and Reigart, 2013). Benzodiazepines such as diazepam can be used to control carbaryl-induced seizures and other CNS effects (IPCS, 1997).

Environmental fate and behavior Carbaryl has an octanol-water partition coefficient (log Kow) of 2.36, is soluble in organic solvents (e.g., dimethyl formamide, acetone), and has low to moderate solubility in water (120 mg L−1 solubility at 20
C) (Gunasekara et al., 2008). The calculated Henry’s law constant of 2.74  10−9 atm m3 g mole−1 and low vapor pressure (0.041 mPa at 23.5
C) (Gunasekara et al., 2008) indicate that surface water volatilization is unlikely. However, carbaryl can be transported in air through volatilized spray drift or by sorption to particulates (USDA, 2019). It has been detected in the air at application sites and at areas remote to application sites (DPR, 2014). Carbaryl undergoes abiotic hydrolysis, photodegradation, and biotic degradation in water (Gunasekara et al., 2008). Photolysis of carbaryl produces 1-naphthol, methylamine and CO2 (DPR, 2014). Both hydrolysis and photolysis occur under aqueous conditions; hydrolysis preferentially occurs at pH 7 and above, and microorganisms enhance the hydrolysis of carbaryl (DPR, 2014). The major photolysis product, 1-naphthol, is further oxidized by sunlight under alkaline conditions to 2-hydroxy-1,4-naphtho-quinone (DPR, 2014). Reported half-lives in canal and river waters range from 4 to 7 days; hydrolysis increases with increasing temperature and alkalinity (Gunasekara et al., 2008). In anaerobic conditions, the carbaryl half-life can be as long as 70 days (USDA, 2019). Although carbaryl is relatively insoluble in water, it has been detected in both surface water and groundwater (DPR, 2014). Carbaryl is the second most frequently found insecticide detected in urban streams (US EPA, 2012). Hydrolysis, photolysis and biotic degradation also occur in soil (Gunasekara et al., 2008). Degradation half-lives in aerobic soils range from 4 to 253 days (US EPA, 2021a); half-lives decrease with increasing pH (USDA, 2019). Carbaryl has a half-life of 72 days in anaerobic soil conditions (USDA, 2019). Estimates for Koc range from 196 to 390, indicating moderate adsorption to soil and potential for groundwater leaching (Gunasekara et al., 2008; PubChem, 2021; USDA, 2019). Carbaryl is not expected to bioaccumulate in aquatic and terrestrial food chains based on the measured Log KOW of 2.36 (Gunasekara et al., 2008). The degradate, 1-naphthol, is expected to be less persistent than carbaryl in soils, but may travel farther in air because it is more volatile (USDA, 2019).

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Ecotoxicology Carbaryl is highly toxic to fish, aquatic invertebrates and non-target insects such as honeybees, and is moderately toxic to birds (DPR, 2014). Species mean acute values (SMAVs, or the geometric mean of acute toxicity values) range from 3.175 mg L−1 for stonefly to 27,609 mg L−1 for catfish (US EPA, 2012). The ten freshwater species most sensitive to carbaryl are in the insecta and crustacea classes; stoneflies are the most sensitive, followed by cladocerans (e.g., Ceriodaphnia dubia and Daphnia carinata) and amphipods (e.g., Gammarus pseudolimnaeus, Hyalella azteca) (US EPA, 2012). Brown trout and lake trout are the most sensitive tested fish species with SMAV of 860 mg L−1and of 988.1 mg L−1, respectively. Threatened/endangered Coho salmon and Chinook salmon have SMAVs of 1654 mg L−1and 2690 mg L−1, respectively; data indicate that tested endangered fish have similar sensitivity to carbaryl as non-endangered fish (US EPA, 2012). Carbaryl is highly toxic to honey bees with an acute contact LD50 of 0.0011 mg bee−1 for the technical formulation and an LD50 of 0.0040 mg/bee for product formulation (USDA, 2019). Oral toxicity follows the same trend, with the technical acute oral LD50 of 0.0001 mg bee−1 10 times more toxic than the formulation LC50 value of 0.0016 mg bee−1 (USDA, 2019). Carbaryl resides have been measured in colonies (average levels of 111 mg kg−1). The 24- and 72-h acute oral LD50 values for the bumble bee are similar to those for the honey bee (USDA, 2019). The acute toxicity (LD50) to birds varies from 16 mg kg−1 to >2000 mg kg−1, with starlings and red-winged black birds the most sensitive tested species (USDA, 2019). A standardized reproduction study using mallard duck established a no-observed-adverseeffect concentration of 300 ppm based on reduced number of eggs produced at a lowest-observed-adverse-effect concentration of 600 ppm. Toxicities to mammals are presented in the Acute and Chronic toxicity sections. Based on estimated risk quotients in 2003, US EPA concluded that carbaryl posed a high risk to aquatic invertebrate species, fish and all-sized mammals from a single outdoor application, and a prolonged risk to birds, aquatic species and all-sized mammals from multiple applications (US EPA, 2003).

Exposure standards and guidelines Carbaryl handlers are required to wear long-sleeved shirts, long pants, shoes, and socks and personal protective equipment (PPE) including chemical-resistant gloves; additional PPE (respirators, aprons, etc.) is required for some formulations (US EPA, 2021c). Restricted entry intervals range from 12 h for most products to 21 days for some agricultural uses (US EPA, 2021c). Recommended threshold and exposure limits for humans include:

• • • • • • •

American Conference of Governmental Industrial Hygienists (ACGIH) Threshold Limit Value (TLV): 0.5 mg m−3 TWA, Inhalable fraction and vapor; [skin] [A4—Not Classifiable as a Human Carcinogen] (ACGIH, 2018). National Institute for Occupational Safety and Health (NIOSH) Recommended Exposure Limit (REL): 5 mg m−3 Time Weighted Average, TWA (NIOSH, 2019). Occupational Safety and Health Administration (OSHA) Permissible Exposure Limit (PEL): 5 mg m−3, TWA (NIOSH, 2019). National Institute for Occupational Safety and Health (NIOSH) Immediately Dangerous To Life or Health Concentration (IDLH): 100 mg m−3 (NIOSH, 2019). United States Environmental Protection Agency (US EPA) Population Adjusted Dose (PAD) (oral): 0.042 mg kg−1 day−1 (acute) (US EPA, 2021a). California Department of Pesticide Regulation acute oral reference dose: 0.01 mg kg−1 (DPR, 2014). European Food Safety Authority Acceptable Daily Intake (ADI): 0.0075 mg kg−1 day−1; EFSA acute reference dose: 0.01 mg kg−1 day−1 (EFSA, 2021).

The US EPA aquatic life ambient water quality criteria, or how much carbaryl can be present in surface water before it is likely to harm plant and animal life, include:

• •

Freshwater: 2.1 mg L−1 for acute exposures, 2.1 mg L−1 for chronic exposures (US EPA, 2012). Estuarine/marine: 1.6 mg L−1 for acute exposures, number not available for chronic exposures (US EPA, 2012).

PubChem and CompTox https://pubchem.ncbi.nlm.nih.gov/compound/6129 https://comptox.epa.gov/dashboard/dsstoxdb/results?search¼DTXSID9020247

Acknowledgment Authors would like to thank Drs. Shelley DuTeaux, Andrew Rubin and Karen Morrison for their helpful discussions and review of this paper.

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References ACGIH (2008) Carbaryl: TLVW Chemical substances. 7th edition documentation Cincinnati, OH: ACGIH. ACGIH (2018) Carbaryl: TLVW Chemical substances. 8th edition documentation Cincinnati, OH: ACGIH. APVMA (2007) The reconsideration of registrations of products containing carbaryl and their approved associated labels, part 1: Uses of carbaryl in home garden, home veterinary, poultry and domestic situations. Final Review Report and Regulatory Decision. Review Summary, vol. 1. Kingston, Australia: APVM. Authority APVM (ed.). Baron RL (1991) Chapter 17: Carbamate Insecticides. In: Hayes WJ and Laws ER (eds.) Classes of Pesticides. San Diego: Academic Press. Blacker AM, Lunchick C, Lasserre-Bigot D, Payraudeau V, and Krolski ME (2010) Chapter 74: Toxicological profile of carbaryl. In: Krieger R (ed.) Hayes’ Handbook of Pesticide Toxicology, 3rd edn. New York: Academic Press. Boronat S, Casado S, Navas JM, and Piña B (2007) Modulation of aryl hydrocarbon receptor transactivation by carbaryl, a nonconventional ligand. The FEBS Journal 274: 3327–3339. Boyd EM and Boulanger MA (1968) Insecticide toxicology. Augmented susceptibility to carbaryl toxicity in albino rats fed purified casein diets. Journal of Agricultural and Food Chemistry 16: 834–838. Branch RA and Jacqz E (1986) Subacute neurotoxicity following long-term exposure to carbaryl. The American Journal of Medicine 80: 741–745. Cambon C, Declume C, and Derache R (1980) Foetal and maternal rat brain acetylcholinesterase: isoenzymes changes following insecticidal carbamate derivatives poisoning. Archives of Toxicology 45: 257–262. Code of Federal Regulations (2021) CFR Title 40, Part 180.517. Carbaryl; Tolerances for Residues. Coppock R and Dziwenka M (2011) Teratogeneses in livestock. In: Gupta R (ed.) Reproductive and Developmental Toxicology. Elsevier Science & Technology. Cranmer MF (1986) Carbaryl. A toxicological review and risk analysis. Neurotoxicology 7: 247–328. Davis AP, Grondin CJ, Johnson RJ, Sciaky D, Wiegers J, Wiegers TC, and Mattingly CJ (2021) Comparative Toxicogenomics Database (CTD): Update 2021. Nucleic Acids Research 49: D1138–D1143. De Jesús Andino F, Lawrence BP, and Robert J (2017) Long term effects of carbaryl exposure on antiviral immune responses in Xenopus laevis. Chemosphere 170: 169–175. Declume C and Benard P (1977) Foetal accumulation of [14C] carbaryl in rats and mice. Autoradiographic study. Toxicology 8: 95–105. Delescluse C, Ledirac N, Li R, Piechocki MP, Hines RN, Gidrol X, and Rahmani R (2001) Induction of cytochrome P450 1A1 gene expression, oxidative stress, and genotoxicity by carbaryl and thiabendazole in transfected human HepG2 and lymphoblastoid cells. Biochemical Pharmacology 61: 399–407. DPR (2014) In: Branch MT (ed.) Carbaryl (1-Naphthyl Methylcarbamate) Occupational and Bystander Risk Characterization Document. Sacramento, CA: Department of Pesticide Regulation. DPR (2019) Initial statement of reasons and public report, Department of Pesticide Regulation, Title 3. In: California Code of Regulations, Amend Section 6400, Expanding Carbaryl Designation as a Restricted Material. Sacramento, CA: DPR. DPR (2021) Pesticide illness surveillance program. In: California Pesticide Illness Query (CalPIQ). Sacramento, CA: Department of Pesticide Regulation. EFSA (2021) The 2019 European Union Report on Pesticide Residues in Food. Authority EFS (ed.), EFSA Journal. Ehrich M, Jortner B, and Padilla S (1995) Comparison of the Relative Inhibition of Acetylcholinesterase and Neuropathy Target Esterase in Rats and Hens Given Cholinesterase Inhibitors. Washington, DC: U.S. Environmental Protection Agency. Gunasekara AS, Rubin AL, Goh KS, Spurlock FC, and Tjeerdema RS (2008) Environmental fate and toxicology of carbaryl. Reviews of Environmental Contamination and Toxicology 196: 95–121. Hodgson E and Rose RL (2007) Human metabolic interactions of environmental chemicals. Journal of Biochemical and Molecular Toxicology 21: 182–186. IPCS (1996) Carbaryl. In: Pesticide Residues in Food: 1996 Evaluations Part II Toxicological. Netherlands: International Programme on Chemical Safety. IPCS (1997) Carbaryl. Geneve: International Programme on Chemical Safety. IPCS (2001) CARBARYL (addendum). Toxicological Evaluations: Pesticide Residues in Food. International Programme on Chemical Safety. Joint FAO/WHO Meeting on Pesticide Residues (JMPR) http://www.inchem.org/documents/jmpr/jmpmono/2001pr02.htm#3.0. Iyer P and Makris S (2010) Chapter 12: Developmental and reproductive toxicology of pesticides. In: Krieger R (ed.) Hayes’ Handbook of Pesticide Toxicology, 3rd edn. New York: Academic Press. Jorsaraei SG, Maliji G, Azadmehr A, Moghadamnia AA, and Faraji AA (2014) Immunotoxicity effects of carbaryl in vivo and in vitro. Environmental Toxicology and Pharmacology 38: 838–844. Judson R, Houck K, Martin M, Richard AM, Knudsen TB, Shah I, Little S, Wambaugh J, Setzer RW, Kothiya P, Phuong J, Filer D, Smith D, Reif D, Rotroff D, Kleinstreuer N, Sipes N, Xia M, Huang R, Crofton K, and Thomas RS (2016) Analysis of the effects of cell stress and cytotoxicity on in vitro assay activity across a diverse chemical and assay space. Toxicological Sciences 153: 409. Km B and Jb B (2010) Chapter 15: Immunotoxicity of pesticides. In: Krieger R (ed.) Hayes’ Handbook of Pesticide Toxicology. Academic Press/Elsevier. Knaak JB, Dary CC, Okino MS, Power FW, Zhang X, Thompson CB, Tornero-Velez R, and Blancato JN (2008) Parameters for carbamate pesticide QSAR and PBPK/PD models for human risk assessment. Reviews of Environmental Contamination and Toxicology 193: 53–212. Koutros S, Harris SA, Spinelli JJ, Blair A, McLaughlin JR, Zahm SH, Kim S, Albert PS, Kachuri L, Pahwa M, Cantor KP, Weisenburger DD, Pahwa P, Pardo LA, Dosman JA, Demers PA, and Beane Freeman LE (2019) Non-Hodgkin lymphoma risk and organophosphate and carbamate insecticide use in the north American pooled project. Environment International 127: 199–205. Laetz CA, Baldwin DH, Collier TK, Hebert V, Stark JD, and Scholz NL (2009) The synergistic toxicity of pesticide mixtures: Implications for risk assessment and the conservation of endangered Pacific salmon. Environmental Health Perspectives 117: 348–353. Lee I, Eriksson P, Fredriksson A, Buratovic S, and Viberg H (2015) Developmental neurotoxic effects of two pesticides: Behavior and biomolecular studies on chlorpyrifos and carbaryl. Toxicology and Applied Pharmacology 288: 429–438. Lucier G, McDaniel O, Williams C, and Klein R (1972) Effects of chlordane and methylmercury on metabolism of carbaryl and carbofuran in rats. Pesticide Biochemistry and Physiology 2: 244–255. Mathur A and Bhatnagar P (1991) A teratogenic study of carbaryl in Swiss albino mice. Food and Chemical Toxicology 29: 629–632. Meeker JD, Ryan L, Barr DB, Herrick RF, Bennett DH, Bravo R, and Hauser R (2004) The relationship of urinary metabolites of carbaryl/naphthalene and chlorpyrifos with human semen quality. Environmental Health Perspectives 112: 1665–1670. Moser VC, McDaniel KL, Phillips PM, and Lowit AB (2010) Time-course, dose-response, and age comparative sensitivity of N-methyl carbamates in rats. Toxicological Sciences 114: 113–123. NIOSH (2019) NIOSH Pocket Guide to Chemical Hazards: Carbaryl. The National Institute for Occupational Safety and Health. NRC (2015) Review of California’s Risk-Assessment Process for Pesticides. Washington, DC: National Research Council of the National Academics. OEHHA (2019) Chemicals Considered or Listed Under Proposition 65: Carbaryl. Sacramento, CA: Office of Environmental Health Hazard Assessment. Oziolor EM, Howard W, Lavado R, and Matson CW (2017) Induced pesticide tolerance results from detoxification pathway priming. Environmental Pollution 224: 615–621. Pan Europe (2006) What Substances Are Banned and Authorized in the EU Market? London, United Kingdom: Pesticides Action Network Europe. PubChem (2021) Compound Summary for CID 6129, Carbaryl. National Center for Biotechnology Information. Roberts JR and Reigart JR (2013) N-Methyl Carbamate Insecticides. In: Recognition and Management of Pesticide Poisonings, 6th edn. Washington, DC: United Stated Environmental Protection Agency. Savitz DA, Arbuckle T, Kaczor D, and Curtis KM (1997) Male pesticide exposure and pregnancy outcome. American Journal of Epidemiology 146: 1025–1036.

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Söderpalm-Berndes C and Önfelt A (1988) The action of carbaryl and its metabolite a-naphthol on mitosis in V79 Chinese hamster fibroblasts. Indications of the involvement of some cholinester in cell division. Mutation Research, Fundamental and Molecular Mechanisms of Mutagenesis 201: 349–363. Tan LF, Sun XZ, Li YN, Ji JM, Wang QL, Chen LS, Bian Q, and Wang SL (2005) Effects of carbaryl production exposure on the sperm and semen quality of occupational male workers. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi 23: 87–90. Tanaka R, Fujisawa S, Nakai K, and Minagawa K (1980) Distribution and biliary excretion of carbaryl, dieldrin and paraquat in rats: effect of diets. The Journal of Toxicological Sciences 5: 151–162. Tanaka R, Fujisawa S, and Nakai K (1981) Study on the absorption and protein binding of carbaryl, dieldrin and paraquat in rats fed on protein diet. The Journal of Toxicological Sciences 6: 1–11. Tang J, Cao Y, Rose RL, and Hodgson E (2002) In vitro metabolism of carbaryl by human cytochrome P450 and its inhibition by chlorpyrifos. Chemico-Biological Interactions 141: 229–241. US EPA (2002) Carbaryl—Report of the Cancer Assessment Review Committee. US EPA (2003) Environmental Fate and Ecological Risk Assessment for the Re-registration of Carbaryl. US EPA (2007a) Carbaryl HED Chapter of the Reregistration Eligibility Decision Document (RED). Washington, DC: US EPA. US EPA (2007b) Guidance on Cumulative Risk Assessment of Pesticide Chemicals That Have a Common Mechanism of Toxicity. Washington, DC: US EPA. US EPA (2012) Aquatic Life Ambient Water Quality Criteria for Carbaryl—2012. Washington, DC: US EPA. US EPA (2015) EDSP Weight of Evidence Conclusions on the Tier 1 Screening Assays for the List 1 Chemicals. Washington, DC: US EPA. US EPA (2016a) Evaluation of Carbaryl and Malathion Human Studies for Their Proposed Application in a Physiologically-Based Pharmacokinetic Model for Risk Assessment. US EPA. US EPA (2016b) Toxicity Forecaster (ToxCast). US EPA. US EPA (2017a) Carbaryl: Draft Human Health Risk Assessment in Support of Registration Review. Washington, DC: US EPA. US EPA (2017b) Carbaryl: Tier I Update Review of Human Incidents and Epidemiology for Draft Risk Assessment. Washington, DC: US EPA. US EPA (2020) Carbaryl (056801) National and State Summary Use and Usage Summary. Washington, DC: US EPA. US EPA (2021a) Carbaryl: Revised Draft Human Health Risk Assessment in Support of Registration Review. Washington, DC: US EPA. US EPA (2021b) ToxCast CompTox Dashboard. US EPA. US EPA (2021c) US EPA ToxCast Screening Library: Carbaryl. US EPA. USDA (2019) Final Human Health and Ecological Risk Assessment for Carbaryl Rangeland Grasshopper and Mormon Cricket Suppression Applications. Riverdale, MD: USDA. Versar I (2018) Final Report: External peer review of EPA’s Physiologically-Based Pharmacokinetic (PBPK) Model for Deltamethrin and Permethrin and PBPK-Pharmacodynamic (PBPK-PD) Model for Carbaryl. Springfield, VA: EPA. Xia Y, Cheng S, Bian Q, Xu L, Collins MD, Chang HC, Song L, Liu J, Wang S, and Wang X (2005) Genotoxic effects on spermatozoa of carbaryl-exposed workers. Toxicological Sciences 85: 615–623. Yearly R and Leonard J (1993) Measurement of pesticides in air during application to lawns, trees and shrubs in urban environments. In: Kd R and Ar L (eds.) Pesticides in Urban Environments: Fate and Significance. Washington, DC: American Chemical Society. Yoon M, Campbell JL, Andersen ME, and Clewell HJ (2012) Quantitative in vitro to in vivo extrapolation of cell-based toxicity assay results. Critical Reviews in Toxicology 42: 633–652. Zhang X, Knaak JB, Tornero-Velez R, Blancato JN, and Dary CC (2010) Chapter 73: Application of physiologically based pharmacokinetic/Pharmacodynamic modeling in cumulative risk assessment for N-methyl carbamate insecticides. In: Krieger R (ed.) Hayes’ Handbook of Pesticide Toxicology, 3rd edn. New York: Academic Press.

Further reading ACGIH (2018) Carbaryl: TLVW Chemical Substances. 8th Edition Documentation Cincinnati, OH: ACGIH. DPR (2014) Carbaryl (1-Naphthyl Methylcarbamate) Occupational and Bystander Risk Characterization Document. Sacramento, CA: Department of Pesticide Regulation. Gunasekara AS, Rrubin AL, Goh KS, Spurlock FC, and Tjeerdema RS (2008) Environmental fate and toxicology of carbaryl. Reviews of Environmental Contamination and Toxicology 196: 95–121. NIOSH (2019) NIOSH Pocket Guide to Chemical Hazards: Carbaryl. U.S. Department of Health and Human Services. USDA (2019) Final Human Health and Ecological Risk Assessment for Carbaryl Rangeland Grasshopper and Mormon Cricket Suppression Applications. Riverdale, MD: USDA. US EPA (2017) Carbaryl: Draft Human Health Risk Assessment in Support of Registration Review. Washington, DC: US EPA. US EPA (2017) Carbaryl: Tier I Update Review of Human Incidents and Epidemiology for Draft Risk Assessment. Washington, DC: US EPA. US EPA (2021) Carbaryl: Revised Draft Human Health Risk Assessment in Support of Registration Review. Washington, DC: US EPA.

Relevant websites http://npic.orst.edu :National Pesticide Information Center. http://www.epa.gov :United States Environmental Protection Agency. http://www.osha.gov :United States Occupational Safety and Health Administration. https://www.cdpr.ca.gov/docs/whs/active_ingredient/carbaryl.htm :Department of Pesticide Regulation. https://pubchem.ncbi.nlm.nih.gov/compound/6129 :NCBI PubChem. https://comptox.epa.gov/dashboard/dsstoxdb/results?search¼DTXSID9020247 :Toxicity Forecaster.

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Carbofuran Siong Fong Sima and Jocephine Jonipb, aFaculty of Resource Science & Technology, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia; bChemsain Konsultant Sdn Bhd, Kuching, Sarawak, Malaysia © 2024 Elsevier Inc. All rights reserved. This is an update of X. Song, Carbofuran, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 673–674, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00108-1.

Chemical profile Introduction Uses/occurrence Exposure (sources, routes and pathways, media, human exposure, typical levels) and exposure monitoring Toxicokinetics (adsorption, distribution, metabolism, excretion) Mechanism of toxicity Immunotoxicity Genotoxicity and carcinogenicity Organ toxicity (pulmonary, Neurotox, Hepatotox, kidney, endocrine disruption, skin, etc.) Interactions Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines Acknowledgment References

513 514 514 514 514 516 516 516 518 518 518 518 522 522 523 523

Abstract This chapter provides an overview on the toxicity of carbofuran including its usage, exposure, toxicity mechanism, degradation and dissipation pathways as well as the regulatory guidelines. Carbofuran is an insecticide banned in 63 countries; however, this pesticide is still commonly used in Asia, Australia and South America. Carbofuran is highly toxic to birds, invertebrates and fish. It is a cholinesterase inhibitor that prevents the breakdown of the neurotransmitter acetylcholine. Carbofuran is mainly degraded via hydrolysis yielding 3-ketocarbofuran and 3-hydroxycarbofuran. Its dissipation is influenced by the environmental factors. The maximum residue level (MRL) of carbofuran in agricultural commodities vary according to countries and crops.

Keywords 3-hydroxycarbofuran; 3-ketocarbofuran; Acetylcholine; Acetylcholinesterase inhibition; Atropine; Carbamylation; Carbofuran phenol; Half-life; Hydrolysis; Pre-harvest interval

Key points

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To provide a broad discussion on carbofuran and its toxicity in various aspects.

Chemical profile

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Name: Carbofuran. Synonyms: 2,3-dihydro-2,2-dimethyl-benzofuranyl-N-methylcarbamate, Furadan, Furadan G, Curaterr, Bay 70143, D 1221, ENT 27164, FMC 10242, NIA 10242, Pillarfuran, and Yaltox. CAS Number: CAS-1563-66-2. Molecular Formula: C12H15NO3 Molecular weight: 221.26. Melting point: 150-152
C. Specific gravity: 1.18 at 20
C/20
C. Vapour pressure: 2  10−5 mmHg at 33
C. Water solubility: 350 mg/L at 25
C.

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Chemical Structure:

Introduction Carbofuran (2,3-dihydro-2,2-dimethyl-benzofuranyl-N-methylcarbamate) is an example of carbamate – a class of synthetic organic pesticide that is used as insecticides, fungicides, nematicides, acaricides, molluscicides and herbicides. Carbofuran is marketed under the trade name of Furadan by FMC Corporation and Curaterr 10 GR, by Bayer among several others. Pure carbofuran is odorless to mildly aromatic white crystalline solid. It is soluble in water and well dissolved in organic solvents.

Uses/occurrence Carbofuran can be applied in granular form or as foliar spray for control of soil-dwelling and foliar-feeding insects including corn rootworms, wireworms, boll weevils, mosquitoes, alfalfa weevil, aphids and white grubs (Kale et al., 2001). It has been applied on sugarcane, sugar beet, maize, paddy, potatoes, corn, soybeans, oil palm and coffee (Otieno et al., 2010; Farahani et al., 2007; Mahrub and Pollet, 1996; Trotter et al., 1991; Bahadir and Pfister, 1987; Rajagopal et al., 1984; Seiber et al., 1978; Siddaramappa et al., 1978). Carbofuran is relatively persistent hence, it has also been employed for pests that are resistant to organophosphate pesticides such as white flies, leaf miners, bees, mealy bugs, scale insects, cockroaches, wasps and aphids. Carbofuran demonstrates high mammalian toxicity and it is highly hazardous to invertebrates and birds (Nicolopoulou-Stamati et al., 2016; Otieno et al., 2010). At an application rate of 0.28–10.9 kg/ha, carbofuran is fatal to fish, wildlife, birds and invertebrates (Eisler, 2000). Carbofuran has been banned in 63 countries including the United States, the European Union, New Zealand, South Korea, Argentina and Canada. Due to its toxicity, it caused the death of millions of birds every year; however, this pesticide is still widely used in Asia, Australia and South America (Tenebaum, 2008; Kitowski et al., 2020).

Exposure (sources, routes and pathways, media, human exposure, typical levels) and exposure monitoring Carbofuran poisoning is often a result of intentional or unintentional exposure through ingestion, inhalation and dermal absorption. It is commonly mistaken as food by birds (Erwin, 1993). In 2018, 34 endangered Andean condors were found dead after consuming carcasses deliberately poisoned with carbofuran for control of livestock predators (Kitowski et al., 2021). Although the use of carbofuran has been banned in the European Union in 2008, incidences of carbofuran poisoning continue to be reported. Carbofuran was detected in the liver of protected white-tailed eagle (Haliaeetus albicilla) and common buzzard (Buteo buteo) in Poland (Kitowski et al., 2020). Intentional/unintentional carbofuran intoxication was likewise recorded in bear, martens, foxes and domesticated animals such as dogs and livestock (Pivariu et al., 2020; Reljic et al., 2012; Novotny et al., 2011; Wang et al., 2007). The lethal dosage of carbofuran vary according to animal species and its exposure pathways as summarized in Table 1. Evidently, carbofuran is less toxic by dermal absorption. Insects and birds are highly sensitive to carbofuran poisoning while bivalve molluscs demonstrate high resistance against this pesticide. The fatal dosage for younger animals is usually lower due to the underdeveloped enzymatic system (Oros and Nagy, 2017). Carbofuran exerts its toxic effects on insects and animals by inhibiting the acetylcholinesterase activity (AChE, a cholinergic enzyme), preventing hydrolysis of acetylcholine (ACh) (California Environmental Protection Agency, 2016). A 26-year-old male died from acute respiratory paralysis due to inhibition of red blood cell AChE activity by 93–99% after carbofuran ingestion (Ferslew et al., 1992). As carbofuran is widely used in agriculture, occupational carbofuran poisoning among farm workers is also very common (Satar et al., 2005; Center for Disease Control and Prevention, 1999; Huang et al., 1998). According to a retrospective study of carbamate poisoning cases in Thailand between 2005 and 2010, a 25.2% from the total of 3183 cases were related to carbofuran (Tongpoo et al., 2015). The occupational poisoning mainly occurs through dermal exposure with symptoms including dizziness, blurred vision, nausea, muscle ache and excessive perspiration with a few complaints of epigastric pain, chest tightness and vomiting. These effects can be rapidly reversed within 2–3 h, after the exposure is removed (Satar et al., 2005; Huang et al., 1998).

Toxicokinetics (adsorption, distribution, metabolism, excretion) Exposure to carbofuran may take place through ingestion, inhalation and skin contact. Via dermal route, carbofuran is rapidly absorbed where the amount penetrating the skin depends on the dosage of exposure. As reported, exposure to 2 mg of carbofuran

Carbofuran Table 1

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Toxicity dosage according to species and exposure routes.

Species Mammals Rats Dogs Guinea pigs Rabbit Sheep Cattle Birds Fulvous ducks Mallard ducks Bobwhite quails Pheasant Chicken Japanese quail House sparrow Fish Rainbow trout Bluegill sunfish Yellow perch African catfish Marine aquatic organisms Dungeness crab Bivalve molluscs Invertebrates Honeybee Houseflies Earthworms Annelid worms

Route

Dosage

References

Oral Dermal Inhalation Oral Inhalation Oral Inhalation Dermal Oral Oral

5 mg/kg (LD50) 120 mg/kg (LD50) 85 mg/m3 (LC50) 19 mg/kg (LD50) 52 mg/m3 (LC50) 9.2 mg/kg (LD50) 0.043 to 0.053 mg/L (LC50) > 1000 mg/kg (LD50) 9 mg/kg (Lethal) 18 mg/kg (Lethal)

Matsumura (1985) Ben-Dyke et al. (1970) Tobin (1970) Kidd and James (1991) Tobin (1970) Eisler (2000) Kidd and James (1991) Kidd and James (1991) Osweiler et al. (1985) Osweiler et al. (1985)

Oral

0.238 mg/kg (LD50) 0.28–0.74 mg/kg (LD50) 12 mg/kg (LD50) 4.15 mg/kg (LD50) 25–39 mg/kg (LD50) 1.3–2.1 mg/kg (LD50) 0.13 mg/kg (LD50)

Smith (1992) Smith (1992) Smith (1992) Smith (1992) Kidd and James (1991) Hill and Camardes (1986)

Oral

0.38 mg/L (LD50) 0.24 mg/L (LD50) 0.147 mg/L (LC50) 0.31 mg/L (LC50)

Smith (1992) Smith (1992) Eisler (2000) Eisler (2000)

Oral

0.19 mg/L (LC50) 3.75–125 mg/L

Eisler (2000) Eisler (2000)

Oral

0.16 mg/bee (LC50) 0.1–1.3 mg/insect (LC50) 2.4 mg/kg (LC50) 11–14 mg/kg (LC50)

Eisler (2000) Eisler (2000) Eisler (2000) Eisler (2000)

evidenced a penetration rate of 1.05 mg/cm2 per hr.; as the concentration increased to 15 mg, a higher rate of 1.52 mg/cm2 per hr. was recorded (Liu and Kim, 2003). In a dermal exposure study involving female mice, the absorption of carbamates, organophosphates, botanicals and chlorinated hydrocarbons were compared; carbofuran was the most rapidly absorbed pesticides among all (Shah et al., 1981). Gammon et al. (2012) further experimented the dermal absorption of carbofuran on human and rat skin over 24 h. The study revealed that at an application of approx. 10 mg/cm2, the absorption by rat skin (1.983 mg/cm2) was about 10 times greater than that by human skin (0.193 mg/cm2). Upon dermal penetration, inhibition of brain and red blood cells AChE was observed within 30 min (Gammon et al., 2012). Based on four suicidal cases of carbofuran ingestion, the fatal concentration of carbofuran in blood is estimated at 0.32 mg/mL (Ameno et al., 2001) however, this lethal level is anticipated to be higher as rapid in vivo metabolism of carbofuran after intake was revealed by Liu et al. (2016). The study concluded that having the concentration of carbofuran in blood between 2.53 and 3.23 mg/mL is important for successful rescue treatment; above this range, carbofuran poisoning is fatal. Toxicity response of AChE inhibition was observed in brain (40%) and red blood cells (55%) of adult rats after 30 min of oral exposure to 0.5 mg/kg of carbofuran. The brain AChE activity recovered after 6 h however it took much longer, between 6 and 24 h, for the red blood cells to restore its activity (Padilla et al., 2007). Carbofuran is hydrolyzed by carboxylesterases or oxidized by cytochrome (CYP) P450 oxidase yielding metabolites of 3-hydroxycarbofuran, 3-ketocarbofuran and other phenol derivatives including 3-hydroxy-7-phenol, 3-keto-7-phenol, 7-phenol (Usmani et al., 2004; Hayes and Law, 2013). These metabolites in their existing state or conjugated with sulfuric acid and glucuronic acid will be excreted through urine and feces (Song, 2014; Usmani et al., 2004). Shormanov et al. (2015) investigated the distribution of carbofuran in rats after oral intake revealing large amount of the pesticide in stomach, intestines, testicles, bladders, ureters and heart. The target organs damaged by carbofuran are mainly brain, liver, muscles and heart (Gupta, 1994). Yulitasari et al. (2021) observed degeneration, necrosis and infiltration of inflammatory cells in liver of mice offspring feeding from lactating mother exposed to carbofuran. Upon absorption into the body, carbofuran can be rapidly hydrolyzed and excreted without accumulation. As demonstrated, rats exposed to 1.2 ppb of aerosol carbofuran excreted 55% of the pesticide after 8 h through respiration (38%), urine (12%) and feces (5%) (Ferguson et al., 1982); typically, within 32 h after exposure, more than 80% of carbofuran can be eliminated.

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Mechanism of toxicity Carbofuran exerts its toxicity by binding to the AChE (acetylcholinesterase) enzyme forming carbamylated-AChE. This prevents the enzyme from breaking down acetylcholine (ACh) into acetate and choline. Acetylcholine is a neurotransmitter synthesized from acetyl co-enzyme A and choline. They can be found in all motor neurons playing crucial roles in numerous physiological functions including muscle movement, blood vessels dilation, memory formation, pain sensation, regulation of cardiac contractions and blood pressure, glandular secretion and etc. ACh is hydrolyzed into choline and acetate by AChE where the free choline is then taken up again by the neuron for synthesis of ACh. When AChE is inhibited, ACh will accumulate at the synapses leading to symptoms such as increased saliva, blurry vision, muscle weakness and cramps depending on the cholinergic receptors involved, whether nicotinic or muscarinic receptors. Nicotinic toxicity will cause muscle weakness and tremors while muscarinic damages lead to excess salivation and respiratory failure (Gupta, 2006). The inhibition of AChE can be restored through dissociation of methylcarbamyl moiety from the enzyme which takes place in a short period of time (Gupta, 1994). Fig. 1 illustrates the mechanism of carbofuran toxicity.

Immunotoxicity The AChE inhibition and over accumulation of ACh could change the signal transduction pathways resulting in inhibition/ activation of immune cells (Dhouib et al., 2016). Carbofuran intoxication has also been found to affect inflammatory responses altering the cell membranes of immunocytes, increasing the risk of cancers and other diseases (Banks and Lein, 2012). The effects of carbofuran on the immune systems have been extensively revealed; this includes significant increase in white blood cells, neutrophils and basophils with decreasing lymphocyte count (Gupta et al., 1982). Carbofuran is also found to disrupt the immune systems by changing the hormone levels. As reported, rats exposed to acute dose of carbofuran (1.5 mg/kg) exhibited significant increase in the levels of progesterone, cortisol, and estradiol at a magnitude of 150–1279% with reduction in testosterone level by 88% (Goad et al., 2004). Hanim Hadie et al. (2013) further evidenced significant increase in thyroid stimulating hormone in rats exposed to a high dosage of 2.4 mg/kg carbofuran. These changes in thyroid stimulating hormone however were not supported in the study by Goad et al. (2004) with exposure dose of 1.5 mg/kg. When carbofuran is metabolized, the inhibition of cytochrome oxidase would lead to the formation of reactive oxygen species that oxidize the unsaturated fatty acids or phospholipids of cell membranes producing aldehydes, ketones, carboxylic acids and polymerization products (Gupta et al., 2000). This reaction of lipid peroxidation induces oxidative stress which is a condition of lacking in antioxidant to scavenge the reactive oxygen species (Lasram et al., 2014; Gupta et al., 2001). Rats exposed to sub-lethal concentration of carbofuran was observed with marked increase in lipid peroxidation along with reduced activities of antioxidant enzymes in brain and heart (Jaiswal et al., 2013; Rai and Sharma, 2007). Carbofuran exposure also stimulates the production of nitric oxide synthase triggering excessive production of superoxide anions which was believed to be linked to suppression of T-cell-mediated immune response (Jeon et al., 2001; Pou et al., 1992). Vitamin C and Bridelia tomentosa leaf extract have been reported with ameliorative effect against carbofuran induced oxidative stress, restoring the activity of antioxidant enzyme (Mondal et al., 2021; Jaiswal et al., 2013).

Genotoxicity and carcinogenicity Carbofuran at 1.08 mM was found to cause in vitro cytotoxicity in cat with maximum DNA damages (Chandrakar et al., 2020). In human blood lymphocytes, in vitro exposure likewise revealed dose-dependent micronuclei formation associated with carbofuran induced oxidative stress (Sharma and Sharma, 2012). Micronuclei are extra-nuclear bodies containing damaged chromosomes or chromosomes that failed to be incorporated into the nucleus after cell division. The genotoxicity and cytotoxicity effects of carbofuran were also reported in ovary cells of Chinese hamster where increased micronuclei and sister chromatid exchange were evidenced with delay in cell growth (Soloneski et al., 2008). The effect of carbofuran and its four metabolites (carbofuranphenol, 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran on DNA was tested on mice exposed to different doses of 0.1, 0.2 and 0.4 mg/kg through intraperitoneal injection. The DNA damaging effects were evaluated using single cell gel electrophoresis (SCGE) assay and micronucleus test. Carbofuran and carbofuranphenol showed negative results in both test and had no obvious toxicity. 3-Hydrocarbofuran and nitrosocarbofuran were positive. 3-Ketocarbofuran could not induce micronucleus formation but caused significant DNA migration in SCGE test. These tests revealed that 3-ketocarbofuran, 3-hydrocarbofuran and nitrosocarbofuran are potential mutagenesis and further research is needed. (Zhou et al., 2005). Naravaneni and Jamil (2005) confirmed the DNA damage caused by carbofuran on human lymphocyte cultures where chromosomal aberrations was shown with increase in comet tail length. Carbofuran is a suspected carcinogen. A questionnaire-based exposure assessment involving 57,311 licensed pesticides applicators and 32,347 of their spouses in Iowa and North Carolina revealed no relationship between carbofuran and cancers specifically lymphatic, colon and prostate cancers. A positive correlation was deduced with lung cancer as the risk for applicators who used the pesticide for >10 years with >10 application days per year was three-fold higher; nevertheless, there was no inferential evidence to support the postulation (Bonner et al., 2004). A more recent study continues to identify a positive correlation between lung cancer

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Fig. 1 Mechanism of carbofuran toxicity. Adapted from Gupta RC (1994) Carbofuran toxicity. Journal of Toxicology and Environmental Health 43(4): 383–418.

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and historical exposure to carbofuran suggesting an odd ratio (OR) of 2.10 (OR > 1.0 implies high odds of lung cancer) (Kangkhetkron and Juntarawijit, 2020). There is evidence that carbofuran can be converted into mutagenic and carcinogenic N-nitroso derivatives in the presence of sodium nitrite (commonly found in human diet) under acidic condition in the stomach. Three nitroso compounds namely nitrosocarbofuran, 3-ketonitrosocarbofuran and 3-hydroxynitrosocarbofuran were found to stimulate chromosome aberrations in Chinese hamster ovary cells with the latter two compounds triggering significantly large sister chromatid exchanges in the cells (Nelson et al., 1981). The formation of nitroso compounds in the stomach is expected to increase the risk of stomach cancer however this hypothesis lacks evidence – too few cases of stomach cancer related to carbofuran for analysis (Bonner et al., 2005). A two-year dietary study involving exposure of carbofuran between 0 and 500 ppm on rats recorded no carcinogenicity on the animals tested hence, the Codex Committee on pesticide residues concluded that carbofuran is unlikely to pose a carcinogenic risk to humans (Gupta, 1994). The US EPA also categorizes carbofuran under the list of non-carcinogenic as the risk is unable to be determined based on currently available information (Jennings and Li, 2017).

Organ toxicity (pulmonary, Neurotox, Hepatotox, kidney, endocrine disruption, skin, etc.) Exposure to carbofuran at 1 mg/kg body weight orally for 28 days in rats recorded significant decrease in AChE activity in cerebral cortex, cerebellum and brain stem (66.6–71.7%) accompanied by elevated lipid peroxidation and reduced glutathione level (Kamboj et al., 2006). The neurotoxic effect is a long-term condition caused by the carbofuran-induced oxidative stress (Fu et al., 2019). Besides, hepatotoxic effects were recorded involving development of lesions in liver of rats exposed to 0–5 mg/kg body weight for 5 weeks (Gbadegesin et al., 2014). Carbofuran also demonstrated nephrotoxicity in rats where significant increase of urea and creatinine level in serum was observed, correlated with the oxidative stress (Kaur et al., 2012). Disruptions in reproductive system were reported in rats exposed to carbofuran; treated female mice showed decreasing healthy follicles and ovary weight while male mice produced abnormal sperm including morphological changes in testes and its enzyme activities. The effects persisted to some extent even after the exposure was ceased (Hadie et al., 2016; Aziz et al., 2008; Baligar and Kaliwar, 2002; Pant et al., 1995).

Interactions Combined or sequential treatments of two or more pesticides often result in additive, synergistic or antagonistic responses. Combined carbofuran and chlorpyrifos (an organophosphate insecticide) treatment on rats at 100 mmol/L and 200 mmol/L, respectively, yielded synergistic effects on oxidative stress; however, the interaction was insignificant at lower dose (Lian et al., 2017). Alachlor (herbicide) combined with carbofuran similarly experienced synergistic responses with growth retardation observed in barley but not in corn. The interactions between alachlor and carbofuran improved the uptake of the herbicide, enhancing its accumulation by delaying the metabolism (Hamill and Penner, 1973a). In addition, sequential application of carbofuran-propanil on rice instigated more serious leaf chlorosis and necrosis on the crops as the interactions enhanced the persistence of the insecticide or the herbicide or both (Rao, 2000; Smith and Tugwell, 2000). Combinations of carbofuran with permethrin or piperonyl butoxide (both insecticides) also recorded synergistic actions against third instar southwestern corn borer (Christian et al., 1986). Interactions of carbofuran with herbicides including alachlor (Hamill and Penner, 1973a), chlorbromuron (Hamill and Penner, 1973b) and butylate (Hamill and Penner, 1973c) are primarily synergistic; in 121 examples of herbicideinsecticide interactions examined, more than 93% revealed synergistic toxicity (Hatzios and Penner, 1985). Mixtures of more than two pesticides including carbofuran likewise evidenced synergistic toxicities on AChE activity (7 carbamates) and genotoxicity on human peripheral lymphocytes (carbofuran/endosulfan/monocrotophos) (Moser et al., 2012; Das et al., 2007).

Clinical management Atropine is one of the most effective antidotes against carbamate poisoning (Eddleston et al., 2008). A medium size Brahminy kite (Haliastur indus) intoxicated by carbofuran was treated with injection Atropine sulfate (0.5 mg/kg body weight Intramuscular (i.m)) and injection Dexamethasone (2 mg i.m) with oral administration of activated charcoal (1 g) as a drench. The bird was given injection Tribivet (B-complex vitamins, 0.5 mL i.m) on the next day and recovered (Sravanthi et al., 2018). Combined treatments with memantine HCl (18 mg/kg) and atropine sulfate (16 mg/kg) prior carbofuran administration in rats was seen to prevent acute poisoning. This treatment strategy offers protection for AChE and accelerates elimination of the pesticide from the body (Gupta and Kadel, 1989). For suicidal carbofuran poisoning, the management procedures typically involve treatment using atropine as the antidote, supported by intubation and benzodiazepines (Klatka et al., 2021; Nguyen et al., 2021).

Environmental fate and behavior Carbofuran has been found in water bodies, soil, air, plants and animals. It is relatively persistent in soil where its degradation could take between 111 and 434 days depending on the soil pH, moisture and temperature (Caro et al., 1975; Lalah et al., 2001). Based on

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the field data, the half-life of carbofuran in soil ranges between 6 and 17 weeks (Caro et al., 1973), some reported longer than 12 months (Szeto and Price, 1991). Carbofuran is highly soluble in water hence it poses risk of pollution and environmental distress toward aquatic organisms. The migration risk of a pesticide into the adjacent groundwater bodies can be indicated based on the groundwater ubiquity score (GUS). Carbofuran is characterized with a GUS index of 4.52 suggesting a high leachability potential (Spadotto, 2002). The adsorption of carbofuran involves transport of carbofuran to the outer layer of soil surface, followed by diffusion of the molecules to the inner active sites where adsorption takes place mainly via electrostatic interactions. The adsorption, described by the distribution coefficient Kd, indicates that at higher initial carbofuran concentration and lower pH, the Kd value decreases signifying a lower adsorption affinity. It is postulated that at lower initial concentrations, the adsorption targets high-affinity sites oppose to low-affinity sites under higher concentrations. Under acidic environment, carbofuran is positively charged fostering electrostatic interactions, increasing the adsorption sites which in turn preventing loss of the pesticide through leaching, volatilization and biodegradation processes (Arias-Estévez et al., 2006). Soil rich of organic matter and clay could further enhance the sorption of carbofuran. Clay minerals is considered one of the best sorbents for pesticides for that it consists of abundant hydroxyl groups with expandable internal surfaces accessible for interactions with water and polar compounds via ligand exchange, hydrogen bonding, Van der Waals mechanisms (Chotzen et al., 2016; Angioi et al., 2005). Whereas in the presence of organic matter with humic substances being the major compounds, carbofuran may interact through ionic exchange, hydrogen bonding, Van der Waals, ligand exchange and hydrophobic mechanisms (Senesi, 1992). Kinetically, the desorption of carbofuran is more rapid than the adsorption (Bermúdez-Couso et al., 2011; Khan et al., 2010). Fig. 2 illustrates the carbofuran adsorption in soil under different conditions: initial concentration, pH, clay minerals and organic matter. Carbofuran is dissipated via various pathways including chemical degradation, volatilization, biotransformation, plant growth dilution and surface runoff. These processes may occur individually or simultaneously, governed by environmental conditions (humidity, rainfall, temperature, UV radiation), plant morphology, pesticide formulation and dosage applied (Fantke and Juraske, 2013). The degradation of carbofuran is highly dependent on pH; the breakdown of carbofuran is much faster in alkaline soils than in acidic soils. The half-life of carbofuran at pH 10 and pH 7 were reported at 1.2 h and 864 h, respectively (Seiber et al., 1978). In alkaline soil, hydrolysis is the major degradation pathway yielding carbofuran phenol as the main metabolite (Yu et al., 1974; Seiber et al., 1978; Talebi and Walker, 1993) whereas in acidic soil, microbial and chemical mechanisms predominate (Farahani et al., 2007; Getzin, 1973). Soil moisture also demonstrated a positive influence on the degradation of carbofuran; the higher the soil moisture, the shorter is the half-life. Under the high moisture condition (100% flooded condition), an anaerobic environment is fostered encouraging hydrolysis reaction and microbial degradation (Benicha et al., 2013; Mojaševic et al., 1996; Parkin and Shelton, 1994; Rajagopal et al., 1984; Getzin, 1973). The degradation of carbofuran is positively correlated with the soil temperature. Sahoo et al. (1993) evidenced that the disappearance of carbofuran was accelerated at 35
C, compared to that at 25
C, with hydrolysis being the major pathway. Parkin and Shelton (1994) reached a similar conclusion comparing the dissipation rate at 30
C and 10
C. Ramanand et al. (1988) revealed that the degradation of carbofuran was only affected when the temperature was higher than 25
C. Numerous studies have also corroborated the ability of soil microorganisms in using carbofuran and its degradation products as a source of carbon and energy (Chanika et al., 2011; Slaoui et al., 2007; Shelton and Parkin, 1991). Actinomyces was found to be responsible for conversion of carbofuran to CO2 (Lalah et al., 2001). At a concentration as low as 0.1 mg/kg carbofuran, it is sufficient to stimulate the growth and activities of bacteria including actinomycetes, fungi, N2-fixing bacteria and phosphate microorganisms. In flooded soil where oxygen is depriving, hydrolysis is the key pathway of carbofuran degradation but subsequent breakdown of carbofuran phenol is facilitated by microorganisms. The effects of pH, moisture and temperature on the degradation of carbofuran, examined using the response surface methodology, revealed that pH is the most dominant factor among the three in controlling the degradation of carbofuran. Rapid breakdown of carbofuran at 98% was recorded in soil at pH 12 while at pH 2, only 68% was attained (Jonip et al., 2019). Carbofuran is characterized with a relatively low vapor pressure of 8.3  10−6 mmHg at 25
C hence hydrolysis is a preferred pathway of degradation over volatilization (Lalah et al., 2001). However, when the temperature increases, the vapor pressure of carbofuran becomes higher and this promotes the loss of carbofuran to the atmosphere via volatilization (Tiryaki and Temur, 2010). In the presence of light, photodegradation takes place and the photometabolites produced differs depending on the solvent system (Raha and Das, 1990). When carbofuran is taken up into plants, animals and insects, the metabolic pathways could diverse as illustrated in Fig. 3, adapted from Metcalf et al. (1968). Primarily, it is oxidized into 3-hydroxycarbofuran and 3-ketocarbofuran, hydrolyzed into various carbofuran phenol derivatives, and finally conjugated with sulfuric acid/glucuronic acid for excretion. Greenhalgh and Belanger (1981) examined the degradation of carbofuran in soil confirming the conversion of carbofuran into 3-hydroxycarbofuran with the maximum attained within 1–7 days and then rapidly declined. The metabolite of 3-ketocarbofuran was rather stable; the maximum level was achieved between 16 and 36 days and thereafter slowly reduced. The metabolite of 3-hydroxycarbofuran has not

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Fig. 2 Adsorption of carbofuran in soil under different conditions: initial concentration, pH, clay minerals and organic matter.

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Fig. 3 Metabolic pathways of carbofuran. Adapted from Metcalf RL, Fukuto TR, Collins C, Borck K, Abd El-Aziz S, Munoz R, and Cassil CC (1968) Metabolism of 2,2-Dimethy1-2,3-dihydrobenzofuranyl-7 N-Methylcarbamate (Furadan) in Plants, Insects, and Mammals. Journal of Agricultural and Food Chemistry 16(2): 300–311, doi:10.1021/jf60156a033.

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been consistently detected in aerobic soils but more often in anaerobic soil (Venkateswarlu and Sethunathan, 1978; Getzin, 1973). Sim et al. (2019) investigated the uptake and dissipation of carbofuran in brinjal and Chinese kale in a field study demonstrating the presence of 3-ketocarbofuran; however, 3-hydroxycarbofuran was undetected. This suggests that 3-hydroxycarbofuran is very unstable concurring the findings of Greenhalgh and Belanger (1981).

Ecotoxicology In plants, carbofuran is taken up, translocated and metabolized. The degradation rate may differ according to plant species. In a field study involving Chinese kale and brinjal, carbofuran was accumulated predominantly in leaves with 3-ketocarbofuran identified at low concentrations (Sim et al., 2019). In Chinese kale, the carbofuran residue and its metabolite were observed to accumulate over time with the maximum concentration attained on Day-3 after application (carbofuran was applied on seedlings of 14 days). The carbofuran residue was then observed to decrease consistently until the level of 0.01 mg/kg was met after 23 days. Sim et al. (2019) concluded that carbofuran was not to be used for Chinese kale as the interval time required for the pesticide to dissipate to a safety level was longer than the anticipated harvesting time. In brinjal plants, carbofuran residue was similarly accumulated and dissipated with leaves demonstrating greater affinity of accumulation than fruits. The pre-harvest interval was 28 days for brinjal treated with single dose of carbofuran at recommended dosage. The pesticide was dissipated to a level below 0.01 mg/kg before the fruit reached its maturity. In soil, the degradation of carbofuran obeys the first order kinetics with the dissipation rate differing by climatic conditions. Field studies showed that the pesticide degradation was much more rapid under tropical climates compared to temperate conditions. Suett (1987) examined the dissipation of carbofuran in sandy loam soil with the plot established in Wellesbourne, UK. Carbofuran applied onto the soil was observed to remain in the field for 2–6 weeks before accelerated loss of the pesticide; this is regarded as the lag phase. The field half-life of carbofuran in soil from varying temperate countries range from 120 days, majority between 40 and 50 days, whereas in tropical/subtropical countries, the half-life is typically between 10 and 20 days. Sim et al. (2019) revealed relatively shorter half-life of 1.24 days for carbofuran in soil under tropical climate, without any lag phase, suggesting enhanced degradation in the conditions of greater solar intensity, higher temperature and rainfall. Carbofuran is relatively susceptible to leaching posing risk of toxicity on aquatic organisms. It may take 1–8 weeks for carbofuran to degrade in water. At a lower water pH, carbofuran is more stable. Carbofuran intoxication in fish exhibits uncoordinated swimming movement along with signs of muscle tetany and hyperemia of gills (Dobšiková, 2003). The LC50 value in fish is postulated at rabbits (8/14) >rats (3/6) >guinea pigs (0/6), but there were no deaths after 6 h at 300–500 ppm (740–1200 mg/m3) in cats, rabbits, or guinea pigs (Thiess et al., 1968).

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Exposure to COS in animals produces serious nervous system effects, with narcotic effects and acute respiratory failure at high concentrations. Rats exposed to COS via inhalation for 4 h showed hypoactivity, lacrimation, breathing difficulties, cyanosis, bleeding from the nose, convulsions, tremors, and behavior abnormalities such as circling (Benson et al., 1995). In a 2-week inhalation study, results showed COS toxicity for the high dose group (450 ppm), but only after at least 6 days of exposure. A diminished weight in females and signs of central nervous system (CNS) dysfunction (ataxia, head tilting, circling, pivoting, prostrate and arched back postures, tremors, loss of muscle control, convulsions, and bulging and dilated eyes) were observed. Depressed red cell counts, slight depression in mean corpuscular volume, and methemoglobinemia were present in both sexes (Monsanto, 1985b). Rats exposed to 200, 300, and 400 ppm of COS for 6 h day−1, 5 days a week, over 12 weeks showed no significant differences in body weight; although reductions in several serum chemistry parameters were noted in exposed males relative to control animals, the effects did not appear exposure related. COS targeted specific neuroanatomical sites in the auditory system, suggesting that decreases in cytochrome oxidase in exposed rats may be involved in the pathogenesis of neuronal injury. These studies demonstrate that this environmental air contaminant has the potential to cause a wide spectrum of brain lesions that are dependent on the degree and duration of exposure (Morgan et al., 2004; Herr et al., 2007). When Fischer 344 rats were exposed to 0 or 500 ppm COS for 1–10 days, 6 h day−1 important gene expression changes occurring in the posterior colliculi after 1 or 2 days of COS exposure that were predictive of the upregulation of genes associated with DNA damage, apoptosis, and vascular mediators. These gene expression findings could be predictive of later CNS lesions caused by COS exposure (Morrison et al., 2009). COS is acutely toxic to rats, with a LD50 of 22.5 mg kg−1, by intraperitoneal injection (Chengelis and Neal, 1980).

Chronic toxicity No information was identified on the chronic reproductive, developmental, or carcinogenic effects of COS in animals. However, COS is the oxidation product of S2C, which has been shown by the US National Institutes of Health to be positive in the strain A mouse lung tumor bioassay. Significant increases in the incidence (tumor-bearing mouse) and frequency (tumors per mouse of lung adenomas) were observed in A/J mice. Chronic exposure to low concentrations of COS may cause damage or irritation to the respiratory tract, including symptoms of rhinitis, pharyngitis, bronchitis, and pneumonitis, and may cause pulmonary oedema, or eye irritation with painful conjunctivitis, photophobia, lacrimation, and corneal opacity. Recovery depends on the length of exposure and the dose. Residual effects during recovery may include coughing, slow pulse, and amnesia. No information regarding the potential carcinogenicity or the developmental or reproductive toxicity of COS in humans was identified. The US Environmental Protection Agency (EPA) and the International Agency for Research on Cancer have not classified COS with respect to potential carcinogenicity. COS is neither genotoxic nor a developmental toxicant but does reversibly impair male fertility. Prolonged, repeated exposure to COS is likely to present similar neurotoxicity hazards to that of the structurally and toxicologically related compound CS2.

Immunotoxicity No studies evaluating the potential immunotoxicity of carbonyl sulfide were located and the knowledge if the immune system is a target of carbonyl sulfide toxicity has not been assessed (Agency for Toxic Substances and Disease Registry-ATSDR, 2016).

Reproductive and developmental toxicity Information on the reproductive toxicity of carbonyl sulfide is limited to a study in which male rats were exposed to inhaled COS prior to mating with unexposed females (Monsanto, 1987). The study reported a decrease in pregnancy rate in unexposed female rats mated with male rats exposed to 182 ppm carbonyl sulfide 6 h/day, 5 days/week for 10 weeks and 6 h/day, 7 days/week for a 3-week mating period. When the males were allowed to recover for 10 weeks prior to mating to unexposed females, no alterations in fertility were observed (Monsanto, 1987). This study identified a NOAEL of 60 ppm. Additional studies are needed to determine the cause of the decreased pregnancy rate and to evaluate whether COS affects female reproduction. No studies evaluating the potential developmental toxicity of COS were located (Agency for Toxic Substances and Disease Registry-ATSDR, 2016). So, developmental toxicity studies including neurodevelopmental toxicity testing are needed since neurotoxicity is a sensitive end point in adults.

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Genotoxicity Genotoxicity testing of COS have been limited to in vivo and in vitro studies by Wang et al. (1999) and an in vitro bacterial mutagenicity study by the National Toxicology Program. Micronuclei were not induced in mouse bone marrow, and chromosomal aberrations were not induced in mouse spermatocytes following acute inhalation or oral exposure, and reverse mutations were not induced in Salmonella typhimurium or Escherichia coli strains (Wang et al., 1999). National Toxicology Program-NTP (2021) reported “weakly positive” results for reverse mutation in S. typhimurium strain TA97, but not in strains TA98, TA100, or TA1535.

Carcinogenicity No information regarding the potential carcinogenicity of COS in humans or animals was identified. The US Environmental Protection Agency (EPA) and the International Agency for Research on Cancer have not classified COS with respect to potential carcinogenicity.

Organ toxicity No studies were located regarding organ toxic effects in humans after inhalation exposure to COS (Agency for Toxic Substances and Disease Registry-ATSDR, 2016). Only the study by Kamstrup and Hugod (1979) examined the respiratory tract in rabbits following inhalation exposure to COS and no morphological alterations were observed in the lungs after continuous exposure to 54 ppm for 7 weeks. In heart, no morphological alterations were observed in the coronary arteries, aortic arch, descending thoracic aorta, or pulmonary arteries (Hugod and Astrup, 1980; Kamstrup and Hugod, 1979) and no myocardial ultrastructural changes (Hugod, 1981) were found in rabbits continuously exposed to 54 ppm COS for 7 weeks. One study examined the potential of carbonyl sulfide to induce hematological alterations. In rats exposed to COS for 11 days, significant increases in methemoglobin levels were observed at  151 ppm; however, the magnitude of the methemoglobin levels in treated animals (1.3–2.3% compared to 0.8–1.0% in controls) was low and was not considered toxicologically relevant. Decreases in erythrocyte count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were observed in female rats exposed to 151 or 253 ppm, but were not observed at 453 ppm or in males. The lack of concentration-response and the finding in only one sex suggest that these alterations may not be related to COS exposure. No alterations in body weight were observed in rats exposed to 453 ppm COS for 11 exposure days (Monsanto, 1985b) or 182 ppm for 13 weeks (Monsanto, 1987). Significant increases in serum cholesterol levels were observed in rabbits continuously exposed to 54 ppm COS for 7 weeks (Kamstrup and Hugod, 1979). However, the alterations were only observed at weeks 1, 6, and 7 and corresponded to a downward fluctuation in control levels. No studies were identified on the potential for COS to disrupt the function of the neuro-endocrine axis (Agency for Toxic Substances and Disease Registry-ATSDR, 2016).

Interactions No studies examining interactions of carbonyl sulfide with other chemicals were located.

Clinical management Following inhalation exposure, the victim should be moved to fresh air immediately. If the victim is not breathing, artificial respiration or cardiopulmonary resuscitation should be given, if necessary. If breathing is labored, the victim should be given oxygen. In case of ocular or dermal contact, the skin or eyes should be flushed with running water immediately. Soap and water may be used for washing exposed skin. If COS is accidentally ingested, medical treatment should be sought immediately. Vomiting should not be induced. Further treatment is symptomatic. Rescuers must prevent exposure by wearing a self-contained breathing apparatus to rescue the victim. Acute or chronic respiratory conditions may be aggravated by overexposure to this gas.

Environmental fate and behavior Most of the releases of COS to the environment are to air, where it is believed to have a long residence time. Its half-life in the atmosphere is estimated to be approximately 2 years. It may be degraded in the atmosphere via a reaction with photochemically

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produced hydroxyl radicals or oxygen, direct photolysis, and other unknown processes related to the sulfur cycle. Sulfur dioxide, a greenhouse gas, is ultimately produced from these reactions. COS is relatively unreactive in the troposphere, but direct photolysis may occur in the stratosphere. Also, plants and soil microorganisms have been reported to remove COS directly from the atmosphere. Plants are not expected to store COS. COS has a high solubility in water and will not readily adsorb to soil particles, sediment, or suspended organic matter. Therefore, COS is expected to volatilize rapidly from soil and water or, depending on volume, concentration, and site-specific characteristics (e.g., soil type, depth to groundwater, temperature, and humidity), may be able to move rapidly through the ground and impact groundwater. COS may be hydrolyzed in water to form H2S and CO2. COS is also actively taken up by some plants and converted to CS2; that is, the atmospheric pathways are reversed, and soils may act as both a net source and a net sink for COS depending on the concentration of COS and the characteristics of the soil. COS is therefore accurately described as a naturally occurring and widely distributed chemical found or produced in the air, soils, live and decomposing vegetation, and food. COS may be released to the atmosphere from deciduous trees, volcanoes, coniferous trees, salt marshes and soils. It may also be released to the environment as a fugitive emission from commercial processes and combustion emissions. If released to air, a vapor pressure of 9.41  103 mm Hg at 25  C (Daubert and Danner, 1989) indicates COS will exist solely as a gas in the atmosphere. Gas-phase COS will be degraded in the atmosphere by reaction with photochemically-produced hydroxyl radicals; the half-life for this reaction in air is estimated to be 22 h (Atkinson et al., 1992). COS did not absorb UV light at environmental wavelengths >290 nm and, therefore, photolysis in the troposphere is negligible (Molina et al., 1981). If released to soil, COS is expected to have very high mobility based upon an estimated Koc of 1 (US EPA, 2012). Volatilization from moist soil surfaces is expected to be an important fate process based upon an estimated Henry’s Law constant of 0.61 atm-cu m mole−1. COS may volatilize from dry soil surfaces based upon its vapor pressure. Biodegradation data in soil or water were not available. If released into water, COS is not expected to adsorb to suspended solids and sediment based upon the estimated Koc. Volatilization from water surfaces is expected to be an important fate process based upon this compound’s estimated Henry’s Law constant. Estimated volatilization half-lives for a model river and model lake are 2 h and 3 days, respectively (Lyman et al., 1990). An estimated Bioconcentration factor (BCF) of 3 suggests the potential for bioconcentration in aquatic organisms is low (Franke et al., 1994). Hydrolysis is not expected to be an important environmental fate process since this compound lacks functional groups that hydrolyze under environmental conditions (pH 5–9). COS is ubiquitous in the atmosphere.

Ecotoxicology In insects, carbonic anhydrase has a key role in toxicity of carbonyl sulfide (Haritos and Dojchinov, 2005). COS is not expected to bioaccumulate in fish or other aquatic organisms since an estimated bioconcentration factor of 11 was calculated in fish. The US EPA (2015) reported that quantitative structure-activity relationship estimates of acute toxicity for fish, daphnid, and algae are greater than 1000 mg L−1.

Other COS is a flammable gas and may be explosive or spontaneously flammable in air under the right conditions. Vapors may ignite at distant ignition sources and flash back. When exposed to fire, humidity, or strong alkalis, COS may form the toxic decomposition products CO and H2S gas. In the presence of strong oxidizers, COS presents a fire or explosion hazard. COS has a vapor density of 2.1 and is therefore heavier than air. Cylinders or tank cars containing COS may rupture violently or rocket under fire conditions. The National Fire Protection Agency flammable limits are as follows: lower—12% by volume, upper—29% by volume, and explosive limits are 12–29%. The Clean Air Act Amendments of 1990 list COS as a hazardous air pollutant generally known or suspected to cause serious health effects. COS is also regulated under the Comprehensive Environmental Response, Compensation, and Liability Act, the Superfund Amendments and Reauthorization Act, and Section 4 of the Toxic Substances Control Act.

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/Carbonyl-sulfide

CompTox URL https://comptox.epa.gov/dashboard/dsstoxdb/results?search¼DTXSID6023949

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Melillo JM and Steudler PA (1989) The effect of nitrogen fertilization on the COS and CS2 emissions from temperature forest soils. Journal of Atmospheric Chemistry 9: 411–417. Molina LT, Lamb JJ, and Molina MJ (1981) Temperature dependent UV absorption cross sections for Carbonyl Sulfide. Geophysical Research Letters 8: 1008–1011. Monsanto (1985a) Initial Submission: Acute Toxicity of Carbon Oxysulfide Administered by Inhalation to Male and Female Sprague-Dawley Rats (Final Report). St. Louis, MO: Monsanto Agricultural Company. Monsanto (1985b) Initial Submission: Two Week Study With Carbonyl Sulfide Administered by Inhalation to Rats With Cover Letter Dated 052892. Monsanto Agricultural Company. Monsanto (1987) One-generation reproduction studies of male albino rats, female albino rats and previously exposed male Sprague-Dawley rats to Carbonyl Sulfide (COS) by inhalation. In: Initial submission: Letter from E.I. Dupont De Nemours & Co to USEPA regarding toxicity studies with Carbonyl Sulfide with cover letter dated 09/01/92. Submitted to the U.S. Environmental Protection Agency under TSCA Section 8ECP, 22-290. EPA88-920008223. OTS0555041. Morgan DL, Little PB, Herr DW, Moser VC, Collins B, Herbert R, Johnson GA, Maronpot RR, Harry GJ, and Sills RC (2004) Neurotoxicity of Carbonyl Sulfide in F344 rats following inhalation exposure for up to 12 weeks. Toxicology and Applied Pharmacology 200: 131–145. Morrison JP, Ton TV, Collins JB, Switzer RC, Little PB, Morgan DL, and Sills RC (2009) Gene expression studies reveal that DNA damage, vascular perturbation, and inflammation contribute to the pathogenesis of Carbonyl Sulfide neurotoxicity. Toxicologic Pathology 37: 502–511. National Research Council (1986) Environmental Tobacco Smoke: Measuring Exposures and Assessing Health Effects. Washington, DC: National Academies Press. National Toxicology Program-NTP (2021) Testing status of Carbonyl Sulfide M950074. https://ntp.niehs.nih.gov/whatwestudy/testpgm/status/ts-m950074.html?utm_source ¼direct&utm_medium¼prod&utm_campaign¼ntpgolinks&utm_term¼ts-m950074. Pellizzari ED, Hartwell TD, Harris BS, Waddell RD, Whitaker DA, and Erickson MD (1982) Purgeable organic compounds in mother’s milk. Bulletin of Environmental Contamination and Toxicology 28: 322–328. Pietri R, Román-Morales E, and López-Garriga J (2011) Hydrogen Sulfide and hemeproteins: Knowledge and mysteries. Antioxidants & Redox Signaling 15: 393–404. Rodgman A and Perfetti TA (2013) The Chemical Components of Tobacco and Tobacco Smoke, 2nd edn. Boca Raton, FL: CRC Press, 2332. Sattler ML and Rosenberk RS (2006) Removal of Carbonyl Sulfide using activated carbon adsorption. Journal of the Air and Waste Management Association 56: 219–224. Sehnert SS, Jiang L, Burdick JF, Risby T, and H. 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Carboxylesterases Somayeh Mojtabavia and Mohsen Aminb, aDepartment of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; bDepartment of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of B. Yan, Carboxylesterases, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 695–698, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00109-3.

Background General structure of CESs Tissue distribution and substrate specificity of human CESs CES inhibitors CES inducers CES inactivators Detoxification reactions and environmental monitoring Interactions among carboxylesterases, organophosphorus, and carbamate insecticides Conclusion References

571 572 572 572 572 573 573 576 576 578

Abstract Carboxylesterases (CESs) belong to the a/b-hydrolase superfamily, responsible for the carboxylester hydrolysis into carboxylic acid and alcohol. CESs are crucial in the biomedical field. They contribute to the delivery of various drugs and conversion of pro-carcinogens into carcinogens. CESs are isolated from diverse sources, such as fungi, bacteria, algae, plants, humans, and animals. Carboxylesterases detoxify organophosphates through a scavenging mechanism, while pyrethroids and carbamates are detoxified through hydrolysis. Carboxylesterases are expressed in different tissues such as gut epithelia, lungs, kidney and liver with various biological activities.

Keywords Carbamates; Carboxylesterase; Detoxification; Hydrolysis; Malathion; Organophosphates; Pyrethrin; Pyrethroids; Pyrethrum; Serine; Xenobiotics

Key points

• • • •

The expression of carboxylesterases is ubiquitous with high levels in various tissues. CESs detoxify and metabolize a wide range of ester- and amide-containing compounds. CESs can be induced and inhibited by both exogenous and endogenous compounds. The enzyme is irreversibly inhibited by organophosphates.

Background Carboxylesterases (CESs, EC 3.1.1.1) are ubiquitous enzymes found in almost all living organisms. These enzymes belong to the a/b-hydrolase superfamily and contribute to the hydrolysis of various amides, carbamates, carbonates, endogenous/xenobiotic esters, and thioesters. Based on amino acid sequence homology, six human CES isoforms (CES1 to CES6) have been identified (Holmes et al., 2010; Satoh and Hosakawa, 2006; Yang et al., 2009). Among these groups, CES1 and CES2 as main isoforms share 48% sequence homology. The CES1 family is split into eight subfamilies from CES1A to CES1H. The main human CES forms, and major isoforms in rats, dogs, and mice, are all members of the CES1A subfamily. A variety of methods have been used to classify carboxylesterases as follows: (1) classification based on substrates (2) cellular localization, (3) mechanistic details as well as active site residues, and (4) molecular structures. These esterases have been classified into families I to XV depending on consensus sequences, GDSL motif, nature of the catalytic site triad, nucleophilic Ser, and substrate specificity (or chain length). Substrate specificity is low in mammalian carboxylesterases. As a result, it appears to be nearly impossible to classify carboxylesterase isozymes solely based on their substrate specificity because individual hydrolases exhibit properties of carboxylesterase or lipase activities. CESs have been isolated from algae, bacteria, animals, plants, humans, fungi, and other sources (Sood et al., 2016). Acid-, cold-, salt-, and thermo-resistant microorganisms have also been reported to produce CESs. Natural esters such as acetylcholine,

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cholesterol esters, phospholipids, triglycerides, and plant cell wall components such as tannin, cutin, and feruloyl esters are hydrolyzed by carboxylesterases. CESs are used in different industries in food processing, biocatalysis, degradation of synthetic materials and biotransformation. Xenobiotics, certain drugs, and endogenous compounds with amide, thioester, and ester bonds are all hydrolyzed by hepatic microsomal carboxylesterases. Therefore, classifying carboxylesterases based on substrate specificity is a difficult task.

General structure of CESs The enzyme’s structure comprises eight stranded b-sheets in the center, surrounded by helices and connecting loops. Ser (S), His (H), and Asp (D) make up the catalytic structure of these enzymes (D). Ser (S) is usually fixed in a pentapeptide motif (GXSXG), in which X can be any residue. This motif creates a nucleophile elbow, a sharp turn in the three-dimensional (3D) structure. This nucleophilic elbow is found at the apex of the sharp turn between alpha-helix and beta-sheet. CESs’ broad substrate specificity is aided by an open, active site as well as a large binding pocket for substrate’s acid moiety. The backbone carbonyl of the residue was three residues above His (H), and it became a critical factor in stabilizing the His (H)-positive sidechain rather than the Asp (D) negative moiety in the catalytic triad (serine, glutamine, and histidine).

Tissue distribution and substrate specificity of human CESs Most metabolic organs, such as the liver, and kidney, have high levels of CES1 and CES2, which suggests that these enzymes protect against xenobiotics (Xu et al., 2016). The liver and adipocytes have high levels of CES1, while the kidney, heart, intestine, lung, monocytes, testis, and macrophages have lower levels. The abundance of CES in human liver cytosol (HLC) and human liver microsomes (HLMs) has been quantified. Furthermore, the secreted forms of CES with high activity levels were discovered in rodent blood, whereas CES1 or CES2 activities were barely detectable in human blood (Singh et al., 2021; Wang et al., 2018). In particular, CES expression profiles in cancer cell lines/tissues were significantly different from those in healthy cells. CES2 is also overexpressed in esophageal squamous carcinoma, kidney adenocarcinoma, thyroid papillary carcinoma, multiple myeloma, and other cancer cell lines. The bioactivation of several cancer prodrugs is significantly correlated with CES2 expression in cancer tissues/cell lines, which is consistent with the anticancer effects of these drugs. These findings suggest that rationally designing prodrugs bioactivated by CES2 will be extremely beneficial in cancer treatment (Xi et al., 2015; Pérez-Aguilar et al., 2015). In comparison to CES1 and CES2, CES3 has received less research. A hydrophobic signal at the N-terminal and a QEDL sequence at the C-terminal of CES3 may help it find its way to the endoplasmic reticulum. It has been discovered in humans’ colon, intestine, liver, placenta, and trachea. CES4A (also known as CES6 & CES8) and CES5A (also known as cauxin & CES7) have received little attention. Because neither CES4A nor CES5A has an endoplasmic reticulum (ER) retrieval signal suggesting they may be secreted from cells. The brain, lungs, and skin (melanocytes) are known to express CES4A, while the brain, lungs, testis, and kidneys are known to express CES5A (Table 1).

CES inhibitors Compared to induction, several therapeutic drugs inhibit CESs. Among these drugs are antihyperlipidemic, anti-psychiatric, antidiabetic, antihypertensive, antiviral, and anticancer drugs. They are also inhibited by pharmaceutical excipients, endogenous compounds like IL-6, oxysterols, as well as fatty acids and chemical materials. Since CESs significantly metabolize the drug, important clinical implications are provided by changing the enzyme activity. Drug efficacy can be decreased, and the incidence of drug-drug interactions (DDIs) may increase when two or more drugs compete for hydrolysis by the same CES enzyme (Hatfield and Potter, 2011). The list of some inhibitors and types of inhibition are shown in Table 2.

CES inducers According to studies, activation of CAR, PPARs, AhR, PXR, hepatocyte nuclear factor-4a (HNF-4a), LXR, or Nrf2 transcriptional pathways could regulate the expression of mammalian CESs. Typically, through nuclear factor erythroid 2-related factor 2 (Nrf2), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator-activated receptors (PPARs), microsomal enzyme inducers (MEIs) affect the target genes. It should be noted that such assessments are concentrated on regulating the rodent Ces genes transcriptionally. Some studies indicated that it is possible to activate Nrf2 by MEIs and induce CES1 in human tumor cells. Furthermore, moderate induction of CES1 and CES2 gene expression in human hepatocytes may be caused by a PXR-activating agent, rifampicin. Future studies are required to explore the association of these signaling pathways with CES expression in rodents and humans. The regulatory effects of some exogenous and endogenous substances were proven on the expression of mammalian CES. Hepatic CE1 expression was induced by treating mice with glucose in vivo because glucose significantly activated the promoter activity of CES1 and incremented the acetylation of histone 3 and 4 in the CES1 chromatin.

Carboxylesterases Table 1

573

Summary of human CES properties.

Name

Isoforms

Human body localization

Subcellular localization

Substrate specificity

Known drug substrates

CES1

4

Liver > lungs  heart, spleen, stomach, testis

Endoplasmic reticulum

Large acyl moiety; small alcohol/ amine/thiol group

CES2

6

Colon, intestine > liver, kidneys, heart, brain, testis

Endoplasmic reticulum

CES3

Up to 8

Liver, colon, intestine, trachea, placenta

CES4A

10

Brain, lungs, skina

Unknown

Unknown

CES5A

4

Brain, kidneys, lungs, testis

Potentially endoplasmic reticulum or Golgi bodies Unknown, potentially secreted Unknown, potentially secreted

Small acyl moiety; large alcohol/ amine/thiol group Unknown

Benazepril, candesartan cilexetil, capecitabine, ciclesonide, cilazapril, clopidogrel, cocaine, dabigatran, delapril, enalapril, flumazenil, fosinopril, heroin, imidapril, mepridine, methylphenidate, moexipril, mycophenolate mofetil, perindopril, oseltamivir, oxybutynin, quinapril, ramipril, rufinamide, sacubitril, sofosbuvir, telotristat etiprate, tenofovir alafenamide, trandolapril Aspirin, capecitabine, cocaine, dabigatran, gemcitabine, heroin, irinotecan (CPT-11), methylprednisolone 21-hemisuccinate, mycophenolate mofetil, tenofovir disoproxil Poor for irinotecan (CPT-11)

Unknown

Unknown

a

Reported to be in the melanocytes of skin.

The expression of hepatic CES1 is also induced by FXR agonist or cholic acid. Then, hepatic TG, plasma cholesterol, and plasma TG levels are reduced. Antioxidants and sensitizers in several cell lines could highly induce CES1. CES1 is induced by antioxidant sulforaphane and sensitizer trinitrobenzene sulfonate (TNBS) through a new element, nuclear factor-E2 related factor-2, in primary hepatocytes and cell lines (human fibrosarcoma cell line HT1080 and Huh7). It was also reported that CES1 and CES2 could be slightly induced by NO1886 (Ibrolipim), a lipoprotein lipase promotion agent in primary cultures of cryopreserved human hepatocytes. Moreover, it was proved that urethane dimethacrylate (UDMA) induced the mRNA expression of CES2 in human dental pulp cells, while the expression of CES1 or CES3 mRNA was not regulated. Furthermore, the protein expression of CES1 and CES2 was reduced by gambogic acid through a dose-dependent mode. Adding gambogic acid significantly reduced the hydrolytic activities of CES1 and CES2 (Wang et al., 2018).

CES inactivators Carbamate compounds were progressed as pharmaceutical agents specifically targeting serine hydrolase supergroup members through covalent binding and modifying serine at the active site. These compounds, as potent inhibitors of acetylcholinesterase (ACE), are extensively utilized for pest control in agriculture and domestic animals. However, several cholinesterase inhibitors that contain the carbamate moiety, such as JZL184 and phenethylcymserine, were found to be CES inhibitors. However, poor isoform selectivity towards different CESs was displayed by all these compounds (Wang et al., 2018). Organophosphate insecticides are ACE inhibitors with toxicity exerted through terminating the nerve impulses via the neurotransmitter acetylcholine metabolism. Some serine hydrolases such as cholinesterases and carboxylesterases (CES1 and CES2) could be inhibited significantly after exposure to organophosphates (Badr, 2020; Bhatt et al., 2020; Wheelock et al., 2005). Organophosphates reacting with CES generate a stable phosphate ester-linked covalently to the catalytic residue (like Ser-221 of CES1) of CES. Several organophosphates are potent irreversible inhibitors of CES with nanomolar level IC50 values such as paraoxon, chlorpyrifos oxon, and bis(4-nitrophenyl)phosphate (BNPP) (Eleršek and Filipic, 2011).

Detoxification reactions and environmental monitoring CESs are members of the a, b-hydrolase group in charge of the detoxification and metabolism of various compounds containing ester and amides. These enzymes contribute to the biotransformation of several drugs and prodrugs, clinically such as the antiplatelet drugs clopidogrel and aspirin, the ACE inhibitors delapril, temocapril, and imidapril, and the antitumor drug

574

Carboxylesterases

Table 2

Inhibitors and inhibition types for CESs.

Inhibitors

CES isoforms

Sources

Inhibition type

Mevastatin Simvastatin

CES1 Recombinant CES1A1 CES1 HLM Recombinant CES2 CES2 HLM CES2 HJM Recombinant CES1A1 CES1 HLM Recombinant CES2 CES2 HLM CES2 HJM CES1 RLM CES1 RJM CES2 HLM CES2 RJM CES1 HLM CES1 RLM CES1 RJM Recombinant CES1A1 CES1 HLM Recombinant CES1A1 CES1 HLM Recombinant CES2 CES2 HLM CES2 HJM Recombinant CES2 CES2 HLM CES2 HJM CES1 CES1 CES CES2 CES1 HLM CES2 HLM CES2 HJM CES2 RJM Recombinant CES1A1 CES1 HLM Recombinant CES1A1 CES1 HLM Recombinant CES2 CES2 HLM CES2 HJM Recombinant CES2 CES2 HLM CES2 HJM Recombinant CES2 Recombinant CES1 Recombinant CES1

Human Human Human Human Human Human Human Human Human Human Human Rat Rat Human Rat Human Rat Rat Human Human Human Human Human Human Human Human Human Human Human Human Rabbit Human Human Human Human Rat Human Human Human Human Human Human Human Human Human Human Human Human Human

Non-competitive Mixed Competitive Non-competitive Non-competitive Non-competitive Mixed Non-competitive Competitive type Non-competitive Competitive Competitive Competitive Competitive Competitive Mixed Uncompetitive Mixed Competitive Non-competitive Mixed Competitive Non-competitive Non-competitive Mixed Competitive Non-competitive Non-competitive Competitive type Non-competitive Non-competitive Non-competitive Competitive Competitive Non-competitive Mixed Competitive Competitive Competitive Competitive Competitive Mixed Non-competitive Mixed Competitive Non-competitive type Competitive type Non-competitive Non-competitive

Troglitazone Fenofibrate Carvedilol Manidipine Nordihydroguaiaretic acid Telmisartan Nitrendipine Diltiazem Verapamil Nelfinavir Tamoxifen Vinblastine Procainamide Physostigmine Sodium lauryl sulfate Polyoxyl 40 hydrogenated castor oil Tween 20 Polyoxyl 35 castor oil Loperamide 27-Hydroxycholesterol Arachidonic acid

irinotecan. The CESs also have critical roles in detoxifying environmental toxicants such as pyrethroids, organophosphates, and carbamates, a major class of insecticides used worldwide and extensively in the USA. Ecological toxicants have several increasing applications worldwide; therefore, the exposure of pyrethroids is a main risk factor in the environment for chronic toxicity to humans and animals. Several lipophilic xenobiotics are removed based on their conversion to water-soluble compounds. Carbamates and pyrethroids are also very apolar compounds. They are accumulated in fatty tissues. The hydrolysis of the carboxylic acid and alcohol is the most operative way to increment the carboxyl ester’s water solubility. Then, producing metabolites have further higher water-solubility, which may be removed in the urine. The chemical structures of pyrethroids and carbamates are related to carboxylic acids. Hence, these compounds can be potentially hydrolyzed by CESs. The mammals’ liver and serum are tissues that have the highest concentration of CESs.

Carboxylesterases

reversible complex

O R1

R1 P

+ OH

O

R1

R2

(+ XH)

X

P O

R2

O

Ser

Ser

Ser AcHE

spontaneous reactivation with hydrolysis

2O

X

dealkylated

+H

R2

phosphorylated AcHE

O P

575

O

–

O

O P R2

OH

(+ ROH)

P

OH

R1

R2

O

Ser Ser

irreversible phosphorylated AcHE Fig. 1 Inhibition of carboxylesterases (CES) and acetylcholinesterase (ACE) by organophosphates. Adapted from Hreljac I, Filipic M (2009) Organophosphorous pesticides enhance the genotoxicity of benzo(a)pyrene by modulating its metabolism. Mutation Research 671: 84–92.

The organophosphorus insecticides of major commercial and toxicological interest are esters or thiols derived from phosphoric, phosphonic, phosphinic, or phosphoramidic acid. The main toxicological effects of organophosphorus are exerted via non-reversible phosphorylation of esterases within the central nervous systems. The inhibition of ACE causes acute toxic effects. An inhibition-based scavenging mechanism yields the detoxification of most organophosphates by CESs. Organophosphates induce toxicity by targeting serine enzymes, particularly ACE, a vital enzyme that terminates neurotransmission of acetylcholine. Structurally, CESs are related to ACE. As ACE, CESs use a catalytic triad for hydrolysis and a serine residue acts as the nucleophile in the triad (Fig. 1). A leaving group “X” is contained in organophosphates (Hreljac and Filipic, 2009). Organophosphorous pesticides enhance the genotoxicity of benzo(a)pyrene by modulating its metabolism. Mut Res 671: 84–92. The enzyme displaces this leaving group via the active-site serine residue by interaction with ACE or CES. Thus, a phosphorylated enzyme complex is formed. The spontaneous reactivation (very slower) or a so-called aging process (permanent inhibition) is undergone by phosphorylated enzymes. Regarding the contribution of CESs in detoxifying the organophosphates, there is an inverse correlation between CES activity and the sensitivity to organophosphorus poisons. Moreover, considerable protection is provided by intravenous administration of purified CESs against the organophosphorus-type compounds’ toxicity like soman, paraoxon, and sarin. Moreover, repeated lower dosing trends of organophosphates trigger tolerance along with the incremented recovery of CES activity. ACE is the main target when inducing acute toxicity. However, toxicity is induced by organophosphates by targeting other proteins like the neuropathy target esterase (NTE). Organophosphorus compounds are detoxified by CESs via hydrolysis along with the scavenging mechanism. For instance, malathion is organophosphorothioate, which comprises a P-S rather than a P-O bond found in organophosphates (Fig. 2) (Buratti and Testai, 2006). Oxidative desulfuration of malathion results in the formation of the corresponding oxon, an organophosphate. Two carboxylic acid ester bonds are contained in the malathion that could undergo hydrolysis by CESs as well. The hydrolysis of malathion prevents oxidative desulfuration, thus representing detoxification (inactivation). Therefore, the relative activity between hydrolysis and desulfuration is the main toxicological determinant of malathion. Neurotoxicity, skin contact toxicity, respiratory toxicity, and reproductive system toxicity are among the reported toxic effects of pyrethroids. The pyrethrins of allethrin and pyrethrum are metabolized by oxidation of the side chain of the acid moiety, hydrolysis of the ester bond playing a less critical role. However, for most other pyrethroids, hydrolysis of carboxylesters is more important for detoxification and degradation than oxidation. Remarkably, CES1 and CES2 had key roles in detoxifying pyrethroids. By CESs, the ester insecticides could be hydrolyzed to low toxic or non-toxic metabolites. In these enzymes, active site amino acids have conserved active participation in catalytic reactions during pyrethroid degradation. A catalytic triad is contained in the catalytic sites of these enzymes. The acylation reactions in the presence of nucleophile are the main biology of pyrethroid hydrolase. The acylation reaction of a serine residue in the enzyme’s active sites yields carboxylesterase catalysis, thus releasing the alcohol from carboxyl ester and producing an acylated intermediate (Fig. 3) (Song et al., 2021). The water molecule acts as a nucleophile and hydrolyzes acylated intermediate via nucleophilic attack mechanism. Each pyrethroid has a different degradation rate with CES due to contrasting substrate affinity. The liver and serum have the highest inclusion in the hydrolysis of pyrethroids. CESs degrades the trans isomers of the pyrethroids faster than the cis isomers. The

576

Carboxylesterases

O

CH3O

CHCOOC2H5

S

CH3O

Cyt P450

CH3O

oxo

P

P CH3O

P

O

CH3O

S

CH3O

CHCOOC2H5

S

Oxonase

DMTP +

CH2COOC2H5

CH2COOC2H5 Malathion

SH

OH

Malaoxon

C2H5COOCH2CHCOOC2H5 Alcohol Carboxylesterases

S (O)

CH3O

+

P CH3O

S

CHCOOH

S (O)

CH3O P

MDA

Ethyl Alcohol

CH2COOC2H5

MMA

CH3O

C2H5OH

+ S

CHCOOH CH2COOH

C2H5OH Ethyl Alcohol

Fig. 2 Major pathways of malathion (MAL) metabolism. (DMPT: dimethyltiophosphate; MMA: malathion (malaoxon)-monocarboxylic acid; MDA: malathion (malaoxon)-dicaroxylic acid). Adapted from that of Buratti FM and Testai E (2006) Malathion detoxification by human hepatic carboxylesterases and its inhibition by isomalathion and other pesticides. Journal of Biochemical and Molecular Toxicology 19: 406–414. doi:10.1002/jbt.20106.

slower degradation of cis isomers may have a role in the high toxicity in mammals as a result of a higher affinity for the Na+-channel compared to the trans isomer. The compounds derived from carbamic acid are probably the insecticides with the broadest range of biocide activities. As stated above, carbamates are vulnerable to CESs-caused hydrolysis. The hydrolysis products are carbamic acid and alcohol instantaneously decomposing to methylamine and carbon dioxide. Furthermore, metabolites caused by oxidation of parent carbamates preserve the carbamic ester bond intact and are also vulnerable to hydrolysis by CESs. It occurs for aldicarb and carbofuran. Compared to the parent compounds, the hydrolysis of oxidized metabolites of these two carbamates is much more pronounced.

Interactions among carboxylesterases, organophosphorus, and carbamate insecticides The toxic acute effects of carbamates are very similar to the acute effects derived from the poisoning of organophosphorus. Organophosphorus can phosphorylate ACE serine residues in an irreversible manner, while the carbamylation of the same serine residue has less stability, with the normal decarbamylation duration as between 30 and 40 min. The mechanism of inhibition of CESs by carbamates is similar to the mechanism of hydrolysis of their natural substrates, which can be observed in Fig. 4A. Firstly, a serine residue is carbamylated by the carbamate, and the release of alcohol moiety. Following carbamylation, a water molecule can reactivate the enzyme, which causes releasing the free enzyme and the carbamic acid. The inhibitory mechanism of CESs by organophosphorus shows similarity to the mechanism of the first phase in the enzyme phosphorylation (Fig. 4B). Nevertheless, in this case, water cannot reactivate the phosphorylated enzyme and regeneration of the free active enzyme is impossible.

Conclusion CESs, as ubiquitous enzymes, have the ability to hydrolyze diverse molecules, including both synthetic and naturally occurring compounds. It can lead to inactivation or activation of the agent. Due to identification of endogenous substrates, it is thought that the enzyme is involved in detoxification processes. Hydrolysis is crucial to detoxification of carbamates, pyrethroids, and

Carboxylesterases

577

Fig. 3 Detailed catalytic mechanism of pyrethroid hydrolase for the degradation of cypermethrin. Histidine, glutamine, and serine (triad at the active site of pyrethroid hydrolase) play a key role in catalysis. The Hydroxyl group of serine makes nucleophilic reactions during enzyme catalysis. Adapted from Song Y-Q, Jin Q, Wang D-D, Hou J, Zou L-W, Ge G-B, Carboxylesterase inhibitors from clinically available medicines and their impact on drug metabolism. Chemico-Biological Interactions 345: 109566.

(A)

XOH

O XOCNHR + HO–Ser–CbE

(B)

H 2O O

RNHC–O–Ser–CbE

H 2O

XOH O

R1O P OH + HO Ser CbE OR2

CO2 + H2NR

O R1O P O Ser CbE OR2

Fig. 4 Inhibition of carboxylesterases (CES) by carbamates (A) and organophosphate (B) insecticides. First step of the inhibition is the carbamylation (A) or phosphorylation (B) of a serine residue of the carboxylesterase, in both cases with releasing of an alcohol (XOH). After carbamylation, a nucleophilic molecule (usually water) attacks the carbamoyl moiety, releasing the free active enzyme plus carbamic acid. The latter instantaneously decomposes to CO2 plus an amine. Water is not able to release the phosphate moiety from the serine of the enzyme, and therefore the enzyme is irreversibly inhibited.

578

Carboxylesterases

organophosphorus. CESs may have some practical uses in elimination of organophosphate toxicity since the ester chemotype is present in numerous drugs with deleterious adverse effects. Further in vivo studies on CES mechanisms of detoxification are conceivable, as CESs can reduce the amounts of promoters that reach the targets in the central nervous system.

References Badr AM (2020) Organophosphate toxicity: updates of malathion potential toxic effects in mammals and potential treatments. Environmental Science and Pollution Research 27(21): 26036–26057. Bhatt P, Bhatt K, Huang Y, Lin Z, and Chen S (2020) Esterase is a powerful tool for the biodegradation of pyrethroid insecticides. Chemosphere 244: 125507. Buratti FM and Testai E (2006) Malathion detoxification by human hepatic carboxylesterases and its inhibition by isomalathion and other pesticides. Journal of Biochemical and Molecular Toxicology 19: 406–414. https://doi.org/10.1002/jbt.20106. Eleršek T and Filipic M (2011) Organophosphorus pesticides-mechanisms of their toxicity. Pesticides—The Impacts of Pesticide Exposure 243–260. Hatfield MJ and Potter PM (2011) Carboxylesterase inhibitors. Expert Opinion on Therapeutic Patents 21(8): 1159–1171. https://doi.org/10.1517/13543776.2011.586339. Holmes R, Wright M, Laulederkind S, et al. (2010) Recommended nomenclature for five mammalian carboxylesterase gene families: Human, mouse and rat genes and proteins. Mammalian Genome 21: 427–441. Hreljac I and Filipic M (2009) Organophosphorous pesticides enhance the genotoxicity of benzo(a)pyrene by modulating its metabolism. Mutation Research 671: 84–92. Pérez-Aguilar B, Vidal CJ, Palomec G, García-Dolores F, Gutiérrez-Ruiz MC, Bucio L, Gómez-Olivares JL, and Gómez-Quiroz LE (2015) Acetylcholinesterase is associated with a decrease in cell proliferation of hepatocellular carcinoma cells. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1852(7): 1380–1387. Satoh T and Hosakawa M (2006) The mammalian carboxylesterases: From molecules to functions. Annual Review of Pharmacology and Toxicology 38: 257–288. Singh A, Gao M, and Beck MW (2021) Human carboxylesterases and fluorescent probes to image their activity in live cells. RSC Medicinal Chemistry 12(7): 1142–1153. Song Y-Q, Jin Q, Wang D-D, Hou J, Zou L-W, and Ge G-B (2021) Carboxylesterase inhibitors from clinically available medicines and their impact on drug metabolism. Chemico-Biological Interactions 345: 109566. Sood S, Sharma A, Sharma N, and Kanwar SS (2016) Carboxylesterases: sources, characterization, and broader applications. Insight Enzyme Research 1: 1–11. Wang D, Zou L, Jin Q, Hou J, Ge G, and Yang L (2018) Human carboxylesterases: A comprehensive review. Acta Pharmaceutica Sinica B 8(5): 699–712. https://doi.org/10.1016/j. apsb.2018.05.005 Epub 2018 Jun 25 PMID: 30245959. PMC6146386. Wheelock CE, Shan G, and Ottea J (2005) Overview of carboxylesterases and their role in the metabolism of insecticides. Journal of Pesticide Science 30(2): 75–83. Xi HJ, Wu RP, Liu JJ, Zhang LJ, and Li ZS (2015) Role of acetylcholinesterase in lung cancer. Thoracic Cancer 6(4): 390–398. https://doi.org/10.1111/1759-7714.12249 Epub 2015 Mar 20 PMID: 26273392. PMC4511315. Xu Y, Zhang C, He W, and Liu D (2016) Regulations of xenobiotics and endobiotics on carboxylesterases: A comprehensive review. European Journal of Drug Metabolism and Pharmacokinetics 41(4): 321–330. Yang D, Pearce R, Wang X, Gaedigk R, Wan YJ, and Yan B (2009) Human carboxylesterases HCE1 and HCE2: ontogenic expression, inter-individual variability and differential hydrolysis of oseltamivir, aspirin, deltamethrin and permethrin. Biochemical Pharmacology 77: 238–247.

Carcinogen classification schemes Helmut Greim, Technical University of Munich, Munich, Germany © 2024 Elsevier Inc. All rights reserved. This is an update of M.A. Kamrin, Carcinogen Classification Schemes, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 699-704, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00370-5.

Introduction UN GHS carcinogen classifications as adopted by the European community US EPA carcinogen classifications Classification based on the 1986 guidelines for carcinogen risk assessment Classification based on the 2005 guidelines for carcinogen risk assessment Carcinogenic to humans Likely to be carcinogenic to humans Suggestive evidence of carcinogenic potential Inadequate information to assess carcinogenic potential Not likely to be carcinogenic to humans Multiple descriptors IARC carcinogen classifications NTP carcinogen classifications Known to be human carcinogen Reasonably anticipated to be human carcinogen OSHA carcinogen classifications Category I potential carcinogens Category II potential carcinogens NIOSH carcinogen classifications ACGIH carcinogen classifications German MAK carcinogen classifications European commission SCOEL carcinogen classifications Outlook References Further reading

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Abstract To rank the relative hazards of chemicals considered to be potential carcinogens, governmental agencies and other bodies have developed a variety of approaches. All of these approaches are based on essentially the same types of carcinogenicity data but apply different scientific judgments to the data resulting in significant variation among the ranking systems. This article provides a summary of the most commonly used systems and the rationales used to place chemicals into the carcinogen categories of each system. Approaches included are the United Nations Globally Harmonized System of Classification and Labeling of chemicals, the classification systems of the EC Scientific Committee on Occupational Exposure Limits, the German MAK Commission, the WHO International Agency for Research on Cancer, the United States Environmental Protection Agency, the US Occupational Safety and Health Administration, the US National Toxicology Program, the US National Institute of Occupational Safety and Health, and the US American Conference of Government Industrial Hygienists.

Keywords Bioassay; Carcinogenesis; Dose–response; Epidemiology; Hazard identification, Risk assessment, Toxicity Testing, The Globally Harmonized System (GHS)

Introduction Several classification schemes have been developed for ranking the relative hazards to humans associated with chemicals that, by one or more criteria, may be considered to be potential carcinogens. The classification schemes are based on scientific judgments that typically take into account all the data available from in vivo animal bioassays, in vitro tests for genetic toxicity, human epidemiology, and structural relationships with other known carcinogens. Classification of a chemical as a carcinogen involves the consideration of many different factors. Classification schemes provide guidance on evaluating and weighting the available

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evidence and placing chemicals into defined categories that can be used to communicate the implications for risk. Factors usually taken into consideration in interpreting the results of an animal bioassay include the following:

• • • • • • • •

Adequacy of experimental design and conduct. Statistical significance of any increase in tumor incidence. Presence or absence of a dose–response relationship and correct dose selection. Nature of tumors (benign or malignant) and relevance of tumor type to humans. Historical control data (incidence and variability) for tumor type. Common (spontaneous) versus uncommon tumors. Number of organs/tissues with tumors. Mechanistic information.

At present, the most commonly used classification schemes are those developed by United States Environmental Protection Agency (US EPA), the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) and the Globally Harmonized System (GHS) developed by the United Nations (UN). The US EPA classification schemes are used for the regulation of chemicals under those laws it administers; for example, Federal Insecticide, Fungicide and Rodenticide Act, and Toxic Substances Control Act as well as by many state regulatory agencies. The IARC Carcinogen Classification is taken into account worldwide and is considered in certain US regulations and laws (e.g., Occupational Safety and Health Administration (OSHA) Hazard Communication Standard). The GHS system is applied in the European Commission and increasingly adopted by other international authorities. Other respected carcinogenic classification schemes include those developed by the European Commission Scientific Committee on Occupational Exposure Limits (EC SCOEL), the German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission), and a number of US organizations including National Institute of Occupational Safety and Health (NIOSH), OSHA, National Toxicology Program (NTP), and American Conference of Government Industrial Hygienists (ACGIH). Each of these schemes is described in the following sections. It should be emphasized that these classification systems are constantly evolving and that changes may occur over time.

UN GHS carcinogen classifications as adopted by the European community1 While there has been an attempt, under UN auspices, to develop a common global classification scheme, it relies on voluntary compliance and agencies in the United States and abroad have been slow to adopt this scheme, whereas the system became mandatory in the European Community. The classification for carcinogens under this scheme, the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) as adopted by the European Community, will be described so it can be compared to other schemes that are in place. Category 1: Known or presumed human carcinogens. A substance is classified in Category 1 on the basis of epidemiological and/or animal data. A substance may be further distinguished as: Category 1A: Known to have carcinogenic potential for humans; classification is largely based on human evidence, or. Category 1B: Presumed to have carcinogenic potential for humans; classification is largely based on animal evidence. The classification in Category 1A and 1B is based on strength of evidence together with additional considerations. Such evidence may be derived from: - Human studies that establish a causal relationship between human exposure to a substance and the development of cancer (known human carcinogen) or - animal experiments for which there is sufficient evidence to demonstrate animal carcinogenicity (presumed human carcinogen). In addition, on a case-by-case basis, scientific judgment may warrant a decision of presumed human carcinogenicity derived from studies showing limited evidence of carcinogenicity in humans together with limited evidence of carcinogenicity in experimental animals. Category 2: Suspected human carcinogen—The placing of a substance in Category 2 is done on the basis of evidence obtained from human and/or animal studies, but which is not sufficiently convincing to place the substance in Category 1A or 1B. based on the strength of evidence together with additional considerations. Such evidence may be from either from limited evidence of carcinogenicity in human studies or from limited evidence of carcinogenicity in animal studies.

US EPA carcinogen classifications The US EPA originally promulgated its carcinogen classification approach in 1986 in its Guidelines for Carcinogens Risk Assessment. However, in 2005, the EPA published a new set of Guidelines for Carcinogen Risk Assessment designed to replace the 1986 1 For further details see ECHA Guidance on the Application of the CLP Criteria Guidance to Regulation (EC) No 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures

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version. This established two different classification systems that both are now in effect. The new guidelines apply only to those compounds that have been newly evaluated or reevaluated since 2005. As a result, carcinogen classifications for some compounds are based on the old system and the new approach is applied to others. Thus, both classification schemes will be described in detail.

Classification based on the 1986 guidelines for carcinogen risk assessment The US EPA, 1986 ‘total-weight-of-evidence’ scheme classifies potential carcinogens into five groups, A–E that indicate the likelihood that they are human carcinogens. These groups are described below. Group A: Human carcinogen—This is reserved for chemicals where there exists clear epidemiological evidence indicating an association between exposure to the chemical and cancer. Group B: Probable human carcinogen—This group is divided into two subgroups, B1 and B2. Group B1 indicates that there is ‘sufficient’ evidence to indicate that the material is an animal carcinogen and that there is ‘limited’ evidence of effects in humans. Group B2 indicates that although there is sufficient evidence in animals, the total weight of evidence for effects in humans is weaker or inadequate. Group C: Possible human carcinogen—classification in this group indicates limited, often marginal evidence of carcinogenicity in animals and no evidence of any effects in humans. Group D: Not classifiable as to human carcinogenicity—This group is used for chemicals for which no data are available. Group E: Evidence of non-carcinogenicity for humans—this group is used for chemicals that show no evidence of any carcinogenicity in at least two adequately conducted animal tests with different species.

Classification based on the 2005 guidelines for carcinogen risk assessment The descriptors of the 2005 Guidelines substitute for the designations of the 1986 Guidelines. They also reflect a change in approach since the descriptors do not stand-alone but instead are incorporated into a narrative that places the descriptors within a discussion of factors that have affected the choice of descriptor. The descriptors are as follows:

Carcinogenic to humans This descriptor indicates strong evidence of human carcinogenicity. It covers different combinations of evidence.

• •

This descriptor is appropriate when there is convincing epidemiologic evidence of a causal association between human exposure and cancer. Exceptionally, this descriptor may be equally appropriate with a lesser weight of epidemiologic evidence that is strengthened by other lines of evidence. It can be used when all of the following conditions are met: (1) there is strong evidence of an association between human exposures and either the cancer or the key precursor events of the agent’s mode of action but not enough for a causal association, (2) there is extensive evidence of carcinogenicity in animals, (3) the mode(s) of carcinogenic action and associated key precursor events have been identified in animals, and (4) there is strong evidence that the key precursor events that precede the cancer response in animals are anticipated to occur in humans and progress to tumors, based on available biological information. In this case, the narrative includes a summary of both the experimental and the epidemiologic information on mode of action and also an indication of the relative weight that each source of information carries, for example, based on human information, based on limited human and extensive animal experiments.

Likely to be carcinogenic to humans This descriptor is appropriate when the weight of the evidence is adequate to demonstrate carcinogenic potential to humans but does not reach the weight of evidence for the descriptor ‘Carcinogenic to Humans.’ Adequate evidence consistent with this descriptor covers a broad spectrum. As stated previously, the use of the term ‘likely’ as a weight of evidence descriptor does not correspond to a quantifiable probability. The examples below are meant to represent the broad range of data combinations that are covered by this descriptor; they are illustrative and provide neither a checklist nor a limitation for the data that might support the use of this descriptor. Moreover, additional information, for example, on mode of action, might change the choice of the descriptor. Supporting data for this descriptor may include the following:

• • • • •

An agent demonstrating a plausible (but not definitively causal) association between human exposure and cancer, in most cases with some supporting biological, experimental evidence, though not necessarily carcinogenicity data from animal experiments; An agent that has tested positive in animal experiments in more than one species, sex, strain, site, or exposure route, with or without evidence of carcinogenicity in humans; A positive tumor study that raises additional biological concerns beyond that of a statistically significant result, for example, a high degree of malignancy, or an early age at onset; A rare animal tumor response in a single experiment that is assumed to be relevant to humans; or A positive tumor study that is strengthened by other lines of evidence, for example, either plausible (but not definitively causal) association between human exposure and cancer or evidence that the agent or an important metabolite causes events generally

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known to be associated with tumor formation (such as DNA reactivity or effects on cell growth control) likely to be related to the tumor response in this case.

Suggestive evidence of carcinogenic potential This descriptor of the database is appropriate when the weight of evidence is suggestive of carcinogenicity; a concern for potential carcinogenic effects in humans is raised, but the data are judged not sufficient for a stronger conclusion. This descriptor covers a spectrum of evidence associated with varying levels of concern for carcinogenicity, ranging from a positive cancer result in the only study on an agent to a single positive cancer result in an extensive database that includes negative studies in other species. Depending on the extent of the database, additional studies may or may not provide further insights. Some examples include:

• •

• •

A small, and possibly not statistically significant, increase in tumor incidence observed in a single animal or human study that does not reach the weight of evidence for the descriptor ‘Likely to Be Carcinogenic to Humans.’ The study generally would not be contradicted by other studies of equal quality in the same population group or experimental system; A small increase in a tumor with a high background rate in that sex and strain, when there is some but insufficient evidence that the observed tumors may be due to intrinsic factors that cause background tumors and not due to the agent being assessed. (When there is a high background rate of a specific tumor in animals of a particular sex and strain, then there may be biological factors operating independently of the agent being assessed that could be responsible for the development of the observed tumors.) In this case, the reasons for determining that the tumors are not due to the agent are explained; Evidence of a positive response in a study whose power, design, or conduct limits the ability to draw a confident conclusion (but does not make the study fatally flawed), but where the carcinogenic potential is strengthened by other lines of evidence (such as structure–activity relationships); or A statistically significant increase at one dose only, but no significant response at the other doses and no overall trend.

Inadequate information to assess carcinogenic potential This descriptor of the database is appropriate when available data are judged inadequate for applying one of the other descriptors. Additional studies generally would be expected to provide further insights. Some examples include:

• • •

Little or no pertinent information; Conflicting evidence, that is, some studies provide evidence of carcinogenicity but other studies of equal quality in the same sex and strain are negative. Differing results, that is, positive results in some studies and negative results in one or more different experimental systems, do not constitute conflicting evidence as the term is used here. Depending on the overall weight of evidence, differing results can be considered either suggestive evidence or likely evidence; or Negative results that are not sufficiently robust for the descriptor, ‘Not Likely to Be Carcinogenic to Humans.’

Not likely to be carcinogenic to humans This descriptor is appropriate when the available data are considered robust for deciding that there is no basis for human hazard concern. In some instances, there can be positive results in experimental animals when there is strong, consistent evidence that each mode of action in experimental animals does not operate in humans. In other cases, there can be convincing evidence in both humans and animals that the agent is not carcinogenic. The judgment may be based on data such as:

• • • •

Animal evidence that demonstrates lack of carcinogenic effect in both sexes in well-designed and well-conducted studies in at least two appropriate animal species (in the absence of other animal or human data suggesting a potential for cancer effects); Convincing and extensive experimental evidence showing that the only carcinogenic effects observed in animals are not relevant to humans; Convincing evidence that carcinogenic effects are not likely by a particular exposure route; or Convincing evidence that carcinogenic effects are not likely below a defined dose range.

A descriptor of ‘not likely’ applies only to the circumstances supported by the data. For example, an agent may be ‘Not Likely to Be Carcinogenic’ by one route but not necessarily by another. In those cases that have positive animal experiment(s) but the results are judged to be not relevant to humans, the narrative discusses why the results are not relevant.

Multiple descriptors More than one descriptor can be used when an agent’s effects differ by dose or exposure route. For example, an agent may be ‘Carcinogenic to Humans’ by one exposure route but ‘Not Likely to Be Carcinogenic’ by a route by which it is not absorbed. Also, an agent could be ‘Likely to Be Carcinogenic’ above a specified dose but ‘Not Likely to Be Carcinogenic’ below that dose because a key event in tumor formation does not occur below that dose.

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IARC carcinogen classifications The International Agency for Research on Cancer (IARC) was established in 1965. In 1971, a resolution has been adopted that IARC should prepare ‘monographs on the evaluation of carcinogenic risk of chemicals to man,’ which became the initial title of the series. In succeeding years, the scope of the program broadened as Monographs were developed for complex mixtures, occupational exposures, physical agents, biological organisms, pharmaceuticals, and other exposures. In 1988, ‘of chemicals’ was dropped from the title, and in 2019, ‘evaluation of carcinogenic risks’ became ‘identification of carcinogenic hazards,’ The distinction between hazard and risk is fundamental. The Monographs identify cancer hazards even when risks appear to be low in some exposure scenarios. This is because the exposure may be widespread at low levels, and because exposure levels in many populations are not known or documented. Details of the principles and procedures of the evaluations are outlined in the Preamble of the IARC Monographs (last update 2021). The following groups for classification of carcinogens are used: Group 1: The agent is carcinogenic to humans. This category applies whenever there is sufficient evidence of carcinogenicity in humans. In addition, this category may apply when there is both strong evidence in exposed humans that the agent exhibits key characteristics of carcinogens and sufficient evidence of carcinogenicity in experimental animals. Group 2A: The agent is probably carcinogenic to humans. This category generally applies when the Working Group has made at least two of the following evaluations, including at least one that involves either exposed humans or human cells or tissues:

• • •

Limited evidence of carcinogenicity in humans, Sufficient evidence of carcinogenicity in experimental animals, Strong evidence that the agent exhibits key characteristics of carcinogens.

If there is inadequate evidence regarding carcinogenicity in humans, there should be strong evidence in human cells or tissues that the agent exhibits key characteristics of carcinogens. If there is limited evidence of carcinogenicity in humans, then the second individual evaluation may be from experimental systems (i.e., sufficient evidence of carcinogenicity in experimental animals or strong evidence in experimental systems that the agent exhibits key characteristics of carcinogens). Additional considerations apply when there is strong evidence that the mechanism of carcinogenicity in experimental animals does not operate in humans for one or more tumor sites. Specifically, the remaining tumor sites should still support an evaluation of sufficient evidence in experimental animals in order for this evaluation to be used to support an overall classification in Group 2A. Separately, this category generally applies if there is strong evidence that the agent belongs, based on mechanistic considerations, to a class of agents for which one or more members have been classified in Group 1 or Group 2A. Group 2B: The agent is possibly carcinogenic to humans. This category generally applies when only one of the following evaluations has been made by the Working Group:

• • •

Limited evidence of carcinogenicity in humans, Sufficient evidence of carcinogenicity in experimental animals, Strong evidence that the agent exhibits key characteristics of carcinogens.

Because this category can be based on evidence from studies in experimental animals alone, there is no requirement that the strong mechanistic evidence be in exposed humans or in human cells or tissues. This category may be based on strong evidence in experimental systems that the agent exhibits key characteristics of carcinogens. As with Group 2A, additional considerations apply when there is strong evidence that the mechanism of carcinogenicity in experimental animals does not operate in humans for one or more tumor sites. Specifically, the remaining tumor sites should still support an evaluation of sufficient evidence in experimental animals in order for this evaluation to be used to support an overall classification in Group 2B. Group 3: The agent is not classifiable as to its carcinogenicity to humans. Agents that do not fall into any other group are generally placed in this category. This includes the case when there is strong evidence that the mechanism of carcinogenicity in experimental animals does not operate in humans for one or more tumor sites the remaining tumor sites do not support an evaluation of sufficient evidence in experimental animals, and other categories are not supported by data from studies in humans and mechanistic studies. An evaluation in Group 3 is not a determination of non-carcinogenicity or overall safety. It often means that the agent is of unknown carcinogenic potential and that there are significant gaps in research. If the evidence suggests that the agent exhibits no carcinogenic activity, either through evidence suggesting lack of carcinogenicity in both humans and experimental animals, or through evidence suggesting lack of carcinogenicity in experimental animals complemented by strong negative mechanistic evidence in assays relevant to human cancer, then the Working Group may add a sentence to the evaluation to characterize the agent as well-studied and without evidence of carcinogenic activity.

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The inclusion of other carcinogens than chemicals broadened the program. For example, in 2011 a possible relationship between mobile phone use and brain cancer has been identified (classification of Group 2B). In 2015 processed meat has been classified as carcinogenic to humans (Group 1) and consumption of red meat as probably carcinogenic to humans (Group 2A). For future priority agents for evaluation by the IARC Monographs program see: https://monographs.iarc.who.int/cards_page/ priorities/.

NTP carcinogen classifications The NTP is responsible for preparing Reports on Carcinogens. The Reports on Carcinogens are mandated by Public Law 95–662 and are for informational purposes only. The listing of a substance in the annual report does not by itself establish that such a substance presents a risk to persons in their daily lives. Clause (I) in subparagraph (4) (A) of Section 301 (b) of the Public Health Service Act requires that a report be published which contains a list of all substances (1) ‘which are either known to be carcinogens or may reasonably be anticipated to be carcinogens,’ and (2) to which a significant number of persons residing in the United States are exposed. As of the 2011 update, for the purpose of Biennial Report on Carcinogens, the classification scheme is outlined below.

Known to be human carcinogen There is sufficient evidence of carcinogenicity from studies in humans, which indicates a causal relationship between exposure to the agent, substance, or mixture, and human cancer.

Reasonably anticipated to be human carcinogen There is limited evidence of carcinogenicity from studies in humans, which indicates that causal interpretation is credible, but that alternative explanations, such as chance, bias, or confounding factors, could not adequately be excluded, or there is sufficient evidence of carcinogenicity from studies in experimental animals, which indicates there is an increased incidence of malignant and/or a combination of malignant and benign tumors (1) in multiple species or at multiple tissue sites, or (2) by multiple routes of exposure, or (3) to an unusual degree with regard to incidence, site, or type of tumor, or age at onset, or there is less than sufficient evidence of carcinogenicity in humans or laboratory animals; however, the agent, substance, or mixture belongs to a well-defined, structurally related class of substances whose members are listed in a previous Report on Carcinogens as either known to be a human carcinogen or reasonably anticipated to be a human carcinogen, or there is convincing relevant information that the agent acts through mechanisms indicating it would likely cause cancer in humans. Conclusions regarding carcinogenicity in humans or experimental animals are based on scientific judgment, with consideration given to all relevant information. Relevant information includes, but is not limited to, dose–response, route of exposure, chemical structure, metabolism, pharmacokinetics, sensitive subpopulations, genetic effects, or other data relating to mechanism of action or factors that may be unique to a given substance. For example, there may be substances for which there is evidence of carcinogenicity in laboratory animals, but there are compelling data indicating that the agent acts through mechanisms which do not operate in humans and would therefore not reasonably be anticipated to cause cancer in humans.

OSHA carcinogen classifications The Occupational Safety and Health Act of 1970 provides the establishment of workplace standards for toxic materials or harmful physical agents which most adequately assures, to the extent feasible, on the basis of the best available evidence, that no employee will suffer material impairment of health or functional capacity even if such employee has regular exposure to the hazard dealt with by such standard for the period of his or her working life. Potential occupational carcinogens regulated under OSHA are classified into two main categories based on the nature and extent of the available scientific evidence: Category I potential carcinogens and Category II potential carcinogens.

Category I potential carcinogens A substance shall be identified, classified, and regulated as a Category I potential carcinogen if, upon scientific evaluation, the secretary determines that the substance meets the definition of a potential occupational carcinogen in (1) humans or (2) a single mammalian species in a long-term bioassay in which the results are in concordance with some other scientifically evaluated evidence of a potential carcinogenic hazard, or (3) a single mammalian species in an adequately conducted long-term bioassay, in appropriate circumstances in which the secretary determines the requirement for concordance is not necessary. Evidence of concordance is any of the following: positive results from independent testing in the same or other species, positive results in short-term tests, or induction of tumors at injection or implantation.

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Category II potential carcinogens A substance shall be identified, classified, and regulated as a Category II potential carcinogen if, upon scientific evaluation, the secretary determines that (1) the substance meets the criteria set forth for Category I, but the evidence is found by the secretary to be only ‘suggestive’; or (2) the substance meets the criteria set forth for Category I in a single mammalian species without evidence of concordance.

NIOSH carcinogen classifications Acting under the authority of the Occupational Safety and Health Act of 1970 (Public Law 91-596), the NIOSH develops and periodically revises recommended exposure limits (RELs) for hazardous substances or conditions in the workplace. These recommendations are then published and transmitted to OSHA for use in promulgating legal standards. NIOSH may identify numerous chemicals that it believes should be treated as occupational carcinogens even though OSHA has not yet identified them as such. Generally, where OSHA has adopted the NIOSH recommendations as OSHA standards, the OSHA permissible exposure limits (PELs) and NIOSH RELs are equal. In cases in which the NIOSH recommendations have not been formally adopted by OSHA, the NIOSH RELs may be different from the OSHA PELs. The NIOSH classification scheme is one of the simplest carcinogen classification schemes; it combines all carcinogens into one category. Within this single category, NIOSH narratively describes the site of the cancer and whether the effect was seen in humans or animals. In determining carcinogenicity, NIOSH uses a classification scheme outlined in 29 CFR 1990.103, which states in part: Potential occupational carcinogen means any substance, or combination or mixture of substances, which causes an increased incidence of benign and/or malignant neoplasms or a substantial decrease in the latency period between exposure and onset of neoplasms in humans or in one or more experimental mammalian species as the result of any oral, respiratory, or dermal exposure, or any other exposure which results in the induction of tumors at a site other than the site of administration. This definition also includes any substance which is metabolized into one or more potential occupational carcinogens by mammals. The NIOSH thresholds for carcinogens were not designed to be protective of 100% of the population. NIOSH usually recommends that occupational exposures to carcinogens be limited to the lowest feasible concentration.

ACGIH carcinogen classifications ACGIH classifies substances associated with industrial processes that are recognized to have carcinogenic or cocarcinogenic potential. In general, the stated classification is intended to provide a practical guideline for the industrial hygiene professional to assist in control of exposures in the workplace. The classification and threshold limit values (TLVs) are not mandated by federal or state regulations, although the ACGIH classifications and values may be considered when standards are adopted by the regulatory agencies. Currently, five categories of carcinogens have been designated by the TLV Committee to recognize the qualitative differences in research results or other data. These five categories are outlined below. A1: Confirmed human carcinogen—The agent is carcinogenic to humans based on the weight of evidence from epidemiologic studies of exposed humans and/or convincing clinical evidence in exposed humans. A2: Suspected human carcinogen—The agent is carcinogenic in experimental animals at dose levels, by route(s) of administration, at site(s), of histologic types(s), or by mechanism(s) that are considered relevant to worker exposure. Available epidemiologic studies are conflicting or insufficient to confirm an increased risk of cancer in exposed humans. A3: Animal carcinogen—The agent is carcinogenic in experimental animals at a relatively high dose, by route(s) of administration, at site(s), of histologic types(s), or by mechanism(s) that are not considered relevant to worker exposure. Available epidemiologic studies do not confirm an increased risk of cancer in exposed humans. Available evidence suggests that the agent is not likely to cause cancer in humans except under uncommon or unlikely routes or levels of exposure. A4: Not classifiable as a human carcinogen—There are inadequate data on which to classify the agent in terms of its carcinogenicity in humans and/or animals. A5: Not suspected as a human carcinogen—The agent is not suspected to be a human carcinogen on the basis of properly conducted epidemiologic studies in humans. These studies have sufficiently long follow-up, reliable exposure histories, sufficiently high dose, and adequate statistical power to conclude that exposure to the agent does not convey a significant risk of cancer to humans. Evidence suggesting a lack of carcinogenicity in experimental animals will be considered if it is supported by other relevant data. Substances for which no human or experimental animal carcinogenic data have been reported are assigned no carcinogen designation by the ACGIH.

German MAK carcinogen classifications Maximale Arbeitsplatzkonzentrationen (MAKs) are the maximum concentrations of chemical substances allowed in the workplace. They are daily 8-h time-weighted average values and apply to healthy adults. These are established by the MAK Commission and the

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carcinogen classifications are used in the derivation of MAK values. A key distinction in this system is whether the chemical is genotoxic or not. The categories are as follows: Category 1: Substances that cause cancer in man and can be assumed to contribute to cancer risk. Epidemiological studies provide adequate evidence of a positive correlation between the exposure of humans and the occurrence of cancer. Limited epidemiological data can be substantiated by evidence that the substance causes cancer by a mode of action that is relevant to man. Category 2: Substances that are considered to be carcinogenic for man because sufficient data from long-term animal studies or evidence from animal studies substantiated by evidence from epidemiological studies indicate that they can contribute to cancer risk. Limited data from animal studies can be supported by evidence that the substance causes cancer by a mode of action that is relevant to man and by results of in vitro tests and short-term animal studies. Category 3: Substances that cause concern that they could be carcinogenic for man but cannot be assessed conclusively because of lack of data. The classification in Category 3 is provisional. Substances for which the available studies have yielded evidence of carcinogenic effects that is not sufficient for classification of the substance in one of the other categories. Further studies are required before a final decision can be made. A MAK or BAT value can be established provided no genotoxic effects have been detected for the substance or its metabolites or the genotoxic effect is not the main effect. Category 3 will be re-evaluated annually to determine whether substances must be reassigned to Categories 1 or 2, whether the database permits their transfer to one of the Categories 4 or 5 or whether they require no classification and can be dismissed completely from the list. Category 4: Substances that cause cancer in humans or animals or that are considered to be carcinogenic for humans and for which a MAK value can be derived. A non-genotoxic mode of action is of prime importance and genotoxic effects play no or at most a minor part provided the MAK and BAT values are observed. Under these conditions, no contribution to human cancer risk is expected. The classification is supported especially by evidence that, for example, increases in cellular proliferation, inhibition of apoptosis, or disturbances in cellular differentiation are important in the mode of action. The classification and the MAK and BAT values take into consideration the manifold mechanisms contributing to carcinogenesis and their characteristic dose–time–response relationships. Category 5: Substances that cause cancer in humans or animals or that are considered to be carcinogenic for humans and for which a MAK value can be derived. A genotoxic mode of action is of prime importance but is considered to contribute only very slightly to human cancer risk, provided the MAK and BAT values are observed. The classification and the MAK and BAT values are supported by information on the mode of action, dose-dependence and toxicokinetic data.

European commission SCOEL carcinogen classifications Similar to the MAK classification system SCOEL has developed a system of carcinogen classifications to support decisions about the occupational exposure limits (OELs) for chemicals in the workplace. The key distinction in this system is whether the chemical is genotoxic or not. The categories are as follows: Group A: Non-threshold genotoxic carcinogens—for low-dose risk assessment the linear non-threshold (LNT) model appears appropriate. Group B: Genotoxic carcinogens for which the existence of a threshold cannot be sufficiently supported at present. In these cases, the LNT model may be used as a default assumption, based on the scientific uncertainty. Group C: Genotoxic carcinogens for which a practical threshold is supported. Group D: Nongenotoxic carcinogens and non-DNA-reactive carcinogens; for these compounds a true (perfect) threshold is associated with a clearly founded NOAEL. Health-based OELs are derived by SCOEL for carcinogens of Groups C and D. If dataset allows, SCOEL performs a risk assessment for carcinogens and/or mutagens placed in Categories A and B. In 2017 the SCOEL activities have been transferred to ECHA, which exclusively uses the GHS classification system.

Outlook Besides evidence from reliable epidemiological studies, classification of carcinogens is often based upon the rodent 2-year bioassays, which, according to the OECD test guideline No. 451 requests inclusion of the maximal tolerated dose (MTD). Since effects at the MTD or at other high doses, rarely, if ever, reflect human exposure scenarios various authors have questioned the predictive value in evaluating carcinogenic compounds (Cohen, 2017; Goodman, 2018; Berry et al., 2019) esp. at MTD levels. Such high dosage, which are very unlikely to occur at relevant human exposure levels, cause major disturbances of homeostasis and saturation of metabolic and reparative pathways with no relevance to human exposure levels. This is explicitly recognized by the US National Toxicology Program (2016), which states that its conclusions on rodent carcinogenicity are relevant only to the conditions of the bioassay under which the respective substance was tested. Current initiatives are directed to reducing animal use in regulatory toxicology and define modes of action, operative in organisms below toxic levels. These include tiered testing strategies founded on new approach methodologies (NAMs) followed by subchronic toxicity testing, by which relevant mode-of-action effects occur and, for non-genotoxic carcinogens, the dose levels below which the key events leading to carcinogenicity are not affected (Cohen, 2017; Goodman, 2018; Felter et al., 2020).

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References Berry CL, Cohen SM, Hayes AW, and Kaminski NE (2019) The NTP 2-year bioassay: Controversies in counting rodent tumors to predict human cancer. Toxicology Research and Application 3. https://doi.org/10.1177/2397847319889535. Cohen SM (2017) The relevance of experimental carcinogenicity studies to human safety. Current Opinion in Toxicology 3: 6–11. Felter SP, Boobis AR, Botham PA, Brousse A, Greim H, Hollnagel HM, and Sauer UG (2020) Hazard identification, classification, and risk assessment of carcinogens: Too much or too little?—Report of an ECETOC workshop. Critical Reviews in Toxicology 50(1): 72–95. Goodman JI (2018) Goodbye to the bioassay. Toxicology Research 7(4): 558–564. National Toxicology Program (2016) 14th Report on Carcinogens. Research Triangle Park, NC: United States Department of Health and Human Services, Public Health Service, National Toxicology Program. https://ntp.niehs.nih.gov/pubhealth/roc/index-1.html. US EPA (1986) Guidelines for Carcinogen Risk Assessment. Risk Assessment Forum, EPA/630/R-00/004: Washington, DC.

Further reading American Conference of Governmental Industrial Hygienists (2001) Documentation of the Threshold Limit Values and Biological Exposure Indices, 7th edn. Cincinnati, OH: ACGIH. Berry Sir CL, Cohen SM, Corton JC, de Camargo JLV, Eisenbrand G, Fukushima S, Greim H, Weber K, Rietjens I, and Strupp C (2023) Letter to the Editors regarding “10% Body weight (gain) change as criterion for the maximum tolerated dose: A critical analysis” Regulatory Toxicology and Pharmacology. in press. Bolt HM and Huici-Montagud A (2008) Strategy of the scientific committee on occupational exposure limits (SCOEL) in the derivation of occupational exposure limits for carcinogens and mutagens. Archives of Toxicology 82(1): 61–64. ECHA (2017) Guidance on the Application of the CLP Criteria. Guidance to Regulation (EC) No 1272/2008 on Classification, labelling and Packaging (CLP) of Substances and Mixtures, Version 5.0, July 2017. https://echa.europa.eu/documents/10162/2324906/clp_en.pdf/58b5dc6d-ac2a-4910-9702-e9e1f5051cc5?t¼1499091929578. Greim H and Reuter U (2001) Classification of carcinogenic chemicals in the work area by the German MAK Commission: Current examples for the new categories. Toxicology 166(1–2): 11–23. National Institute for Occupational Safety and Health (2020) NIOSH Pocket Guide to Chemical Hazards. Washington, DC: US Department of Health and Human Services, Public Health Service. Occupational Safety and Health Administration (1996) Identification, Classification, and Regulation of Potential Occupational Carcinogens. Subtitle B—Regulations Relating to Labor, Chapter XVII. Title 29 Code of Federal Regulations (CFR) (1990). Washington, DC: Occupational Safety and Health Administration. US EPA (2005) Guidelines for Carcinogen Risk Assessment. Risk Assessment Forum, EPA/630/P-03/001B: Washington, DC.

Relevant websites http://www.acgih.org-American :Conference of Governmental Industrial Hygienists. http://osha.gov/dsg/hazcom/ghs.html :Guide to Globally Harmonized System of Classification and Labeling of Chemicals. https://publications.iarc.fr/Book-And-Report-Series/Iarc-Monographs-On-The-Identification-Of-Carcinogenic-Hazards-To-Humans :IARC Monographs on the Identification of Carcinogenic Hazards to Humans. https://monographs.iarc.who.int/cards_page/preamble-monographs/ :Preamble IARC Monographs on the Identification of Carcinogenic Hazards to Humans. http://www.cdc.gov :National Institute for Occupational Safety and Health. https://ntp.niehs.nih.gov/index.cfm :National Toxicology Program. http://www.osha.gov :Occupational Safety and Health Administration. http://www.epa.gov :US Environmental Protection Agency. https://onlinelibrary.wiley.com/doi/book/10.1002/3527600418 (up to 2019) and https://series.publisso.de/en/pgseries (from 2020) :The MAK Collection for Occupational Health and Safety.

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Carcinogen-DNA adduct formation and DNA repair Madiha Khalid and Mohammad Abdollahi, Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran © 2024 Published by Elsevier Inc. This is an update of A. Weston, M.C. Poirier, Carcinogen–DNA Adduct Formation and DNA Repair, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 705–712, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00593-5.

Carcinogen-DNA adduct formation and DNA repair Exogenous carcinogens and DNA adduct formation Alkylating agents Aromatic amine Heterocyclic amines Mycotoxins Polycyclic aromatic hydrocarbons Reactive oxygen species Mechanism of DNA repair Direct DNA repair Nucleotide excision repair Base excision repair DNA mismatch repair Homologous recombination repair Non-homologous DNA end-joining Conclusion References

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Abstract Numerous chemicals that have the potential to be reactive or inactive chemically operate as exogenous carcinogens and have a propensity to bind covalently to DNA in order to damage DNA. DNA adducts are a distinctive kind of DNA damage caused by the covalent bonding of a chemical moiety to DNA. Since these excess products are not removed by the cell, they may cause mutation and eventually the emergence of disease and cancer. Some categories of exogenous carcinogens that cause DNA damage include alkylating chemicals, aromatic amines, heterocyclic amines, mycotoxins, polycyclic aromatic hydrocarbons, and reactive oxygen species. Even some of these damages have been linked to the emergence of diseases, aging, and even cancer. Single and double strand breaks in DNA can permanently alter its structure. The mechanisms of DNA repair rely on a number of metabolic pathways that must all be active simultaneously in order to restore DNA structure. The mechanism of DNA repair are direct DNA repair, nucleotide excision repair, base excision repair, mismatch repair, homologous recombination repair, and non-homologous end joining.

Keywords Base excision repair; Carcinogen; DNA adduct; DNA mismatch repair; DNA repair; Homologous recombination repair; Nucleotide excision repair

Key points

• • • • •

DNA adducts are a distinctive kind of DNA damage caused by the covalent bonding of a chemical moiety to DNA. Some categories of exogenous carcinogens that cause DNA damage include alkylating chemicals, aromatic amines, heterocyclic amines, mycotoxins, polycyclic aromatic hydrocarbons, and reactive oxygen species. Some of the DNA damages have been linked to the emergence of diseases, aging, and even cancer. The mechanisms of DNA repair rely on a number of metabolic pathways that must all be active simultaneously in order to restore DNA structure. The mechanism of DNA repair are direct DNA repair, nucleotide excision repair, base excision repair, mismatch repair, homologous recombination repair, and non-homologous end joining.

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Carcinogen-DNA adduct formation and DNA repair When negatively charged chemical components of DNA bind covalently to an active chemical species, such as an electrophilic positively charged chemical moiety, additional cellular products known as DNA adducts are generated. Such DNA adducts are not removed by the cell, which could lead to cancer and mutation. Both epigenetic and mutational processes can lead to cancer (Trosko and Chang, 1978). Somatic mutations, such as point mutations, deletions, gene duplications, or recombination, as well as large chromosomal alterations, have a considerable impact on the initiation of carcinogenesis. On the other hand, it is thought that suppressed mutations are modulated by genes, leading to tumorigenesis and includes stages of initiation, promotion, progression, and metastasis. Therefore, carcinogenesis is based on the initiation phase of mutagenesis occurring before or concurrently with tumorigenesis (Trosko and Chang, 1978; Council NR, 1983; Carlberg and Velleuer, 2021). Mutagenic carcinogens cause defects in DNA replication, or repair mechanism, or DNA damage or unplanned DNA synthesis (Council NR, 1983). DNA damage is believed to be essential but not necessary for tumorigenesis, as other events must also occur. DNA adduct formation and mutagenesis are thought to cause changes in gene expression that result in clonal expansions of cells lacking in growth control, or tumors, which take a long time to manifest.

Exogenous carcinogens and DNA adduct formation Exogenous carcinogens can be chemically reactive or inactive. Inactive or inert carcinogens need metabolic activation to become reactive intermediate metabolites, which is a vital step in the initiation of carcinogenesis, while highly reactive carcinogens interact with DNA directly. Following is a discussion of a few groups of exogenous carcinogens.

Alkylating agents There are numerous exogenous and endogenous sources of alkylating agents. Industrial chemicals, environmental pollutants, naturally occurring compounds, chemotherapeutic drugs, and experimental carcinogens are examples of exogenous sources (La et al., 2010). Examples of chemotherapeutic alkylating agents are (a) triazenes and hydrazines, (b) nitrosoureas, (c) nitrogen mustards, (d) oxazaphosphorines, (e) alkyl alkane sulfonates, and (f ) ethylene imines or aziridines (Chiorcea-Paquim and Oliveira-Brett, 2023). These electrophiles interact with the ring nitrogen and extracyclic oxygen atoms of DNA bases to generate covalent adducts, which can then cause aberrant base pairing, DNA strand breakage, or cross-linking of DNA strands (Chiorcea-Paquim and Oliveira-Brett, 2023).

Aromatic amine Smoke from cigarettes and cooked foods are two common sources of carcinogenic aromatic amines (Kadlubar, 1990). The active metabolite, N-hydroxy arylamines, which is produced during the liver metabolism of aromatic amines by cytochrome P-450IA2, can react covalently with urothelial DNA at acidic pH levels while passing through the urinary bladder and can also generate protein adducts with hemoglobin. Smoking is linked to higher amounts of carcinogen-DNA adducts in the human bladder, with 4-amino-biphenyl (ABP) being a prominent product. Additionally, glucuronyl transferases in the liver can conjugate N-hydroxy arylamines, enabling biliary transport to the colon lumen, where beta-glucuronidases can regenerate the aglycone. Additionally in the colon mucosa, acetyltransferases can O-acetylate the N-hydroxy metabolite in order to further activate it, hence raising the risk of colorectal cancer (Kadlubar, 1990).

Heterocyclic amines Cooking meat results in the production of heterocyclic amines, which increase the risk of digestive and reproductive cancer (Kang et al., 2022). Two mutagenic and carcinogenic heterocyclic amines produced during routine cooking are the amino-alpha-carbolines 2-amino-9H-pyrido [2, 3-b]indole (AlphaC) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAlphaC) (Frederiksen, 2005). Similarly, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), a heterocyclic aromatic amine generated in cooked beef, pose a risk for human prostate cancer and bladder cancer (Le Marchand, 2021). Phases I and II drug metabolizing enzymes catalyze the formation of a highly electrophilic intermediate called nitrenium from heterocyclic amines, which interacts with cellular macromolecules to generate DNA adducts and cause mutations and malignancies in a variety of biological systems (Wang et al., 2019; Turesky, 2002).

Mycotoxins Mycotoxins are extensively distributed in nature, include many fungal compounds such ochratoxins, hydrazine, and aflatoxins. Aspergillus fungus are the primary producers of the Aflatoxin B1 (AFB1) and Sterigmatocystin (STE). The most hazardous substance is AFB1, and its metabolites exhibit a range of biological activities, such as acute toxicity, teratogenicity, mutagenicity, and

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carcinogenicity. Epoxidation, DNA adduction, inflammation, and oxidative stress all contributed to the development of hepatocellular carcinoma (Cao et al., 2022). Grain and grain-based items, cheese, coffee, spices, and beer all contain STE. It is an AFB1 biogenic precursor, and the two share a number of structural and biological characteristics. STE-induced toxicity includes altering immune system function and activating several signaling pathways, as well as causing oxidative stress, mitochondrial malfunction, apoptosis, and cell cycle arrest. Additionally, STE was found to be genotoxic because it can cause DNA damage by forming DNA adducts (Zingales et al., 2020).

Polycyclic aromatic hydrocarbons PAHs are a wide class of chemical compounds composed of two or more fused benzene rings assembled in various ways. These are mostly generated when organic materials like coal, oil, natural gas, wood, and their byproducts are burned. PAHs are also produced by fires, grilled food, and tobacco smoke (Kim et al., 2013; Louro et al., 2022). PAHs are toxic, mutagenic, and carcinogenic. Topical PAHs activate aryl hydrocarbon receptor and promote the enzymatic generation of reactive intermediates that may cause protein and/or DNA adducts formation (Sousa et al., 2022; Das and Ravi, 2022). The primary PAH that has been frequently identified in the air, surface water, soil, sediments, cigarette smoke, smoked and grilled food products is benzo[a]pyrene (B[a]P). Cytochrome P450 converts this xenobiotic into the carcinogenic metabolite 7b,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), which forms DNA adducts and results in mutations and malignant changes. Additionally, B[a]P has pro-oxidative potential, is teratogenic, neurotoxic, and epigenotoxic, and affects animal fertility (Bukowska et al., 2022).

Reactive oxygen species Endogenous DNA damage arises from reactive intermediates of oxygen reduction that attack the bases or the deoxyribosyl backbone of DNA (Marnett, 2000). Alternatively, Reactive oxygen species, reactive nitrogen species, and DNA reactive aldehydes are generated as a result of the lipid peroxidation of polyunsaturated fatty acids and are significant contributors to carcinogenesis (Nair et al., 2007). Malondialdehyde (MDA), 4-hudroxy-2-nonenal (HNE), acrolein and crotonaldehyde are the most prevalent reactive Table: Exogenous carcinogens and mechanism of DNA adduct formation. Exogenous carcinogens

Examples

Source

Mechanism

1.

Alkylating agents

Triazenes, hydrazines, nitrosoureas, nitrogen mustards, oxazaphosphorines, alkyl alkane sulfonates, ethylene imines (Chiorcea-Paquim and Oliveira-Brett, 2023)

Industrial chemicals, environmental pollutants, naturally occurring compounds, chemotherapeutic drugs, and experimental carcinogens (La et al., 2010)

2.

Aromatic amines

N-hydroxy arylamines, 4-amino-biphenyl (Kadlubar, 1990)

Cigarette smoke and cooked foods (Kadlubar, 1990)

3.

Heterocyclic amines

Amino-alpha-carbolines 2-amino-9H-pyrido [2, 3-b]indole, 2-amino-3-methyl-9H-pyrido[2,3-b] indole (Frederiksen, 2005), 2-Amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (Le Marchand, 2021)

Cooked meat (Frederiksen, 2005; Le Marchand, 2021)

4.

Mycotoxins

Aflatoxin B1, sterigmatocystin (Cao et al., 2022; Zingales et al., 2020)

Aspergillus fungus (Cao et al., 2022; Zingales et al., 2020)

5.

Polycyclic aromatic hydrocarbons

Benzo[a]pyrene (B[a]P) (Bukowska et al., 2022)

Coal, oil, natural gas, wood, fires, grilled food, and tobacco smoke (Kim et al., 2013; Louro et al., 2022)

Alkylating agents interact with the ring nitrogen and extracyclic oxygen atoms of DNA bases to generate covalent adducts, which can then cause aberrant base pairing, DNA strand breakage, or cross-linking of DNA strands (Chiorcea-Paquim and Oliveira-Brett, 2023). Liver metabolism of aromatic amine generate N-hydroxy arylamines by cytochrome P-450IA2, can react covalently with urothelial DNA at acidic pH to form carcinogen-DNA adduct (Kadlubar, 1990) Phases I and II drug metabolizing enzymes catalyze the formation of a highly electrophilic intermediate called nitrenium from heterocyclic amines, which interacts with cellular macromolecules to generate DNA adducts (Wang et al., 2019; Turesky, 2002) Epoxidation, DNA adduct formation, inflammation, and oxidative stress (Cao et al., 2022; Zingales et al., 2020) Cytochrome P450 converts (B[a]P) into 7b,8a-dihydroxy-9a,10aepoxy-7,8,9,10-tetrahydrobenzo[a] pyrene, which forms DNA adducts (Bukowska et al., 2022)

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electrophile products of lipid peroxidation that damage DNA either by interacting directly with DNA bases or by producing more reactive bifunctional intermediates (Kawai et al., 2004).

Mechanism of DNA repair DNA damage affects its chemical structure in a number of ways, including dimerization, deamination, and formation of covalent addition products with activated, aromatic, or alkyl chemicals or species, as well as the production of oxyradicals because of normal metabolic activity. Single and double strand breaks are examples of these modifications to the DNA structure. Several of these DNA damage types have the potential to permanently alter DNA sequence, and some of them have even been linked to the onset of diseases, aging, and even cancer (Weston and Poirier, 2005). The mechanisms of DNA repair involve a number of metabolic pathways that require the simultaneous occurrence of numerous gene products to restore DNA structure. Cell cycle restriction point genes carry out the entire DNA repair process, and these complexes typically include a damage sensor, a damage eliminator, and a polymerase or patch synthesizer, and a ligase. It is known that more than 150 genes contribute to DNA repair via many pathways (Weston and Poirier, 2005). Mechanism of DNA repair are direct DNA repair (DDR), nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), homologous recombination repair (HRR), and non-homologous end joining (NHEJ).

Direct DNA repair DDR provides cells efficient and simple means to reverse covalent DNA adducts among the various repair pathways. Processes involving DNA photolyases, alkyltransferases, and dioxygenases carry out DDR (Yi and He, 2013). Cyclobutane pyrimidine dimers (CPDs) and pyrimidine pyrimidines (6-4) photoproducts are the principal DNA lesions caused by UV exposure (6–4 PPs). DNA photolyases uses blue and near UV light to repair such type of DNA damage by using a process that involves an electron transfer from FADH- to the UV induced lesion, a dimer splitting step, and a final electron transfer from the pyrimidine monomer radical back to FADH, thereby regenerating FADH- (Brettel and Byrdin, 2010). Some alkylating agents are commonly utilized as chemotherapeutics. O6-alkylguanine-DNA alkyltransferases and members of the AlkB family of dioxygenases repair N-alkylated lesions that prevent Watson-Crick pairings (Yi and He, 2013; Fu et al., 2012).

Nucleotide excision repair NER is the primary method to deal with DNA double strand distortion, through the involvement of several enzymes functioning sequentially or as molecular complexes. Global genome repair (GGR) and transcription-coupled repair (TCR) are NER subpathways. Generally, the three steps of NER mechanism are: (a) initial damage recognition and verification, (b) dual incisions and excision products release, and (c) gap filling, repair synthesis and DNA ligation. NER removes damaged nucleotides by making two incisions on either side of the lesion, releasing a tiny single-stranded DNA fragment of 22–30 nucleotides, as in the case of a mammalian cell (Canturk et al., 2016). The recovery of double strand DNA is then accomplished through repair synthesis and ligation (Lehmann, 2011). The DNA damage-binding protein 2 (DDB2/XPC) and DNA damage-binding protein 1 (DDB1) complex, is the DDB complex, that initially detects lesions through the GGR mechanism, Then, DDB and XPC are released prior to the dual incisions stage, and XPC, TF11H, XPA and RPA, XPG, and XPF are sequentially recruited (Scrima et al., 2011; Fischer Eric et al., 2011). While in TCR, inhibited polymerases II function as the damage sensor, recruiting CSB, CSA and UVSSA, which together activate TF11H. However, for both sub-pathways, XPG and XPF perform the 30 and 50 incisions, creating about 26 nucleotide-long single-stranded DNA fragments with damage that are released in association with TFIIH and XPG. DNA polymerases and ligases close the resulting gaps to restore complete double stranded DNA (Zhang et al., 2022).

Base excision repair Similar to NER, BER uses a number of enzymes that either work in sequence or as molecular complexes to repair the damage. To identify and remove the appropriate base changes, BER uses certain glycosylases. This results in apurinic/apyrimidinic (AP) sites, which are then processed by APE1 endonuclease and other BER factors (Drohat and Maiti, 2013; Lindahl, 2016; Fortini et al., 1999). The BER pathway can repair a few damages since only 11 glycosylases have been found from the human genome (Kumar et al., 2020), and each one can only recognize a small fraction of lesions with identical structural features (Zharkov and Grollman, 2005). While NER provides an adaptable repair mechanism that can detect a wide range of structurally heterogeneous base modifications and adducts. In BER, a glycosylase such as hOgg1 or UDG detects and eliminates a lesion, creating an apurinic site. Next, an endonuclease breaks the damaged strand and a polymerase synthesizes a patch. Finally, DNA ligase I or DNA ligase III finishes the ligation. The patch size in BER can be either short, made up of one nucleotide, or long, made up of 2–10 nucleotides. DNA ligase II performs DNA ligation for short BER, which is polymerase dependent, whereas DNA ligase I performs DNA ligation for long BER, which is related with proliferating cell nuclear antigen (Weston and Poirier, 2005).

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DNA mismatch repair MMR is essential for preserving genomic stability. When DNA repair processes place a conventional base in the wrong place opposite to a non-complementary partner, they generate nucleotide mismatches. Base-base mismatches and insertion/deletion mispairs caused by DNA replication and recombination are corrected by MMR mechanisms such as purine to purine or pyrimidine to pyrimidine (G-T or A-C) during transitions or purine to pyrimidine or pyrimidine to purine (C-C, T-T, C-T, A-A, G-G, A-G) during transversions. Meiosis disorders, sterility in mammalian systems, genome-wide instability, and predisposition to certain malignancies, including hereditary non-polyposis colorectal cancer, are all linked to MMR defects (Weston and Poirier, 2005; Li, 2008). Both NER and MMR include the degradation of a large section of the damaged strand, followed by the synthesis of 50 and 30 patches using the undamaged strand as a template, and finally ligation to finish the repair (Weston and Poirier, 2005). The important human MMR proteins include homologs of MutS (Drummond et al., 1995), MutL (Li and Modrich, 1995), EXO1 (Tishkoff et al., 1998), single strand DNA binding protein RPA (Ramilo et al., 2002; Lin et al., 1998), proliferating cellular nuclear antigen (PCNA) (Gu et al., 1998; Johnson et al., 1996; Umar et al., 1996), DNA polymerase d (Longley et al., 1997), and DNA ligase 1 (Li, 2008; Zhang et al., 2005), which play a key role in recognizing mismatches and initiating repairs (Kunkel and Erie, 2005). An MSH1MSH2-MSH6-PMS1 or MSH1-MSH2-MSH6-PMS2 repair protein complex that simultaneously anchors to the mismatch and the closest unmethylated adenine in the GATC recognition sequence recognizes the DNA mismatch. In order to facilitate the activity of a DNA polymerase in replication of the repair patch, PCNA serves as a sliding clamp and erode the entire sequence between the mismatch and the GATC recognition sequence. After that, a DNA ligase I combines with polymerase to finish the repair process (Weston and Poirier, 2005).

Homologous recombination repair Multiple cellular activities, including ionizing radiation, oxidative and mechanical stress, can result in double strand breaks. The two unique processes for repairing DNA double strand breaks are HRR and NHEJ. HRR is essential for the preservation of telomeres, chromosomal segregation, replication fork preservation, and genome integrity. It also helps to remove harmful lesions, including double-stranded breaks and interstrand crosslinks from chromosomes (San Filippo et al., 2008). The repair procedure is initiated by a number of HRR sensors, including DNA-protein kinases, ATM, and ATM linked to RAD3. Likewise a number of DNA repair proteins, including RAD-51, -52, -54, RAD51-B, -C, -D, XRCC-2, -3, and BRCA1, -2 participate in the repair mechanism. The MRN complex, also known as the Nibrin complex with RAD50/MRE11, causes synchronous recision of both strands, while the RAD51, RAD51B, C, and D proteins set up the single-stranded DNA segments for sister chromatic exchange and the homologous duplex invasion. An undamaged duplex DNA sequence serves as a template to finish the polymerization through the repair of Holliday junctions, DNA pairing, and gap filling in the damaged homolog. Repair mechanisms provide crossover events and non-crossover events with an equal chance to occur. Nevertheless, HRR is frequently carried out without a crossover event, a method that is more conservative in limiting genomic alterations (San Filippo et al., 2008; Sung and Klein, 2006).

Non-homologous DNA end-joining The other important repair method for DNA double strand breaks is NHEJ. It contributes to immunological diversity by re-ligating the RAG1 and RAG2 cleavage products. NHEJ, in contrast to HRR, does not require a genetic DNA sequence homolog since repair takes place without replicating an undamaged template. For NHEJ, nucleases are required to repair damaged DNA, polymerases are needed to add new DNA, and ligases are required to maintain the integrity of the DNA strands (Lieber, 2010). A deletion mutation will also happen if the original sequence can’t be restored by ligating two blunt ends together through a process referred as “illegitimate.” When single-stranded overhanging segments are present, some degradation may take place to produce a blunt end before ligation (ligase IV). Alternately, the XRCC4 ligase IV complex may recruit polymerase m and polymerase l to fill the gap (Weston and Poirier, 2005; Lieber, 2010).

Conclusion A variety of exogenous carcinogens have the capacity to bind covalently to DNA and create DNA adducts. As a result of such additional cellular products not being eliminated by the cells, they cause mutation, which in turn results in disease and even cancer. Alkylating compounds, aromatic amines, heterocyclic amines, mycotoxins, polycyclic aromatic hydrocarbons, and reactive oxygen species are some types of exogenous carcinogens that damage DNA. However, many DNA repair mechanisms, including direct DNA repair, nucleotide excision repair, base excision repair, mismatch repair, homologous recombination repair, and non-homologous end joining, rely on a number of metabolic pathways that must all be active at the same time in order to restore DNA structure.

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Carcinogenesis Catarina V Jota Baptistaa,c, Ana I Faustino-Rochaa,c,d, Fernanda Seixasb,e, and Paula A Oliveiraa,b, aCentre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Inov4Agro, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal; bDepartment of Veterinary Sciences, UTAD, Vila Real, Portugal; cDepartment of Zootechnics, School of Sciences and Technology, University of Évora, Évora, Portugal; dComprehensive Health Research Center (CHRC), University of Évora, Évora, Portugal; eVeterinary and Animal Research Center (CECAV), UTAD, Vila Real, Portugal © 2024 Elsevier Inc. All rights reserved. This is an update of M.C. Botelho, J.P. Teixeira, P.A. Oliveira, Carcinogenesis, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 713–729, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00371-7.

Introduction Overview Nomenclature of cancer (neoplasia) Tissue changes associated with carcinogenesis Hyperplasia and preneoplastic lesions Metaplasia and dysplasia Anaplasia - a hallmark of malignancy Staging and grading of cancers Molecular basis of cancer Multistep genetic model of carcinogenesis Proto-oncogenes and oncogenes Tumor suppressor genes Acquisition of mutations Growth factors, hormones, and signal transduction Telomeres and telomerase Heredity and cancer: Family cancer syndromes The immune system and cancer Operational phases and theoretical aspects of carcinogenesis Initiation Promotion Progression Metastization Exogenous factors influencing carcinogenesis Chemical and physical agents and lifestyle factors Infectious agents and inflammation Identification of carcinogenic agents Molecular epidemiology of cancer Conclusion References Further reading

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Abstract Cancer is a complex disease with multiple causes. Many intrinsic and extrinsic factors influence the development of cancer. Intrinsic or host factors include age, sex, genetics, immune system, metabolism, and hormones. Extrinsic factors are divided in different groups, as physical (different types of non-ionizing and ionizing radiations); chemical (as some mineral or organic substances); and biological (produced by some living organisms, for instance, some plants, virus, bacteria or fungi). Intrinsic and extrinsic factors can interact with one another to influence the development of cancer. In this article, we will discuss all the varied aspects of research that will ultimately lead to the prevention of cancer in man.

Keywords Anaplasia; Cancer; Carcinogenesis; Dysplasia; Epigenetics; Hyperplasia; Initiation; Metaplasia; Metastasis; Mutation; Neoplasia; Oncogene; Progression; Promotion; Transduction; Tumor

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00124-X

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Providing a summary and promoting a better understanding of the carcinogenesis. Giving a multidisciplinary and current approach to tumorigenesis, namely on its nomenclature, molecular and cellular biology and histopathology. Co-relating the aspects of the biology and development of tumors with current diagnostic and therapeutic strategies. Illustrating the developed ideas with well-studied tumor lesions and recent scientific discoveries.

Introduction Cancer research have been suffering a remarkable evolution in the last decades, contributing to continuous change in the way we study, prevent, diagnose or treat cancer; and this knowledge progression is definitely not slowing down. Thus, it becomes crucial to periodically revise the vast information on the subject. The aim of this chapter is to address the different aspects of carcinogenesis by providing an up-to-date overview of the scientific knowledge available. From nomenclature to the molecular basis of carcinogenesis, we managed to give a complete and consistent approach on the subject, illustrating the presented scientific facts with specific examples. Overall, this chapter presents all the aspects that may be considered in cancer prevention.

Overview Cancer, or neoplasia, is a complex disease with multiple causes and influence factors. Even thought the global cancer death rates continue to decline, incidence rates are leveling off among men and slightly increasing among women, all over the world. Many intrinsic and extrinsic factors interfere in cancer development. Intrinsic or host factors include age, sex, genetics, immune system, metabolism, and hormones. Extrinsic factors are divided in different groups, as physical (different types of non-ionizing and ionizing radiations); chemical (as some mineral or organic substances); and biological (produced by some living organisms, for instance, some plants, virus, bacteria or fungi). Intrinsic and extrinsic factors interact one another to influence tumorigenesis. Besides its considerable physical and emotional suffering, cancer is associated an enormous cost in the healthcare systems, in lost productivity and medical and research expenditures. Considerable effort continues to be exerted by clinicians and researchers to understand this complex and multifactorial disease so that strategies can be developed to decrease or prevent its occurrence. Current regulatory guidelines have been crafted to reduce exposure to agents identified as potentially capable of causing cancer (Coleman, 2018). During the past 45 years of cancer research, much information has been generated indicating that cancer is a multistep and progressive disease. This is supported by several research fields and studies, as epidemiology and population genetics, morphological and clinical studies, as well as experimental investigations in laboratory animals. Studies of biopsy and autopsy tissue samples from humans and animals, particularly experimental animal models of carcinogenesis, have provided important information about this multistep process at phenotypical levels. Then, molecular biological analyses have confirmed that neoplasias arise from the clonal expansion of a single transformed cell and that during its evolution, it accumulates epigenetics and nonlethal genetic damage, particularly in genes that regulate cell growth, apoptosis and DNA repair. Finally, immunological studies have allowed the comprehension of the complex cross-talk between cell tumors and the multiple cells present in tumor microenvironment (intratumoral fibroblasts, intratumoral adypocytes, intratumoral endothelial cells and pericytes, and mainly immune and inflammatory cells), conditioning and modulating its growth and development. The process of carcinogenesis may take months in experimental laboratory animals and years in humans. The identification of this process early in its natural history evolution enhances the success of surgical or therapeutical intervention strategies in termination the disease. By the time a neoplasia has progressed to the malignant stage and spread throughout the body, even radiation, chemotherapy or immunomodulation combined with surgery are unlikely to result in clinical cure (Klaunig, 2020). The process of carcinogenesis is schematically summarized in Fig. 1.

Nomenclature of cancer (neoplasia) The word “Cancer” comes from the Latin, but it is originally Greek. It is a derived term for “crab,” that describes the clinical appearance or infiltrative behavior of these abnormal growths (Zachary and McGavin, 2011), and because of the way as cancer often adheres to any body part, almost like the crab. Carcinogenesis is narrowly defined as the production of carcinoma but is more commonly used in the broadest possible sense to indicate generation of neoplasias that are new and typically abnormal growths, generally uncontrolled, and becoming progressively more serious with time. Neoplasia means ‘new growth’ and two important terms that relate the clinical behavior and growth characteristics of neoplasias are (1) benign and (2) malignant, whose features are listed in Table 1. Basically, benign neoplasias are normally slow-growing circumscribed, localized growths frequently amenable to surgical removal with a low probability of

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Fig. 1 Process of carcinogenesis depicted schematically showing the postulate pathway in which accumulation of genetic damage leads to malignant.

Table 1

Comparative features of benign and malignant neoplasias.

Effect

Host effect

Injury on the host

Benign

Little; not generally lethal

Usually negligible Usually slow but may compress or obstruct vital tissue

Malignant

Usually fatal if not treated

Can kill the host by Rapid growth, destruction of vital escapes normal tissue and body control dissemination mechanisms

Growth rate

Extent and mode of Metastasis growth

Microscopic features

Cytologic features

Usually encapsulated; expansive

No metastasis Well Mitoses uncommon; (remains localized differentiated nuclear and cellular at site of origin) cells resemble isomorphism normal tissues inconspicuous nucleolus Infiltrative growth Metastasis are Poor Mitoses are numerous Capsule absent or common differentiation and abnormal; incomplete and anaplasia anisokaryosis and (pseudocapsule) anisocytosis hyperchromatic; nucleolus

recurrence, that do not metastasize. Malignant neoplasias have usually a more aggressive growth, are locally invasive, may metastasize (spread to other organs), and may be difficult to delimit during surgical excision. Regarding the distinction between (1) tumor and (2) cancer, tumor broadly refers to any organ enlargement or swelling. Although tumor is a Celsus’ cardinal sign of inflammation, it is often used as synonymous of neoplasia, as many neoplasias are associated to inflammation. Cancer refers to malignant neoplasia. Unfortunately, layperson frequently use tumor and cancer interchangeably alike without qualifying whether it is a benign or malignant process. Neoplasias are classified based on (1) the cell or tissue of origin and (2) biological characteristics. There are two basic cell types that can originate neoplasias: mesenchymal and epithelial cells (Fig. 2). Mesenchyme (tissues derived from the embryonic mesoderm) includes the connective tissue, blood and lymphatic vessels, muscles, cartilage and bones. Epithelial cells line the internal and external surfaces of the body, and origins major organs of the body, such as liver and lungs. Most epithelial tissues are derived from the endoderm and ectoderm germ layers of the embryo. There are general guidelines used in the nomenclature of neoplasias. A benign epithelial neoplasia originating from a glandular tissue, or a tumor derived from nonglandular epithelium but exhibits a tubular growth pattern, is called an ‘adenoma.’ One or more qualifiers are added to indicate the tissue of origin and/or morphological features as in hepatocellular adenoma, thyroid follicular adenoma, or renal tubular cell adenoma. A exophytic (“growing outward”) tumor originating from an lining or covering that shows warty projections is denominated as papilloma; when papilloma grows inside a ductus can be referred as intraductal papilloma. In general, benign mesenchymal neoplasias are designated by attaching the suffix -oma to the name of the cell type from which the tumor originates, as in meningioma (meninges), hemangioma (blood vessels), and fibroma (fibroblasts).

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Fig. 2 Tissue types associated with neoplasm names. Table 2

Selected nomenclature of neoplasia.

Tissue Epithelium Squamous Transitional Glandular Liver cell Glands Connective tissue Adult fibrous Embryonic Cartilage Bone Fat Muscle Smooth muscle Skeletal muscle Endothelium Lymph Blood Lymphoreticular Thymus Lymph nodes Hematopoietic Bone marrow Neural tissue Nerve sheath Astrocytes

Benign neoplasia

Malignant neoplasia

Squamous cell papilloma Transitional cell papilloma

Squamous cell carcinoma Transition cell carcinoma

Hepatocellular adenoma Adenoma (mammary, salivary, etc.)

Hepatocellular carcinoma Adenocarcinoma (mammary, salivary, etc.)

Fibroma Myxoma Chondroma Osteoma Lipoma

Fibrosarcoma Myxosarcoma Chondrosarcoma Osteosarcoma Liposarcoma

Leiomyoma Rhabdomyoma

Leiomyosarcoma Rhabdomyosarcoma

Lymphangioma Hemangioma

Lymphangiosarcoma Hemangiosarcoma

Not recognized Not recognized

Thymoma Lymphoma (malignant lymphoma)

Not recognized

Leukemia

Benign peripheral nerve sheath tumor Not recognized

Malignant peripheral nerve sheath tumor Astrocytoma

Malignant epithelial neoplasias are called ‘carcinomas’ and qualified by its histogenetic. Thus, malignant skin neoplasias composed predominantly of squamous cells, are called squamous cell carcinomas; if mainly formed by basal cells are classified as basal cell carcinomas. Malignant mesenchymal neoplasias are called ‘sarcomas.’ Examples of the latter include osteosarcoma, a malignant bone neoplasia; fibrosarcoma, a malignant neoplasia of the fibroblasts; and leiomyosarcoma, a malignant neoplasia of the smooth muscle tissue. A cancer composed of cells of unknown tissue origin, is designated as undifferentiated malignant tumor. Nomenclature for several neoplasias is presented in Table 2.

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Neoplasia nomenclature has numerous exceptions. Though some neoplasias name have the suffix -oma they are always malignant, as the thymoma (also called malignant thymoma or thymic sarcoma), the lymphoma (malignant lymphoma or lymphosarcoma), and the melanoma (malignant melanoma). Moreover, the suffix -blastoma highlights the indifferentiation of the cells, malignancies in primitive or precursor cells, as the retinoblastoma or the nephroblastoma. On the other hand, some neoplasias are named for their physical attributes such as pheochromocytoma (in Greek, “phios” means dusky, “chroma” means color). In addition, some neoplasias may be denominated by the name of the person first describing the lesion, and examples such as Hodgkin’s lymphoma, Burkitt lymphoma, Wilms’ tumor (nephroblastoma), Kaposi’s sarcoma or Ewing sarcoma, among others, have persisted to date. Neoplasias composed of mixtures of cells are named accordingly; examples include fibroadenoma, adenosquamous carcinoma, and carcinosarcoma, mixed carcinoma of the dog’s mammary gland or the metaplastic carcinoma of the breast. Moreover, there are several tissue alterations that are not usually neoplastic in origin but have names with the suffix -oma: hamartomas (a disorganized aggregate of normal indigenous cells that represent faulty differentiation during embryonic development) and choristomas (focal collections of normal but ectopically located tissue due to developmental malformation, such as islands of pancreatic cells in the wall of the stomach). Finally, localized overgrowths of excess of skin with a fibrovascular axis (acrochordon) or excess inflammation and granulation tissue, such as on vocal cord or external auditory canal inflammatory polyps, are clinically recognized as pseudotumors but they are not neoplastic growths (Berman, 2005; Berman, 2004; Damjanov, 2009).

Tissue changes associated with carcinogenesis Hyperplasia and preneoplastic lesions Proliferative lesions, which may be classified as hyperplasia, metaplasia, dysplasia, benign neoplasia, or malignant neoplasia, represent continuous changes with considerable overlap biological and molecular features rather than discrete morphologic entities (Fig. 3). The classification of lesion as preneoplastic, benign neoplasia, or malignant neoplasia depends on the lesion’s most prominent morphologic and behavioral features. All neoplasias are derived from the clonal proliferation of a single initiated cell, a genetically mutated but phenotypically normal cell. Usually at some point early in the clonal expansion, the proliferating cells become phenotypically distinguishable from the surrounding normal tissue and are classified as ‘preneoplastic.’ Preneoplasia refers to an increase in proliferative lesions that lead to accumulation of genotypic errors and phenotypic changes that confer the cell adaptative and selective advantages, leading to tumor development. Although not all neoplasias exhibit a morphological recognizable preneoplastic change, in those instances in which alterations are confirmed, their occurrence documents that there is a response to tissue insults. Examples of preneoplastic lesions are presented in Table 3. Preneoplastic lesions have the capacity and propensity to reversibility. In some instances, a preneoplastic lesion represents the clonal expansion of a cell that has sustained genetic damage. A benign neoplasia is generally a localized expansive growth that compresses adjacent non-neoplastic (“normal”) tissue but is usually not immediately life threatening unless it physically interferes with normal function, for example, by blocking the intestinal tract or compressing vital areas in the brain or heart. A benign neoplasia, the clonal expansion of cells that have suffered some genetic mutations is further along the spectrum of changes that may precede the development of malignant neoplasia. In experimental carcinogenesis animal models, malignant neoplasias are frequently observed, arising from or within a benign neoplasia. Features of benign neoplasias are listed in Table 1.

Preneoplastic changes

Normal

Adenoma

Fig. 3 Morphologic continuum of carcinogenesis. Table 3

Examples of presumptive preneoplastic lesions.

Tissue

Presumptive preneoplastic lesion

Mammary gland

Atypical epithelial hyperplasia Lobular hyperplasia with atypia Atypical hepatocellular hyperplasia Oval cell proliferation Atypical tubular hyperplasia Increase in keratinocytes Hyperplastic nodules Atypical acinar cell nodules Aberrant crypt foci Squamous metaplasia

Liver Kidney Skin Pancreas Colon Bladder cancer

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Malignant neoplasias are rapidly growing, locally invasive proliferations that destroy surrounding tissues and are thus life threatening. They also may spread to distant organs in the body, mostly via the blood and lymphatic vessels. When precursor lesions are present prior to or concomitant with malignant neoplasia, it is probable that the malignancy is a consequence of the same factors that produced the precursor lesions. Malignant neoplastic features are listed in Table 1 (Brambilla et al., 2003; Feo, 2011; Kumar et al., 2014; Preneoplastic Changes, 2011; Zachary and McGavin, 2011).

Metaplasia and dysplasia In addition to hyperplasia, several qualitative cytological features allow the morphologic classification of the spectrum of proliferative lesions that may be observed in the process of carcinogenesis: metaplasia and dysplasia. Metaplasia is the substitution, in the post natal life, of a fully differentiated cell type to another fully differentiated cell; although most of the times reversible, metaplasia predisposes to certain forms of neoplasia. A classic example is the replacement of the normal respiratory epithelium of airways by squamous epithelium (Fig. 4) in cases of chronic lung irritation in tobacco smokers. While the squamous epithelium is believed to provide functional protection against the irritant properties of the smoke, the loss of the ciliated columnar epithelium results in increasing lung inflammation. When the irritative factor is removed, the squamous epithelium is replaced by normal ciliated columnar epithelium. Exceptionally, some metaplastic lesions, once detected, evolves to a tumor lesion, as the intestinal metaplasia of the stomach, associated with a Helicobacter pylori infection (Giroux and Rustgi, 2017). Dysplasia is defined as abnormal growth of a tissue with respect to shape, size, proliferation and organization of the cells. Normal cell-to-cell orientations are disorganized or disrupted, and cells show cellular and nuclear pleomorphism, overcrowding, and increased mitosis (Fig. 4). When present, dysplasia may be associated with chronic irritation, may coexist with metaplasia, and can be seen during neoplastic transformation. It is considered a preneoplastic change precursor of malignant transformation, and may be referred to as “carcinoma in situ” (Kumar et al., 2014).

Fig. 4 Epithelial tissue changes associated with neoplastic evolution.

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Anaplasia - a hallmark of malignancy Anaplasia is a qualitative alteration of cellular differentiation. Anaplastic cells may bear little, if any, resemblance to non-neoplastic (normal) cells. The lack of differentiation is associated with morphologic changes such as loss of cell polarity, cellular and nuclear pleomorphism, nuclear crowding and increased mitotic rate. This feature is considered a hallmark of malignancy (Haschek et al., 2010).

Staging and grading of cancers In human oncology, the experience from multiple years of observation on the clinical evolution of many cancers has strengthened the predictivity of histological grades and clinical staging in prognostication. The purpose of grading and staging a neoplasia is to predict its biological behavior and to help establish an appropriate therapeutic regimen. Grading is an evaluation of morphologic microscopic characteristics based on the extent of cellular anaplasia and the degree of proliferation. Generally, neoplasias with a high degree of anaplasia, associated with specific growth patterns, and high mitotic rate, some of which may be abnormal mitosis, are given a high grade of malignancy. Most grading schemes categorize neoplasias into one to three or four grades of malignancy. Staging of a cancer, which is independent of grading, is a clinical classification based on the extent of the cancer growth and its dissemination on the body. It provides quick prognostic information, and may influence the choice of appropriate therapy. Criteria used for staging neoplasias include the size of the primary neoplasia, the degree of invasion of the surrounding (“normal”) tissues, whether the cancer has spread to local lymph nodes, or to distant tissues. Thus, it is apparent that staging will have a large influence on the therapeutic approach. A small and localized breast cancer would most likely be treated by nodulectomy and possibly radiation therapy, whereas a large, infiltrative breast cancer would more likely be treated by mastectomy. If the cancer has spread to lymph nodes or distant sites, more aggressive therapy is implemented (Hinck and Näthke, 2014; Hortobagyi et al., 2018). The ultimate fate of cells or proliferative tissue masses is influenced by the amount of sustained genetic damage. Cells with minimal DNA damage may persist in a latent form, indistinguishable from surrounding normal cells. If such a latent cell sustains additional damage even long after the initial insult, it may then progress further along the pathway to malignancy (Fig. 1). As additional genetic damage occurs, the altered cell population expands and eventually leads to irreversible uncontrolled growth that may or may not be corrected by aggressive medical intervention.

Molecular basis of cancer Multistep genetic model of carcinogenesis Genetically, the multistage process involves the activation of growth-enhancing proto-oncogenes, inactivation of growth-inhibitory tumor suppressor genes, silencing of apoptotic genes and/or DNA repair genes, as well as epigenetic events that alter gene expression, genetic polymorphisms and methylation (Table 4). Cancer cells frequently contain mutations in multiple genes as well as large chromosomal abnormalities. Since their discovery, in 1989, more than 100 protooncogenes and about 15 tumor suppressor genes have been identified. Proto-oncogenes were first discovered in cancer-causing animal viruses that carried them. Intense study of these viruses, particularly by Varmus and Bishop in the 1970s, resulted in the discovery that some endogenous animal genes had been picked up by virus ancestors and incorporated into the viral genome (denominated viral oncogenes or V-onc). Soon thereafter a number of these proto-oncogenes were identified in both the animal and human genomes and later found to play a role in cancer development (Bister, 2015; Varmus, 1988). Table 4

Genetic and epigenetic events involved in cancer.

Proto-oncogenes (growth-enhancing) Growth factors

PDGF-B, FGF, sis

Growth factor receptors Signal transduction Nuclear regulatory proteins Cell cycle regulators Tumor suppressor genes (growth-inhibiting) Cell surface molecules Regulate signal transduction DNA repair, cell cycle Apoptosis genes DNA repair genes Epigenetic events

EGFR, CSF ras, abl myc, fos Cyclins and cdks TGF-bR NF1 p53, Rb, BRCA1 Bcl-2, Bcl-x, Bax, bad, Bcl-xS HNPCC, XP Methylation

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Fig. 5 Multistep aspects of human colon carcinogenesis.

A widely accepted multistep model of carcinogenesis proposed by Fearon and Vogelstein in 1990 serves as the framework for studies in carcinogenesis (Fig. 5). By studying multiple benign and malignant colonic neoplasias from individuals with multiple tumors, they found that benign neoplasias harbored mutations in genes such as APC, ras, and p53, and that there were frequently multiple mutations per neoplasia, particularly on malignant ones. The model describes a progressive acquisition of mutations, and it is believed the total accumulation of mutations (at least five to seven) rather than the order is important in the carcinogenic process. New evidence has been published to further refine this model (Fearon and Vogelstein, 1990). Recently, it has been proposed that some neoplasias are dependent on the continued activation or overexpression of a particular oncogene for maintaining malignant behavior. Others have found that some neoplasias are ‘hypersensitive’ to the inhibitory effects of specific tumor suppressor genes. These findings suggest that the multistage process of carcinogenesis is not simply a summation of individual effects of cancer genes but that some individual cancer genes can override the others (referred to by some as the ‘Achilles heel of cancer’), and they offer new strategies for the cancer prevention and therapy.

Proto-oncogenes and oncogenes Among the estimated 25,000 genes in the mammalian genome, there are about 100 genes that are classified as proto-oncogenes because activation of these genes to oncogenes appears to be an essential event for the development of many, if not all, cancers. In fact, oncogenes were first discovered by studying genetic alterations in cancers. The term oncogene activation indicates a quantitative or qualitative alteration in the expression or function of the proto-oncogene. The proto-oncogenes have essential function in the mammalian genome, mainly as cell cycle and cell differentiation regulators. These genes are highly conserved in evolution which is evidenced by structurally and functionally similar genes in yeast, earthworms, animals, and humans. Since their normal function is to control how a tissue grows and develops, if they do not function properly, abnormal growth and development may occur leading to neoplasia (so, they are called oncogenes). The appearance (phenotype) and function of a tissue is a consequence of which genes are actively producing their programmed product, typically a protein, which in turn affects the structure and function of the cells comprising a given tissue. All somatic cells in

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Fig. 6 Intrinsic and extrinsic factors modulating specific gene expression and its effect on tissue phenotype and function.

the body inherit the same complement of maternal and paternal genes. The reason that some cells form liver and produce products such as albumin, while other cells form kidney tubules and excrete substances from the body is a consequence of which genes are expressed in those cells. Liver cells do not express several critical genes that are important in kidney function, and vice versa. Specific gene expression and its effect on tissue phenotype and function are modulated by several intrinsic and extrinsic factors (Fig. 6). Since the function of proto-oncogenes is to control cell growth, proliferation, and differentiation, inappropriate expression of these genes due to mutation (oncogenes) will result on abnormal tissue proliferation and growth, promoting tumorigenesis. Oncogenes can be activated by several different mechanisms e.g., retroviral transduction, chromosomal translocation, gene amplification, point mutation, promoter/enhancer insertion, or decreased methylation of promoters. Once activated, an oncogene will be either inappropriately expressed (e.g., production of an altered message and protein) or overexpressed (e.g., production of too much of a normal message and protein), contributing to the neoplastic multistep process. Examples of activated or amplified oncogenes detected in human and animal neoplasias are listed in Tables 5 and 6, respectively. For some cancers, the frequency of oncogene activation is relatively high, while for other cancers, the activation of known oncogenes is uncommon. Identification of specific alterations in oncogenes in certain cancers represents a first step in determining the molecular basis of cancer and can lead to the development of tailored molecular therapeutic strategies. Experimental evidences indicate that oncogene activation can be early critical events in carcinogenesis, and experimental studies with known chemical carcinogens show that they produce specific alterations in certain oncogenes, reflecting the manner in which the carcinogen chemically affects DNA (Kontomanolis et al., 2020).

Table 5

Examples of human neoplasias associated with activated or amplified oncogenes.

Oncogene

Type of human neoplasia

H-RAS

Squamous cell carcinoma Urinary bladder carcinoma Lung carcinoma Acute myelogenous leukemia Lung adenocarcinoma Colon carcinoma Ovarian carcinoma Gastric carcinoma Renal cell carcinoma Acute myelogenous leukemia Pancreatic ductal adenocarcinoma Acute myelogenous leukemia Chronic myelogenous leukemia Chronic myelogenous leukemia Breast carcinoma Salivary gland adenocarcinoma Small cell lung carcinoma Burkitt’s lymphoma Neuroblastoma

K-RAS

N-RAS ABL ERBB2 MYC N-MYC

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Examples of animals neoplasias associated with activated oncogenes.

Oncogene

Type of animalneoplasia

H-ras

Hepatocellular adenoma and carcinoma Harderian gland adenoma Mammary carcinoma Skin squamous cell carcinoma Lung adenoma and adenocarcinoma Pancreatic carcinoma Hepatocellular carcinoma Leukemia Lymphoma Fibrosarcoma Neuroblastoma, Mammary carcinoma Lymphosarcoma Leukemia

K-ras

N-ras Raf ERBB2 Abl C-myc

Tumor suppressor genes Tumor suppressor genes, originally called antioncogenes, control cell growth and differentiation, so their function suppress the development of cancerous growth. While oncogenes must be activated to promote cancer, tumor suppressor genes must be inactivated or lost for cancer to develop. It has been shown that loss or mutation of both alleles must occur in order to silence these genes. A well-known and extensively studied tumor suppressor gene is the retinoblastoma gene (RB-1). In hereditary retinoblastoma, an affected child is born with deletions of one allele of chromosome 13 containing the RB-1 gene. A second mutation event leading to a loss or alteration of the remaining RB-1 allele occurs while retinal cells are undergoing growth during development, and the ocular retinoblastoma, frequently present in both eyes, will occur early in life. Loss or alteration of both copies of this tumor suppressor gene is sufficient to cause retinoblastoma. Although named for the disease in which it was discovered, alterations in the RB-1 gene have been detected in breast, lung, prostate, and bone cancers (Dyson, 2016).

Acquisition of mutations The rate of mutation has been intensely studied in the carcinogenic process. Mutations in cellular DNA can arise during normal cell replication by infidelity in DNA replication (mispairing) as well as by chromosomal deletions, amplifications, or rearrangements. Considering mispairing in nucleotide bases alone, it is estimated that spontaneous mispairing during normal cell replication can occur with a frequency of approximately 1.4  10−10 nucleotide bases per cell division. Since there are nearly 1016 cell divisions per human lifespan and 2  109 nucleotide base pairs per genome, a total of 2.8  1015 mispairings could occur over a lifetime ((1.4  1010)  (2  109)  1016). If each mispair led to a mutation that resulted in a cancer, a typical human would have billions of cancers in one average lifetime. Since such high estimates of mutation frequency are clearly in excess of what is observed, it is clear that evolutionary barriers on multicellular organisms (cell cycle arrest, apoptosis, limits to the number of cell divisions, cell adhesion, and asymmetric cell division) explains why cancer is remarkably rare. There are efficient mechanisms to repair DNA damage, thereby precluding successive accumulation of critical mutations. Cell proliferation is also critical for ‘fixing’ DNA damage, since without cell division there will be no inheritance of DNA errors. The cell has relatively efficient mechanisms to repair damage prior to cell division. In a rapid proliferating tissue, cell division can occur before the cell amend DNA errors, leading to increasing risk to develop cancer. While all of the above underscore the importance of cell proliferation in carcinogenesis, neoplasia does not occur exclusively or necessarily in tissues that have high proliferation rates. Consequently, other important mechanistic factors influence the complex process of carcinogenesis. In 1994, Loeb et al. proposed that neoplastic cells likely have a higher mutation rate than normal cells (approximately 2  107 per gene per cell division) and thereby this genomic instability increases the likelihood of neoplastic cells acquiring further mutations conducive to neoplasia. This is referred to as the ‘mutator phenotype’ (Fig. 7). It suggests that early mutation in stability genes (i.e., DNA repair, mismatch repair, DNA replication, or chromosome maintenance) will lead to the mutator phenotype and

Fig. 7 Mutator phenotype model.

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further mutations contribute to tumor promotion and progression. Others argue that the mutation rate is similar between neoplastic and normal cells but the higher proliferation rate of neoplastic cells leads to mutation accumulation. The healthy debates continue to feed our quest to prevent and cure the neoplastic process (Loeb and Loeb, 2000).

Growth factors, hormones, and signal transduction While alterations in cellular DNA are critical in carcinogenesis, some cancer-causing agents, particularly those that are not genotoxic, play a major role in cancer development by indirectly influencing gene expression and growth control by altering signal transduction. While the pivotal role of hormones in the orchestration of tissue growth and development has been appreciated for decades, the recent discovery of polypeptide growth factors has added to our knowledge a complex constellation of control mechanisms that regulate normal cell growth and may cause pathogenic effects when signaling mechanisms are disturbed. Both hormones and growth factors bind to specific cellular receptors triggering cascades of intracellular signaling transducers mediators that regulate the expression of certain genes, affecting cell crosstalk and functions, as cellular proliferation. These cascades of intracellular reactions, sometimes referred to as signal transduction, are the processes whereby external stimulus triggers intracellular biochemical cascades that act as direct transcriptional regulators (activators or repressors) of specific genes. A simplified depiction of the interaction of hormones and growth factors in cell signaling is presented in Fig. 8. This concept is perhaps best exemplified by the process whereby a normal hormone stimulates a tissue to grow. An example is breast development and milk production in response to the hormone prolactin. In this example, prolactin binds to a specific prolactin receptor on the external surface of the cell, which, in turn, triggers a biochemical change inside the cell membrane via molecules that are attached to the external receptor and pass through the cell membrane. This triggers a signaling cascade that mediates the activation of specific genes that initiate breast cells proliferation and milk secretion. The signaling pathways are highly interactive with numerous positive (signal-sending) and negative (signalblocking) feedback loops. An appropriate balance between these loops is necessary for the proper functional response to the initial stimulus, and when disrupted may cause multiple diseases or even cancer. Some forms of cancer development are believed to be facilitated by dysregulation in one or more signal transduction pathways. Thus, exposure to certain agents may potentially affect the balance of positive and negative feedback loops in one or more signal transduction pathways turning cells more susceptible to stimuli that promote growth. An example is the nongenotoxic skin tumor promoter phorbol ester, which activates protein kinase C (PKC), a multifunctional protein kinase family that is involved in controlling the function of other proteins, playing important roles in several signal transduction cascades, many of which are critical cell regulators. Treatment of initiated mouse skin with phorbol ester activates PKC, resulting in the development of benign

Fig. 8 Simplified depiction of the interaction of hormones and growth factors with cellular signal transduction.

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and malignant skin neoplasias. The complexity and pivotal importance of the signal transduction pathways help explain why multiple types of agents influence carcinogenesis, why multiple steps are involved in the carcinogenic process, and why different cancers are so heterogeneous. Signal transduction involves shifts in intracellular ion fluxes for elements such as sodium, potassium, and calcium. It also often involves activation of PKC, enzymes that phosphorylate many proteins that may be important in mitosis. Part of the signal transduction cascade involves increased expression of cyclic adenosine monophosphate, now recognized as a mitogenic signal, and activating one or more cellular proto-oncogenes. Current research demonstrates that increasing numbers of proto-oncogenes and growth factors are integral parts of the signal transduction pathways and, when altered, influence the development of cancer by subverting signal transduction (Bafico and Aaronson, 2003; Griner and Kazanietz, 2007).

Telomeres and telomerase Telomerase activation appears to be a critical component of the immortalization process in neoplastic cells, and it may provide the basis for new therapeutic targets. Telomeres are specialized structures at the ends of chromosomes, and telomerase is the enzyme that maintains the length of the telomeres. During each round of cell division, there is a loss of a small number of nucleotides, causing progressive erosion of genetic material at the end of each chromosome: so, as a normal cell divides, the telomeres shorten and telomerase is inactive. After a certain number of divisions, the shortened telomeres signal the cell to cease dividing and the cells become ‘senescent’ or perhaps will die by apoptosis. Germ cells and some neoplastic cells have sustained function of the telomerase enzyme, which helps maintain lengthening of the telomeres and promote continued replication. Tumors having an increased telomerase activity suggest a direct effect, but it is only part of the story. For example, p53 is activated by telomerase and in the absence of p53 these cells fail to undergo apoptosis and go on to proliferate (Corey, 2009; Jafri et al., 2016).

Heredity and cancer: Family cancer syndromes That certain cancers occur in greater frequency within families represents primary empirical evidence for susceptibility based on some hereditary element. Some genetic predispositions exist for cancers of unknown etiology, while interactions between genetic susceptibility and environmental factors are probably responsible for a large proportion of human cancers. Hereditary predispositions include DNA repair deficiencies, inability to detoxify carcinogens, and germline loss or mutations of critical genes. Examples of genetic predispositions to cancer are listed in Table 7 and include neurofibromatosis, retinoblastoma, breast cancer, and colon adenomatous. In many of these instances, one event in the carcinogenic process is believed to be an inherited germline mutation in the DNA. Another inherited anomaly, a mutation in DNA repair genes causes inability to repair ultraviolet light-induced DNA damage in individuals with the condition Xeroderma pigmentosum, causing high sensitivity to sunlight exposure and a high incidence of skin neoplasia even at young ages. Individuals bearing DNA repair genes mutation have high risk to develop all cancer types as these repair systems are essential for the maintenance of genome integrity in the face of replication errors, environmental insults, and the cumulative effects of age. However, the majority of genetic damage associated with carcinogenesis is acquired either Table 7

Examples of genetic predisposition to cancer development.

Genetic predisposition

Associated cancer

Germline deletion on chromosome 13 Germline deletion on chromosome 11

Retinoblastoma Osteosarcoma Nephroblastoma Hepatoblastoma Rhabdomyosarcoma Adrenal carcinoma Breast or ovarian cancer

Germline mutation in BRCA1 or BRCA2 Li–Fraumeni syndrome Von Hippel–Lindau disease Von Recklinghausen’s disease Familial dysplastic nevi Xeroderma pigmentosa-defective ability to repair damaged DNA Ataxia-telangiectasia Familial adenomatous polyposis

Soft tissue sarcomas Breast cancer Brain hemangiomas Retina hemangiomas Fibrosarcoma Neuroma Pheochromocytoma melanoma Cutaneous squamous cell carcinoma Leukemia Lymphoma Stomach carcinoma Colon adenocarcinoma

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in utero or from environmental and/or lifestyle factors to which individuals are exposed. Even for those individuals with a hereditary predisposition to neoplasia, additional DNA damage is necessary to lead ultimately to its development. Environmental factors that may increase the risk of cancer development in genetically predisposed individuals include exposure to radiation and agents that stimulate cellular proliferation. Experimental systems to study genetic susceptibility to cancer are critically needed to assess the role of gene-environmental interaction in the development of human cancer (Axilbund et al., 2011). For some cancers in genetically predisposed individuals, the data are consistent with an association between malignant neoplasia and biallelic genetic alteration, and this is supported by studies of tumor suppressor genes, which prevent the development of neoplasia. Alteration or loss of a single tumor suppressor gene allele is usually insufficient to allow the development of a neoplasia. In other words, the remaining functional tumor suppressor gene copy is sufficient to prevent the development of neoplasia; if it is lost or altered, however, neoplasia can develop. This situation occurs in hereditary childhood retinoblastoma, a malignant neoplasia of the retinal cells of the eye. Susceptible individuals inherit a partial loss of one copy (one allele) of chromosome 13, where the RB-1 is located, and acquire an alteration or loss of the remaining RB-1 allele during early development. The affected child subsequently develops retinoblastoma, often within the first 2 years of life (Fabian et al., 2018).

The immune system and cancer The proper functioning of the immune system is evidenced by recovery from common childhood diseases such as mumps and chicken pox. A properly functioning immune system recognizes the foreignness of the agents responsible for these diseases, responds to and eliminates the foreign agents, and confers long-term immunity to subsequent infection by the same or similar agents. It has been proposed that cancer cells are recognized as foreign and that the immune system functions to eliminate such cells before they are transformed into large, malignant neoplasias. This process involves elaboration of antibodies that bind to the cancer cells and activate processes whereby the cancer cells are killed. In addition, specific cells of the immune system, such as cytotoxic T lymphocytes, natural killer cells, and macrophages, have mechanisms to recognize and eliminate foreign cells. The process of immune surveillance is facilitated when the cancer cells express surface antigens that are recognized as foreign. The development of malignant disease might be seen as a failure of immune surveillance and associated to immunoediting. During the escape phase of immunoediting there is an immunosuppressive microenvironment that promotes tumor growth, survival, invasion and drug resistance. Exposure to agents that depress the normal functioning of the immune system can lead indirectly to neoplasia by permitting early persistence and development of recently emergent cancer cells. Once a neoplasia has reached a critical size and growth rate, it may not be possible for even a properly functional immune system to effectively eliminate the neoplastic cells, due to immune tolerance. The pharmacologic manipulation of the immune system can ameliorate cancer patients. This is the case of immunotherapy, where the use of BCG (Bacillus Calmette Guerin) in bladder cancer patients, to achieve a non-specific immune system stimulation, is a classical example. More recently new precision medicine techniques based on personalized biological therapies, as the T-cell transfer therapies (CAR T-cell therapy; TIL therapy), or dendritic cell-based vaccines are being tailored successfully in some tumor types, bringing new hope to control immunotolerance induced by tumor cells (Guallar-Garrido and Julián, 2020; Zhang et al., 2017).

Operational phases and theoretical aspects of carcinogenesis In addition to being complex, the process of carcinogenesis is typically long and tumors becomes clinically apparent just on late stages of its natural history. While perturbations in cellular DNA are essential to carcinogenesis, they alone are not sufficient to cause. Thus, in some experimental situations, a few minutes of exposure to a carcinogen is sufficient to result ultimately in cancer, whereas in other situations, exposure to the same carcinogen will not result in cancer unless there is additional experimental manipulation. Smokers illustrate this principle since many, but not all, ultimately develop lung cancer. In other experimental studies, simultaneous administration of a carcinogen and a second agent may enhance, reduce, or block the carcinogenic process depending on the agent employed. These and other carcinogenesis studies have elucidated some of the mechanisms and factors that influence carcinogenesis, delimited some of the specific stages in the multistep process, and continually reminded us of the complexity of this disease process. Multistep experimental models of carcinogenesis are useful in defining events in the neoplastic process; provide the foundations for current operational descriptions and hypotheses of the biological mechanisms of carcinogenesis (Fig. 1); are available for many organs including the skin, liver, urinary bladder, lung, intestine, mammary gland, prostate, and pancreas; and frequently are derived from chemical carcinogenesis studies on laboratory animals. The operational phases of carcinogenesis include initiation, promotion, progression and metastization (Fig. 9) (Gatenby and Vincent, 2008; Stewart, 2019).

Initiation Carcinogenesis may initiate by the action of biological, physical or chemical agents that cause a non lethal, permanent, DNA error on the cell. This DNA irreversible change may ultimately cause tumor transformation, if the mutated cell do not repair the DNA damage. The initiated cell is phenotypically normal, but genotypically different from the other “normal” cells and the capacity for autonomous growth may remain latent for weeks, months, years or decades. Direct-acting carcinogens are electrophilic reactive and

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Fig. 9 The four operational phases of carcinogenesis (initiation, promotion, progression and metastization). Adapted from Siddiqui, I.A., Sanna, V., Ahmad, N., Sechi, M. and Mukhtar, H. (2015) Resveratrol nanoformulation for cancer prevention and therapy. Annals of the New York Academy of Sciences. 1348. https://doi. org/10.1111/nyas.12811.

interact directly with nucleophilic cellular regions, as the DNA, to produce the damage while indirect-acting carcinogens must be bioactivated by the cell (metabolic activation) to produce an electrophilic reactive intermediates that interacts with DNA exerting genotoxic damage. The majority of damaged cells have the ability to repair the damaged DNA over a period of days or weeks; however, if a cell undergoes cell division prior to repair the DNA damage, the DNA error becomes ‘fixed,’ is no longer reparable, and is inherited by all subsequent daughter cells. The operational phase of initiation is relatively short and may occur within hours or days. In contrast, the promotion and progression of an initiated cell to a fully malignant neoplasia is a multistep, long process requiring months in animals and years/decades in humans. As most initiators are genotoxic, a battery of short term mutagenicity tests in bacteria and cell culture systems has evolved to identify chemicals with genotoxic properties, as the Ames Test. Once identified, such chemicals should be rigorously regulated to prevent human exposure. This approach is considered prudent because of the irreversible nature of the changes that occur during initiation. Indeed, it is generally believed that even a single molecule of a mutagenic agent is sufficient to damage DNA irreversibly. Thus, for practical purposes, there is no threshold or safe level of exposure to a mutagenic agent. Features of initiation are listed in Table 8. Initiators interact with host cellular macromolecules and nucleic acids in specific patterns. Some agents have both initiating and promoting activities (see below) and can induce neoplasias rapidly and in high yield when there is repeated or high-level exposure: these agents are complete carcinogenic agents. Cancer stem cells are a subpopulation of neoplastic cells responsible for tumor initiation and growth, and clone heterogeneity. There are rare cells with indefinite potential for self-renewal that drive tumorigenesis. Like other stem cells, they are able to develop signaling pathways during initiation and propagation. In most cases, these cells are responsible for radio and chemotherapy resistance and have a high plasticity, presenting various functional and phenotypic appearances. This diversity allows an adaptation to distinct environments and tissues and is remarkably important in the therapy and prognosis of the patient (Bajaj et al., 2019; Walcher et al., 2020).

Table 8

Salient features of initiation and promotion of neoplasia.

Initiators/initiation • Effect is irreversible • Only one exposure may occur • Multiple exposures may be additive • Initiated cell have no morphological change • Agents are considered carcinogens • Agents are mutagenic • No measurable threshold dose • Must occur before exposure to the promoter • Does not result in neoplasia unless promoter is subsequently applied • Number of initiated cells dependent on dose of initiator • Initiated cell is an adult stem cells (denominated as cancer stem cell) Promoters/promotion • Nonadditive • Agents not capable of initiation • Reversible, epigenetic action • Modulated by diet, hormones, environment, and other factors • Measurable threshold dose • Measurable maximal response • Agents not considered carcinogens but may be cocarcinogens • Exposure is effective after the initiator • Agents are usually not mutagenic • Prolonged and repeated exposure is usually required • Progression

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Promotion Promotion is classically considered a stage on the multistep carcinogenic process in which specific agents, known as promoters, enhance the development of pre-neoplastic stages and neoplasia by providing initiated cells with selective growth advantages over the surrounding normal cells. The features of promotion are listed in Table 8. The promoter activity on the initiated cell is epigenetic, reversible but cumulative (dose-dependent effect), requiring multiple exposure of the initiated cell to the promoter agent to produce cancer. Promoters have no effect when the organism has not been previously exposed with an initiator. Promoters are often specific for a particular tissue or species due to their interaction with receptors that are present in different amounts in different tissue types. Radiation, dietary foods or contaminants, environmental toxins, multiple medical drugs, virus and other biological agents, may act as promoters. The temporal sequence of promoter exposure is critical for the definition of promotion. The agent must act after initiation and enhance the neoplastic process to be considered a promoter. If an agent is given simultaneously with an initiator and results in enhancement of development of neoplasias, it is regarded as a cocarcinogen rather than a promoter. While some promoters are cocarcinogenic (e.g., phorbol esters), not all promoters (e.g., phenobarbital and phenol) possess cocarcinogenicity and, conversely, not all cocarcinogens are promoters. Under these same conditions of simultaneous administration, a diminution in the neoplasia response is considered evidence of anticarcinogenic activity. Several rodent liver tumor promoters, which are active when administered after a variety of initiators, prevent or delay the development of liver neoplasias when added to diets along with an active carcinogen. While upper and lower thresholds have been demonstrated experimentally for promoters, some consider that, in an absolute sense, it is statistically impossible to prove or disprove the existence of thresholds for promoters for much the same reasons that this cannot be done for initiators. One can never be certain that an apparent no-effect level would, indeed, be without effect if a sufficiently large enough number of animals were used. Promoters include agents such as drugs, plant products, and hormones that do not directly interact with host cellular DNA (are not genotoxic) but somehow influence the expression of DNA (epigenetic effect). Experimental evidence suggests that regulation of gene expression is unique to the nature of the promoter agent. Some promoters are believed to produce their effect by interaction with receptors in the cell membrane, cytoplasm, or nucleus (e.g., hormones, dioxin, phorbol ester, and polychlorinated biphenyls). Alternatively, promoting agents may exert their effect through their molecular orientation at cellular interfaces. Other promoters may selectively stimulate DNA synthesis and enhance cell proliferation in initiated cells, thereby giving them a selective growth advantage over surrounding normal cells; or may induce neoplasia inducing cycles of necrosis and repair (cell proliferation). Promoters appear to have a relatively high tissue specificity. Thus, phenobarbital functions as a promoter for rodent liver neoplasia but not urinary bladder neoplasia. Saccharin, on the other hand, promotes urinary bladder neoplasia but not liver neoplasia in the rat. Similarly, 12-o-tetradecanoylphorbol-13-acetate (phorbol ester) is a potent skin and forestomach neoplasia promoter in the laboratory rodent, but has no appreciable activity in the liver. Other agents, such as the antioxidants 3-t-butyl4-methoxyphenol and 2,6-di-t-butyl-4-methoxyphenol, may act as promoters in one organ and antipromoters in another and have no effect in a third organ. Thus, the practical definition of a promoter must include the designation of the susceptible tissue (Rao et al., 1984; Matsuoka et al., 1990). Tumor promotion may be modulated by several factors such as age, sex, diet, hormone balance and genetic polymorphisms. The correlation of increased rates of breast cancer in women following a ‘Western’ lifestyle has implicated meat and fat consumption as playing an important role in breast cancer development. Experimental demonstration of the role of a high-fat diet in the promotion of mammary cancer in rats exposed to the mammary carcinogen dimethylbenzanthracene has been documented. Similarly, bile acids, as modulated by fat consumption, are known promoters of rat liver carcinogenesis and human colorectal cancer. Age and sex-associated modulations in hormonal levels of estrogens, progesterone, and androgens have been implicated as potential promoters of breast cancer on the basis of epidemiological studies in humans. Experimental studies have repeatedly shown that these hormones, in addition to pituitary prolactin, promote mammary cancer in rats initiated with carcinogens (Nguyen et al., 2018; Zhao et al., 2013).

Progression Progression is the part of the multistep neoplastic process associated with the development of the cell into a biologically malignant cell population. Progression is frequently used to signify the stages whereby a benign proliferation becomes malignant or, alternatively, where a neoplasia develops from a low grade to a high grade of malignancy. During progression, neoplasias show increased invasiveness, develop the ability to metastasize, and show genetic instability, and alteration on biochemical, metabolic, and morphologic characteristics. Tumor cell heterogeneity is an important feature of tumor progression, and includes production of antigenic and protein product variants, ability to secrete angiogenic and growth factors, emergence of chromosomal variants, epithelial-mesenchymal transition and development of metastatic capability, alterations in metabolism, and a decrease in sensitivity to radiation or chemotherapy. The development of intraneoplastic diversity may result from increasing genetic instability. Alternatively, the heterogeneity observed in tumor progression may be generated by epigenetic, regulatory mechanisms that are a part of the process of promotion. More than likely, genetic and epigenetic events subsequent to initiation operate in a non-mutually exclusive manner during progression, possibly in an ordered cascade of latter events superimposed upon earlier events. The most plausible mechanism of progression invokes the notion that, during the process of tumor growth, there is a natural selection that favors enhanced growth of subpopulations (clones) of the neoplastic cells. In support of this mechanism is increased

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phenotypic heterogeneity observed in malignant but not in benign neoplasia. Presumably, a variety of subpopulations arises, and it is only a matter of time before the emergence of a subpopulation with a more aggressive biological characteristics or at least an accelerated growth advantage. This can be observed occasionally during experimental hepatocarcinogenesis when a phenotypically distinguishable carcinoma can be observed arising within an existing adenoma. Distinction between tumor promotion and tumor progression is not readily discernible in the routine histopathologic evaluation of neoplasias and may be somewhat academic. What is believed to distinguish progression from promotion is the presence of structural genomic alterations in the former and their absence in the latter. Both structural genomic changes and biochemical changes associated with tumor progression cannot be defined by conventional histopathology. Established and emerging technologies centered on histochemistry, immunocytochemistry, in situ hybridization, identification of activated oncogenes, loss of tumor suppressor genes, gene expression, proteomic and metabolomic profiling offer promise to distinguish various stages of progression in the evolution from benign to malignant neoplasias (Arvelo et al., 2016).

Metastization Metastization is defined as the spread of cancer cells from the primary site to other parts of the body mainly through the bloodstream or the lymph system, but also through coelomic cavities or contiguity (anatomic proximity). Nowadays, cancer metastasis is no longer interpreted as a linear cascade of events, but rather as a series of synchronous and partly coincident processes. For metastasis occurrence, many mechanisms are required: cell migration, angiogenesis, matrix degradation, evasion of host immune system and metastatic colonization (homing). Initially, metastasis is highly influenced by the complex tissue microenvironment. Interactions between cancer cells, immune cells, endothelial cells, stromal fibroblasts as well as changes in tissue oxygen tension and the structure of the adjacent extracellular matrix (ECM) represent some of those influence factors. The hypoxia-inducible factor (HIF) is a potent angiogenic factor, and can switch on ameboid cell migration as the oxygen tension oscillates, stimulating mutual signaling between mesenchymal stem cells and cancer cells which leads to the metastatic phenotype. Tumor-associated macrophages (TAMs) can be stimulated by cancercell-secreted lactate to start angiogenesis. Colonization of different tissues by disseminated tumor cells is a very inefficient process. Relatively high numbers of circulating tumor cells (CTCs) are detected in cancer patient’s blood, but fewer metastasis are clinically detectable. After arresting in the vascular bed, a successfully metastasized cell has to arrive, subsist, and adapt in a new tissue microenvironment that may or may not be compatible with survival. That is the main reason why metastatic lesions are primarily detected in select organ sites (bone, liver, lung, and brain) but rarely in others (kidney, heart, and stomach). The preference of cancer subtypes for different tissues as a metastatic site or homing is not entirely understood yet. There are multiple theories to justify the homing process, and distinct approaches are trying to identify the processes and factors involved in tumor colonization. For instance, the metastasis suppressor RARRES3 facilitates breast cancer tropism to the lung by increasing the lung parenchyma’s cellular invasion. Circulating growth factors and cytokines as well as microRNA exosomes, have been described to contribute to the modification of premetastatic niches, and changes on tumor microenvironment and drug resistance. Furthermore, animal model studies have contributed to understanding their effects on increasing vascular permeability and inducing alterations on the metastatic site’s resident cells. Recent reports have also suggested the role of specific integrin receptors on the exosomes in tissue tropism. Thus, metastatic colonization is not merely an outgrowth of cancer cells from the primary organ, but a group of complex interactions between disseminated cancer cells and the different tissue microenvironments of the organism (Arvelo et al., 2016; Seyfried and Huysentruyt, 2013; Suhail et al., 2019).

Exogenous factors influencing carcinogenesis Important exogenous factors that contribute to induction of cancer include natural and synthetic chemicals, environmental exposures to ultraviolet and medical radiation, diet and lifestyle, and infectious agents such as viruses, parasites, and bacteria. Evidence for a causal association between exogenous factors and neoplasia is derived from epidemiologic studies, analysis of occupationally exposure common cancers, and animal models.

Chemical and physical agents and lifestyle factors Many chemicals that cause cancer interact directly with and alter DNA or are metabolized to chemical derivatives capable of doing so. Exposure to carcinogens can occur in certain occupational settings. Association of hepatic angiosarcomas with occupational exposure to vinyl chloride, pulmonary mesotheliomas with exposure to asbestos fibers, and leukemia with benzene are well-known examples. Exposure to other carcinogenic agents may occur in the diet or as a consequence of certain lifestyle practices such as cigarette smoking associated with pulmonary cancer, and high animal fat diets linked to breast and colon cancer. Strong associations have been made between exposure of light-skinned individuals to ultraviolet radiation and skin cancer. Exposure to natural or occupational ionizing radiation, X-rays, and medical radioisotopes, medical drugs and chemotherapeutic drugs have also been associated with human neoplasia. Examples include leukemias in radiologists and atom bomb victims, lung cancer in uranium mineworkers, and thyroid and breast cancer following diagnostic or therapeutic use of radiation, and bladder cancer in paint or rubber industry workers (Charbotel et al., 2014; Mundt et al., 2017).

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Infectious agents and inflammation Viral, parasitic, and bacterial infections have been linked to cancer (Table 9). DNA viruses such as Epstein-Barr, hepatitis B, hepatitis C, papillomaviruses, Kaposi sarcoma herpes virus and RNA viruses, such as human T-cell leukemia virus type I and human immunodeficiency virus have been implicated in causing cancer in humans and are listed as ‘known-to-cause-cancer’ in humans by the International Agency for Research on Cancer (IARC). In man, the liver fluke, Opisthorchis viverrini, is associated with the development of cholangiocarcinomas of the liver and the blood fluke, Schistosoma haematobium, with carcinoma of the urinary bladder. There is evidence that chronic Helicobacter pylori infection of the stomach in humans not only is related to gastrointestinal ulcers, but also may be linked to gastric carcinoma or lymphoma development (van Tong et al., 2017). For oncogenic viruses, the viral or host genes generally drive the neoplastic process while for some agents there appears to be an association between biological and other physical or chemical carcinogenetic mechanisms, as in the chronic inflammation and nitric oxide (NO) production in the development of cancer. When DNA viruses infect cells, the viral DNA inserts itself wholly or partially into the genome of the infected cell. It appears that such integration of viral DNA into the mammalian genome is sometimes sufficient to cause neoplastic transformation of the infected cell, which is accompanied by the production of new proteins essential for the neoplastic process. RNA viruses associated with neoplasia are chiefly represented by the retroviruses. RNA Table 9

Viruses and parasites causally related to neoplasias.

DNA viruses Virus

Type of neoplasia

Species

Herpes

Lymphosarcoma

Herpes simplex 2 Papillomaviruses

Cervical carcinoma Papillomas

Human papillomavirus

Warts Epidermoid carcinoma Cervical carcinoma Hepatocellular carcinoma Hepatocellular carcinoma

Chicken Monkey Rabbit Human Cow Rabbit Horse Dog Human

Woodchuck hepatitis virus Hepatitis B virus Epstein-Barr Hepatitis B & C Papillomaviruses T-cell leukemia virus type I

Woodchuck Human

RNA viruses Virus

Type of neoplasia

Species

Human T cell leukemia virus (HTLV-I and -II) Avian erythroblastosis virus

T cell lymphoma

Human

Leukemia Sarcoma Leukemia Sarcoma Leukemia Sarcoma

Chicken

Abelson leukemia virus Hervey sarcoma virus Feline sarcoma virus

Mouse Rat Cat

Parasites Parasite

Type of neoplasia

Species

Schistosoma haematobium Opisthorchis viverrini Spirocerca lupi

Bladder Cholangiocarcinoma Sarcoma

Human Human Dog

Bacteria Bacteria

Type of neoplasia

Species

Helicobacter pylori

Stomach carcinoma

Human

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viruses possess an enzyme called reverse transcriptase, which is capable of forming a DNA copy of the viral RNA when the virus infects a host cell. This DNA ultimately inserts itself into the host genome in much the same way as DNA viruses do, possibly resulting in the development of neoplasia (Lambert, 2009; Zella and Gallo, 2021). The role of inflammation in cancer development is being intensively studied. There are a number of chronic inflammatory conditions, infectious and noninfectious, in humans and animals associated with an increasing risk of cancer, and there are many investigators examining the role of NO and oxygen radical damage to DNA or other cellular processes, such as cell proliferation and apoptosis. NO induces p53, prevents apoptosis in cells such as endothelium, promotes angiogenesis, and inhibits DNA-repair activities - all processes that might provide a selective advantage to neoplastic cell growth (Singh et al., 2019).

Identification of carcinogenic agents There are two methods utilized to identify potential human carcinogens, the most direct of which is based on retrospective epidemiological studies in human populations using existing historical records associated with known cases of neoplasia. These records include death certificates where cause of death is indicated; hospital records; responses to questionnaires that document environmental or work-associated exposure to potential carcinogenic agents; and studies of neoplasia in culturally, ethnically, or religiously distinctive human populations. Association of cigarette mesotheliomas and exposure to chemicals and bladder cancer was the result of such retrospective epidemiological work. Prospective epidemiological studies identify a given population of individuals who agree to be monitored for several years to permit identification of potential carcinogenic factors associated with neoplasias that may occur (Loomis et al., 2018). Another method used to identify potential human carcinogens involves testing known chemicals and agents in experimental animals. Such tests have been referred to as animal bioassays and are typically conducted using rodents (mainly rats and mice) exposed to high doses of the suspect agent for a large portion of their lifespan (typically 2 years). If such agents are observed to produce neoplasia in the experimental animals, the agent is regarded as a potential human carcinogen. In countries worldwide, legal requirements mandate that all new chemical agents and drugs be tested in animal bioassays to determine whether they cause cancer in the test animals. Additionally, since the mild-1960s in the United States, the National Cancer Institute and currently the National Toxicology Program have collectively conducted animal bioassays on more than 500 chemical agents to assess their potential to cause cancer. Interpretation of results from human epidemiological studies and animal bioassays to identify carcinogenic agents has often proved difficult and controversial. Humans are rarely exposed to only one potential cancer-causing agent in their lifetime, and the amount and duration of that exposure may be difficult or impossible to quantify rigorously. Long latency intervals may occur between exposure to a potential carcinogen and ultimate development of neoplasia, making accurate assessment of cause and effect almost impossible. Despite such limitations, epidemiological studies that clearly show an association between a given chemical exposure or lifestyle habit with an enhanced rate of a specific cancer are regarded as the most relevant method for identification of human carcinogens. While animal bioassays have proved useful for the identification of agents that can cause cancer in the laboratory rodent, they only identify an agent as potentially hazardous to human health. Additional facts and factors must be considered in classifying such an agent as a likely human carcinogen (Ashby, 1997; National Research Council (US) Committee on Comparative Toxicity of Naturally Occurring Carcinogen, 1996; Conte et al., 2022). The current approach for assessing the scientific relevance of either epidemiological or animal bioassay results to human health risk involves a ‘weight-of-evidence’ procedure in which national and international panels of expert scientists from several disciplines examine all available information on the suspect agent in making their assessment. Included in this analysis are the strength of the epidemiological evidence, the dose-response curve of the animal response, comparative species metabolism and ability to extrapolate between species, likely mechanism of cancer induction for the agent in question, its genotoxicity, the amount of the agent in the environment, and the number of people potentially exposed to it (Madia et al., 2020). Based on this type of analysis, so far 121 agents have been classified as known human carcinogens by the IARC (some of which are in Table 10) and 89 more agents have been designated as probable human carcinogens. The 14th US Health and Human Services Annual Report on Carcinogens lists 62 known human carcinogens and 135 substances that are reasonably anticipated to be human carcinogens.

Molecular epidemiology of cancer The molecular epidemiology of cancer is the study of molecular alterations, primarily mutations, in investigating the etiology of cancer, as well as, identifying individual cancer risk. The possibility of identifying cancer-causing agents based on the occurrence of predictable molecular alterations that are found in the neoplasia is intriguing. It is based on the hypothesis that there are carcinogen-specific patterns of mutations that reflect direct interactions of carcinogens with cancer genes. For example, lung and colon cancers from smokers tend to have a specific mutation in the ras oncogene or p53 tumor suppressor gene (i.e., mostly a G-T nucleotide base substitution) and this mutation is likely due to the direct interaction of the carcinogen in smoke benzo(a)pyrene with DNA (Rivlin et al., 2011). Such chemical-specific mutational profiles (or ‘molecular signatures’) have been used to support a causal association between particular genetic events in tumors and a specific carcinogen, such as neoplasias associated with exposure to radon, aflatoxin B1, vinyl chloride, and the nitrosamines (Tables 11 and 12). The strongest evidence for linkage between a cancer-causing agent and a specific type of neoplasia is that of the CC-TT double base changes observed in skin neoplasias of both humans and animals. This mutation is consistent with the predicted UV-induced damage of dipyrimidine dimers. In liver tumors

Carcinogenesis Table 10

Some selected agents or mixtures for which there is sufficient evidence of carcinogenicity in humans.

Organic compounds 2-Napthylamine 4-Aminobiphenyl Aflatoxin B1 Analgesics containing phenacetin Azathioprine Benzene Benzidine Betel quid with tobacco Bis(chloromethyl)ether Chlorambucil Chlornaphazine Chloromethyl methyl ether Cyclophosphamide Diethylstilbestrol Melphalan Methyl-CCNU MOPP (and other combined therapies) Mustard gas Myleran Thiotepa Tobacco products and tobacco smoke Treosulfan Vinyl chloride Soots, tars, and oils Coal tar pitches Coal tars Mineral oils, untreated and mildly treated Shale oils Soots Hormones Diethylstilbestrol Estrogens Oral contraceptives Metals Arsenic compounds Chromium compounds Nickel and nickel compounds Fibers Asbestos Erionite Talc-containing asbestos fibers Other 8-Methoxypsoralen + UV radiation

Table 11

Molecular signatures of malignant human cancers.

Human Exposure

Neoplasia type

Predominant mutation (nucleotide base changes)

Cigarette smoke

Lung carcinoma Colon Lung carcinoma Lung carcinoma Hepatocellular carcinoma Skin carcinoma

K-ras, codons 12 and 13 (G-T) K-ras, codons 12 and 13 p53, multiple codons (G-T) p53, codon 249 (G-T) p53, codon 249 (G-T)

Hepatic angiosarcoma Hepatocellular carcinoma

p53, codon 249 (A-T)

Radon Aflatoxin B1 Ultraviolet light Vinyl chloride

p53, dipyrimidine sites (CC-TT)

K-ras, codons 12 and 13 (G-A)

615

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Carcinogenesis Table 12

Molecular signatures of malignant rodent cancers.

Rodent Exposure

Neoplasia type

Predominant mutation (nucleotide base changes)

Methylnitrosourea Aflatoxin B1 Diethylnitrosamine Ultraviolet light Vinyl chloride

Mammary carcinoma (R) Lung carcinoma (M) HCC (M) Skin carcinoma (M) HCC (R)

K-ras, codon 12 (G-C) K-ras, codon 12 (G-C) H-ras, codon 61 (A-G) p53, dipyrimidine sites (CC-TT) H-ras, codon 61 (A-T)

HCC, hepatocellular carcinoma; M, mouse; R, rat.

from persons living in geographic areas with a high exposure to aflatoxin B1, there is a frequent mutation at the third nucleotide pair of codon 249 in the p53 gene, suggesting the mutation is chemical specific and imparts a specific growth or survival advantage to the mutated liver cells. Animal studies have confirmed that there are certain chemical-specific mutational profiles in neoplasias; however, there are many examples where the mutational profile varies by strain (Table 13), species, dose, or dosing regiment. For example, diethylnitrosamine, a strong, cross-species hepatocarcinogen, will induce liver neoplasias in mice, rats, and rainbow trout, but the frequency and type of ras mutation in the neoplasia vary widely, and the mutations are not simply a reflection of direct DNA interaction (Table 14). In some studies, in vitro mutation assays were poor predictors of liver tumor mutation profiles in the mouse. In this complex process, carcinogens might also be influencing by events such as DNA repair, oxidative DNA damage, methylation, cell death, proliferation, and/or a hypermutable state. Molecular epidemiologic studies aiming identify individual’s risk of developing cancer have found that persons with germline mutations in cancer genes (i.e., BRCA1 or BRCA2) or genetic variations (genetic polymorphisms) of carcinogen metabolizing enzyme activities (i.e., cytochrome P450s or glutathione-S-transferases) or DNA repair capacities can present increased risk of tumorigenesis in their lifetime. High throughput analyses to examine single nucleotide polymorphisms (SNPs) are being used to search for biomarkers of cancer risk in individuals, and some of this information is being used to establish preventive measures to decrease carcinogenic risk (Boffetta and Islami, 2013; Chen and Hunter, 2005).

Table 13 Sensitivity to liver tumor development and H-ras codon 61 mutations in spontaneous hepatocellular tumors of various strains of mice. Sensitivity

High Intermediate Low

Strain

Codon 61 mutation

Frequency

C3H B6C3F1 CD-1 C57BL

23/89 183/333 9/36 5/34

(26%) (56%)a (25%) (15%)a

AAA

CGA

CTA

17 106 8 0

3 50 1 1

3 21 0 4

a Occasional mutations in other codons of H- and K-ras. Adapted from Maronpot RR, Fox T, Malarkey DE, Goldsworthy TL (1995) Mutations in the ras proto-oncogene: clues to etiology and molecular pathogenesis of mouse liver tumors. Toxicology 101: 125–156.

Table 14 Species and strain comparisons of mutational profiles induced by diethylnitrosamine (DEN). Animal

CD-1 mouse C3H mouse B6C3F1 mouse C57BL mouse F344 rat Rainbow trout

Frequency of ras mutations

13/25 54/114 63/239 2/59 0/19 6/7

(52%) (26%) (26%) (2%) (0%) (86%)

Type

H- and N-ras H-ras H-ras H-ras K-ras K-ras

Nucleotide base substitutions C-A

A-G

A-T

A-C

G-A

12 28 16 0 0 0

1 24 32 1 0 0

0 2 15 0 0 0

0 0 0 1 0 0

0 0 0 0 0 6

Data adapted partly from Maronpot RR, Fox T, Malarkey DE, Goldworthy TL (1995) Mutations in the ras protooncogene: clues to etiology and molecular pathogenesis of mouse liver tumors. Toxicology 101: 125–156.

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Conclusion All of life is a balancing act of good versus evil and production versus destruction. Similar balancing factors are evident in carcinogenesis where regulatory mechanisms for tissue proliferation are balanced against those for cellular differentiation. It is well established that carcinogenesis requires the accumulation of multiple alterations in the genome of the affected cells. At the genetic level, two opposing classes of genes, oncogenes, and tumor suppressor genes, as well as apoptotic and DNA repair genes, have been implicated in carcinogenic process. In addition, the development of cancer is influenced by host factors such as age, sex, diet, nutrition, general health status, and inherited predispositions for cancer and by complex positive and negative intracellular signaling mechanisms. Treatment of cancer is based on our understanding of the mechanistic underpinnings of the carcinogenic process and attempts to shift the balance of critical factors in favor of patient survival. The probability of developing cancer is directly proportional to the intensity, route, and duration of exposure to cancer-causing factors, as well as genetic susceptibility. Public health strategies are based on the premise that reduction or prevention of exposure to cancer-causing factors will decrease the incidence of cancer.

See also: Carcinogen classification schemes; Carcinogen-DNA adduct formation and DNA repair; Cell proliferation; Chromosome aberrations; Epidemiology; Immune system; International Agency for Research on Cancer; Ionizing radiation toxicology; Mechanisms of toxicity; Molecular toxicology: Recombinant DNA technology; Mouse lymphoma assay; Toxicity testing in the 21st century: Approaches to implementation

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Kontomanolis EN, Koutras A, Syllaios A, Schizas D, Mastoraki A, Garmpis N, Diakosavvas M, Angelou K, Tsatsaris G, Pagkalos A, Ntounis T, and Fasoulakis Z (2020) Role of oncogenes and tumor-suppressor genes in carcinogenesis: A review. Anticancer Research 40(11): 6009–6015. International Institute of Anticancer Research. 10.21873/anticanres.14622. Kumar V, Abbas A, and Aster J (2014) Robbins & Cotran Pathologic Basis of Disease, 9th edn. Philadelphia: Elsevier. Lambert P (2009) Oncogenic viruses. Encyclopedia of Microbiology, pp. 421–429. Elsevier Inc. https://doi.org/10.1016/B978-012373944-5.00308-4. Loeb KR and Loeb LA (2000) Significance of multiple mutations in cancer. Carcinogenesis 21(3): 379–385. Oxford Academic. https://doi.org/10.1093/carcin/21.3.379. Loomis D, Guha N, Hall AL, and Straif K (2018) Identifying occupational carcinogens: An update from the IARC Monographs. Occupational and Environmental Medicine 75(8): 593–603. https://doi.org/10.1136/OEMED-2017-104,944. Madia F, Worth A, Whelan M, and Corvi R (2020) Carcinogenicity assessment: technical and political challenges. European Journal of Public Health 30(Supplement_5). https://doi.org/ 10.1093/eurpub/ckaa165.148. Matsuoka A, Matsui M, Miyata N, Sofuni T, and Motoi I (1990) Mutagenicity of 3-tert-butyl-4-hydroxyanisole (BHA) and its metabolites in short-term tests in vitro. Mutation Research/ Genetic Toxicology 241(2): 125–132. https://doi.org/10.1016/0165-1218(90)90115-I. Mundt KA, Dell LD, Crawford L, and Gallagher AE (2017) Quantitative estimated exposure to vinyl chloride and risk of angiosarcoma of the liver and hepatocellular cancer in the US industry-wide vinyl chloride cohort: Mortality update through 2013. Occupational and Environmental Medicine 74(10): 709–716. https://doi.org/10.1136/oemed-2016-104,051. National Research Council (US) Committee on Comparative Toxicity of Naturally Occurring Carcinogens (1996) Carcinogens and anticarcinogens in the human diet: A comparison of naturally occurring and synthetic substances. In: Methods for Evaluating Potential Carcinogens and Anticarcinogens, vol. 4. Washington, DC: National Academies Press, USA. Nguyen TT, Ung TT, Kim NH, and Do Jung Y (2018) Role of bile acids in colon carcinogenesis. World Journal of Clinical Cases 6(13): 577–588. Baishideng Publishing Group Co. 10. 12998/wjcc.v6.i13.577. Preneoplastic Changes (2011) Encyclopedia of Cancer, p. 2977. Springer Berlin Heidelberg. https://doi.org/10.1007/978-3-642-16,483-5_4723. Rao MS, Lalwani ND, Watanabe TK, and Reddy JK (1984) Inhibitory Effect of Antioxidants Ethoxyquin and 2(3)-tert-Butyl-4-hydroxyanisole on Hepatic Tumorigenesis in Rats Fed Ciprofibrate, a Peroxisome Proliferator. Cancer Research 44(3): 1072–1076. Rivlin N, Brosh R, Oren M, and Rotter V (2011) Mutations in the p53 tumor suppressor gene: Important milestones at the various steps of tumorigenesis. Genes and Cancer 2(4): 466–474. Impact Journals, LLC. https://doi.org/10.1177/1947601911408889. Seyfried TN and Huysentruyt LC (2013) On the origin of cancer metastasis. Critical Reviews in Oncogenesis 18(1–2): 43–73. https://doi.org/10.1615/CritRevOncog.v18.i1-2.40. Singh N, Baby D, Rajguru J, Patil P, Thakkannavar S, and Pujari V (2019) Inflammation and cancer. Annals of African Medicine 18(3): 121–126. https://doi.org/10.4103/aam. aam_56_18. Stewart BW (2019) Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks. Tumor Site Concordance and Mechanisms of Carcinogenesis. https://www.ncbi. nlm.nih.gov/books/NBK570326/. Suhail Y, Cain MP, Vanaja K, Kurywchak PA, Levchenko A, Kalluri R, and Kshitiz (2019) Systems biology of cancer metastasis. Cell Systems 9(2): 109–127. Cell Press. https://doi.org/ 10.1016/j.cels.2019.07.003. van Tong H, Brindley PJ, Meyer CG, and Velavan TP (2017) Parasite infection, carcinogenesis and human malignancy. eBioMedicine 15: 12–23. Elsevier B.V. https://doi.org/10.1016/ j.ebiom.2016.11.034. Varmus H (1988) Retroviruses. Science 240(4858): 1427–1435. https://doi.org/10.1126/science.3287617. Walcher L, Kistenmacher AK, Suo H, Kitte R, Dluczek S, Strauß A, Blaudszun AR, Yevsa T, Fricke S, and Kossatz-Boehlert U (2020) Cancer stem cells—Origins and biomarkers: Perspectives for targeted personalized therapies. Frontiers in Immunology 11: 1280. Frontiers Media S.A. https://doi.org/10.3389/fimmu.2020.01280. Zachary JF and McGavin D (2011) Pathologic Basis of Veterinary Disease, 5th ed. Mosby. Zella D and Gallo RC (2021) Viruses and bacteria associated with cancer: An overview. Viruses 13(6): 1039. https://doi.org/10.3390/V13061039. Zhang C, Liu J, Zhong JF, and Zhang X (2017) Engineering CAR-T cells. Biomarker Research 5(1): 1–6. BioMed Central Ltd. https://doi.org/10.1186/s40364-017-0102-y. Zhao Y, Tan YS, Aupperlee MD, Langohr IM, Kirk EL, Troester MA, Schwartz RC, and Haslam SZ (2013) Pubertal high fat diet: Effects on mammary cancer development. Breast Cancer Research 15(5). https://doi.org/10.1186/bcr3561.

Further reading Benigni R (2012) Alternatives to the carcinogenicity bioassay for toxicity prediction: are we there yet? Expert Opinion on Drug Metabolism & Toxicology 8(4): 407–417. Columbano A, Feo F, and Pani P (eds.) (1991) Chemical Carcinogenesis 2: Modulating Factors. New York: Springer. D’Amato G and Patarca R (1998) General biological aspects of oncogenesis. Critical Reviews in Oncogenesis 9(3–4): 275–373. PMID: 10201632. Diori Karidio I and Sanlier SH (2021) Reviewing cancer’s biology: An eclectic approach. Journal of the Egyptian National Cancer Institute 33: 32. Doktorova TY, Pauwels M, Vinken M, Vanhaecke T, and Rogiers V (2012) Opportunities for an alternative integrating testing strategy for carcinogen hazard assessment? Critical Reviews in Toxicology 42(2): 91–106. Fois SS, Paliogiannis P, Zinellu A, Fois AG, Cossu A, and Palmieri G (2021) Molecular epidemiology of the main druggable genetic alterations in non-small cell lung Cancer. International Journal of Molecular Sciences 22(2): 612. MDPI AG. Grice HC and Ciminera JL (eds.) (1988) Carcinogenicity: The Design, Analysis, and Interpretation of Long-Term Animal Studies. New York: Springer-Verlag. Hsu C-H and Stedeford T (eds.) (2010) Cancer Risk Assessment: Chemical Carcinogenesis, Hazard Evaluation, and Risk Quantification. New Jersey: John Wiley & Sons, Inc. Monk D (2010) Deciphering the cancer imprintome. Briefings in Functional Genomics 9: 329–939. Nowsheen S, Aziz K, Kryston TB, Ferguson NF, and Georgakilas A (2012) The interplay between inflammation and oxidative stress in carcinogenesis. Current Molecular Medicine 12(6): 672–680. Oliveira PA, Colaço A, Chaves R, Guedes-Pinto H, De-La-Cruz PLF, and Lopes C (2007) Chemical carcinogenesis. Anais da Academia Brasileira de Ciências 79: 593–616. Pavanello S and Lotti M (2012) Biological monitoring of carcinogens: Current status and perspectives. Archives of Toxicology 86(4): 535–541. Penning TM (ed.) (2011) Chemical Carcinogenesis. New York: Springer Science þ Business Media. Peters JM, Shah YM, and Gonzalez FJ (2012) The role of peroxisome proliferator-activated receptors in carcinogenesis and chemoprevention. Nature Reviews. Cancer 12(3): 181–195. Sharma S, Kelly TK, and Jones PA (2010) Epigenetics in cancer. Carcinogenesis 31: 27–36. Simic MG, Grossman L, and Upton AC (eds.) (1986) Mechanisms of DNA Damage and Repair: Implications for Carcinogenesis and Risk Assessment. New York: Plenum Press. Wallace TA, Martin DN, and Ambs S (2011) Interactions among genes, tumor biology and the environment in cancer health disparities: examining the evidence on a national and global scale. Carcinogenesis 32: 1107–1121. Waters MD, Jackson M, and Lea I (2010) Characterizing and predicting carcinogenicity and mode of action using conventional and toxicogenomics methods. Mutation Research 705: 184–200.

Relevant website http://monographs.iarc.fr/ :Important Monograph Series.

Cardiovascular system EC Bowdridgea,b, E DeVallancea,b, KL Garnera,b, JA Griffitha,b, PA Stapletonc, S Hussaina,b, and TR Nurkiewicza,b, aWest Virginia University School of Medicine, Morgantown, WV, United States; bCenter for Inhalation Toxicology (iTOX), Morgantown, WV, United States; c Rutgers University-Ernest Mario School of Pharmacy University, Piscataway, NJ, United States © 2024 Elsevier Inc. All rights reserved. This is an update of P.A. Stapleton, T.L. Knuckles, V.C. Minarchick, G. Gautam, T.R. Nurkiewicz, Cardiovascular System, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 730–747, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00985-4.

Introduction Cardiovascular homeostasis The heart The heart as a pump Cardiac muscle Impulse conduction Intrinsic modulators of cardiac activity Pathologic changes in the heart Agents causing morphologic changes The blood vessels Blood flow Pathological changes Artery wall structure Endothelial damage Atherosclerosis and arteriosclerosis Metals Primary amines Cigarette smoke Nicotine Electronic cigarettes Conclusion/summary/outlook References Further reading

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Abstract Cardiovascular toxicology is the study of chemicals that cause untoward effects on the heart or vasculature. This is an ongoing course of study because the toxicants that humans are exposed to is constantly changing, however many of the mechanisms of toxicity remain largely unchanged over hundreds of years. Therefore, this chapter introduces the concept of cardiovascular homeostasis, and how fundamental anatomical and physiological elements make this possible. Lists of common toxicants that target cardiac and vascular mechanisms, and examples of how such agents alter cardiovascular health are discussed. Upon completion of this chapter, the reader should have a general understanding of how certain toxicants influence cardiovascular health, and to distinguish between therapeutic dose/benefit vs untoward outcome.

Keywords Arteriole; Artery; Capillary; Cardiovascular; Endothelium; Homeostasis; Mechanisms; Smooth muscle; Toxicology; Vein; Venule

Key points

• • • •

The cardiovascular system is composed of the heart, arteries, arterioles, capillaries, venules, and veins. Cardiovascular homeostasis is critical to maintaining the stability of an organism. Pesticides, complex mixtures, pollutants, metals, inorganic and organic chemicals, as well as commonly prescribed therapeutic and abused drugs can all be cardiovascular toxicants. Toxicants can cause cardiac dysfunction by affecting cardiac output, pathological and/or morphological changes, blood flow or endothelial damage.

Encyclopedia of Toxicology 4th Edition

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Introduction The central tenet of toxicology was stated by the sixteenth-century physician Paracelsus: “All substances are poisons. There is none that is not a poison. The right dose differentiates a poison and a remedy.” This tenet has withstood the repeated tests of science and time; and over 500 years later, firmly remains the foundation for all toxicological considerations. The scope of this chapter encompasses the toxicities of several classes of chemicals on the cardiovascular system (CVS). These include drugs (therapeutic, and those commonly abused), pesticides, organic and inorganic chemicals, metals, pollutants, and complex mixtures (e.g., combustion exhaust, cigarette smoke, vaping, etc.). The CVS consists of the heart and the vasculature (arteries, arterioles, capillaries, venules, and veins). The lymphatics, while a critical component for normal cardiovascular function, are commonly not included in CVS discussions for a variety of reasons (as it is a parallel system focused on interstitial fluids). Therefore, this chapter focuses on the heart and vasculature because they are either the primary targets, or the major site of effect/consequence after exposure to the toxicants described herein. It begins by defining cardiovascular homeostasis, followed by cardiac anatomy and physiology. Next are examples of toxicants that can alter ion movement, muscle function, and thus, cardiac output. The second part of the chapter begins with a description of the anatomy and physiology of vasculature, followed by examples of specific toxicants. A list of cardiotoxic agents and their mechanisms of toxicity is presented in Table 1. A listing of vasculotoxic agents and related compounds is presented in Table 2. Neither the contents Table 1 General mechanisms of cardiotoxicity; and cardiotoxicity of key pharmaceutical agents, naturally occurring substances used as pharmaceuticals, and selected industrial agents. Mechanism

Cellular perturbations

Organ manifestations

" [Ca2+]i # Conduction velocity # Na+ channel activity # Conduction velocity # K+ channel activity # Repolarization " Action potential duration # L-type Ca2+ channel activity

Positive inotropic effect Proarrhythmic Proarrhythmic

General mechanisms of cardiotoxicity Interference with ion homeostasis Inhibition of Na+/K+ ATPase Na+ channel blockade K+ channel blockade Ca2+ channel blockade

Altered coronary blood flow Coronary vasoconstriction or obstruction Ischemia/reperfusion injury Oxidative stress

Organellar dysfunction Sarcolemmal injury Sarcoplasmic reticulum dysfunction Mitochondrial injury

Apoptosis

Oncosis

Agents

# Ca2+-induced-Ca2+ release # AV conduction Ischemia (ATP depletion, intracellular acidosis) Oxidative stress, " [Ca2+]i intracellular pH change Lipid peroxidation DNA damage Mitochondrial dysfunction, Altered [Ca2+]i homeostasis Altered membrane integrity Altered [Ca2+]i homeostasis ATP depletion Cytochrome c release Altered mitochondrial [Ca2+]i homeostasis Cellular shrinkage Sarcolemmal blebbing Chromatin condensation Redistribution of membrane phospholipids DNA fragmentation Cellular swelling Sarcolemmal blebbing Chromatin clumping Mitochondrial swelling Cardiotoxic manifestations

Proarrhythmic Negative inotropic effect Negative chronotropic effect Bradycardia Myocardial infarction Cardiac myocyte death Cardiac remodeling Cardiac myocyte death Cardiac myocyte death

Cardiac myocyte death Cardiac myocyte death Cardiac myocyte death

Cardiac myocyte death

Cardiac myocyte death

Proposed mechanisms of cardiotoxicity

Arrhythmic—a drug or substance that alters the natural rhythm of the heart; inotropic—toxicants that increase (positive) or decrease (negative) the force of heart muscle contraction; chronotropic—toxicants that increase (positive) or decrease (negative) the number of beats per minute of the heart; and blockade—substances that inhibit receptor function.

Table 2

Cardiotoxicity of key pharmaceutical agents. Category

Adenosine

Antiarrhythmic drugs

Class I (disopyramide, encainide, flecainide, lidocaine, mexiletine, moricizine, phenytoin, procainamide, propafenone, quinidine, tocainide) Class II (acebutolol, esmolol, propranolol, sotalol) Class III (amiodarone, bretylium, dofetilide, ibutilide, quinidine, sotalol)

Antiarrhythmic drugs Antiarrhythmic drugs Antiarrhythmic drugs

Class IV (diltiazem, verapamil)

Antiarrhythmic drugs

Cardiac glycosides (digoxin, digitoxin) Ca2+ sensitizing agents (adibendan, levosimendan, pimobendan)

Inotropic drugs and related agents Inotropic drugs and related agents

Catecholamines (dobutamine, epinephrine, isoproterenol, norepinephrine)

Inotropic drugs and related agents

Bronchodilators (albuterol, bitolterol, fenoterol, formoterol, metaproterenol, pirbuterol, procaterol, salmeterol, terbutaline) Appetite suppressants (amphetamines, fenfluramine, phentermine)

Inotropic drugs and related agents

Anthracyclines (daunorubicin, doxorubicin, epirubicin)

Antineoplastic drug

5-Fluorouracil Cyclophosphamide

Antineoplastic drug Antineoplastic drug 4Hydroxy-cyclophosphamid (metabolite) Antibacterial drugs Decreased [Ca2+]i

Inotropic drugs and related agents

Antibacterial drugs

Fluoroquinolones (grepafloxacin, moxifloxacin, sparfloxacin)

Antibacterial drugs

Tetracycline Chloramphenicol Amphotericin B

Antibacterial drugs Antibacterial drugs Antifungal drug

Flucytosine

Antifungal drugs 5-Fluorouracil metabolite Antiviral drugs

Physiological endpoints Hypertension Cardiomyopathy Ventricular tachycardia Decreased energy production, heart failure Cardiac arrest Atrial arrhythmia Arrhythmia

b-adrenergic receptor blockade K+ channel blockade QTc interval prolongation Ca2+ channel blockade

Bradycardia, cardiac arrest Arrhythmia Ventricular tachycardia Bradycardia Weakened ventricular contraction Arrhythmia Arrhythmia Decreased ventricular filling during relaxation, arrhythmia Tachycardia Ischemia, myocardial infarction (MI) Decreased energy production, heart failure Arrhythmia Heart failure Heart failure Tachycardia (acute) Heart failure (chronic) Tachycardia, pulmonary hypertension Arrhythmia Valvular insufficiency Decreased cardiac output Heart failure Cardiomyopathy Heart failure Arrhythmia Arrhythmia Heart failure Weakened ventricular contraction

Inhibition of Na+, K+-ATPase, increased [Ca2+]i Increased Ca2+ sensitivity, inhibition of phosphodiesterase b1-adrenergic receptor activation Coronary vasoconstriction Mitochondrial dysfunction Increased [Ca2+]i Oxidative stress Apoptosis Nonselective activation of b1-adrenergic receptors Increased serotonin? Na+ channel blockade? Altered [Ca2+]i balance Oxidative stress Mitochondrial injury Apoptosis Coronary vasospasm? Altered ion balance

K+ channel blockade QTc interval prolongation K+ channel blockade QTc interval prolongation Decreased [Ca2+]i Decreased [Ca2+]i Ca2+ channel blockage? Na+ channel blockade? Increased membrane permeability? Coronary vasospasm?

Arrhythmia Ventricular tachycardia Arrhythmia Ventricular tachycardia Weakened ventricular contraction Weakened ventricular contraction Weakened ventricular contraction Arrhythmia

Mitochondrial injury

Cardiomyopathy

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Aminoglycosides (amikacin, gentamicin, kanamycin, netilmicin, streptomycin, tobramycin) Macrolides (azithromycin, clarithromycin, dirithromycin, erythromycin)

Prominent cardiac effects Decreased conductivity Altered [Ca2+]i balance Oxidative stress Mitochondrial injury K+ Hyperpolarization Decreased refractory period Na+ channel blockade

Cardiovascular system

A. Cardiotoxic agents Agents Ethanol

Arrhythmia, cardiac arrest

(Continued )

Table 2

(Continued)

Centrally acting drugs Centrally acting drugs

Phenothiazine antipsychotic drugs (chlorpromazine, thioridazine)

Centrally acting drugs

Other antipsychotic drugs (clozapine)

Centrally acting drugs

General inhalational anesthetics (enflurane, desflurane, halothane, isoflurane, methoxyflurane, sevoflurane)

Centrally acting drugs

Other general anesthetics (propofol)

Centrally acting drugs

Cocaine

Local anesthetics

Ca2+ channel blockade Altered Ca2+ balance, b-adrenergic receptor sensitization Ca2+ channel blockade Altered Ca2+ balance, b-adrenergic receptor sensitization Na+ channel blockade Sympathomimetic effects Coronary vasospasm Altered Ca2+ balance Mitochondrial injury Oxidative stress Apoptosis Na+ channel blockade

Other local anesthetics (bupivacaine, etidocaine, lidocaine, procainamide)

Local anesthetics

Astemizole, terfenadine

Antihistamines

Rapamycin, tacrolimus

Immunosuppressants

Cisapride

Miscellaneous drugs

Methylxanthines (theophylline)

Miscellaneous drugs

Sildenafil Radiocontrast agents (diatrizoatemeglumine, iohexol)

Miscellaneous drugs Miscellaneous drugs

Inhibition of phosphodiesterase Apoptosis

Interleukin-2 (Aldesleukin) Interferon-g (Actimmune)

Naturally occurring substances Naturally occurring substances

Selected industrial agents Toluene (paint products)

Increased nitric oxide synthase expression Increased nitric oxide synthase expression Altered ion balance

Solvents

Carbon tetrachloride Chloroform

Halogenated hydrocarbons Halogenated hydrocarbons

Decreased parasympathetic activity Increased adrenergic sensitivity Altered ion balance Decreased parasympathetic activity Increased adrenergic sensitivity

K+ channel blockade QTc interval prolongation Altered Ca2+ balance K+ channel blockade QTc interval prolongation Altered Ca2+ balance, Inhibition of phosphodiesterase

Decreased energy production, heart failure Decreased energy production, heart failure Arrhythmia, cardiac arrest Ventricular tachycardia Bradycardia, arrhythmia Arrhythmia Ventricular tachycardia Tachycardia, weakened ventricular contraction Ventricular tachycardia Ventricular tachycardia Myocarditis Weakened ventricular contraction Decreased cardiac output Arrhythmia Weakened ventricular contraction Decreased cardiac output Arrhythmia Arrhythmia Tachycardia, strengthened ventricular contraction, and increased blood pressure Ischemia, MI Decreased cardiac output Cardiomyopathy Heart failure Arrhythmia Cardiac arrest Bradycardia Arrhythmia Ventricular tachycardia Decreased cardiac output Heart failure Arrhythmia Ventricular tachycardia Increased cardiac output Tachycardia Arrhythmia Arrhythmia Heart failure Arrhythmia Cardiac arrest Weakened ventricular contraction Cardiomyopathy Arrhythmia Arrhythmia Arrhythmia Weakened ventricular contraction

Cardiovascular system

Tricyclic antidepressants (amitriptyline, desipramine, doxepin, imipramine, protriptyline) Selective serotonin reuptake inhibitors (fluoxetine, citalopram)

Inhibition of mitochondrial DNA polymerase and synthesis Inhibition of mitochondrial ATP synthesis Altered ion balance (Ca2+, K+, Na+) QTc interval prolongation Ca2+ channel blockade Na+ channel blockade QTc interval prolongation Ca2+ channel blockade? QTc interval prolongation QTc interval prolongation

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Nucleotide analog reverse transcriptase inhibitors (stavudine, zalcitabine, zidovudine)

Table 2

(Continued)

Chloropentafluoroethane

Halogenated hydrocarbons

Altered ion balance

1,2-Dibromotetra-fluoromethane e.g., Acetone, methyl ethyl ketone

Halogenated hydrocarbons Ketones

Cadmium, cobalt, lead

Heavy metals

Altered coronary blood flow Decreased parasympathetic activity Increased adrenergic sensitivity Altered ion balance Metal ions are taken up by the cell leading to altered cellular structure Altered Ca2+ balance

(Barium, lanthanum, manganese, nickel) B. Vasculotoxic agents Agents Industrial and environmental agents Allylamine b-Aminopropionitrile Boron Butadiene Carbamylhydrazine Carbon disulfide

Ca2+ channel blockade Sources

Prominent Vascular Effects

Physiological Endpoints

Acrolein and hydrogen peroxide

Smooth muscle cell injury and resultant proliferation Damage to connective tissue Increase in microvascular permeability Blood vessel tumors Tumors of pulmonary blood vessels Endothelial injury

Atherosclerosis

Endothelial damage, clot formation, edema Endothelial permeability, edema Vasoconstriction, increased permeability, edema Arterial lesion in the lung, intimal thickening

Blood clots, atherosclerosis, ischemia, MI Pulmonary edema Impaired vision, blindness

Endothelial damage

Pulmonary fibrosis

Hemorrhage; clot formation Vasoconstriction

Blood clots, hypotension, ischemia Renal failure

Blood vessel inflammation Vasospasm Vasospasm Vasospasm Vasoconstriction

Ischemia, stroke, MI Ischemia, stroke, MI Angina Gangrene Localized ischemia

Synthetic precursor Fumigant/solvent

Antibiotics/antimitotics Cyclophosphamide

Environmental

4-Hydroxy-cyclophosphamid (metabolite)

Pulmonary edema, atherosclerosis

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5-Fluorodeoxyuridine Gentamicin Vasoactive agents Amphetamine Dihydroergotamine Ergonovine Ergotamine Epinephrine

Synthetic precursor

Aortic lesion, atherosclerosis, aneurysm Pulmonary edema, hemorrhage Ischemia, cancer Cancer Impaired vision, blindness, blood clot, ischemia, MI Atherosclerosis Cell membrane damage, vasodilation, oxidative Hypotension, cardiomyopathy stress Impaired q venous blood flow Impaired venous return, hemorrhage, necrosis Hemoglobin methylation Decreased O2 carrying capacity, hypotension Vascular smooth muscle cell proliferation Atherosclerosis Vasoconstriction Acute renal failure Edema Pulmonary edema, hemorrhage Platelet activation Cerebral/pulmonary blood clots Damage to endothelial and smooth muscle cells Pulmonary hypertension, venous occlusion Reduced enzyme function Atherosclerosis Blood vessel tumors Cancer, portal hypertension

Cardiovascular system

Glycerol Hydrogen fluoride Paraquat Pyrrolidine alkaloids Organophosphate pesticides Vinyl chloride Gases Carbon monoxide Nitric oxide Oxygen Ozone

Arrhythmia Weakened ventricular contraction Arrhythmia Cardiac hypertrophy Arrhythmia

Chlorophenoxy herbicides Dimethylnitrosamine Dinitrotoluenes

Decreased cardiac output Arrhythmia Ischemia/MI Arrhythmia

(Continued )

Table 2

(Continued)

Chloroquine Fructose Iodoacetates Anticoagulants Warfarin Clopidorgrel bisulfate (plavix) Radiocontrast dyes Metrizamide; metrizoate Cyanoacrylate adhesives 2-Cyano-acrylate-n-butyl Miscellaneous Aminorex fumarate Oral contraceptives Penicillamine Talc and other silicates Tetradecylsulfate Na Nonsteroidal antiinflammatories (NSAIDs) Cyclooxygenase-2 inhibitors (Vioxx and Celebrex) Aspirin

Vascular damage, occlusion, loss of blood vessel number Vascular damage, occlusion, loss of blood vessel number Vascular damage, occlusion, loss of blood vessel number Vascular damage, occlusion, loss of blood vessel number

Ischemia, angina Coronary artery disease Hypertension, atherosclerosis, aneurysm Ischemia, hypertension Ischemia, angina Impaired vision, blindness Impaired vision, blindness Impaired vision, blindness Impaired vision, blindness

Edema Clot formation

Hemorrhage Blood clot, embolism, ischemia

Coagulation; cell death

Blood clots, kidney failure

Cell adhesion

Blood clots

Intimal and medial thickening Clot formation Vascular lesion Clot formation Clot formation

Pulmonary hypertension Blood clots, ischemia, stroke, MI Kidney failure Blood clots Deep-vein thrombosis, pulmonary embolism

Platelet aggregation, clot formation Inhibition of platelet aggregation

Embolism, ischemia, MI Hemorrhage

Cardiovascular system

Norepinephrine Metabolic affectors Alloxan

Tobacco

Vasospasm, endothelial damage Intimal proliferation, vascular occlusion Systemic endothelial and smooth muscle cell dysfunction Reduced smooth muscle responsiveness Vasospasm, endothelial damage

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Histamine Methysergide Nicotine Nitrites and nitrates

Cardiovascular system

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of this chapter nor the material in the tables is intended to be exhaustive or fully inclusive. The agents noted here serve as common or representative cardiotoxic and vasculotoxic agents. There are detailed articles in specialty texts that discuss these and other agents more fully. An updated bibliography is presented under “further reading” and “additional reading.”. Where applicable, it is important to keep in mind that the toxicological manifestation associated with the chemicals, drugs and medical devices, must not overshadow their related health and therapeutic benefit.

Cardiovascular homeostasis Homeostasis is the regulation of an internal environment in order to maintain stability. The preeminent French physiologist Claude Bernard introduced the notion that the blood (and lymph fluid), which bathe all mammalian tissues, constitute the milieu interne or internal milieu of the greater organism. The CVS functions primarily to transport nutrients (oxygen, glucose) and wastes (carbon dioxide) to and from (respectively) all tissues in the body. Equally important CVS functions include cellular communication/feedback, temperature regulation, fluid and ion balance, and host defense. Hence, cardiovascular homeostasis refers to the regulation of these collective functions, which serve to maintain the internal milieu; thereby, maintaining the stability of the organism. It is these functions that are directly and indirectly impacted by exposure to various toxicants. Therefore, an understanding of the impacted components or tissues is necessary first if we are to comprehend the consequences of toxicant exposure on human health (Penn and Murphy, 2005; Stapleton et al., 2014).

The heart The heart as a pump To maintain cardiac homeostasis, blood must flow continuously throughout the body. Blood flow is proportional to blood pressure. Blood pressure is generated by heart muscle contractions, or pumping. The mammalian heart (Fig. 1) is a dual pump (left and right) that normally operates through a tightly controlled conduction of electrical impulses that ultimately produce cardiac contractions in a continuous rhythm. This creates two independent circuits: the pulmonary and systemic circuits. Each pump consists of two connecting chambers – an atrium and a ventricle – which contract in sequence to provide pressure (or force) via their concerted pumping action. The atria function primarily as reservoirs for blood between contractions, whereas the ventricles are responsible for pumping the blood through the circuits, have thick muscular walls, and are located beneath the thinner-walled atria. The atria also force the last volume of blood into the ventricle necessary for more efficient pumping. In a normal cardiac cycle, the atria contract first, and the ventricles contract second (while the atria relax). To ensure one-way flow during alternating contractions, the heart is equipped with specialized valves. The atrioventricular (AV) valves prevent backflow of blood into the atria during ventricular contraction (systole), and the aortic/pulmonary (semilunar) valves prevent backflow of blood into the ventricles during ventricular relaxation (diastole). During systole, the two ventricles develop pressure and eject blood into the pulmonary artery and aorta. At this time the AV valves are closed and the semilunar valves are open. The semilunar valves are closed and the AV valves are open during diastole. The right atrium receives blood flowing from the systemic venous system via the superior and inferior vena cava. This blood initially passes passively through the right AV orifice directly into the right ventricle. An atrial contraction then propels a small volume of additional blood into the right ventricle (mentioned above). A ventricular contraction closes the AV valve allowing blood now to be propelled past the pulmonary valve into the pulmonary circuit. As blood flows through the pulmonary vasculature, carbon dioxide in the venous-return blood is exchanged for oxygen (gas exchange occurs down concentration gradients via passive diffusion) so that the blood pumped through the next (systemic) circuit to the rest of the body will be properly oxygenated. The left atrium receives freshly oxygenated blood from the pulmonary vasculature via the pulmonary vein. Again, blood traverses the AV orifice until an atrial contraction provides complete filling of the ventricle and closes the AV valve. The strong contraction of the thick-muscled left ventricle now opens the aortic valve, allowing blood to flow into the systemic circulation (under high pressure) via the aorta. In the absence of injury and/or disease, the heart is a very efficient, durable, and reliable pump. In the 80-year life span of a person, and at a contraction rate of 72 beats per minute, a heart will beat 3.000,000,000 times. Two major features of the heart contribute to its unique characteristics: the nature of the heart muscle and the specialized electrical conduction system of the heart (Penn and Murphy, 2005; Stapleton et al., 2014).

Cardiac muscle There are three distinct types of muscle tissue in vertebrates: striated, smooth, and cardiac. Striated, or skeletal muscle is attached, at least at one end, to the skeleton via tendons. This muscle type is often referred to as the voluntary muscle, as it can be consciously controlled by the somatic nervous system. Smooth muscle is usually arranged in sheets or layers in tubular systems, such as arteries and veins (see Section “Blood vessels”), the gastrointestinal and respiratory tracts, and the genitourinary tracts. The activities of the smooth muscles are not under conscious control; rather they are coordinated by the autonomic (involuntary) nervous system. The cardiac muscle comprises the bulk of the heart wall proper; and small amounts are found in the superior vena cava and pulmonary vein. The cardiac muscle is not under conscious control; it has an automaticity center which is influenced by the autonomic nervous system (see Section “Impulse conduction”). In the heart, cardiac muscle cells are joined in a network of fibers and are connected by

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Cardiovascular system

(A) Aorta Ligamentum arteriosis Pulmonary semilunar valve Superior vena cava Left pulmonary arteries and veins

Right pulmonary arteries

Left atrium Mitral valve Chordae tendineae

Right atrium

Intercalated disk

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Papillary muscles

Cusp of right AV (tricuspid) valve Right ventricle

Left ventricle Inferior vena cava Descending aorta

Striated myocardial cells are joined by intercalated disks.

(B)

SA node Left atrium

Bundle of His

Right atrium Left ventricle

AV node Right ventricle

Right and left bundle branches

Purkinje fibers

Fig. 1 (A) Anatomy of the heart. (B) Conduction system of the heart. (A) Reproduced from Carroll RG (2006) The Heart. Elsevier’s Integrated Physiology 66; (B) Reproduced from Goldman L and Ausiello D (2008) Electrocardiography. Cecil Medicine, 23rd edn., 230.

gap junctions, which facilitate the conduction of electrical impulses through the cardiac muscle network. This is referred to as a functional syncytium. In addition to cardiac myocytes, there are specialized cardiac conducting cells that initiate, attenuate, or accelerate the electrical impulses for coordinated contraction of the cardiac network. Proportionally speaking, cardiac myocytes make up 99% of the contracting heart mass, whereas the conducting cells make up 1%. Despite their small percentage, numerous toxicants have robust effects on these conducting cells, and this results in significant myocyte dysfunction that can result in fatalities (Penn and Murphy, 2005; Stapleton et al., 2014).

Impulse conduction The specialized electrical conduction system of the heart allows for the synchronous contraction of the left and right sides of the heart and the sequential contraction of the atria and ventricles (Fig. 1B). Electrical impulses most quickly arise in the spontaneously

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firing cells of the sinoatrial (SA) node commonly called the ‘pacemaker.’ The SA node is located at the junction of the superior vena cava and the right atrium. A wave of depolarization (see below) originating at the SA node is conducted first to the cells of the right atrium, then to the cells of both atria, finally converging on a second group of specialized cells – the cells of the AV node. These cells act as a conduit for the original impulse from the SA node to the AV node, which lies at the junction of the median wall of the right atrium and the septum separating the two ventricles. From the AV node, the impulse wave next passes into the ventricular conduction system – the bundle of His and Purkinje fibers – located within the ventricular septum, which allows for the depolarization of ventricular muscle (Penn and Murphy, 2005; Stapleton et al., 2014). If a microelectrode is inserted into a resting muscle or nerve cell (termed ‘excitable tissue’), an electrical potential difference will be recorded across the membrane of that cell. In the case of cardiac muscle cells, this resting potential is −90 mV (intracellular relative to extracellular). In other words, the cell membrane is electrically polarized with the inward facing surface of the membrane having a net negative charge with respect to the outer facing surface of the membrane. This polarity is maintained primarily by the presence of extracellular positively charged ions and intracellular negatively charged proteins. The flux of ions through active (requiring cellular energy) and passive (concentration-driven) processes is responsible for changes in electrical potential. In the resting cardiac muscle cell, the concentration of potassium ions (K+) is higher inside the cell than outside, while sodium ions (Na+) are at a much higher concentration outside the cell than inside. Cellular energy is required to maintain the appropriate resting state distributions of the different ions across the cell membrane. In the case of K+ and Na+ ions, there is a cell membrane pump, which requires energy derived from the hydrolysis of the terminal phosphate group from adenosine triphosphate (ATP). The associated enzyme responsible for this hydrolysis is the Na+–K+ ATPase. When an electrical stimulus is received by a cardiac muscle cell, voltage-gated channels in the cell membrane open allowing Na+ to diffuse down its concentration and electrical gradients into the cell. This influx of positive charge causes the cell membrane to become ‘depolarized’ (i.e., to have less negative charge). As depolarization proceeds, the membrane may reach the threshold potential (−70 mV for most cardiac muscle cells). Any further depolarization results in a phenomenon known as the action potential, which completely depolarizes the cell. At the peak of the action potential, the inside of the cell actually becomes positive relative to the outside (+30 mV). The cell membrane then repolarizes relatively slowly and reaches the −90 mV resting potential before it can respond to another electrical impulse. The wave of depolarization moves very rapidly across the membrane of an individual cardiac muscle cell. In addition, the wave of action potentials is propagated to adjacent cells via the specialized gap junctions. This propagation allows for the complete depolarization of most cells in the network, thus initiating the contraction of the heart muscle as a group. Cardiac muscle cells predominantly display a fast response action potential (Fig. 2), and cells in the atria and ventricles exhibit a rapid conduction velocity due to the gap junctions. The depolarization–action potential–repolarization process is divided into five phases. Phase 0 begins when the threshold potential has been reached. At this time, many ‘fast’ Na+ channels in the cell membrane open allowing an inrush of Na+ ions to initiate the action potential. At the end of phase 0, the cell is completely depolarized. Toward the end of phase 1 and the start of phase 2, the Na+ influx begins to decrease, as does the membrane potential. During the relatively long (200–300 ms) phase 2 plateau, calcium (Ca2+) and Na+ ions enter through ‘slow’ membrane channels. Movement of ions through these ‘slow’ channels only takes place after the membrane potential has dropped to approximately −55 mV, that is, after the ‘fast’ Na+ ion current has ceased. While these ‘slow’ inward currents occur, there is also a slow outward movement of K+ ions which keeps the plateau relatively steady. The Ca2+ influx of phase 2 triggers the process known as excitation–contraction coupling, in which the myosin thick filaments slide past the thin actin filaments in the contractile unit of the muscle known as the sarcomere. This process requires energy and involves activation of a myosin ATPase that hydrolyzes ATP. The released energy is utilized to form cross-bridges between the actin and myosin molecules. Both the velocity and the force of contraction are dependent on the amount of Ca2+ ions that reaches the site of contraction. Within the resting muscle cell, Ca2+ is sequestered in a compartment called the

Ito

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+30

K+

Ca2+

Ica.L

0

–30

Na –60

–90

+

INa

K+

IK

200 msec

K+ IK1

Fig. 2 The principal ionic movements during the different phases of the action potential in a cardiac muscle cell. Reprinted with permission from De Canterina R et al. (2003). Antiarrhythmic effects of omega-3 fatty acids: From epidemiology to bedside. American Heart Journal 146(3): 240–430. http://www.sciencedirect. com/science/article/pii/S0002870303003272.

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sarcoplasmic reticulum. During the action potential, Ca2+ and Na+ ions that enter the cell cause depolarization of the sarcoplasmic reticulum membrane, resulting in the release of large amounts of Ca2+, which are needed for effective contraction of the sarcomere. Between contractions, Ca2+ is once again sequestered in the sarcoplasmic reticulum so that the actin–myosin interaction is not overly prolonged. During the long duration of the plateau phase, a new action potential cannot be initiated because the ‘fast’ Na+ channels are inactivated or refractory to further electrical stimulation. During phase 3, membrane permeability to K+ increases and the ‘slow’ Ca2+ and Na+ channels become inactive. The ensuing efflux of K+ ions allow for repolarization of the membrane until the normal resting potential is reached (phase 4). In contrast, conduction velocity is slow in muscle fibers at the SA and AV nodes. Unlike the majority of cardiac muscle cells, these pacemaker cells have an unstable resting potential (approximately −60 mV) due to a cell membrane alteration that allows Na+ ions to leak into the cell without a concurrent K+ ion efflux. This Na+ leakage reduces the membrane potential allowing even more Na+ ions to move into the cell. In addition to the inward Na+ movement, there is also an inward Ca2+ flow which causes the pacemaker cells to have a more positive resting potential. Finally, the cell produces an action potential at approximately −40 mV. This phenomenon is called spontaneous diastolic depolarization. The overall effect is that pacemaker cells initiate waves of depolarization that move across the heart causing the muscle to contract. As noted previously, this phenomenon occurs 72 times per minute (more or less depending on autonomic nervous system stimulation, periods of stress, or physical activity). The SA node is responsible for this rate as it depolarizes the fastest. The other nodes and components of the cardiac conduction may also drive depolarizations, only slowly. The purpose of this redundancy is to ensure pacemaker activity in the heart (to support cardiac homeostasis). The waves of electrical activity may be recorded in an electrocardiogram (ECG), which displays the net electrical changes relative to where the recording electrodes are placed on the surface of the body (Penn and Murphy, 2005; Stapleton et al., 2014).

Intrinsic modulators of cardiac activity The heart responds constantly to hormonal and nervous system signals. Sympathetic nervous system terminals releasing norepinephrine are found in cardiac cells of the atria and ventricles. This allows for reflex regulation of heart muscle contractility. Sympathetic innervation is also present to the SA node and AV junction, where norepinephrine release acts to increase heart rate (enhanced phase 4 depolarization) and also to increase conduction velocity by reducing the AV junction impedance to conduction. Sympathetic innervation also occurs down to the resistance arterioles functionally increasing total peripheral resistance upon stimulation. Parasympathetic innervation is provided by cranial nerve 10, the vagus nerve, to the SA node and the AV junction. These fibers release acetylcholine, which slows SA node activity (decreasing the rate of phase 4 depolarization) and decreases conduction throughout the AV junction (Penn and Murphy, 2005; Stapleton et al., 2014). A practical example of normal nervous system regulation of cardiovascular activity is the processes of blood pressure regulation via baroreceptors located in the aortic arch and carotid arteries. Baroreceptors sense mechanical deformation when these arterial walls stretch and relax. These receptors send impulses to the cardiovascular regulatory sensors in the medulla (nucleus tractus solitarii) where dual reflex impulses are generated that reduce sympathetic signaling and increase parasympathetic outflow (vagus nerve) that ultimately decrease heart rate contractility, peripheral vascular resistance, and venous return. This results in a decreased blood pressure. Conversely, a decrease in blood pressure will decrease vagal stimulation in favor of sympathetic input. The sympathetic reflex is characterized by increases in heart rate, myocardial contractility, venous return, peripheral vascular resistance, and cardiac output. In addition, the sympathetic response can be produced by the release of naturally occurring catecholamines (epinephrine and norepinephrine) from the medulla of the adrenal gland (Penn and Murphy, 2005; Stapleton et al., 2014).

Pathologic changes in the heart The major pathologic changes that occur in the heart are associated with effects on heart rate, contractility of heart muscle, or electrical conduction. Regarding heart rate changes, an arrhythmia, as the name indicates, is a loss of rhythm and here refers to an irregularity of the heartbeat. Two of the more common forms are tachycardia, which is an abnormally rapid heartbeat, and fibrillation, which is a rapid twitching of the muscle fibrils. Either of these can occur in the atria or the ventricles. Agents that alter ion levels and fluxes and alter aspects of impulse transmission can produce arrhythmias. The most common site of arrhythmic impulse generation is the SA node. If depolarization after an action potential is accelerated or delayed anywhere within the heart, an aberrant action potential can be triggered and result in an arrhythmia (Penn and Murphy, 2005; Stapleton et al., 2014). Still other pathologic changes are associated with effects on the force of contraction. The heart muscle exhibits a higher rate of oxygen consumption and a greater energy requirement than many other tissues. Thus, impaired contraction can result from interference with any of the major cycles critical for proper energy metabolism or from processes that interfere with delivery or utilization of the optimum levels of oxygen. For example, if blood flow through the coronary arteries is occluded, as occurs during atherosclerosis, compromising blood flow and leading to a decreased delivery of oxygen to the heart muscle. This decrease in blood flow, reducing vital nutrient delivery, is termed ischemia. When the heart becomes ischemic, a patient may feel significant discomfort as heart or chest pain, termed angina. When this occurs acutely and to the point of cell (cardiac myocyte) death, a myocardial infarction (MI) may result, leading to devitalization of a segment of the heart musculature. Even if death does not occur, there will likely be a decrease in the force or efficiency of contraction of the heart muscle. ‘Recreational’ use of psychoactive drugs

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(e.g., amphetamines, cocaine) can result in profound and sudden cardiovascular responses including increases in blood pressure and heart rate due to acute catecholamine release in response to the drugs. These effects can be life threatening in individuals with underlying, and possibly previously unknown, cardiovascular problems including coronary artery disease, high blood pressure, or cerebrovascular disease (Penn and Murphy, 2005; Stapleton et al., 2014). Cells with high-energy requirements, such as cardiomyocytes, have large numbers of organelles called mitochondria, which produce and supply ATP. Enzymes are organic catalysts that interact with specific substrate molecules to help speed up chemical reactions. The ATPases are enzymes that catalyze the hydrolysis of ATP with its attendant release of energy, which is made available for cellular processes. The myosin ATPase involved in muscle contraction was mentioned above and ATPases involved in the energy-driven pumping of ions including Na+, K+, and Ca2+ were mentioned above and are noted again below. During oxidative metabolism of organic substrates, the process of electron transport to molecular oxygen in mitochondria is coupled to oxidative phosphorylation, which yields ATP. Some poisons and anticancer drugs, such as cyanide and doxorubicin, interfere with electron transport and/or uncouple phosphorylation. This causes a direct decrease in the amount of energy available to the heart muscle and results in reduced contractility (Penn and Murphy, 2005; Stapleton et al., 2014). As noted above, the inward Ca2+ ion movement is vital for the contraction of the cardiac muscle. This inward movement is blocked by Ca2+ antagonists, such as cobalt and barium, and is stimulated by catecholamines. Increased Ca2+ influx leads to increases in the intracellular level of cyclic adenosine monophosphate (AMP), a compound that helps mediate numerous metabolic responses within cells. This, in turn, leads to increased availability of Ca2+ ions for interaction with the contractile proteins. The same effect can be achieved by increased levels of free Ca2+ ions outside of the cells or increased levels of cyclic AMP within cells, as is seen with the vasodilating drug papaverine. Another mechanism for increasing intracellular Ca2+ levels in cardiac cells involves the cardiac glycoside drugs, for example, digitalis from the foxglove plant. This drug inhibits the ATPase that pumps Na+ ions out of cells. This results in elevation of Na+ ion levels inside the cell, which in turn leads to increases in intracellular Ca2+ ion levels and therefore increased rate and strength of contraction. Toxins that increase the permeability of the cardiac muscle cell membrane to the Na+ ion, for example, the marine compound, ciguatoxin or the Columbian frog poison active agent, batrachotoxin, have a similar effect. On the contrary, agents that decrease membrane permeability to Na+ ions will depress myocardial contractility. Included here are a diverse group of compounds including tetrodotoxin, from the Japanese pufferfish; the shellfish-derived poison, saxitoxin; and polyethylene glycol, the active ingredient in many antifreeze preparations. Local anesthetics such as lidocaine and procaine depress the fast inward Na+ ion current, the slow Ca2+ ion inward current, and the K+ ion outward current. They tend to slow the heart rate and the force of contraction; thus, they are commonly used as antiarrhythmic drugs (Penn and Murphy, 2005; Stapleton et al., 2014). There are compounds that interfere with the regular activity of Ca2+ ions in cardiac cells, either by replacing them (as is the case with a number of heavy metals) or by altering the flux of Ca2+ ions across the cell membrane. Among metals, lanthanum, manganese, and nickel all block Ca2+ channels in the cell membrane. Both barium and cobalt ions antagonize endogenous Ca2+ ion levels and tend to shorten the action potential. Lead ions have multiple effects, including displacement of Ca2+ and interference with Ca2+ ion availability, energy metabolism, and ATP synthesis in heart muscle cells. Among organic chemicals, the opium derivative, papaverine, also blocks slow Ca2+ ion channels. Cardiotoxins such as cobra venom and bacterial endotoxins both interfere with Ca2+ ATPase activity; but endotoxin also depresses Ca2+ uptake by heart muscle cells (Penn and Murphy, 2005; Stapleton et al., 2014). Drugs prescribed to alleviate one set of medical problems can have striking and sometimes fatal effects on the cardiac system. Antipsychotics derived from phenothiazine, including chlorpromazine, depress myocardial contractility and cardiac output. Chlorpromazine can also impair cardiac reflex mechanisms and cause a focal myocardial necrosis. Cyclophosphamide, an anticancer agent, also causes myocardial necrosis as well as changes in ECG patterns. Another anticancer agent, doxorubicin, can produce cardiomyopathies with subsequent congestive heart failure. Severe dysrhythmias and some cases of sudden death have been reported. Overdoses of the tricyclic antidepressants, for example, amitryptaline, can result in severe cardiotoxicity, probably due to anticholinergic activity. At high doses, the antidepressant imipramine will depress contractility, lower heart rate, and depress cardiac output. Cardiac arrest may also occur. Some antibiotics, including gentamycin and neomycin, depress Ca2+ ion uptake and therefore reduce contractility of the cardiac muscle. Although the sympathetic system transmitters, the catecholamines, are essential for maintenance of normal myocardial contractility, it has been long recognized that when administered at higher than normal levels for extended periods of time, they can lead to severe myocardial necrosis (Penn and Murphy, 2005; Stapleton et al., 2014). In addition to inducing direct cardiac toxicity, many drugs increase the time between depolarization and repolarization of the left and right ventricles. This increase in time is referred to as QT interval prolongation (time from the start of the Q wave to the end of the T wave on an ECG) (Fig. 3). Common classes of drugs that induce QT interval prolongation are the tricyclic antidepressants, fluoroquinolones among others (Table 1). QT interval prolongation is a sensitive biomarker for risk of developing a fatal arrhythmia, torsades de pointes (a.k.a. polymorphic tachycardia). In addition to being a measurement for risk of developing sudden cardiac death, QT prolongation, has been used as an important screening tool for the evaluation of pharmaceutical drugs in development (Penn and Murphy, 2005; Stapleton et al., 2014). Profound cardiotoxic responses can result from inhalation of a number of halogenated alkanes. These are low molecular weight hydrocarbons with some or all of the hydrogen atoms being substituted by halogens, usually chlorine or fluorine. These agents depress heart rate, contractility, and electrical conduction. The effects are generally more pronounced as the number of halogen atoms increases. Some of these compounds have the additional and profound effect of sensitizing cardiac muscle cells to catecholamines. In humans without preexisting cardiac disease, the effects of most of these compounds are reversible, although

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R T

P

J

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PQ QRS QT

Fig. 3 EKG schematic. Reproduced from Farkas A et al. (2004) How to measure electrocardiographic QT interval in the anaesthetized rabbit. The Journal of Pharmacological and Toxicological Methods 50(3): 175–185. http://www.sciencedirect.com/science/article/pii/S0002870303003272.

chronic exposure may cause some irreversible damage. As would be expected, the older halogenated hydrocarbon anesthetics such as halothane and enflurane had similar effects (Penn and Murphy, 2005; Stapleton et al., 2014). In contrast, low-pressure fluorocarbons, such as trichlorofluoromethane, can be particularly toxic. In most cases, the levels generally encountered in the environment are too low to have any major lasting effect and even at relatively high levels (up to 15%) fatalities are rarely recorded. However, at levels much above this, for example, over 20%, tragic results can ensue. Among people who inhale these agents from closed bags to ‘get high,’ fatalities can result because the levels of these agents in the bags can reach 35–40% (Penn and Murphy, 2005; Stapleton et al., 2014).

Agents causing morphologic changes A number of cardiotoxic compounds have been listed to this point, including some that interfere with Na+/K+ ATPases; increase Na+ or Ca2+ influx; or depress myocardial function by replacing Ca2+, decreasing Na+ permeability, or altering contractility. These agents produce toxic responses in the heart muscle often resulting in death, but do so without causing any major morphologic changes in the heart. Other cardiotoxic compounds produce characteristic morphologic lesions in the heart muscle. There are a few basic types of such pathologic alterations, such as toxic myocarditis and MI. Chemicals, which produce toxic myocarditis cause cell damage and, ultimately, cell death. Whether or not they produce damage acutely or chronically is generally a function of the toxicant dose. The acute myocarditis is characterized by edema (accumulation of excess fluid), inflammatory cell responses, and multiple regions of cardiac cell death. However, the inflammatory response will be attenuated or may be absent if the toxic agent suppresses the immune system, for example, drugs given to prevent rejection of transplanted organs (Penn and Murphy, 2005; Stapleton et al., 2014). MI in the heart arises from a sudden insufficiency or local arrest of the blood supply to the heart that can result in necrosis of a region of the heart. In advanced arteriosclerosis, occlusion of the major arteries supplying the heart muscle with blood can result in an MI. Even in the absence of arteriosclerosis, MIs can result, for example, from amphetamine abuse, which produces severe inflammations of critical arteries (i.e., an arteritis). Intravenous drug use can cause infective endocarditis (an inflammation of the internal lining of the heart), which can lead to vessel occlusion with an embolus, thus resulting in an MI. Cocaine abuse can result in ventricular tachycardia (i.e., rapid heartbeat) and fibrillation, MI, and sudden death. At higher doses, cocaine can increase the levels of catecholamines, ultimately resulting in increased Ca2+ ion activity, accelerated heartbeat, arrhythmias, etc. Chemicals that antagonize Ca2+ ion movement through Ca2+-specific membrane channels prevent the ventricular arrhythmias induced by cocaine. Gross MI can result from toxic exposures to carbon monoxide, nitrates, ergot derivatives, and some potent anticancer drugs (see above) (Penn and Murphy, 2005; Stapleton et al., 2014). Media attention and Food and Drug Administration (FDA) guidelines have focused on the cardiovascular effects of appetite-suppressant drugs and nutraceuticals or herbal medicines. In 1997, the antiobesity drugs fenfluramine and dexfenfluramine were withdrawn from the United States sales market due to convincing correlations made between drug usage and cardiac valvular abnormalities. Since then, the deleterious morphologic effects of these drugs have been described as valvular encasement and/or endocardial fibrosis. These lesions can have life-threatening consequences, including progressive aortic valvular regurgitation. The mechanism underlying the pathogenesis of these lesions remains unclear. There appears to be a correlation with increased serotonin levels in the blood and endocardial fibrosis; thus, there is speculation that these drugs may increase serotonin levels or may increase sensitivity of tissues to serotonin. In addition, the dietary supplement ephedra (ma huang) has been implicated in cases of MI due to coronary artery vasoconstriction and sudden cardiac death; thus, in 2004, the FDA issued a warning about ephedra with a ban on its sale and use in the United States (Penn and Murphy, 2005; Stapleton et al., 2014). Another type of gross morphologic lesion in the heart muscle is hypersensitivity myocarditis. This is an inflammatory response that is the most common type of heart disease associated with therapeutic drug use. There are five primary clinical criteria for

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diagnosis of this condition: (1) previous use of the drug(s) without deleterious incidents; (2) no apparent relationship between the size of the drug dose and the hypersensitivity response; (3) clinical symptoms consistent with responses to allergens or infectious disease agents; (4) independent confirmation of immunologic responses; and (5) persistence of the symptoms as long as drug use is continued. Histologically, there is infiltration of the heart muscle with numerous types of white blood cells and this is associated with local regions of lysis of the cardiac muscle cells. However, gross fibrosis and extensive regions of myocardial necrosis are usually absent. Among the drugs that have been reported to elicit this response are the antibiotics penicillin, streptomycin, ampicillin, tetracycline, and sulfadiazine (Penn and Murphy, 2005; Stapleton et al., 2014).

The blood vessels The second part of the CVS is composed of the blood vessels, which are an extensive series of tubular conduits of varying diameters. All but the narrowest of these vessels have a complex wall structure (see below). One major group of vessels, the arteries, distributes blood under various degrees of pressure to all parts of the body. A second major group of vessels, the veins, returns the blood to the heart. With the exception of the pulmonary artery, which brings blood from the heart to the lungs, the arteries carry blood that is more oxygenated than the blood in their venous counterparts. The large- and medium-sized arteries and veins share the same general structure, although the thicknesses of specific cell layers as well as the cell density within layers can vary considerably (Penn and Murphy, 2005; Stapleton et al., 2014).

Blood flow Despite the system’s vital function of transporting blood throughout the body, it would be overly simplistic to view the vascular system as merely a series of pipes of varying diameter. When the left ventricle contracts to deliver blood to the aorta, the largest artery in the body, not only is the blood pressure generated at contraction relatively high, but it is also maintained at a moderately high pressure between contractions of the heart. If the arteries were a set of rigid pipes, the pressure in the artery system would fall to zero between contractions. The fact that this does not occur is due chiefly to the presence of numerous elastic layers (composed of the protein elastin) in the largest arteries. As the heart contracts, the blood pumped into these large arteries causes the elastin in the walls to stretch. Following contraction, the semilunar valves close (see description of valves above) and the walls of the elastic arteries contract passively to maintain pressure within the system until the ventricles fill and contract once again. Overall, this expansion and passive contraction is termed vascular compliance. There are large, elastic arteries, which function primarily to maintain the pressure within the arterial system during diastole, the resting phase of heart contraction. This allows for steady flow to downstream capillaries, despite the inconsistent pressure highs (systole) and lows (diastole) at each heartbeat. There are also muscular arteries, which function primarily to distribute the blood throughout the body to organs and tissues, each of which may require different amounts of blood. To help ensure that appropriate volumes of blood are delivered on demand, the size of the lumen (the space through which the blood flows) of the muscular arteries must be regulated quickly and reliably. Any delay or interruption to this arteriole regulation or delivery would be termed vascular dysfunction and could lead to tissue or organ damage downstream. This is accomplished systemically via innervation by sympathetic fibers of the autonomic nervous system and by local vasoactive chemical signals at the tissue level (Penn and Murphy, 2005; Stapleton et al., 2014). Because capillary walls are thin (to permit diffusion) the blood that is delivered to them must be delivered under reduced pressure. This is accomplished by the arterioles, which combine relatively muscular walls with a narrow lumen. The arterial blood pressure is a function of cardiac output and the total peripheral vascular resistance, which is primarily a function of the degree of normal tension (tone) of the smooth muscle cells in the arteriolar walls. Arteriolar tone is a unitless value with 100% tone representing complete vessel closure/constriction, and 0% representing a maximally dilated vessel. Broadly speaking, arterioles typically maintain a partial state of tone (40–60%) to be able to maintain tissue homeostasis, by constricting or dilating as needed. Tone is under the influence of many intrinsic and extrinsic factors such as the autonomic nervous system (almost exclusively sympathetic activity), circulating hormones (catecholamines), tissue metabolic state (oxygen and carbon dioxide), temperature, and physical forces (blood flow shear stress). If tone increases above the normal range and remains so for extended periods of time, hypertension (high blood pressure) can result (Penn and Murphy, 2005; Stapleton et al., 2014). From the capillaries, blood flows first into the narrowest members of the venous system, the collecting venules, and then into the muscular venules, whose diameter is approximately twice as large and whose walls contain one or two layers of smooth muscle cells. Blood then flows into progressively larger veins, first to the small and then to the medium-sized veins. Veins that are located deep within tissue tend to have thinner, less muscular walls than do superficial veins. The final set of veins to receive blood before it is delivered back to the heart are the inferior and superior vena cava. The outermost cellular layer in these veins is considerably thicker and the innermost layer is considerably thinner than those of the aorta, the first artery leaving the heart. Another difference between arteries and veins is that the latter have a more extensive vasa vasorum, an arterial blood supply to the vessel wall. Since venous blood is relatively poorly oxygenated, veins require supplemental oxygenation supplied by the vasa vasorum. Because venous blood is under low pressure, the vasa vasorum can penetrate closer to the innermost layer of the vein without being occluded by compressive pressures in the wall (Penn and Murphy, 2005; Stapleton et al., 2014).

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Pathological changes Approximately 90% of the pathologic alterations seen in veins are associated with one of three conditions: deep-vein thrombosis, which often appears following acute MI, thrombotic strokes and/or major surgery; varicose veins, which usually arise secondary to sustained increases of venous pressure; and superficial thrombophlebitis, which occurs in humans with varicose veins as well as in some females after pregnancy. A few venotoxic responses to exogenous (i.e., from outside the body) agents are noted below; however, the great majority of vasculotoxic agents have their effects on the arteries. Therefore, only a description of the artery wall structure is presented below along with listings and selected descriptions of agents that damage the arteries. The majority of these vasculotoxic agents affect vascular compliance or arteriole regulation, resulting in an inappropriate distribution of blood flow. There are many normal physiological processes, which also effect vascular compliance and may compound toxicological exposure, including aging, hypertension, and atherosclerosis. Additionally, exposure to toxicological agents (e.g., carbon disulfide or methysergide; see Table 2 for additional examples) may compound existing disease or hasten disease progression (Penn and Murphy, 2005; Stapleton et al., 2014).

Artery wall structure There are three principal layers (tunics) that have been identified in the wall of large- and medium-sized arteries (Fig. 4). The outermost layer, the tunica adventitia, is composed of connective tissue cells as well as extensive deposits of the proteins collagen and elastin. The adventitia in muscular arteries is approximately one-half the thickness of the middle layer, the media. In muscular arteries, the media is composed primarily of layers of smooth muscle cells. The principal extracellular protein component is elastin.

Valve Endothelium Tunica intima

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Endothelium Basement membrane Circular internal elastic fibers Tunica media Tunica adventitia Arteriole

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Fig. 4 Comparison of typical artery and typical vein. Reprinted with permission from Cotran R, Kumar V, and Collins T (eds.) (1999) Robbins Pathologic Basic of Disease, 6th edn. Saunders, Philadelphia, PA; Carroll RG (2006) Vascular system. Elsevier’s Integrated Physiology 78.

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In elastic arteries the tunica media also predominates, but in this case, there are many layers of elastin with smooth muscle cells between the layers. The media is separated from the adventitia by a prominent elastic layer, the external elastic lamina. The adventitia of elastic arteries is thinner than that of muscular arteries. In large arteries, a vasa vasorum will also be present. The innermost layer of the artery wall is the tunica intima, which is separated from the media by the internal elastic lamina. In photomicrographs, the inner elastic lamina appears fenestrated (pores that allow cells or otherwise nonpermeable materials to exit the vascular lumen). This may serve as a relatively low-resistance pathway for migration of smooth muscle cells into the intima from the media, a process thought to be involved in the development of atherosclerotic plaques (see below). A single layer of endothelial cells (see below) borders the intima at the lumenal surface (Penn and Murphy, 2005; Stapleton et al., 2014). The media is the most heterogeneous in composition and the most variable in size of the three major coats of the artery. The predominant cell type in the media is the smooth muscle cell. Although some subtle differences in both appearance and behavior have been noted between smooth muscle cells in the intima versus those in the media, it is still not clear whether this is due to the presence of more than one type of smooth muscle cell, or to differing microenvironments in these two adjacent regions of the artery wall (Penn and Murphy, 2005; Stapleton et al., 2014). Atherosclerosis is a major cause of death in most industrial societies. The characteristic lesion of this disease, the atherosclerotic plaque, is found in the intima of large- and medium-sized arteries, thereby thickening the vessel, effecting vascular compliance, and the ability to efficiently distribute blood. An additional problem with advanced plaques is that thrombus formation is likely to occur in regions of plaque rupture. The combination of the two events can lead to partial or even total occlusion of major arteries. If this occurs in one or more of the coronary arteries, a serious or even fatal MI may result. A discussion of arteriosclerosis and exogenous agents that can modulate this condition is presented below (Penn and Murphy, 2005; Stapleton et al., 2014). There are a large variety of compounds that evoke toxic responses within the arterial intima, several of which will be discussed below. These compounds are of interest not only because of their cardiovascular side effects but also by gaining an understanding of the mechanism(s) whereby these agents act in living organisms, new insights into the complexities of the arterial intima may be revealed. Currently, heart disease and stroke are the leading causes of death in the United States and they account for nearly 900,000 deaths per year. These deaths can be contributed to many factors including both cardiotoxic and vasculotoxic agents; however, the major (and largely unavoidable) vasculotoxic agent that is associated with these diseases is tobacco smoke, which will be discussed later (Penn and Murphy, 2005; Stapleton et al., 2014).

Endothelial damage Maintenance of the integrity of the single layer of endothelial cells that lines all of the vascular system is critical for normal vessel function. The intact endothelium is a dynamic system. It acts as a permeability barrier, preventing access of blood-borne contaminants to intimal cells. The intact endothelium also prevents adherence of white blood cells and thrombi; produces and secretes a wide range of growth regulatory molecules; and maintains vascular tone by releasing molecules that modulate dilation and constriction of blood vessels crucial for the appropriate distribution of blood. Endothelial injury or dysfunction is often considered a first-step of atherosclerosis. Endothelial cells are capable of oxidizing low-density lipoprotein (LDL), which is primarily responsible for transporting cholesterol through the blood to tissues. Oxidized LDL can injure the endothelium directly, produce molecules that allow specific types of white blood cells to adhere to the endothelial surface, and attract inflammatory cells to the inner surface of the artery. Presently, the prevailing view is that these events are critical to the early stages of atherosclerotic plaque formation. Since the structural and metabolic integrity of endothelial cells is vital to normal arterial function, and agents causing damage to endothelial cells might be present in the blood at any time, there must be efficient processes available to repair the endothelium and maintain its integrity should it become damaged (Penn and Murphy, 2005; Stapleton et al., 2014). Blood vessels of similar anatomical structure have distinct responses to chemical stress depending on the organ system with which they are associated. This may be due to subtle differences at the cellular and subcellular levels between similar cells and/or to local responses to different stimuli, for example, due to specific hormone receptors or patterns of innervation. Consider the blood–brain barrier, which prevents many potentially toxic blood-borne agents from reaching the brain. If the metabolic status of the endothelial cells in vessels at the brain level is altered, one result can be a disruption of the tight junctions between the endothelial cells, with a resulting increase in permeability. As a result, the brain, which normally is shielded from a number of toxic agents, may now be exposed to them. Lack of oxygen or markedly reduced local blood flow (ischemia) will lead to swelling of the endothelial cells and a widening of the junctions. Chemicals, including alcohols and surfactants, that solubilize lipids, which are an important component of cell membranes, can also impair the barrier. Lead ions interact with sulfhydryl (–SH) groups that are critical to the functioning of many endothelial cell enzymes and structural proteins. Lead ions thus produce damage to the endothelial cells in blood vessels supplying the brain well before the typically recognized damage to nervous system cells is recognized. Chemicals that raise osmotic pressure, such as solutions of high salt or the alcohol, mannitol, can cause endothelial cells to shrink, thereby causing the tight junctions between the cells to separate (Penn and Murphy, 2005; Stapleton et al., 2014). The liver is the organ largely responsible for detoxification of xenobiotic (foreign biological) chemicals and partly, as a consequence, is also constantly at risk for damage by toxic chemicals. One such chemical, the carcinogen dimethylnitrosamine, first induces the proliferation of endothelial cells, followed by increased formation of vascular connective tissue, and, ultimately, total venous occlusion. Plant toxins of the pyrrolizidine alkaloid family, including monocrotaline, can produce identical effects. Monocrotaline, which enters the body in a nontoxic form, is metabolized to its toxic form(s) by the liver. In addition to liver

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damage, this agent causes structural remodeling of blood vessels in the lung and a resultant increase in pulmonary arterial pressure. This effect is similar to the chronic pulmonary hypertension from which many people suffer (Penn and Murphy, 2005; Stapleton et al., 2014). For some updated literature resources on endothelial function and damage, refer to the “Further Reading” section.

Atherosclerosis and arteriosclerosis Atherosclerosis is a physiological term to describe the progressive thickening of the arteries that compromises lumen size (thus decreasing the open area available for appropriate blood flow). Whereas arteriosclerosis (literally ‘artery hardening’) is the general term used to describe stiffening that can occur for a variety of reasons in arteries of all sizes. From a clinical perspective, the atherosclerotic plaque or lesion is of great interest (Fig. 5). It is the principal lesion associated with human thrombosis leading to myocardial and cerebral infarction, which are the primary causes of death and CVD remains the largest disease burden in the world (Roth et al., 2020). Plaque development is complex, involving processes as diverse as cell proliferation, cell death, synthesis and deposition of a variety of extracellular macromolecules (e.g., collagens, elastin, proteoglycans), lipid accumulation, and mineralization. The plaque typically appears in the arterial intima with a variety of associated cell types, including smooth muscle cells, macrophages, lymphocytes, platelets, and endothelial cells. From a mechanical perspective, the thickening leading to the eventual hardening of the arteries (arteriosclerosis) decreases their elasticity, which directly compromises vascular compliance, and results in an increased cardiac workload (for each beat), ineffective vascular pressure regulation, and altered blood flow distribution. Plaque formation has been classified as a problem of proliferation, degeneration, an inflammatory process, a response to injury, and a process related to benign tumor formation. There is considerable clinical and experimental evidence that supports each of these views (Penn and Murphy, 2005; Stapleton et al., 2014). Additionally, it has recently been shown that fine particulate matter in air pollution can contribute to the development of atherosclerosis (Tian et al., 2021). Although in most cases atherosclerosis does not manifest as a clinically serious condition until well into middle age or beyond, it is a disease that begins early in life. Autopsy studies on US soldiers killed during the Korean War revealed that many already had arterial deposits characteristic of the early stages of atherosclerosis. More recent studies on children through people in the third decade of life have confirmed and expanded these findings. The good news is that while there are genetic factors which may predispose an individual to develop atherosclerosis, there is considerable evidence that individual choices and lifestyle decisions can play a large role in preventing, or at least mitigating, the early onset of clinical symptoms of this disease. Further, results from a

Endothelial cells Basement membrane

Tunica adventia

Internal elastic membrane Smooth muscle cells External elastic membrane Connective tissue

Tunica media

Tunica intima

Fig. 5 Diagrammatic representation of the main components of the vascular wall. Reprinted with permission from Cotran R, Kumar V, and Collins T (Eds.) (1999) Robbins Pathologic Basis of Disease. 6th edn. Philadelphia, PA: Saunders; Mulroney SE, Myers AK (2009) The peripheral circulation. Netter’s Essential Physiology 126. New York: Saunders. http://www.netterimages.com/image/21407.htm.

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limited number of laboratory animal studies suggest that it may even be possible to reverse the clinical course of the disease (Penn and Murphy, 2005; Stapleton et al., 2014). Since aging is inevitable, there are three areas where lifestyle modification can have profound effects on moderating development of clinically significant atherosclerosis. In addition to exercise, the two areas most amenable to change are diet and smoking. There is strong epidemiological evidence associating elevated levels of serum cholesterol with increasing risk of atherosclerosis and subsequent heart attacks. As noted above, LDL is primarily responsible for transporting cholesterol and its esters through the bloodstream to the tissues. Oxidized LDL can damage vessel wall cells, including endothelial cells. Oxidized LDL can act as and generate a chemoattractant, which attracts monocytes to the endothelial surface and possibly help mediate passage of monocytes across the endothelium where they may differentiate into tissue macrophages. Monocyte-derived macrophages act as scavengers to help remove harmful molecules such as oxidized LDL. When normal control mechanisms are dysfunctional, macrophages filled with oxidized LDL can become foam cells, which are critical to the formation of early stage atherosclerotic plaques. Studies on experimental animals as well as humans have shown that reduction in levels of plasma cholesterol and LDL can lead to significant widening of the arterial lumen. There is evidence that probucol, a drug originally used for its plasma cholesterol-lowering capability, may function primarily as an antioxidant protecting the integrity of LDL (Penn and Murphy, 2005; Stapleton et al., 2014). Relaxation of blood vessels appears to be at least partially under the control of endothelial cells and their secreted products, especially endothelium-derived relaxation factor (more specifically, nitric oxide). Oxidized LDL directly inhibits the endothelial cell-associated vessel relaxation. The generation of increased reactive oxygen species in association with elevated levels of blood cholesterol has also been reported. One of these reactive oxygen species, superoxide (O2−), may interact with nitric oxide within the artery wall, preventing endothelium-dependent vasodilation. In addition, a common product of the reaction between nitric oxide and superoxide is peroxynitrite (ONOO−). This highly reactive molecule may act to stimulate lipoprotein oxidation, which, as noted above, is regarded as an early step in the development of atherosclerotic plaques (Penn and Murphy, 2005; Stapleton et al., 2014). Oxidants arise from two sources. The first, which is internal, is related to various metabolic processes, including respiration, phagocytic activity to destroy bacteria- and/or virus-infected cells, and, paradoxically, attempts to detoxify foreign substances. In the process of carrying out the latter activity, toxic oxidant by-products can be produced. The second source is external. While the potential protective effects of dietary components and supplements, for example, vitamins, are still being debated, it is reasonable to conclude that decreasing exposure to oxidants from external sources would be beneficial not only in reducing chances of premature atherosclerosis, but also of other diseases, including cancer. By far the most common, avoidable, and dangerous source of external oxidants is cigarette smoke, which is considered a principal contributor to one-quarter of all heart disease cases, one-third of all cancers, and  480,000 premature deaths in the United States every year. As economies of developing countries expand and as cigarette smoking becomes more popular throughout the world, health problems associated with cigarette smoking will increase rapidly.

Metals A number of metals that cause kidney damage act on arteries supplying blood to this organ. Elevated levels of cadmium are associated with hypertension, at least in animal studies. Cadmium has also been implicated in thickening of the wall of arterioles and deposition of fibrotic tissue in capillaries in the testes as well as the kidneys. Agents that chelate cadmium can reverse many of these effects, as can elevation of body levels of zinc. It appears that cadmium and zinc are antagonistic and that maintenance of a cadmium/zinc ratio within fairly well defined limits may be important in preventing cadmium-associated vessel wall changes. Three other metal ions that have been implicated in damage to vessel walls are mercury, chromium, and arsenic. Mercury, which interferes with protein –SH groups, may cause vasoconstriction of preglomerular vessels in the kidney. Inorganic arsenic (iAs) exposure at low to moderate levels has been reported by have significant effects on cardiovascular disease (CVD) (Al-Forkan et al., 2021; Xu et al., 2020; Moon et al., 2017). Arsenic, though an unlikely contributor to blood vessel damage on a worldwide level, represents a striking example of how local environmental alterations can have profound effects on a large portion of a population. On the southwest coast of Taiwan, the artesian well water consumed by the local population has high levels of arsenic and about one out of every 100 people suffer from blackfoot disease. In late stages of this disease, extremities can become gangrenous, leading to spontaneous or surgical amputation of extremities. People suffering from this disease exhibit much higher levels of both peripheral vascular disease and cardiovascular disease. The mechanism of the action of arsenic on the blood vessels remains unclear. For some updated literature resources, refer to the list under “Further Reading” section of the chapter.

Primary amines Cardiotoxicity of primary amines (epinephrine, norepinephrine, isoproterenol) was noted earlier, and has been recognized for nearly 100 years. The vascular toxicity of these and related compounds has also recently been recognized. The effects seem to focus on medial cells of the artery wall, rather than on adventitial or endothelial cells. Early changes include loss of medial cells, mineralization, and loss of elastic fibers. Later there is a compensatory proliferation of intimal cells. The vascular toxicity of two related compounds is particularly striking. One of these compounds, allylamine, will be discussed in the end of this chapter. The

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second is b-aminopropionitrile (b-APN), which is the active agent in the toxic sweet pea, Lathyrus odoratus. Consumption of flour derived from this plant results in lathyrism, a condition often seen in children and young adults residing in Algeria, Ethiopia, and parts of India. Sudden death can result because of rupture of aortic aneurysms, which are ballooned and weakened segments of the artery wall. The toxicity of b-APN has been related to its inhibition of an enzyme which normally cross-links collagen and elastin in large elastic arteries, including the aorta, thereby strengthening them (Penn and Murphy, 2005; Stapleton et al., 2014).

Cigarette smoke Cigarette smoke is composed of active smoke, the smoke coming from the mouth end of the cigarette and breathed in by the smoker; and passive smoke (second-hand smoke; environmental tobacco smoke) which is composed mostly of the smoke coming off the burning end of the cigarette plus a small percentage of exhaled smoke. Active and passive smoke contain many constituents in common, but often in strikingly different concentrations. Among the more than 4000 different chemicals that have been identified in cigarette smoke, prominent candidates that have been considered as vasculotoxic agents include carbon monoxide and various carcinogens. In addition to interfering with transport of well-oxygenated blood, carbon monoxide may cause endothelial cell damage directly, although the mechanism is not clear. Another major class of potential, vasculotoxins in cigarette smoke is the carcinogens. Most of these are found in the tar condensate fraction of cigarette smoke. Some, including benzo(a) pyrene, are well-known carcinogens that are found in other environmentally prominent substances including coal tar derivatives, charcoal-broiled meat, and automobile exhaust. Other smoke carcinogens include the nitrosamines, some of which are tobaccospecific. Both benzo(a)pyrene and the parent nitrosamines require metabolic activation to become carcinogenic. The enzymes involved in these processes are members of the cytochrome P-450 system. During the course of detoxifying these agents so that they ultimately can be excreted readily, one or more toxic and possibly carcinogenic metabolites may be generated. Compounds such as benzo(a)pyrene induce the appearance of the cytochrome P-450 system enzymes, and smokers are constantly exposed to the P-450 inducers. Generation of endothelial cell-damaging agents during the metabolism of benzo(a)pyrene derived from cigarette smoke has been shown to be a possible mechanism to explain the initiation of atherosclerotic plaques. Oxidants derived from cigarette smoke can damage lipids, an important constituent of cell membranes, as well as cellular macromolecules, including DNA. There is some evidence that cigarette smoke may cause damage to artery wall cell DNA in animal models; however, if such damage does occur it would provide independent support for the view that DNA alterations are characteristic of atherosclerotic plaques in animal models of the disease as well as in humans. In related experimental animal studies, the chemical allylamine caused both myocardial lesions and vascular fibrosis. Allylamine toxicity is thought to be mediated via metabolism of this compound to the reactive aldehyde, acrolein, which is also a prominent component of cigarette smoke. Studies with cultured artery wall cells indicate that the primary arterial effect of allylamine is on the smooth muscle cells. Proliferation of intimal smooth muscle cells in response to allylamine exposure results in activation of a specific cellular DNA sequence, the H-ras oncogene, which is implicated in the development of certain forms of cancer. This lends further support to the contention that there may be molecular similarities between the development of the lesions of atherosclerosis and of cancer (Penn and Murphy, 2005; Stapleton et al., 2014). One of the problems researchers have faced in identifying specific health-threatening components of cigarette smoke is that while at moderate to high concentrations many of these agents can be toxic, in many cases the individual concentrations of these factors in cigarette smoke are likely too low to be able to account individually for the toxic and disease-promoting effects of cigarette smoke. The US Environmental Protection Agency sidestepped this problem in 1992 by declaring environmental tobacco smoke, with its thousands of components, to be a human class A carcinogen. The American Heart Association has classified environmental tobacco smoke as an environmental poison and as a major preventable cause of cardiovascular disease. Regarding environmental tobacco smoke, estimates provide evidence that as many as 69,000 excess heart disease deaths annually in the United States can be attributed directly to involuntary exposure to cigarette smoke. For a detailed estimates of tobacco related mortality from different diseases, readers can refer to the Center for Disease Control datasets available at – https://www.cdc.gov/tobacco/data_statistics/fact_ sheets/health_effects/tobacco_related_mortality/index.htm. In support of these estimates, a number of laboratories have reported that inhalation of sidestream cigarette smoke accelerates arteriosclerosis in different experimental model systems of the disease. Since epidemiological and autopsy evidence strongly support the view that atherosclerosis begins as early as childhood, the experimental results with environmental tobacco smoke suggest that involuntary exposure of children to tobacco smoke may accelerate plaque development. The insidious nature of involuntary exposure to environmental tobacco smoke is further emphasized by recent findings in a mouse model of atherosclerosis. Male mice exposed only in utero to environmental tobacco smoke develop accelerated atherosclerosis as adults, even in the absence of a high fat diet. Additionally, Mourino et al. (2023), reported that increased postnatal serum cotinine concentrations, the predominant metabolite of nicotine, had greater influence on adolescent’s cardiometabolic risk compared to the prenatal period. It was further postulated that these associations may be sex-specific. This study further reinforced the ongoing need for public health interventions to minimize the exposure of children to passive smoke (i.e., second-hand smoke or environmental tobacco smoke). In the United States, where studies show that many children are less active physically and have poorer diets than children growing up a few of generations ago, involuntary exposure to second-hand smoke may well represent a major additional risk factor for the development of atherosclerosis. Fortunately, extensive epidemiologic evidence from both cancer and heart disease studies indicates that as the time since cessation of smoking increases, the chances of dying prematurely from either disease decrease.

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Thus, the vasculotoxic effects of cigarette smoke, both active and passive, may be largely reversible (Penn and Murphy, 2005; Stapleton et al., 2014).

Nicotine Tobacco smoke has been associated with various cardiovascular diseases. However, the use and dependency on tobacco products stems from a need to reach a level of nicotine that is consistent with the user’s addiction. The introduction of nicotine cannot be thought of as benign and the use of nicotine (e.g., e-cigarettes, nicotine patch, nicotine gum, nasal sprays, inhalers, lozenges, or smokeless tobacco products) should also be considered to have negative cardiovascular health consequences, though the extent of these health consequences is currently unclear. Acutely, nicotine can stimulate the release of catecholamines through activation of the sympathetic nervous system, thereby increasing blood pressure and heart rate; while chronic use has been shown to have multiple cellular targets that alter cardiac and vascular molecular pathways promoting hypertension, cardiovascular disease, and altering cardiac rhythm. Nicotine has also been implicated in abdominal aortic aneurysm both through indirect mechanisms (e.g., hypertension) and direct alterations leading to vascular smooth muscle cell damage and aortic remodeling. Taken together, the movement of patients from tobacco use to nicotine replacement therapies, but more importantly nicotine cessation, should be considered paramount to prevent the cardiovascular effects associated with nicotine use (Penn and Murphy, 2005; Stapleton et al., 2014).

Electronic cigarettes The use of electronic cigarettes (e-cig) has increased exponentially over the last 10 years. Specifically, women using during their pregnancies has increased significantly due to their false promotion as being safer than traditional tobacco cigarettes. While the use of traditional cigarettes has decreased (from 42% to 14%), up to 7% of women reported vaping during their pregnancies. The health consequences of e-cig exposure on fetuses either pre- or postnatally is unknown. Propylene glycol (PG) and vegetable glycerin (VG) are the two main bases used in e-cig juice with the ratio of these two constituents being customizable to individual users. Recently, studies have shown PG/VG exposure, without the addition of nicotine, disrupts normal lung function in mice underpinning the importance of defining the physiological effects of these two substances. These two chemicals are the main components of e-cig liquid, the health effects resulting from alterations in the PG/VG ratio are currently understudied. Maternal exposures to toxicants during critical periods of gestation can affect important aspects of fetal development, such as organogenesis, angiogenesis, and neural development leading to fetal health deficits. Understanding how e-cig use affects overall health and cardiac function, in addition to maternal e-cig use during gestation affects fetal health, is paramount because very little is known about the health consequences of short- and long-term exposures. Based on their comprehensive literature review and data analysis, Damay et al. (2022) reported presence of nicotine, PG, particulate matters, heavy metals, and flavoring agents in e-cigs. It was also reported that e-cig exposure is linked to atherosclerosis progression based on the molecular mechanism that lead to reactive oxygen species (ROS) formation, endothelial disfunction, and inflammation. However, Damay et al. (2022) recommended that further research is required to determine the precise mechanism responsible for this link, and clinical evidence that supports these findings.

Conclusion/summary/outlook Through hundreds of years, and countless agents, studies and trials, the fundamental principle of toxicology remains the balance between a poison and a remedy. That is, the difference between an untoward outcome and positive therapeutic application of compounds must be considered. This chapter is not meant to associate a ‘good’ or ‘bad,’ but to open awareness to all toxicological considerations, and encourage special consideration for cardiovascular impacts.

See also: Amphetamines; Arsenic; Batrachotoxin; Blood; Chemicals of environmental concern; Chromium; Cocaine; hERG (human ether-a-go-go related gene); Mercury; Tetrodotoxin; Tobacco

References Al-Forkan M, Wali FB, Khaleda L, et al. (2021) Association of arsenic-induced cardiovascular disease susceptibility with genetic polymorphisms. Scientific Reports 11: 6263. https:// doi.org/10.1038/s41598-021-85780-8. Damay VA, Lesmana R, Akbar MR, Lukito AA, Tarawan VM, Martha JW, and Nugroho J (2022) Electronic cigarette and atherosclerosis: A comprehensive literature review of latest evidences. International Journal of Vascular Medicine 2022: 4136811. https://doi.org/10.1155/2022/4136811. PMID: 36093338. PMC9453087. Moon KA, Oberoi S, Barchowsky A, Chen Y, Guallar E, Nachman KE, Rahman M, Sohel N, D’Ippoliti D, Wade TJ, James KA, Farzan SF, Karagas MR, Ahsan H, and Navas-Acien A (2017) A dose-response meta-analysis of chronic arsenic exposure and incident cardiovascular disease. International Journal of Epidemiology 46(6): 1924–1939. https://doi.org/ 10.1093/ije/dyx202. Erratum in: Int J Epidemiol. 2017 Jun 1; 47(3): 1013.

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Mourino N, Pérez-Ríos M, Yolton K, Lanphear BP, Chen A, Buckley JP, Kalkwarf HJ, Cecil KM, and Braun JM (2023) Pre-and postnatal exposure to secondhand tobacco smoke and cardiometabolic risk at 12 years: Periods of susceptibility. Environmental Research: 115572. Penn A and Murphy G (2005) Cardiovascular system. In: Penn A and Murphy G (eds.) Encyclopedia of Toxicology, 2nd edn, vol. 1, pp. 467–485. Elsevier Inc. © 2005. Roth G, Mensah G, Johnson C, et al. (2020) Global burden of cardiovascular diseases and risk factors, 1990–2019. Journal of the American College of Cardiology 76(25): 2982–3021. https://doi.org/10.1016/j.jacc.2020.11.010. Stapleton PA, Knuckles TL, Minarchick VC, Gautam G, and Nurkiewicz TR (2014) Cardiovascular system. Encyclopedia of Toxicology (Third Edition, 2014), vol. 1, pp. 730–747., ISBN:9780123864550. https://doi.org/10.1016/B978-0-12-386454-3.00985-4. Tian M, Zhao J, Mi X, Wang K, Kong D, Mao H, and Wang T (2021) Progress in research on effect of PM2.5 on occurrence and development of atherosclerosis. Journal of Applied Toxicology 41(5): 668–682. Xu L, Mondal D, and Polya DA (2020) Positive association of cardiovascular disease (CVD) with chronic exposure to drinking water arsenic (As) at concentrations below the WHO provisional guideline value: A systematic review and meta-analysis. International Journal of Environmental Research and Public Health 17(7): 2536. https://doi.org/10.3390/ ijerph17072536. Erratum in: Int J Environ Res Public Health. 2020 Dec 02; 17(23). PMID: 32272785. PMC7178156.

Further reading A detailed list of bibliographic resources based on specific chemicals, stressors, toxicologic agent and their relationship with broader cardiovascular health outcome is provided below. Particulate matters and cardiovascular Atkinson RW, Kang S, Anderson HR, Mills IC, and Walton HA (2014) Epidemiological time series studies of PM2.5 and daily mortality and hospital admissions: A systematic review and meta-analysis. Thorax 69(7): 660–665. https://doi.org/10.1136/thoraxjnl-2013-204492. Epub 2014 Apr 4. PMID: 24706041. PMC4078677. Cai Y, Zhang B, Ke W, Feng B, Lin H, Xiao J, Zeng W, Li X, Tao J, Yang Z, Ma W, and Liu T (2016) Associations of short-term and long-term exposure to ambient air pollutants with hypertension: A systematic review and meta-analysis. Hypertension 68(1): 62–70. https://doi.org/10.1161/HYPERTENSIONAHA.116.07218. Epub 2016 May 31. PMID: 27245182. Cao L, Wang L, Wu L, Wang T, Cui X, Yu L, Diao R, and Mao H (2021) Particulate matter and hypertensive disorders in pregnancy: Systematic review and meta-analysis. Public Health 200: 22–32. https://doi.org/10.1016/j.puhe.2021.08.013. Epub 2021 Oct 13. PMID: 34653738. Chen J and Hoek G (2020) Long-term exposure to PM and all-cause and cause-specific mortality: A systematic review and meta-analysis. Environment International 143: 105974. https://doi.org/10.1016/j.envint.2020.105974. Epub 2020 Jul 20. PMID: 32703584. Domínguez-Rodríguez A, Báez-Ferrer N, Abreu-González P, Rodríguez S, Díaz R, Avanzas P, and Hernández-Vaquero D (2021) Impact of desert dust events on the cardiovascular disease: A systematic review and meta-analysis. Journal of Clinical Medicine 10(4): 727. https://doi.org/10.3390/jcm10040727. PMID: 33673156. PMC7918944. Faridi S, Brook RD, Yousefian F, Hassanvand MS, Nodehi RN, Shamsipour M, Rajagopalan S, and Naddafi K (2022) Effects of respirators to reduce fine particulate matter exposures on blood pressure and heart rate variability: A systematic review and meta-analysis. Environmental Pollution 303: 119109. https://doi.org/10.1016/j.envpol.2022.119109. Epub 2022 Mar 8. PMID: 35271952. Farhadi Z, Abulghasem Gorgi H, Shabaninejad H, Aghajani Delavar M, and Torani S (2020) Association between PM2.5 and risk of hospitalization for myocardial infarction: A systematic review and a meta-analysis. BMC Public Health 20(1): 314. https://doi.org/10.1186/s12889-020-8262-3. PMID: 32164596. PMC7068986. Franchini M, Mengoli C, Cruciani M, Bonfanti C, and Mannucci PM (2016) Association between particulate air pollution and venous thromboembolism: A systematic literature review. European Journal of Internal Medicine 27: 10–13. https://doi.org/10.1016/j.ejim.2015.11.012. Epub 2015 Nov 27. PMID: 26639051. Hall KC and Robinson JC (2019) Association between maternal exposure to pollutant particulate matter 2.5 and congenital heart defects: A systematic review. JBI Database of Systematic Reviews and Implementation Reports 17(8): 1695–1716. https://doi.org/10.11124/JBISRIR-2017-003881. PMID: 31021973. PMC6707530. Heo S, Son JY, Lim CC, Fong KC, Choi HM, Hernandez-Ramirez RU, Nyhan K, Dhillon PK, Kapoor S, Prabhakaran D, Spiegelman D, and Bell ML (2022) Effect modification by sex for associations of fine particulate matter (PM2.5) with cardiovascular mortality, hospitalization, and emergency room visits: Systematic review and meta-analysis. Environmental Research Letters 17(5): 053006. https://doi.org/10.1088/1748-9326/ac6cfb. Epub 2022 May 16. PMID: 35662857. PMC9162078. Huang F, Wang P, Pan X, Wang Y, and Ren S (2020) Effects of short-term exposure to particulate matters on heart rate variability: A systematic review and meta-analysis based on controlled animal studies. Environmental Pollution 256: 113306. https://doi.org/10.1016/j.envpol.2019.113306. Epub 2019 Oct 3. PMID: 31733955. Jaganathan S, Jaacks LM, Magsumbol M, Walia GK, Sieber NL, Shivasankar R, Dhillon PK, Hameed SS, Schwartz J, and Prabhakaran D (2019) Association of long-term exposure to fine particulate matter and cardio-metabolic diseases in low- and middle-income countries: A systematic review. International Journal of Environmental Research and Public Health 16(14): 2541. https://doi.org/10.3390/ijerph16142541. PMID: 31315297. PMC6679147. Jilani MH, Simon-Friedt B, Yahya T, Khan AY, Hassan SZ, Kash B, Blankstein R, Blaha MJ, Virani SS, Rajagopalan S, Cainzos-Achirica M, and Nasir K (2020) Associations between particulate matter air pollution, presence and progression of subclinical coronary and carotid atherosclerosis: A systematic review. Atherosclerosis 306: 22–32. https://doi.org/ 10.1016/j.atherosclerosis.2020.06.018. Epub 2020 Jul 5. PMID: 32682146. Khosravipour M, Safari-Faramani R, Rajati F, and Omidi F (2022) The long-term effect of exposure to respirable particulate matter on the incidence of myocardial infarction: A systematic review and meta-analysis study. Environmental Science and Pollution Research International 29(28): 42347–42371. https://doi.org/10.1007/s11356-022-18986-6. Epub 2022 Mar 30. PMID: 35355187. Lederer AM, Fredriksen PM, Nkeh-Chungag BN, Everson F, Strijdom H, De Boever P, and Goswami N (2021) Cardiovascular effects of air pollution: Current evidence from animal and human studies. American Journal of Physiology. Heart and Circulatory Physiology 320(4): H1417–H1439. https://doi.org/10.1152/ajpheart.00706.2020. Epub 2021 Jan 29. PMID: 33513082. Liu X, Lian H, Ruan Y, Liang R, Zhao X, Routledge M, and Fan Z (2015) Association of exposure to particular matter and carotid intima-media thickness: A systematic review and metaanalysis. 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Zhao R, Chen S, Wang W, Huang J, Wang K, Liu L, and Wei S (2017) The impact of short-term exposure to air pollutants on the onset of out-of-hospital cardiac arrest: A systematic review and meta-analysis. International Journal of Cardiology 226: 110–117. https://doi.org/10.1016/j.ijcard.2016.10.053. Epub 2016 Oct 25. PMID: 27806308. Chemical and environmental exposure and cardiovascular Cosselman KE, Navas-Acien A, and Kaufman JD (2015) Environmental factors in cardiovascular disease. Nature Reviews Cardiology 12(11): 627–642. Guo X, Song Q, Wang H, Li N, Su W, Liang M, Sun C, Ding X, Liang Q, and Sun Y (2022) Systematic review and meta-analysis of studies between short-term exposure to ambient carbon monoxide and non-accidental, cardiovascular, and respiratory mortality in China. Environmental Science and Pollution Research International 29(24): 35707–35722. https://doi.org/10.1007/s11356-022-19464-9. Epub 2022 Mar 7. PMID: 35257337. Navas-Acien A, Sanchez TR, Mann K, and Jones MR (2019) Arsenic exposure and cardiovascular disease: Evidence needed to inform the dose-response at low levels. Current Epidemiology Reports 6: 81–92. Sekhotha MM, Monyeki KD, and Sibuyi ME (2016) Exposure to agrochemicals and cardiovascular disease: A review. International Journal of Environmental Research and Public Health 13(2): 229. https://doi.org/10.3390/ijerph13020229. PMID: 26901215. PMC4772249. Signorelli SS, Oliveri Conti G, Zanobetti A, Baccarelli A, Fiore M, and Ferrante M (2019) Effect of particulate matter-bound metals exposure on prothrombotic biomarkers: A systematic review. Environmental Research 177: 108573. https://doi.org/10.1016/j.envres.2019.108573. Epub 2019 Jul 2. PMID: 31323394. Other exposure and cardiovascular Dillon DT, Webster GD, and Bisesi JH Jr (2022) Contributions of biomass/solid fuel burning to blood pressure modification in women: A systematic review and meta-analysis. American Journal of Human Biology 34(1): e23586. https://doi.org/10.1002/ajhb.23586. Epub 2021 Mar 1. PMID: 33645874. Ma Z, Cao X, Chang Y, Li W, Chen X, and Tang NJ (2021) Association between gestational exposure and risk of congenital heart disease: A systematic review and meta-analysis. Environmental Research 197: 111014. https://doi.org/10.1016/j.envres.2021.111014. Epub 2021 Mar 11. PMID: 33716027. Patsouras MD and Vlachoyiannopoulos PG (2019) Evidence of epigenetic alterations in thrombosis and coagulation: A systematic review. Journal of Autoimmunity 104: 102347. https://doi.org/10.1016/j.jaut.2019.102347. Epub 2019 Oct 11. PMID: 31607428. Additional reading list Acosta D (2019) Cardiovascular Toxicology. Target Organ Toxicology Series. , 4th edn. CRC Press. The book is divided into four parts and 16 chapters. Part I Introduction includes two chapters (Chapter 1: Advances in Cardiovascular Toxicology; and Chapter 2: Pathobiology of Myocardial Ischemic Injury). Part II – Methods include three chapters (Chapter 3: Nonclinical Safety Assessment of the Cardiovascular Toxicity of Drugs and Combination Medical Devices; Chapter 4: Novel Approaches in the Evaluation of Cardiovascular Toxicity and Chapter 5: Cardiovascular Dynamics in Conscious Primates). Part III – Cardiotoxicity includes eight chapters (Chapter 6: Cardiovascular Toxicity of Antimicrobials; Chapter 7: Cardiotoxicity of Anthracyclines and Other Antineoplastic Agents; Chapter 8: Catecholamine-induced Cardiomyopathy; Chapter 9: Adverse Effects of Centrally Acting Drugs on the Cardiovascular System; Chapter 10: Adverse Effects of Drugs on Electrophysiological Properties of the Heart; Chapter 11: Cardiovascular Effects of Steroidal Agents; Chapter 12: Cardiotoxicity of Industrial Chemicals and Environmental Pollutants; and Chapter 13: Adverse Effects of Cigarette Smoking on the Cardiovascular System). Part IV - Vascular Toxicity includes three more chapters (Chapter 14: Overview of Vascular Toxicology; Chapter 15: Pathobiology of the Vascular Response to Injury; and Chapter 16: The Arterial Media as a Target of Injury by Chemicals). Campen MJ (2019) Toxic responses of the heart and vascular system. In: Klaassen CD (ed.) Casarett & Doull’s Toxicology: The Basic Science of Poisons, 9th edn. McGraw Hill. https:// accesspharmacy.mhmedical.com/content.aspx?bookid¼2462§ionid¼202674948. McQueen CA (ed.) (2018) Cardiovascular toxicology. Comprehensive Toxicology, vol. 13. (Major Reference Works); Editor in Chief. available at: https://www.sciencedirect.com/ referencework/9780081006016/comprehensive-toxicology#book-info. Cox LA Jr (2020) Target sites: Cardiovascular. Information Resources in Toxicology, pp. 529–532. Available at. https://www.sciencedirect.com/science/article/pii/ B9780128137246000517. Glass C and Witzum J (2001) Atherosclerosis: The road ahead. Cell 104: 503–516. Libby P (2003) Vascular biology of atherosclerosis: Overview and state of the art. The American Journal of Cardiology 91(3A): 3A–6A.

Relevant websites https://www.springer.com/journal/12012/ :Cardiovascular Toxicology Journal. https://www.springer.com/journal/12012/editors :Cardiovascular Toxicology Journal.

Careers in toxicology Mary Beth Genter, University of Cincinnati, Cincinnati, OH, United States © 2024 Elsevier Inc. All rights reserved. This is an update of M.B. Genter, S.J. Borghoff, B.J. Eidemiller, A. Woolley, Toxicology, Education and Careers, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 721–726, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00441-3.

Introduction Why consider a career in toxicology? Challenges Opportunities Competitive salaries and professional advancement What do toxicologists do? Research Product safety evaluation Teaching Public service and regulatory affairs Clinical, forensic, and analytical toxicology Occupational toxicology Who employs toxicologists? Industry Contract research organizations Academia Government Consultants Non-profit research Preparing for a career in toxicology Overview Undergraduate and graduate training Planning Selection of an appropriate toxicology program Financial assistance Recent efforts to enhance training of PhD toxicologists Further training Continuing education in toxicology Certification in toxicology Summary References Further reading

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Abstract Toxicology is the study of harmful effects of agents on people, animals, other living organisms, and the environment. Toxicology education requires not only a solid background in the fundamental principles of the field, but also an understanding of bench work and its possible translation for human benefit as well as its use in risk assessment. Employment opportunities are available in multiple job sectors, including academia, the chemical and pharmaceutical industries, contract research organizations, as consultants, and in various governmental and non-profit organizations. Training in toxicology should be broad in both the science and skills that impact effective experimental design, as well as the use, interpretation and communication of toxicology data, and decision-making processes.

Keywords Certification in toxicology; Computational toxicology; High-throughput methods; Postdoctoral training in toxicology; Risk assessment; Toxicology careers; Toxicology education; Toxicology job opportunities; Toxicology salaries; Toxicology training programs

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Career opportunities in toxicology Toxicology education and training opportunities Certification opportunities in toxicology Core competencies in toxicology

Introduction Toxicology is the study of harmful effects of agents on people, animals, other living organisms, and the environment. The field of toxicology provides the opportunity to contribute to the well-being of current and future generations and the environment. Toxicology is a multidisciplinary science that includes cutting-edge fields related to biology (such as genetics, genomics, and molecular biology) and chemistry, with elements of physical and computational sciences. As a result, individual training in toxicology can take on many facets, resulting in a wide variety of career opportunities. With the growing understanding of the impact of chemicals on humans and the environment, there is the accompanying realization that assessment of the hazards and risks associated with the use of (and exposure to) chemicals is central to the health of all living things.

Why consider a career in toxicology? Challenges Chemicals are an essential component of the high standard of living that we enjoy. With every technological advance comes benefits, but also potential hazards and risks. The challenge to toxicologists is to ensure that products or by-products of modern living do not unnecessarily endanger human or environmental health. Toxicologists conduct studies that predict potential impacts or develop means to reduce damage when an adverse response is detected. Thus, the toxicologist has the reward of contributing to the protection of health of people, animals, and the environment.

Opportunities A wide variety of career opportunities exists in toxicology in many employment sectors, including government, industry, academia, not-for-profits, and consulting. Toxicologists participate in basic research studying mechanisms by which chemicals exert their toxic or cancer-causing effects. Toxicologists may apply state-of-the-art techniques in molecular biology, genomics, chemistry, and the biomedical sciences to understand dose-response relationships, critical endpoints, target tissues, and mechanisms of toxicity. Many toxicologists work in the biotechnology, chemical, pharmaceutical, and consumer products industries to develop and evaluate new cosmetic and personal care product ingredients, drugs, and medical devices, and to test and ensure that their products and workplaces are safe. Regulatory toxicologists work in government at the local, state and federal levels to ensure that chemicals are produced, used, and safely released into the environment. Data from laboratory studies can be used as the basis for risk assessments to evaluate levels of exposure that are predicted to be without adverse effects for the majority of exposed individuals (Fig. 1). In performing a risk assessment, critical elements that are evaluated are the target organs and types of damage a chemical might

Fig. 1 The intersection of risk assessment and risk management. Based on National Research Council (2009) Science and Decisions: Advancing Risk Assessment. Washington, DC: The National Academies Press. doi: 10.17226/12209. Accessed 3/10/2022.

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produce (hazard identification); the doses or concentrations of an agent producing the toxic effects (dose-response assessment); and evaluation of levels of exposure, including both environmental, workplace, or internal doses of an agent (exposure assessment). The output of these steps is a risk characterization, which often is the point at which regulators step in and use these results to set regulatory levels for contaminants in drinking water, ambient air, workplace settings, etc. (National Research Council, 2009). Toxicologists, often in partnership with industrial hygienists, are also involved with monitoring both water and air for levels of specific chemicals, physical hazards (e.g., noise, radiation, heat), and biological agents known to cause adverse health effects to people and the environment. In academia, toxicologists train future toxicologists and other health professionals, and may also conduct research to understand the mechanisms by which chemicals cause toxic effects in living organisms. Clinical toxicologists help to diagnose patients with diseases caused by toxic substances, including overdoses and drug reactions, and often work in hospitals or poison control centers. Forensic toxicologists help establish the cause of death or identify important toxicity clues that can be used to solve a crime, whereas the occupational toxicologist conducts studies to understand conditions of chemical exposure or work practices that may place the worker at unacceptable risks.

Competitive salaries and professional advancement Biotechnological research and development demonstrate higher employment growth than in many fields, and the need for toxicologists is high and the discipline provides the potential for good income. Salaries are especially competitive for the toxicologist with advanced training and certification. Specific information on salaries for toxicologists in various workplace settings and with various levels of experience and education can be found in the Tenth Triennial Toxicology Salary Survey, which was completed by members of various toxicology professional societies (Sullivan and Gad, 2020). Seventy-seven percent of those responding to the tenth triennial salary survey reported salaries exceeding $120,000 per year, 26% of the respondents made more than $200,000 per year, and 62% reported bonuses, stock options, and commissions in addition to their salary (Sullivan and Gad, 2020).

What do toxicologists do? Research Research in toxicology is generally conducted in various specialty areas such as carcinogenesis, reproductive and developmental toxicology, neurotoxicology, immunotoxicology, respiratory toxicology, dermal toxicology, endocrine toxicology, genetic toxicology, and environmental toxicology. An evolving area of study is the toxicology of mixtures, which is important because humans are seldom exposed to single chemicals, so methods to assess and understand interactions between/among multiple chemical exposures are urgently needed. The concept of epigenetics has recently expanded into the field of toxicology to include studies of how chemicals may impact biological and toxic responses by altering gene expression without changing the nucleotide sequence of that gene. Given that true epigenetic responses can have transgenerational effects, interest in this area of toxicology has grown tremendously in recent years (e.g., Skinner and Nilsson, 2021). Another emerging challenge for toxicologists involves nanomaterials (materials with at least one dimension less than 100 nm). Use of nanomaterials (often composed of single metals, mixtures of metals, or various configurations of elemental carbon) is rapidly expanding in applications including medical applications, as well as use in electronics, health care and consumer products, and beyond. Given the extremely small size and unique physicochemical properties of nanomaterials, special challenges have arisen, from understanding bioavailability and distribution of nanomaterials in living organisms, to protecting workers who manufacture and formulate nanomaterials from potential exposures and health risks. A challenge to all toxicologists is to use research models that accurately predict outcomes for human exposure to potential toxicants, products, and drugs. Some researchers study the effects of substances on living organisms, working with various systems ranging from whole organisms (in vivo) to isolated cell suspensions or cell cultures (in vitro). Although certain animal models are established for toxicology testing, these organisms are often physiologically very different from humans. An important area of research is determining the appropriate test methodology to use in specific situations and how results in these models translate to protect human and environmental health. The US Environmental Protection Agency (US EPA) recently estimated that little to no toxicity data exist for approximately 40,000 active substances, thus necessitating the development and use of reliable high-throughput assays. In addition, due to ethical concerns, as well as the high cost and low throughput nature of in vivo studies, strategies such as computational toxicology are being refined. Computational toxicology, which is a rapidly evolving and expanding field of toxicology, uses approaches such as in vitro and in silico assays, as well as quantitative structure activity relationships (QSARs) tools in lieu of in vivo assays. Finally, microphysiological systems, often called “organs on a chip” are allowing toxicologists to “build” organ systems, such as a miniaturized cardiovascular system, and test materials in a more physiologically realistic setting, compared to cell culture systems involving one cell line, or simple co-culture systems.

Product safety evaluation Toxicologists in product safety evaluation are continuously developing better ways to evaluate the potential adverse effects of drugs, chemicals, and physical agents and to determine the doses at which adverse responses occur. Many industries employ toxicologists

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in the role of study director to evaluate and monitor the safety of their products. For drugs, food additives, cosmetics, agricultural chemicals, and other classes of chemicals, federal laws require that manufacturers provide adequate testing of products before they are approved for use. Tests to determine whether a chemical has the potential to cause cancer, birth defects, reproductive effects, neurological toxicity, or other adverse effects are commonly conducted by the manufacturer or in contract research labs. Toxicologists involved in product safety evaluation have the responsibility to ensure that these tests are designed, conducted, analyzed, and interpreted in a scientifically sound manner. Data derived from these studies are reviewed by toxicologists in various regulatory agencies such as the United States Food and Drug Administration (US FDA), or the US EPA or the European Chemicals Agency (ECHA) to ensure that the products will not present an unreasonable risk to human health or the environment.

Teaching Toxicologists employed in colleges, universities, and medical centers are frequently involved in teaching courses in toxicology and pharmacology. Many colleges and universities are developing new courses at both the undergraduate and graduate levels to provide students with a background in the science of toxicology. While some academic institutions do not have a specific graduate program in toxicology, they still may invite toxicologists to participate in curriculum development and teach in basic programs such as chemistry and biology. Thus, opportunities exist to teach toxicology in small colleges as well as major universities. In order to enhance critical thinking and communication skills, as well as provide “real world” toxicology experience, partnerships between academic institutions and industry, government agencies, and policy makers are increasingly important and encouraged.

Public service and regulatory affairs There has been tremendous growth in public awareness of chemical hazards over the past two decades, which has resulted in the passage of many laws governing the production, use, and disposal of chemicals. Many local, state, and federal agencies employ toxicologists to assist in the development and enforcement of their laws. An increasingly important area of toxicology is public communication of chemical risks. Toxicologists employed by regulatory agencies may be called on to examine the scientific basis for regulatory actions or to assist in communicating to the public the reasons regulatory actions are or are not taken in particular situations. There are many private consulting firms with expanding expertise in toxicology that can provide such services to local and state health departments, public utilities, and private industries. Thus, many employment opportunities in the private sector are available to the toxicologists interested in assisting public agencies and private industries in resolving many public health and environmental problems.

Clinical, forensic, and analytical toxicology Clinical toxicologists are health professionals concerned with diseases caused by exposure to toxic agents such as drugs and other chemicals, including accidental and intentional overdoses. Generally, clinical toxicologists are physicians, pharmacologists, and veterinarians who receive specialized clinical training in toxicology. Forensic toxicologists may interact with clinical toxicologists to establish analytical chemical methods for the detection of toxic agents in tissue samples from poisoned patients. Research performed by clinical and forensic toxicologists has led to the recognition of new chemical hazards and the development of novel therapies for poisoning. Clinical and forensic toxicologists may be employed in health departments, industry, and other settings in which health professionals are employed. Analytical toxicologist often have a strong background in organic and inorganic chemistry and are integral in the development and validation of new, more sensitive methods to detect and quantitate various chemicals in many different materials.

Occupational toxicology Occupational toxicologists conduct studies to understand the conditions of chemical exposure and develop work practices that reduce health risks to the worker. They often work closely with industrial hygienists and are most frequently employed in industry, academia, and government. Their efforts are focused on obtaining knowledge of the relationship between workplace exposure to a chemical, the health effects that are of concern to workers, and setting exposure limits to avoid health effects at the workplace.

Who employs toxicologists? Industry The Tenth Triennial Toxicology Salary Survey, with respondents from over 30 countries (most respondents were in the United States, Canada, Japan, and western Europe), revealed that 44% of the toxicologists are employed in some industrial position. Product development, product safety evaluation, and regulatory compliance generate a large job market for toxicologists. Within the industry sector, the pharmaceutical industry was the largest employer (66% of those working in industry), followed by the chemical, consumer product, medical devices, food or food ingredients, and petroleum, industries. These industries employ toxicologists holding bachelors, masters, and doctoral degrees (Sullivan and Gad, 2020).

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Contract research organizations Contract Research Organizations (CROs), which are rapidly expanding worldwide, employ approximately 13.5% of the toxicologists who responded to the respondents to the Tenth Triennial Toxicology Salary Survey (Sullivan and Gad, 2020). Scientists in these organizations plan and conduct studies for other pharmaceutical and biotech organizations.

Academia Academic institutions account for 16% of all employed toxicologists, based on responses to the Tenth Triennial Toxicology Salary Survey. Most academic toxicologists who responded to the survey have advanced degrees and are employed at public research universities (Sullivan and Gad, 2020).

Government The broad category of “government” (including federal government, military, state, and local governments) accounts for approximately 11.5% of toxicologists (Sullivan and Gad, 2020). Although most government jobs are with federal regulatory agencies such as the US FDA and US EPA, state governments and local governments may employ toxicologists with masters or doctoral degrees. Many toxicologists employed by the federal government are involved in the development and enforcement of laws related to the toxicity of materials. In addition, a number of federal agencies employ toxicologists to conduct both basic and applied research in toxicology, including the following agencies in the US: US EPA; the US FDA’s research laboratory at the National Center for Toxicological Research (NCTR); National Institute of Environmental Health (NIEHS); National Institute for Occupational Safety and Health (NIOSH); US Centers for Disease Control and Prevention (CDC), and US Consumer Products Safety Commission (CPSC).

Consultants Toxicology consultants, whether working independently or as a member of a consulting firm, represent about approximately 12% of the toxicologists responding to the Tenth Triennial Toxicology Salary Survey (Sullivan and Gad, 2020). In the consulting field, experienced individuals provide professional guidance and advice to local public agencies, industries, and attorneys involved in issues with toxic chemicals. In some cases, toxicology consultants also have law or business degrees to provide highly-specialized specific expertise.

Non-profit research Non-profit research institutions provide opportunities for research in toxicology to just over 1% of the toxicologists responding to the Tenth Triennial Toxicology Salary Survey (Sullivan and Gad, 2020). Numerous public and private research foundations employ toxicologists to conduct research on specific problems of industrial or public concern. Toxicologists at all levels of experience and education may find employment with these research foundations.

Preparing for a career in toxicology Overview Toxicology training programs and research centers are growing around the world as emerging economies realize the value of the discipline in protecting the health of citizens, workers, and in the production of safe products for the world market. Some toxicologists are qualified at the BS/BSc or MS/MSc level but many of these also have (or are studying for) specific toxicology certifications or have extensive work experience. Even possession of a PhD in toxicology may not be a sufficient proof of training and expertise. This is true of toxicologists working at higher levels, such as consultants, regulatory toxicologists working for government agencies, and others whose output affects the assessment of toxicological risks to humans and the environment. Some toxicologists have human or veterinary medical training; in many cases, graduate and professional studies have been reinforced by an interval of postdoctoral research with a mentor. Even then, specific types of experience and credentials are needed for successful employment in some sectors. These qualifications may include membership in professional societies, and the various certifications in toxicology. A limited number of colleges and universities confer undergraduate degrees in toxicology. For undergraduates who do not graduate from these undergraduate programs, careful course selection as an undergraduate will enhance graduate education opportunities in toxicology. Many toxicology program admissions committees expect prior coursework in areas such as biochemistry, cell biology, chemistry, genetics, statistics, and other coursework that is regarded as foundational for success in a graduate toxicology curriculum. For individuals who have already received an advanced degree such as a PhD, MD, or DVM in areas other than toxicology, careers can be focused toward toxicology through postdoctoral, clinical, or research training.

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Undergraduate and graduate training Planning Particularly in the United States, individuals with degrees at the BS or MS levels may work in toxicology at CROs and other industries at the technical support level. In Europe, although many study directors in CROs are BS/BSc level toxicologists with appropriate on-the-job training, there is increasing recognition that specialist MS/MSc qualifications are desirable. The breadth of career choices and opportunities for advancement are generally much greater for those with post-baccalaureate degrees. Acceptance into graduate programs in toxicology generally requires a strong academic record and some research experience, but not necessarily undergraduate toxicology coursework. Most graduate toxicology programs have specific prerequisites for admission. The primary requirement is a baccalaureate degree in a relevant field of study such as biology, chemistry, environmental health, or other science-related field. Laboratory courses in chemistry and biology are an important part of an undergraduate education and help develop research skills. Additional upper level courses in cell biology, biochemistry, genetics, and/or physiology will often increase the competitive advantage for graduate school admissions. As the ability to be an effective communicator becomes increasingly important for toxicologists, coursework in scientific writing and public speaking is also useful, as well as mathematical and statistical skills. Individual graduate programs should be consulted to determine specific admission requirements. In addition to a strong academic record, basic laboratory research experience enhances the likelihood of admission to a graduate program in toxicology. Cooperative work–study programs enhance those skills by placing students in a research setting during the semester. Summer internships in a research laboratory provide another approach to gain or enhance laboratory skills. Research internships can provide undergraduate science majors with a stimulating research experience in toxicology. Internships may be available in academic and industrial research laboratories across the country. In Europe, entrance to toxicology as a career follows a broadly similar path to that in the United States. For those who come to toxicology without a PhD, there are an increasing number of professional qualifications that can be achieved with 5 years experience and records of appropriate training. More information can be found via the website of Federation of European Toxicologists & European Societies of Toxicology (EUROTOX, n.d.; http://www.eurotox.com) and of various national societies of toxicology in Europe. Some students obtain pharmacy/pharmacology training first and then do graduate work in toxicology. In the US, some universities train physician-scientists, who earn both MD and PhD degrees; these professionals are well prepared to work in basic and clinical research. Similarly, students may obtain a veterinary degree followed by postdoctoral toxicology training; toxicologic pathologists supply particular expertise that is in high demand.

Selection of an appropriate toxicology program Numerous academic institutions in the United States and abroad support graduate training in toxicology. Identifying a graduate training program and mentor most appropriate for a particular individual requires some advanced planning. First, individuals should establish a potential career plan. To aid in this process, many students complete an Individual Development Plan (IDP), such as that provided by American Association for the Advancement of Science (AAAS, 2021). Toxicology is divided into many subspecialties, such as neurotoxicology, chemical carcinogenesis, genomics, teratology, inhalation toxicology, nanotoxicology, computational modeling, and risk assessment. Although such a choice early in the education process does not commit a student to this direction, careful assessment helps in deciding which programs are most likely to meet an individual student’s goals. Potential applicants are encouraged to communicate with toxicologists in local universities, industries, and governmental agencies to help them select a training program and decide on a future career direction. Details of the specific requirements of toxicology graduate programs can be obtained by referring to individual university websites. Most PhD programs in toxicology require some prior laboratory experience. Most applications to toxicology graduate programs require official college transcripts, a letter of intent, and letters of recommendations. The letter of intent is written by the applicant and describes why the individual wants to be admitted into a particular graduate program, as well as the individual’s general career goals. Recommendations are generally from individuals who know the applicant on a professional or academic level. Examples of appropriate references are research mentors, professors, advisors, and employers. Many toxicology programs, but not all, also require Graduate Record Examination (GRE) scores. The graduate curriculum for a doctorate in toxicology often includes courses in cell biology, biochemistry, physiology, anatomy, histology, pathology, pharmacology, bioinformatics, and statistics. The academic program can also include topics such as analytical methods, carcinogenesis, mutagenesis, teratogenesis, comparative toxicology, molecular mechanisms of toxicology, organ-specific toxicity, risk assessment, computational methods, and/or risk communication. Some programs may also include coursework in statistics, computer science, computational modeling, immunology, and pharmacokinetics. In the United States, the PhD candidate is typically required to conduct a program of original scholarly research that generally extends over a period of two or more years. Another requirement for completion of a PhD program at most US institutions is the writing of a dissertation. This document is written by the student and typically includes an introduction or literature survey, a statement of the hypothesis underlying the dissertation research, methods, results, and a discussion. In keeping with the tradition of the doctorate degree, defense of the graduate research dissertation is expected.

Financial assistance In the United States, financial assistance is often available through research and teaching assistantships, fellowships, traineeships, and grants. Unlike the experience of graduate students in some disciplines, students of toxicology often receive stipend and tuition support and indeed, many toxicology programs are committed to supporting all of the graduate students that they accept. Inquiries should be made to the prospective institution, program, and mentor to determine the availability of grants and financial aid.

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Recent efforts to enhance training of PhD toxicologists Recent efforts to better align toxicology curricula with employer needs have been undertaken under the leadership of the Society of Toxicology (SOT, n.d.; https://www.toxicology.org/). A needs assessment survey conducted by SOT found that, while many toxicology programs are heavily focused on genetics and molecular biology endpoints, with less emphasis on whole animal models, the latter studies are still necessary and critical for human health risk assessment efforts. Survey respondents also pointed out that while many newly graduated PhDs in toxicology may be prepared for academic careers, they might lack some desirable skills for careers in other job sectors that employ toxicologists. Furthermore, a major area of consensus of an Education Summit, held in October of 2011 and sponsored by SOT and the National Institute for Environmental Health Sciences (NIEHS), was a set of Core Competencies that should ideally serve as the basis for toxicology education (Table 1). In addition, much research occurs in teams (often interdisciplinary teams), so project management and interpersonal skills are also strengths. Participants and speakers at the Education Summit discussed shrinking federal funds for training toxicologists, and also noted that new graduates lacked some desirable skills, particularly in preparation for employment in industry or in the field of risk assessment. These factors, taken together, renewed interest for academia–industry and academia–government partnerships to share the training of toxicologists and also produce graduates with a better training portfolio for entry into nonacademic positions as toxicologists (Barchowsky et al., 2012). Useful guidelines have also been published by EUROTOX that outline expectations of a European Registered Toxicologist (ERT), both in terms of curriculum and in suggested course work. Although the guidelines are oriented toward the taking of defined courses, with examination results, they also acknowledge that training can be on the job (http://www.eurotox.com).

Further training European and US PhD or MD students who are interested in a career at an advanced level in toxicology may seek additional specialized postdoctoral training. This training may last from 2 to 4 years beyond the doctoral degree, and typically involves an in-depth, independent research project under the mentorship of an established toxicologist. Most institutions that offer a doctoral degree in toxicology or support advanced research in toxicology have postdoctoral programs; some postdoctoral may also be found in government agencies, pharmaceutical and chemical companies, and not-for-profit organizations. Individuals who have not received training in a graduate program in toxicology can enter a career in toxicology at the time of postdoctoral training. Since a postdoctoral fellowship usually has highly specific requirements, students with specialized training in fields such as molecular biology, genetics, computational modeling, chemical engineering, medicine, and many others may find a toxicology-related research topic that requires their specialty.

Continuing education in toxicology Toxicology is not a stagnant field, and many toxicologists regularly seek opportunities to stay current in the field in multiple ways, such as by attending toxicology-focused conferences, continuing education courses, webinars, and seminars sponsored by universities and toxicology-focused organizations. Certain certifications in toxicology require demonstration of continuing education in toxicology. Career development and progression in the field may also require demonstration of knowledge and expertise in emerging technologies in toxicology and emerging related scientific disciplines. To fulfill this need, professional organizations serving toxicologists regularly offer continuing education opportunities for their members, as well as non-members who attend their annual meetings. With the need for global continuing education on an ongoing basis, professional organizations are also developing continuing education opportunities throughout in the form of webinars that can be readily accessed without the need to attend a conference in person. Table 1

Core competencies for the “Total Toxicologist.”

Fundamentals of toxicology Advanced principles of toxicology Pathophysiology Anatomy and physiology Applied systems biology Biochemistry Molecular genetics Regulatory frameworks Experimental design Communication skills Critical thinking skills Data and statistical analysis Reproduced with publisher’s permission from Barchowsky A. et al. (2012) The toxicology education summit: Building the future of toxicology through education. Toxicological Sciences 127(2): 331–338.

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Certification in toxicology Certification in toxicology is conferred by several organizations, worldwide, and is intended to objectively demonstrated competence in toxicology in a rigorous public arena (Brock et al., 2009). Key features of the certifications conferred by the American Board of Toxicology, EUROTOX, and the Japanese Society of Toxicology are summarized below. The American Board of Toxicology (ABT), Inc. was incorporated in 1979 and administered its first certification examination in 1980 (abtox.org). The individuals passing this exam, as well as those who have certified subsequently, earn the right to use the credential “Diplomate of the American Board of Toxicology,” or “DABT.” The process of becoming a DABT begins with an evaluation of the candidate’s eligibility, as determined by a subcommittee of the ABT, using a list of eligibility standards. Those individuals who are deemed eligible and are active in the practice of toxicology then take a multiple-choice examination. DABT status is conferred on those individuals passing this exam. Recertification is required every 5 years after an individual is initially certified, and this process includes demonstration of eligibility, as evidenced by continued active practice of toxicology, professional development/continuing education activities, and beginning in 2017, interpretation of selected literature. There is often a monetary benefit of certification in toxicology, which was identified by comparing the salaries of individuals with the same numbers of years of experience in toxicology, with and without ABT certification (Gad and Sullivan, 2016). As individuals gain increasing experience in toxicology, the next step is often recognition by the Academy of Toxicological Sciences (ATS). Certification by ATS involves a peer-review process and recertification every 5 years (acadtoxsci.org/). Individuals receiving this recognition typically are more senior toxicologists who may have served as officers in professional toxicology organizations, or have served on grant reviewing boards, or national and international toxicology committees. The certification process in Europe is somewhat different. The designation “European Registered Toxicologist” (ERT) is broadly equivalent to the Diplomate status with the ABT. The requirements are very similar, except that there is no examination. There is a requirement for two senior toxicologists from different organizations to act as referees; these individuals must provide extensive justification for inclusion of the candidate on the register of the national society (http://www.eurotox.com). In Japan, registration as a Diplomate of the Japanese Society of Toxicology (DJST) is only open to members of that Society. Eligibility for DJST is based on a point system, and to be eligible, the candidate must have three consecutive years of membership in JSOT, must demonstrate appropriate experience in the practice of toxicology, and must demonstrate a high level of activity in publishing in toxicology journals, attending national and international toxicology meetings, and participating in JSOT-sponsored educational courses. An examination is part of the process, and recertification is required every 5 years. The International Union of Toxicology (IUTOX), the international association composed of national and regional toxicology societies, has undertaken a study of the various ways that expertise of toxicologists is recognized in order to produce a compendium to allow global comparison of certifications via the Toxicology Recognition Task Force Matrix (TRTFM, n.d.) Results of surveys by the Task Force can be found at the TRTFM website: https://www.iutox.org/trtf.asp.

Summary Toxicology is a growing field, with a worldwide presence. Toxicology education occurs at the undergraduate level, graduate level, and in postdoctoral training in various formats. Individuals with degrees from bachelor level to doctoral level of scientists can be employed in toxicology, and many toxicologists have additional degrees in clinical or veterinary medicine. Career opportunities in toxicology range from employment in large pharmaceutical organizations, to chemical companies, to governmental agencies, contract research organizations, consulting firms, etc. Toxicologists may wish to seek certification in toxicology to assure employers and clients of their competency in the field, and these certifications can be attained from various organizations world-wide.

References AAAS (2021). https://myidp.sciencecareers.org/. Accessed 3/10/2022. Barchowsky A, Buckley LA, Carlson GP, Fitsanakis VA, Ford SM, Genter MB, et al. (2012) The toxicology education summit: Building the future of toxicology through education. Toxicological Sciences 127(2): 331–338. Brock WJ, Woolley AP, and Sugimoto T (2009) Certification in toxicology: An international perspective of risk:benefit. International Journal of Toxicology 28(3): 147–150. EUROTOXFederation of European Toxicologists & European Societies of Toxicology. http://www.eurotox.com. Accessed 3/10/2022. Gad SC and Sullivan DW Jr. (2016) Ninth triennial toxicology salary survey. International Journal of Toxicology 35(2): 243–251. https://doi.org/10.1177/1091581816630296. National Research Council (2009) Science and Decisions: Advancing Risk Assessment. Washington, DC: The National Academies Press. https://doi.org/10.17226/12209. Accessed 10 March 2022. Skinner MK and Nilsson EE (2021) Role of environmentally induced epigenetic transgenerational inheritance in evolutionary biology: Unified evolution theory. Environmental Epigenetics 7(1): dvab012. https://doi.org/10.1093/eep/dvab012. Society of Toxicology. https://www.toxicology.org/. Accessed 3/10/2022. Sullivan DW and Gad SC (2020) Tenth triennial toxicology salary survey. International Journal of Toxicology 39(3): 189–197. https://doi.org/10.1177/1091581820910378. Toxicology Recognition Task Force Matrix (TRTFM). https://www.iutox.org/trtf.asp. Accessed 3/10/2022.

Further reading Academy of Toxicological Sciences. acadtoxsci.org. Accessed 3/10/2022.

Catecholamines Bracha Gurwitz and Sidhartha D Ray, Department of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S. Othumpangat, Catecholamines, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 748–750, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00271-2.

Chemical profile Introduction Uses Environmental fate and behavior Routes and pathways Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Animal Human In vitro Chronic toxicity Human Clinical management Ecotoxicology Conclusion References Further reading

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Abstract Catecholamines are endogenous compounds and are synthesized in the brain, the adrenal medulla, and by some sympathetic nerve fibers. Catecholamine is the name of a group of compounds that contain a catechol nucleus and an amine group. Examples are dopamine, norepinephrine, and epinephrine (aka adrenalin), and non-mammalian compounds such as octopamine. Epinephrine stimulates both the alpha- and beta-adrenergic systems. Epinephrine is called adrenaline and norepinephrine is called noradrenaline. Commercially available self-injectable epinephrine (aka Epipen®) has saved numerous lives over the decades. Catecholamines are water-soluble and could bind to plasma proteins. Dopamine is extensively metabolized in the liver resulting in inactive metabolites (75% of the dose) and norepinephrine (active, 25% of the dose) in the adrenergic nerve terminals. Dopamine is widely distributed in the body but does not cross the blood-brain barrier to a substantial extent. Dopamine is linked with our brain’s reward center. Dopamine is frequently used in critically ill newborn infants for treatment of shock and cardiac failure. Dopamine toxicity damages the neurons. Catecholamine metabolites and their conjugates are excreted in urine. Each compound has its own synonyms.

Keywords Catecholamines; Dopamine; Epinephrine; Neurons; Neurotransmitter; Norepinephrine; Sympathomimetic; Tyrosine

Key points

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Catecholamine is the name of a group of compounds that contain a catechol nucleus (a benzene ring with two adjacent hydroxyl substituents) and an amine group. This group includes the mammalian neurotransmitters, such as dopamine, norepinephrine, and epinephrine, and nonmammalian compounds such as octopamine. Dopamine is widely distributed throughout the central nervous system and is involved in the control of movement. Dopamine is abundant in meats, dairy products, and soy. Amino acid phenylalanine is the precursor for all catecholamines including dopamine. The amount of dopamine that can be made is limited by the activity of the first enzyme in the synthesis chain—tyrosine hydroxylase. Catecholamines are considered non-genotoxic, but they are reproductive toxins. Their effects may vary in humans and experimental animals. Epipen® contains epinephrine (adrenaline), which is used in emergencies to treat very serious allergic reactions on exposure to insect stings or bites, foods, drugs, chemicals, or other substances. Epinephrine acts very fast in this situation to improve

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breathing, stimulate heartbeat, raise a rapidly declining blood pressure, reverse hives, and reduce swelling of the face, lips, and throat. Dopamine is frequently used in critically ill newborn infants for treatment of shock and cardiac failure. DA (and serotonin and endorphins) is also known as happy hormone because of its connection to reward center of our brain.

Chemical profile

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Name: Dopamine Chemical Abstracts Service Registry Number: 51-61-6. Synonyms: Pyrocatechol; 4-(2-Aminoethyl) pyrocatechol; 3-Hydroxytyramine; 3,4-Dihydroxyphenethylamine; 4-(2-Aminoethyl)-1,2-benzenediol; Dopastat. Molecular Formula: C8H11NO2

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Name: Epinephrine Chemical Abstracts Service Registry Number: 51-43-4. Synonyms: Benzyl alcohol, Adrenalin, Epirenamine, Methylaminoethanolcatechol, Vasotonin. Molecular Formula: C9H13NO3

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Name: Norepinephrine Chemical Abstracts Service Registry Number: 51-41-2. Synonyms: 4-(2-Amino-1-hydroxyethyl)-1,2-benzenediol; a-(Aminomethyl)-3,4-dihydroxybenzyl alcohol; 2-Amino-1-(3,4-dihydroxyphenyl)ethanol; 1-(3,4-Dihydroxyphenyl-2-aminoethanol); Noradrenaline. Molecular Formula: C8H11NO3

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Introduction Catecholamines are endogenous neurotransmitters or hormones. Dopamine and norepinephrine are in the monoamine class. They are synthesized in the brain, the adrenal medulla, and by some sympathetic nerve fibers. Dopamine biosynthesis begins with phenylalanine as the precursor, and proceeds sequentially through tyrosine, DOPA, and then dopamine. Tyrosine hydroxylase serves as the rate-limiting enzyme in this pathway. Another important enzyme is DOPA decarboxylase, which decarboxylates DOPA to form dopamine. The same enzyme acts on both naturally occurring DOPA and L-dopa (levodopa), the Parkinson’s disease medicine. Catecholamines are formed from dopamine mediated by the enzyme dopamine beta-hydroxylase, and epinephrine is formed from norepinephrine by enzyme phenylethanolamine N-methyltransferase. Parkinson’s disease is one of the most common neurodegenerative disorders and is characterized by the selective loss of dopaminergic neurons in the substantia nigra. Dopamine is widely distributed throughout the CNS and is involved in the control of movement. Dopamine is abundant in meats, dairy products, and soy. The amount of dopamine that can be made is limited by the activity of the first enzyme in the synthesis chain—tyrosine hydroxylase. Cells that use dopamine as a neurotransmitter are referred to as dopaminergic. Norepinephrine is an important neurotransmitter in both the CNS and the sympathetic part of the autonomic nervous system. The hormone epinephrine acts together with the sympathetic nervous system to initiate the body’s quick response to stressful stimuli (Othumpanagat, 2014). Mammalian monoamines are fascinating compounds, because they act as neurotransmitters and as regulatory molecules that strive to maintain homeostasis, but that may become part of the problem in some diseases. For example, the well-known neurotransmitters adrenaline & noradrenaline of sympathetic neurons are secreted by the adrenal gland to maintain homeostasis. However, patients with adrenal gland tumor can land into serious symptoms due to the overproduction of these monoamines. Dopamine (DA) is more known as a neurotransmitter, although it also acts as a compound that helps in maintaining homeostasis. DA does not cross blood brain barrier. Imbalances in dopamine neurotransmission and alterations of brain circuits where dopamine is a key factor are involved in a variety of neurological and neuropsychiatric disorders, from alcohol/drug addiction to schizophrenia. Five receptors have been characterized for DA (D1, D2, D3, D4 and D5), and these belong to the superfamily of G protein-coupled receptors (GPCRs). Coincidentally, antipsychotics precisely act on the dopamine system. Although the exact mechanisms remain obscure, several studies show a dopamine link between the gut and the CNS someway linked to the immune system. Studies on primary in vitro cultures of melanocytes exposed to different doses of epinephrine, norepinephrine, and dopamine concluded DA to be more toxic than epinephrine and norepinephrine (Pubchem: NorEpinephrine, n.d.; Talwar, 2022).

Uses Catecholamines are sympathomimetic drugs. Dopamine and norepinephrine are used as vasopressors (antihypotensives). Catecholamines are water-soluble and are 50%-bound to plasma proteins and can always be detected in the circulating blood. Epinephrine stimulates both the alpha- and beta-adrenergic systems, causes systemic vasoconstriction and gastrointestinal relaxation, stimulates the heart, and dilates bronchi and cerebral vessels. It is also used as a vasoconstrictor, cardiac stimulant, or bronchodilator to counter allergic reaction, anesthesia, and cardiac arrest. Epinephrine is used to treat severe allergic (anaphylactic) reactions because it can prevent or minimize the effects of histamine. It is also an antiglaucoma agent. Epinephrine is also commonly used treat emergent priapism (Pubchem: Epinephrine, n.d.; Fatih et al., 2020). The use of catecholamines is also being investigated as a treatment for beta-blocker toxicities. Catecholamines are supposed to provide a survival benefit and improve hemodynamics (Rotella et al., 2020). Dopamine is frequently used in critically ill newborn infants for treatment of shock and cardiac failure (Pubchem: Dopamine, n.d.). The role of Catecholamines is implicated in numerous stress-related and other psychiatric disorders (Ross and Van Bockstaele, 2020). Dopamine agonists are used to treat depression, and medications which increase norepinephrine is first line in the treatment for ADHD (Fatih et al., 2020).

Environmental fate and behavior Routes and pathways Catecholamines are mostly administered by intravenous injection or infusion. They have a very short half-life when circulating in the blood and are easily soluble in water. Epinephrine is available in nebulized racemic dosage form for inhalation. Intoxication from catecholamine usually results from iatrogenic overdoses, accidental intravenous administration, and the injection of solution intended for nebulization. High concentrations of dopamine present inside of a cell than there are vesicles to store can lead to oxidative stress and cause damage or death of the cell. It is thought that dopamine overload causes biochemical damage to cellular mitochondria, that provide the cell with all the energy it requires to function, resulting in death of the cell. Catecholamines produced circulatory changes that reversed propofol anesthesia in animal models (Fig. 1).

Toxicokinetics Epinephrine is well absorbed after oral administration but is rapidly inactivated in the gut mucosa. When catecholamines were intravenously injected or infused, the onset of drug effect is rapid (within 5 min for dopamine and 3–10 min for epinephrine) and

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Dopamine Synthesis & Metabolism 1

2

L-Tyrosine

Dopamine

DOPA decarboxylase Aromatic L-amino acid decarboxylase

Tyrosine hydroxylase

O2 Tetrahydropbiopterin

3

L-Dihydroxyphenylalanine

CO2

H2O dihydropbiopterin

H2O dehydroascorbic acid

5

Epinephrine (Adrenaline)

4

Norepinephrine (Noradrenaline)

O2 ascorbic acid

Phenylethanolamine N-Methyltransferase

S-Adenosyl methionine

Homocysteine

Fig. 1 The best understood biosynthetic pathway to dopamine synthesis within the CNS. Dopamine is primarily synthesized in dopamine-producing neurons (dopaminergic neurons) within the brain, with small amounts of dopamine also being produced in the medulla of the adrenal glands. In the classical pathway, the direct metabolic precursor of DA is L-dihydroxyphenylalanine [2] (levodopa or L-DOPA) which is synthesized either directly from tyrosine [1], a non-essential amino acid or indirectly from an essential amino acid, phenylalanine. L-phenylalanine forms L-tyrosine in the liver (by the enzyme phenylalanine hydroxylase—not shown) in the presence of oxygen, iron, and tetrahydrobiopterin as cofactors. Tyrosine produced in the liver is then transported into the dopaminergic neurons within the brain. This is followed by the conversion of L-tyrosine [1] into L-DOPA [2] through hydroxylation at the phenol ring by the enzyme tyrosine hydroxylase. Next, L-DOPA is converted into dopamine (3,4-dihydroxyphenethylamine) through decarboxylation by the enzyme DOPA decarboxylase in the pre-synaptic terminal. DOPA decarboxylase is also known as aromatic-L-amino acid decarboxylase due to its action on all naturally occurring aromatic L-amino acids. Furthermore, under specific conditions, dopamine can also be synthesized by a minor pathway, in which L-tyrosine is converted into p-tyramine (mediated by aromatic-L-amino acid decarboxylase), with subsequent hydroxylation to dopamine by the enzyme CYP2D6 which is found in the substantia nigra of human brain. DA is metabolized after reuptake into dopaminergic neurons or glial cells. For further biotransformation, please refer to the mechanisms of toxicity section of this article. The following steps are straightforward which involves formation of norepinephrine [4] and epinephrine [5] (as indicated in the figure). Courtesy original artwork provided by Burd E, Gurwitz B and Ray S (2022).

the duration of drug effect is short (10 min for dopamine, 1 or 2 min for norepinephrine, and 15 min to hours for epinephrine depending on route of administration). Exogenous catecholamines in the circulation are rapidly and efficiently taken up by adrenergic neurons. Catecholamines are metabolized by monoamine oxidase (MAO), which is localized largely in the outer membrane of neuronal mitochondria, and by catechol-O-methyl transferase (COMT), which is found in the cytoplasm of most animal tissues, particularly the kidneys and the liver. The primary metabolites of dopamine are homovanillic acid and dihydroxyphenylacetic acid (75%) and norepinephrine (25%), whereas metabolites of epinephrine and norepinephrine are vanillylmandelic acid and 3-methoxy-4-hydroxyphenethyleneglycol. Catecholamine metabolites and their conjugates are excreted in urine.

Mechanism of toxicity Catecholamines are sympathomimetic drugs. These drugs increase heart rate and cardiac output and may produce cardiac arrhythmias. Administration of norepinephrine also results in increased peripheral vascular resistance. Both effects may cause serious systemic hypertension, which may cause cerebral hemorrhage. Reduced hepatic and renal blood flow may cause tissue ischemia, increase glycolysis, and serum lactic acidosis. In very high doses, a paranoid state may be induced. Recent studies have demonstrated that norepinephrine may enhance or inhibit immune function under certain conditions. Increased levels of catecholamines can also increase fat lipolysis and reduce adipogenesis. Production of reactive oxygen species and formation of quinone during the metabolism of dopamine are likely involved in dopamine toxicity. Numerous in vitro and in vivo studies concerning dopamine-induced neurotoxicity have been reported in the last two decades. The reactive oxygen species generated in the enzymatic oxidation or auto-oxidation of excess dopamine-induced neuronal damage and apoptotic cell death. Dopamine and its metabolites containing two hydroxyl residues exert cytotoxicity in dopaminergic neuronal cells mainly due to the generation of highly reactive dopamine quinones which are dopaminergic neuron-specific cytotoxic molecules. Dopamine oxidation generates to 5,6-indolequinone, dopamine-O-quinone, and aminochrome. Roles of 5,6-indolequinone and dopamine-O-quinone in the degenerative process in Parkinson’s diseases remains obscure because both are unstable metabolites. Therefore, aminochrome remains the prime suspect in Parkinson’s disease because it stabilizes the neurotoxic protofibrils of alpha-synuclein, induces mitochondrial dysfunction leading to oxidative stress, and the degrades vital proteins via lysosomal and proteasome systems. It has also been proposed that dopamine quinones may irreversibly

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alter protein function through the formation of 5-cysteinyl-catechols on the proteins. Overall, it is believed that the melanin-synthetic enzyme tyrosinase in the brain may rapidly oxidize excess amounts of cytosolic dopamine and L-dopa and prevent slowly progressive cell damage by auto-oxidation of dopamine, thus maintaining dopamine levels (Latif, 2021). More work is needed to firmly establish all these effects in in vivo setting. The induction of ER stress is now believed to be a unique effect of catecholamines. There are multiple pathways by which catecholamines regulate ER stress, and pathways are cell-type specific. For instance, norepinephrine treatment failed to evoke ER stress in human fibroblasts, while it did evoke ER stress in other cells. Inducing ER stress can be advantageous at certain instances, such as during an acute burn injury. However, sustained high levels of catecholamines can cause hypermetabolism and metabolic dysfunction (Abdullahi et al., 2020). Recent research suggests that catecholamines are implicated in tumor growth. VEGF upregulation, which is the mediator of angiogenesis, was observed in ovarian cancer cells after exposure to norepinephrine. In addition, the stromal invasiveness of the ovarian cancer cells increased, linking the role of catecholamines in the role of tumor growth and metastasis (Melhem and Conzen, 2011).

Acute and short-term toxicity Animal Overdose of catecholamines may result in animal death. In test animals, there is evidence that death is the result of respiratory arrest caused by hypertension following overdose of epinephrine. Old research performed on laboratory rats, rabbits, mice, and dogs resulted in death, internal bleeding and pulmonary congestion when exposed to very high doses of Dopamine (Rotella et al., 2020). Catecholamines were shown to exert developmental and reproductive toxicity (Aydın and Tugcu, 2021). Some studies have reported toxic effects of catecholamine overdose (epinephrine + Nor-epinephrine) in rats. This combo exposure protocol provided important clinical insights into the pathogenesis of catecholamine storm in vivo. Combination exposure led to tachycardia (ventricular cardiomyopathy), pulmonary edema (plain injury), higher expression of Troponin T and connexin43. Rats injected with NE had a lower survival rate than those injected with E (Lu et al., 2020). For dopamine: LD50 oral mouse ¼ 1460 mg/kg and LD50 oral rats ¼ 1780 mg/kg; For Epinephrine LD50 SC or dermally rat ¼ 62 mg/Kg; For Norepinephrine: LD50 oral mouse ¼ 20 mg/kg (Pubchem: NorEpinephrine, n.d.).

Human At high infusion rates of dopamine, ventricular arrhythmias, and hypertension may occur. Nausea, vomiting, and angina pectoris are occasionally seen. Gangrene of the extremities may occur in patients with profound shock given large doses of dopamine for long periods of time. Norepinephrine may cause dose-related hypertension (sometimes indicated by headache), reflex bradycardia, increased peripheral vascular resistance, and decreased cardiac output. High doses of norepinephrine (in excess of 8–12 mg of base per min) cause intense vasoconstriction, which results in ‘normal’ blood pressure but decreased tissue perfusion. Local necrosis may result from perivascular infiltration and angina, mesenteric ischemia, and peripheral ischemia. Epinephrine may cause dose-related restlessness, anxiety, tremor, cardiac arrhythmias, palpitation, hypertension, weakness, dizziness, and headache. A sharp rise in blood pressure from over-dosage of epinephrine may cause cerebral hemorrhage and pulmonary edema. High catecholamine levels in blood are also associated with stress, due to psychological or environmental stressors.

In vitro Primary cultures of melanocytes exposed to different doses of epinephrine, norepinephrine, and dopamine observed DA to be more toxic than epinephrine and norepinephrine. Persistent exposure to dopamine resulted in decreased cell proliferation and adhesion potential with concomitant apoptosis. Gene expression changes also confirmed the weak adhesion and survival potential of cells under the toxic effects of dopamine (Talwar, 2022).

Chronic toxicity Human Prolonged use and repeated injection of epinephrine may lead to tolerance and local necrosis. Prolonged use of norepinephrine may cause edema, hemorrhage, focal myocarditis, necrosis of the intestine, or hepatic and renal necrosis. It may also cause plasma volume depletion, which may result in perpetuation of the shock state or recurrence of hypotension when the drug is discontinued. High levels of catecholamines may be also due to the low levels of monoamine oxidase A (MAO-A). MAO-A is one of the enzymes responsible for degradation of these neurotransmitters, and thus balance the levels of catecholamines. Catecholamine-secreting tumors, such as the phaeochromocytoma (PC) or paragangliomas can lead to fatal multiorgan dysfunction secondary to catecholamine-induced hypertension or hypotension and cardiovascular collapse. A catecholamine crisis is defined as acute and severe hemodynamic instability and can occur in a patient with PC, with the most common complication being cardiomyopathy, or more specifically, Takotsubo cardiomyopathy. Evidence suggests that this specific form of

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cardiomyopathy is the result of hyperstimulation of the cardiac sympathetic nerve terminals secondary to increased catecholamine levels, specifically NE levels. (Casey et al., 2017). Findings in laboratory rats with PC suggests that the cardiopulmonary toxicities of Norepinephrine is greater than that of Epinephrine (Rotella et al., 2020).

Clinical management Basic and advanced life-support measures should be utilized as necessary. Treatment is directed at ameliorating tachycardias, shock, cardiac arrhythmias, systemic hypertension, pulmonary edema, and lactic acidosis. In the case of severe toxicity, administration of a rapidly acting a-adrenergic blocking drug such as phentolamine may be considered. Glutathione is a scavenger for dopamine oxidation intermediates, and it may provide complete protection against dopamine-mediated toxicity. In treatment of PC and paragangliomas, the most important consideration is the hemodynamic management, as during the removal of the tumor, the abrupt decrease in catecholamines can cause a hypertensive crisis. Adrenergic a-blockers are typically used before resection of the tumor, and during surgery, to provide blood pressure control. General anesthesia is preferred over spinal anesthesia because spinal anesthesia is not sufficient in protecting against the hemodynamic changes caused by catecholamine release during tumor resection. There is no specific treatment for catecholamine-induced cardiomyopathy, but it should include lowering sympathetic activity by means sympathetic antagonists and diuretics administration (Jia et al., 2017). As is discussed above, COMT (catechol-O-methyl transferase) is involved in dopamine degradation; therefore, inhibitors of COMT (entacapone and tolcapone) offer therapeutic options of preserving endogenous dopamine levels, by reducing its breakdown (Song et al., 2021).

Ecotoxicology Toxicity of catecholamines in a ciliated protozoan Tetrahymena pyriformis has been reported in study. Catecholamines exhibited moderate acute toxicity to the protozoans. Dopamine showed toxic potential Effective Concentrations (EC10) of 0.63 ppm in T. pyriformis and a higher concentration of dopamine inhibited the synthesis of adrenalin in these protozoans.

Conclusion Catecholamines (CAs) are essential biogenic amines that are present in extremely small amounts in human body as neurotransmitters or hormones; however, in the brain, they act at the highest levels of mental function. CAs modulate myriad of functions such as processing of associations, integration of thought processes with movement and speech, emotional tone or affect, mood, appetite, arousal, and sleep/wakefulness state, but unfortunately, these functions have not been modeled in experimental animals. Most studies are based on humans. CAs are short-lived signaling molecules in plasma, with a half-life between 10 s and 100 s. Approximately one half of the CAs circulate in plasma, but plasma concentrations fluctuate widely, and their role in specific mental disorders remains obscure. Human performance and behavior are heavily influenced by the levels of CAs. Under normal stress and arousal, elevated CA output associated with improved performance, whereas under conditions of high levels of stress, additional increase of these substances may cause decline in mental functioning and abnormal behavior. Commercially available self-injectable epinephrine (aka Epipen®) has saved numerous lives over the decades (Table 1). Table 1

List of catecholamines used in medicine.

Medication

Brand name

Indications and uses

Norepinephrine Epinephrine

LevophedW AdrenalinW, AllerjectW, AnapenW, ArticadentW, AstracaineW, Auvi-QW, CitanestW, Citanest ForteW, EmeradeW, EpipenW, LignospanW, MarcaineW, Marcaine With EpinephrineW, OctocaineW, Octocaine With EpinephrineW, OrablocW, ScandonestW, SensorcaineW, Sensorcaine With EpinephrineW, SeptanestW, SeptocaineW, SymjepiW, UltacanW, UltracaineW, VivacaineW, XylocaineW, Xylocaine With EpinephrineW, ZorcaineW

Blood pressure control during hypotensive states Allergic reactions, to restore cardiac rhythm, nasal congestion, glaucoma, and asthma

Dopamine Carbidopa Dipivefrin Metaproterenol Dobutamine Methyldopa Isoprenaline Levodopa Racepinephrine

DuodopaW, DuopaW, LodosynW, ParcopaW, RytaryW, SinemetW, StalevoW

IsuprelW DuodopaW, DuopaW, InbrijaW, ParcopaW, ProlopaW, RytaryW, SinemetW, StalevoW AsthmanefrinW

Hemodynamic imbalances, poor perfusion of vital organs, low cardiac output, and hypotension In combination with levodopa for the symptomatic treatment of idiopathic Parkinson’s disease and other conditions associated with parkinsonian symptoms Chronic open-angle glaucoma. Bronchospasms, asthma, and COPD Cardiac decompensation Hypertension and hypertensive crisis Bradycardia and heart block Parkinson’s disease Asthma

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See also: Mode of action in toxicology; Occupational exposure limits; Monoamine oxidase inhibitors; Estrogens II: Catechol estrogens

References Abdullahi A, Wang V, Auger C, et al. (2020) Catecholamines induce endoplasmic reticulum stress via both alpha and beta receptors. Shock 53(4): 474–480. https://pubmed.ncbi.nlm. nih.gov/31232864/. Aydın A and Tugcu G (2021) Toxicological assessment of epinephrine and norepinephrine by analog approach. Food and Chemical Toxicology 118: 726–732. https://pubmed.ncbi. nlm.nih.gov/29913233/. Casey RT, et al. (2017) Management of an acute catecholamine-induced cardiomyopathy and circulatory collapse: A multidisciplinary approach. Endocrinology, Diabetes & Metabolism Case Reports 2017: 17-0122. Fatih O, Muhammed CT, _llbey KO, and Ebru K (2020) The role of biogenic amines in nutrition toxicology: Review. International Journal of Nutrition 5(1): 21–29. Jia X, Guo X, and Zheng Q (2017) Perioperative management of paraganglioma and catecholamine-induced cardiomyopathy in child—A case report and review of the literature. BMC Anesthesiology 17(1): 142. Latif S (2021) Dopamine in Parkinson’s disease. Clinica Chimica Acta 522: 114–126. https://pubmed.ncbi.nlm.nih.gov/34389279/. Lu WH, et al. (2020) Norepinephrine leads to more cardiopulmonary toxicities than epinephrine by catecholamine overdose in rats. Toxics 8(3): 69. https://pubmed.ncbi.nlm.nih.gov/ 32947820/. Melhem A and Conzen S (2011) Connecting environmental stress to cancer cell biology through the neuroendocrine response. In: Nriagu JO (ed.) Encyclopedia of Environmental Health, pp. 822–827. Burlington: Elsevier. Othumpanagat S (2014) Catecholamines. In: Encyclopedia of Toxicology, 3rd edn, 748–750. https://www.sciencedirect.com/science/article/pii/B9780123864543002712. Pubchem: Dopamine (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/Dopamine Pubchem: Epinephrine (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/5816 Pubchem: NorEpinephrine (n.d.) https://pubchem.ncbi.nlm.nih.gov/compound/439260 Ross JA and Van Bockstaele EJ (2020) The role of catecholamines in modulating responses to stress: Sex-specific patterns, implications, and therapeutic potential for post-traumatic stress disorder and opiate withdrawal. European Journal of Neuroscience 52(1): 2429–2465. Rotella J, Greene SL, Koutsogiannis Z, et al. (2020) Treatment for beta-blocker poisoning: A systematic review. Clinical Toxicology 58(10): 943–983. https://pubmed.ncbi.nlm.nih.gov/ 32310006/. Song Z, et al. (2021) Different catechol-o-methyl transferase inhibitors in Parkinson’s disease: A Bayesian network meta-analysis. Frontiers in Neurology 12: 707723. https://pubmed. ncbi.nlm.nih.gov/34630283/. Talwar S (2022) Dopamine toxicity contributes to melanocyte loss via melanocytorrhagy: An in vitro study. International Journal of Dermatology. https://doi.org/10.1111/ijd.16166.

Further reading Brunton LL, et al. (2018) Goodman & Gilman’s the Pharmacological Basis of Therapeutics. New York: McGraw Hill Medical. Clausius N, Born C, and Grunze H (2009) The relevance of dopamine agonists in the treatment of depression. Neuropsychiatrie 23(1): 15–25. Melhem A and Conzen S (2011) Connecting environmental stress to cancer cell biology through the neuroendocrine response. In: Nriagu JO (ed.) Encyclopedia of Environmental Health, pp. 822–827. Burlington: Elsevier. Nelson LS, et al. (2019) Goldfrank’s Toxicologic Emergencies. New York, N.Y: Mc Graw Hill Education. Ross JA and Van Bockstaele EJ (2020) The role of catecholamines in modulating responses to stress: Sex-specific patterns, implications, and therapeutic potential for post-traumatic stress disorder and opiate withdrawal. European Journal of Neuroscience 52(1): 2429–2465.

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CCA-treated wood Ayesha Rahman Ahmed, Department of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, WA, United States © 2024 Elsevier Inc. All rights reserved. This is an update of C. Barton, CCA-Treated Wood, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 751–752, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00272-4.

Chemical profile Background Uses/occurrence Exposure and exposure monitoring Toxicokinetic Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Eye: Dust may irritate eyes Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines References

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Abstract Chromated copper arsenate (CCA) is a chemical preservative that protects wood from rotting due to insects and microbial agents. CCA has been used to pressure treat lumber since the 1930s. Since the 1970s, the majority of the wood used in residential settings was CCA-treated wood. CCA is a registered chemical pesticide that is subject to U.S. Environmental Protection Agency’s (EPA’s) regulation. The pesticide registration for CCA was modified as a result of a voluntary agreement reached on February 12, 2002 between the registrants and EPA, to transition to a new generation of preservatives for most non-industrial applications. That agreement permitted the use of CCA for all existing registered uses until December 31, 2003, and the continued sale and distribution of CCA-treated wood treated by the label. After January 1, 2004, following label amendment, CCA was permitted and continues to be sold to treat wood only for commercial, industrial and non-residential uses. The exposure to CCA is intensified by hand-to-mouth behavior, which is well-documented among children. The exposure to CCA is direct contact with wood as arsenic in CCA-treated wood can be dislodged. The exposure through contaminated soil through leaching of the CCA from wood into surrounding soil is well documented. The exposure through incomplete fixation leads to arsenic, chromium, and copper toxicity causing skin irritation, and increasing risk of chronic health effects. The acute and chronic health effect is mainly due to the arsenic and chromium toxicity.

Keywords CCA; Chromated copper arsenate; Pressure treated; Treated wood; Wood preservative

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00375-4

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Glossary

Chromated copper arsenate (or CCA) A chemical preservative that protects wood from rotting due to insects and microbial agents.

Key points

• • • • • •

CCA is an inorganic arsenical used as a wood preservative. In February 2002, the Environmental Protection Agency (EPA) announced a voluntary phase-out by industry of most residential uses of this arsenic-based wood preservative. CCA exposure is mainly through ingestion and inhalation, and to a lesser extent skin absorption, that poses risks to both human health and the environment. Children face especially high risks from exposure since they take in more pesticides relative to body weight than adults. EPA Carcinogen Assessment Group has classified inorganic arsenic as a Group A carcinogen. The form of chromium (hexavalent) found in CCA has also been found by EPA to be a known human carcinogen.

Abbreviations

CCA Chromated copper arsenate CPSC Consumer Product Safety Commission EPA U.S. Environmental Protection Agency

Chemical profile The composition of CCA products is usually described in terms of the mass percentages of its component compounds including chromium trioxide or chromic acid (CrO3), cupric oxide or copper (II) oxide (CuO), and arsenic pentoxide (As2O5).

• • • • •

Name: Copper Arsenate, Chromated Synonyms (Chromated copper arsenate): CCA-C, Copper Arsenate, Chromated, HSDB 7705, Arsenic acid (H3AsO4), copper(2+) salt (2:3), chromated Chemical Abstract Service Registry Number (Chromated copper arsenate): 37337-13-6 Molecular Formula (Chromated copper arsenate): As2CrCuO9 Chemical Structure (Chromated copper arsenate):

Source: https://pubchem.ncbi.nlm.nih.gov; https://w.chemicalbook.com/ChemicalProductProperty_US_CB71325163.aspx

Background In varying ratios, arsenic pentoxide, chromic acid, and cupric oxide are used for chromated copper arsenate (CCA) formulations to protect the wood from rotting. The copper is a preservative and protect the wood from the attack of bacteria and fungi, arsenic is an insecticide and chromium acts as a binder of arsenic and copper to the wood surface (Wang and Chiu, 2008). The American Wood-Preservers Association (AWPA, 1998) has typically classified CCA treatment solutions as A, B or C based on the ratio of the metal oxides (Shamim et al., 2008). The requirement for American Wood-Preservers’ Association (AWPA’s) Preservative Standard CCA-Type A, B and C composition are shown below (ASTM, 1995; Cox, 1991; Katz and Salem, 2005).

CCA-treated wood

Composition in percentage

Type A (min-max)

Type B (min-max)

Type C (min-max)

Arsenic pentoxide (As2O5) Chromic acid (CrO3) Cupric oxide (CuO)

16.4 (14.6–19.7) 65.5 (59.4–69.3) 18.1 (16.0–20.9)

45.1 (42–48) 35.3 (33–38) 19.6 (18–22)

34 (30–38) 47.5 (44.5–50.5) 18.5 (17–21)

659

CCA treated wood is also referred to as a pressure-treated wood where lumber is loaded into a horizontal cylinder. The cylinder door is sealed, and a liquid solution containing CCA is pumped in. The pressure in the cylinder is then raised, forcing the CCA into the wood. At the end of the process, the excess treatment solution is pumped back into a storage tank for reuse. The CCA solution is toxic. Therefore, it can be applied only by U.S. Environmental Protection Agency (EPA) certified pesticide operators (Barton, 2014). EPA conducts registration reviews to ensure that products can carry out their intended function without creating unreasonable risks to human health and the environment. EPA implemented additional mitigation measures to protect workers who apply chromated arsenicals (epa.gov website).

Uses/occurrence CCA is a chemical mixture (pesticides) registered by the EPA for use as a wood preservative. It has been demonstrated to protect the wood from dry rot, fungi, molds, termites, and other pests that can threaten the integrity of wood products. CCA-treated wood is most commonly used in outdoor settings as it is relatively strong, durable, and maintains its structural integrity 10–20 times longer than untreated wood. More than 90% of all outdoor wooden structures are made with CCA-treated lumber. The common use of CCA-treated wood were highway noise barriers, signposts, utility poles, and retaining walls (Barton, 2014). Until December 31, 2003, the CCA-treated wood was commonly used around homes for decks, walkways, fences, gazebos, boat docks, and playground equipment. From January 2004 onwards, EPA and the lumber industry agreed to limit the use of CCA-treated wood in the residential construction. This agreement was intended to protect the health of humans and the environment by reducing exposure to the CCA-treated wood. As a result of this decision, CCA-treated wood can no longer be used to construct residential structures. On February 22, 2002, EPA in a Federal Register notice (Federal Register Vol. 67, No 36) announced the receipt of voluntary requests from registrants to cancel certain CCA products and amend other CCA registrations by terminating certain uses under section 6(f ) (1) of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). The registrants phased out all uses of CCA products except the treatment of wood products that fall under the AWPA Standards, based on the 2001 and 2002 editions of the AWPA Standards, listed in the text of the approved label amendments. The wood treated with CCA can still be used for commercial, industrial, and non-residential use including commercial wood poles, posts, shakes, shingles, permanent foundation support beams, pilings, and other wood products permitted by approved labeling (epa.gov website).

Exposure and exposure monitoring The human exposure to the toxic metals in CCA-treated wood may occur from direct contact with the wood (dermal sorption), inhalation of wood dust, or the contaminated water and soil containing CCA leachate (Gordon et al., 2002; Stilwell and Gorny, 1997). The wood treated with CCA is not classified as hazardous as CCA supposedly ‘fixes’ to the wood in a way that makes the chemical insoluble and somewhat leach resistant. However, some of the metals is not ‘fixed’ and leaching of the copper, chromium, and arsenic could occur, due to the free unbound metal on the surface of the treated wood. The leaching of copper, chromium, and arsenic into the water and soil has been widely investigated by laboratory and field studies, and although contradictory in results and interpretations, these studies indicated that leaching of the metals depend on many factors including pH, salinity of water body, temperature, moisture content of the treated wood, wood type, wood texture, pH, type, and organic content of soil (Shamim et al., 2008). The wood products treated with CCA were found to have effect on both the human health and environment due to the high level of arsenic, copper, and chromium in treated wood. The chemicals not only migrate from treated wood into surrounding soil over time but may also be dislodged from the wood surface upon contact with skin. The amount and rate at which arsenic leaches vary considerably depending on numerous factors, such as local climate, the acidity of rain and soil, age of the wood product, and the quantity of the applied CCA. The leaching may also occur with the newer structures which might decrease with time. Since excessive exposure to arsenic is hazardous to health, precautions should be taken to decrease exposure. The reapplication of the seal depending upon wear and weathering of the wood may prevent the migration of arsenic from the wood. The young children are at increased risk of exposure to CCA because they tend to spend more time playing outdoors, and they have frequent hand-to-mouth activities. When playing on playground equipment or decks built with CCA-treated wood, children could be exposed to CCA by touching the CCA leachate on the wood surface with their hands and then inadvertently ingesting the CCA on their hands through hand-to-mouth activity. The children may also be exposed to CCA in contaminated soil when playing under these structures by touching the contaminated soil with their hands and then placing them in their mouths. The amount of

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CCA leached on the surface of the wood depends upon the type of wood and the age of the structure. The amount ingested is also dependent upon the frequency of hand-to-mouth activity. It is recommended to wash hands thoroughly after contact with treated wood, especially before eating and drinking; and ensure that food does not come into direct contact with any treated wood. The workers should wear gloves when handling wood, wear goggles and dust-mask when sawing and sanding. It is also recommended to never burn CCA-treated wood (Barton, 2014).

Toxicokinetic The working solution of CCA used for the wood treatment contains chromium in the hexavalent [Cr(VI) or Cr + 6] oxidation state and Arsenic (As) in the pentavalent [As(V) or As+5] oxidation state. After the fixation of chemicals in the treated wood, the chromium is present predominantly in the trivalent [Cr(III)] form while arsenic remains pentavalent [As(V)] (Nico et al., 2006). The LD50 of the hexavalent chromium in microorganisms were found to be in the range of 50 mg/kg and 5 mg/kg for 48 h. The 48 h LC50 in the aquatic invertebrate were found to be in the range of 66 mg/L to 64 mg/L and 17.6 to 249 mg/L for fish (Morais et al., 2021). In humans, the chronic toxic effects of hexavalent chromium include irritation and sensitization of the skin, irritation and corrosion of the mucous membranes of the respiratory system and cancer of the lungs (Katz and Salem, 2005). The toxicity of trivalent chromium compounds is two to three orders of magnitude less than that of the hexavalent chromium compounds (Katz and Salem, 1993). The 48 h LC50 for acute copper toxicity in aquatic invertebrates ranges from 5 mg/ l to >10 mg l−1. In humans and other mammals, the high concentration of copper is toxic, although it is essential for normal human metabolism (Katz and Salem, 2005). The arsenic LC50 values range from a 0.5 mg l−1 to 47.9 mg l−1 for the aquatic invertebrates. In Mallard ducks, the acute LD50 is 300 mg/ kg. For humans, the acute lethal oral arsenic dose is reported to be 1–2.5 mg/ kg (Cox, 1991). Arsenic released by the CCA-treated wood at the playground and outdoor equipment is reported to persist as surface residue on the wood. The arsenic on the CCA-treated wood is found in the range of 2-45 mg cm−2. This raises concern for the exposure in children playing on CCA-treated playgrounds. The estimated arsenic mass in children was reported to be in the range of 0.2–2 mg per day (Stilwell and Gorny, 1997; Shalat et al., 2006). The Pilot study by Shalat et al. showed that the arsenic was dislodgeable on children’s hands which could contribute to safety concern (Shalat et al., 2006).

Mechanism of toxicity There is no strong evidence that interactions among the components of CCA will result in a marked increase in toxicity when compared to the individual toxicity of arsenic, chromium, or copper. The components of CCA affect some of the same organs of the body and may have additive toxicity to those organs (ATSDR, 2002). The chromium in its hexavalent form, Cr+6 or Cr(VI) is a known dermal sensitizer. The hexavalent Cr (Cr[VI]) is present in the CCA treatment solution and leaches from wood much more readily than trivalent Cr (Cr[III]) which is the predominant form after fixation occurs (Song et al., 2006). Cr(III) can be oxidized to Cr(VI) in water or soil under certain conditions after leaching from wood. In aqueous media, arsenic typically exists as arsenate, As(V) and arsenite, As(III). Both As(III) and As(V) are stable and toxic. As(III) is more poisonous and more mobile than As(V) (Zheng et al., 2015). The pentavalent arsenic [As(V)] having dermal and inhalation route exposure is a target for the non-cancer and cancer risks. As(V) has shown acute, sub-chronic, and chronic toxicity with the dermal exposure. Copper is an important micronutrient and is toxic in the free ionic state above trace level (Hingston et al., 2001).

In vitro toxicity data The relationship studies between the exposure to Cu and carcinogenicity is very limited. In vitro study by Ohgami et al. have demonstrated the increased carcinogenic risk by co-exposure to Cr and As in the normal human lung (BEAS-2B) and carcinoma (A549) cell lines (Ohgami et al., 2015). In colony survival studies, increased cytotoxicity (p < 0.05) occurred in V79 cells treated with CCA wood dust (351 +/− 77 mg/mL, mean +/− SE) compared with control wood dust (883 +/− 91 mg/mL). Gordon et al. demonstrated that CCA-treated wood dust is cytotoxic in vitro and induces an increase in the steady-state expression level of metallothionein mRNA in V79 cells. (Gordon et al., 2002).

Acute and short-term toxicity Animal There is a considerable amount of acute toxicity data in animals for the arsenic, chromium, and copper toxicity, when administered alone, as could be seen in the Tables 1 and 2. In acute toxicity animal studies, administration of chromium (VI) (as chromic acid) by the oral, dermal, and inhalation routes resulted in significant acute toxicity as measured by lethality. Earlier studies have shown that the mixture of the three compounds, Na2Cr2O7, CuSO4 and Na3AsO4 used in CCA had higher risk of nephrotoxicity in male Wistar rats when compared to the individual components (Mason and Edwards, 1989). The co-administration of these compounds

CCA-treated wood Table 1

661

The following is the acute toxicity data of arsenic acid (75%) (USEPA, 2008).

Study Type

Results

Toxicity category

Acute Oral

Mouse LD50 ¼ ♂141 mg/kg ¼ ♀ 160 mg/kg M + F ¼ 150 mg/kg Rat LD50 ¼ ♂ 76 mg/kg ¼ ♀ 37 mg/kg M + F ¼ 52 mg/kg Rabbit LD50 ¼ ♂ 1750 mg/kg ¼ ♀ 2300 mg/kg Mouse LC50 ¼ ♂1.153 mg/L ¼ ♀ 0.79 mg/L M + F ¼ 1.040 mg/L Rabbit 3/6 animals died by day 7. The 3 surviving animals were sacrificed on day 9 because of severe ocular irritation and corrosion. Rabbit At 30 min, all animals showed moderate to severe erythema and slight to severe edema. All animals died before the 24-h observation. Guinea Pig Not a Sensitizer

II

Acute Oral

Acute Dermal Acute Inhalation

Primary Eye Irritation Primary Skin Irritation Dermal Sensitization

I

II II

I I Not Applicable

Revised CCA Risk Assessments (RED Case 0132), Environmental Protection Agency. https://www.regulations.gov/document/EPA-HQ-OPP-2003-0250-0053.

Table 2

The following is the acute toxicity data of chromium (VI) (USEPA, 2008).

Study Type/[Substance Tested]

Results

Toxicity Category

Acute Oral/Rat [Chromic Acid, 100% a.i.]

LD50 ¼ ♂ 56 mg/kg ¼ ♀ 48 mg/kg M + F ¼ 52 mg/kg LD50 ¼ ♂ >48 mg/kg ¼ ♀ 48 mg/kg M + F ¼ 57 mg/kg LC50 ¼ ♂ 0.263 mg/L ¼ ♀ 0.167 mg/L M + F ¼ 0.217 mg/L Corrosive (data waiver) Corrosive (data waiver) Strong sensitizer

I

Acute Dermal/Rabbit [Chromic Acid, 100% a.i.] Acute Inhalation/Rat [Chromic Acid, 100% a.i.] Primary Eye Irritation [Various Cr(VI) compounds] Primary Dermal Irritation [Various Cr(VI) compounds] Dermal Sensitization/Guinea Pig [Various Cr(VI) compounds]

I I I I Not Applicable

Revised CCA Risk Assessments (RED Case 0132), Environmental Protection Agency. https://www.regulations.gov/document/EPA-HQ-OPP-2003-0250-0053.

intraperitoneally produced a significant increase in the acute toxicity relative to that of the high-dose component alone in the male Wistar rats. The low doses of CCA constituents (Na2Cr2O7: 5 mg/kg, CuSO4: 5.9 mg/kg and Na3AsO4: 25 mg/kg), and high doses of CCA constituents (Na2Cr2O7: 35 mg/kg, CuSO4: 23.5 mg/kg and Na3AsO4: 90 mg/kg) showed that CCA may present a greater acute toxicity hazard than its individual constituents.

Human The acute toxic effect in humans is a result of inhalation or ingestion of arsenic or arsenic contaminated substances. The symptoms include pain, eye irritation, nausea, vomiting, diarrhea, characteristic skin lesions, decreased production of red and white blood cells, abnormal heart function, blood vessel damage, liver and/or kidney damage, and impaired nerve function causing a “pins-andneedles” feeling (ATSDR, 1989). In cases of extreme exposure, arsenic is fatal with a lethal dose of 25 mg arsenic per kg of body weight (Cox, 1991). The symptoms of acute poisoning from chromium (IV) include severe redness and swelling of the skin (Agency For Toxic Substances And Disease Registry, n.d.).

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CCA-treated wood

Chronic toxicity The toxic properties of many compounds of arsenic and chromium are known and extensively revised, but relatively limited information is available regarding the toxicology of CCA (Katz and Salem, 2005).

Animal Studies on rats show increased fetal mortality, cleft pallet and increased ratio of male to female offspring (Agency, 1981; National Research Council (US) Committee on Medical and Biological Effects of Environmental Pollutants, 1977). In addition, birth defects have been observed in animals exposed to chromium (VI) (Agency For Toxic Substances And Disease Registry, n.d.). In addition, the teratogenic effects of chromium (VI) have also been detected in exposed animals (Casalegno et al., 2015; Dartsch et al., 1998). Few studies have concluded that CCA and its compounds per se show hepatic and renal changes in mice models (Matos et al., 2009a, b, 2010, 2020). More recently, Takahashi et al. reported on the toxic effects of As and/or Cr on the hematopoietic, gastrointestinal, hepatic, and renal systems of Wistar Hannover rats exposed to 40 and 80 mg/kg/day (Takahashi et al., 2018). Chronic toxicity of arsenic has been studied in many animal body systems; some of the health effects are specific to the exposure, whereas most of the effects are systemic in nature. As indicated in reports from global health authorities and WHO (Morais et al., 2021), arsenic exposure causes damage to the mucous membranes, peripheral and central nervous systems, neuronal network, and hearing capacity. In addition, arsenic poisoning has been associated with the suppression of the immune system, as well as with increased fetal mortality in rats (Baker et al., 2018).

Human Exposure through incomplete fixation: Pressure treated wood frequently fails to be fully dried before leaving production facilities. This can cause the CCA preservative to not fully fix to the wood. Workers have been exposed to massive quantities of arsenic, chromium and copper, causing skin irritation, and increasing risk of chronic health effects (Sheet, 2003). Chronic effects of arsenic exposure have been seen in many body systems. Although some health effects are exposure specific, most are systemic and can result from any root. Arsenic poisoning damages mucus membranes, and it produces peripheral nervous system disturbances and degeneration and hearing loss (Cox, 1991; Morais et al., 2021). In addition, research links exposure with immune system suppression, leaving victims more vulnerable to other ailments (Cox, 1991). Children face particular risk from exposure to arsenic as a exposure to CCA treated wood in the park (Shalat et al., 2006; Lew et al., 2010; Barraj et al., 2007; Zhou and Xi, 2018). A Thailand Health Research Institute study showed an inverse relationship between the levels of arsenic found in children’s hair and their height. This relationship was significant for both high and low arsenic accumulations. This study represents defining data on low-level arsenic exposure’s effect on the growth of children (Siripitayakunlit et al., 2000). The exposure of workers to CCA-coated timber promotes arsenic accumulation via inhalation with potential health risks (Cocker et al., 2006). Human exposure to hexavalent chromium may occur through inhalation, ingestion and absorption by dermal contact (Baranowska-Dutkiewicz, 1981). Chromium also poses long-term health threats. Studies of chromium (VI) from industrial emissions have found it to be highly toxic due to strong oxidation characteristics and ready membrane permeability (Zhang et al., 2022) 23. Cr (VI) has been known to cause damage to kidneys and liver. Skin contact with certain chromium (VI) compounds can cause skin ulcers. Chronic human exposure to hexavalent chromium results in irritation and rashes on skin, and corrosion and irritation in the airways of the respiratory system, causing damage to the mucous membranes and development of lung cancer (Morais et al., 2021; Wang et al., 2019; Cox, 1991). The results of the studies on chromium (VI) released from industrial emissions have shown it to be highly toxic in nature due to its very strong oxidative properties and smooth membrane permeability. Chromium (VI) has been found to show nephrotoxicity and hepatotoxicity (Morais et al., 2021). The toxicity in humans includes itching, burning rashes, neurological symptoms, and breathing problems that have been associated with handling unmarked chromated arsenical wood preservatives, including contact with the sap draining from wood treated with chromated arsenical wood preservatives. Treated wood has been reported as a potential source of dermal and inhalation exposure leading to dermatitis and the development of film on the teeth. “Ruined” nerves in feet and legs believed to be from sawdust and fumes from cutting and routing chromated arsenical wood preservatives treated lumber have also been reported (USEPA/Office of Prevention, 2008).

Immunotoxicity CCA has sensitizing activity. This type of solvent can influence the results of sensitization assays evaluating metals. Depending on wood species, it may cause respiratory sensitization and/or irritation. Symptoms can include irritation, redness, scratching of the cornea, and tearing. Dermal sensitization. Respiratory sensitization, and Exposure to wood dusts can result in hypersensitivity and development of contact dermatitis. The primary irritant dermatitis resulting from skin contact with wood dusts consist of erythema, blistering, and sometimes erosion and secondary infections occur. May cause eczema-like skin disorders (dermatitis) (Fukuyama et al., 2008; United Nations Environment Programme, World Health Organization and International Labour Organisation (1981)).

CCA-treated wood

663

Reproductive and developmental toxicity This product is not expected to cause reproductive or developmental effects.

Genotoxicity No human data available for genotoxicity of CCA treated wood. No component of this product present at levels greater than or equal to 0.1% is identified as a mutagen by OSHA.

Carcinogenicity The human lung normal (BEAS-2B) and carcinoma (A549) cells exposure to Cr, Cu and As at the molar ratio in a representative CCA-treated wood revealed that the untreated wood ash and CCA-treated wood ash both presented a carcinogenic risk (Ohgami et al., 2015). Carcinogenic to humans as Wood/Wood dust is carcinogenic to humans, arsenic pentoxide is carcinogenic to humans, while trivalent chromium is not classifiable as to carcinogenicity to humans. EPA’s Carcinogen Assessment Group classified inorganic arsenic as a Group A carcinogen. Arsenic ingestion and inhalation has been reported to increase the risk of cancer, especially in the liver, bladder, kidney and lung (ATSDR, 1989). The form of chromium (hexavalent) found in CCA has also been found by EPA to be a known human carcinogen. An EPA “Product Matrix” on “Wood Preservatives” states that “inorganic arsenic compounds have been shown to cause cancer in humans (Agency. and seahome/housewaste/house/woodpre.htm., Copyright, 1996 by Purdue Research Foundation, West Lafayette, Indiana 47907, n.d.).”

Organ toxicity Small amounts of arsenic can be transferred from CCA-treated wood to skin from touching CCA-treated wood surfaces (ATSDR; Toxicological Profile for Arsenic. Atlanta, 2007). Overall, the components of CCA affect some of the same organs of the body, and may have additive toxicity to those organs (Katz and Salem, 2005). Skin: Handling may cause splinters. Prolonged contact with treated wood and/or treated wood dust, especially when freshly treated at the plant, may cause irritation to the skin. Abrasive handling or rubbing of the treated wood may increase skin irritation. Some wood species, regardless of treatment, may cause dermatitis or allergic skin reactions in sensitized individuals. May cause eczema-like skin disorders (dermatitis).

Eye: Dust may irritate eyes Ingestion: Not likely due to the form of the product. Ingestion of dusts generated during working operations may cause nausea and vomiting. Inhalation: Wood dust: May cause nasal dryness, irritation and mucostasis. Coughing, wheezing, sneezing, sinusitis and prolonged colds have also been reported. Depending on wood species may cause respiratory sensitization and/or irritation. Symptoms can include irritation, redness, scratching of the cornea, and tearing. Airborne treated or untreated wood dust may cause nose, throat, or lung irritation and other respiratory effects.

Clinical management The chromated copper arsenate leaches from wood and into surrounding soil, where it can contaminate groundwater and potentially cause toxic chemical exposure for the public. In addition, people who work with treated wood, such as construction workers and carpenters, can be exposed to high levels of CCA. Exposure to chromated copper arsenate can lead to arsenic poisoning and, in cases of extremely high exposure, death. The symptoms of arsenic poisoning include sore throat, Irritated lungs, nausea, vomiting, abnormal heart rhythm, numbness in the extremities, darkening of the skin, decreased production of red and white blood cells. Arsenic is also known to cause cancer. Currance et al. have provided the immediate first aid, basic treatment and advanced treatment for arsenic and related compounds toxicity (Currance et al., 2007).

Environmental fate and behavior The earlier studies have reported the release of chromated copper arsenate in the environment through waste streams and leaching into soils. The treated lumber products have included decks, playsets, picnic tables, landscaping timbers, residential fencing, patios,

664

CCA-treated wood

and walkways/boardwalks. If released to soil through leaching from treated wood, most leaching takes place in the first few days and the extent and rate of leaching being highest for copper and lowest for chromium. If water is highly acidic, the leaching rates and amounts of leachates increase. Generally, in soil and water, the amounts of metals released are in the order of Cu > As > Cr. In some recent cases it has been shown that the order of release rates are: As>Cu > Cr. In all cases, the amounts of chromium released is least of the three metals. Numerous studies conducted on bioaccumulation in various aquatic organisms have also been carried out over a period of time. A number of these species have shown a degree of bioaccumulation and toxic effects have been observed. The studies were conducted under varying conditions and very few studies reported depuration rates. The overall robust fate assessment cannot be made, as the studies were conducted under different laboratory or field conditions, which were not standardized. Hence, while the exposure and hazards of these metals on humans, plants, aquatic organisms may be determined, a complete fate assessment is not possible (Stilwell and Gorny, 1997; Stilwell, 1998; Solo-Gabriele and Townsend, 1999).

Ecotoxicology The arsenicals in CCA pose serious ecological threats. Many aquatic organisms are extremely sensitive to arsenic exposure, which can result in serious health effects and even death at relatively low levels. Arsenic bioconcentrates in aquatic organisms in fresh water organisms up to 17 times background levels, and in marine oysters 350 times background levels (ATSDR. Toxicological Profile for Arsenic. Washington, 1993). Because of bioaccumulation, low levels of arsenic pose devastating threats to larger animals including top predators that eat organisms exposed to arsenic. The previous studies have shown that high quantities of arsenic leaching from CCA-treated wood may bioaccumulate. This is especially true in soils and water with slightly acidic pH (ATSDR. Toxicological Profile for Arsenic. Washington, 1993). The copper in CCA can be toxic to aquatic life. The LC50 for aquatic invertebrates and fish ranges from 5 micrograms (mg) per liter to 100,000 mg/L (Hodson et al., 1979). Effects on aquatic invertebrates include decreased feeding and egg production and impairment of certain behaviors, such as the ability of clams to burrow (Sheet, 2003). In addition, fish growth, spawning and survival are all affected by the presence of copper. Salmon have been known to head back downstream without spawning due to high copper concentration. Gill lesions, kidney damage, and diabetes like symptoms in a variety of fish species were also observed in association with copper concentrations (Sheet, 2003) .

Exposure standards and guidelines Chromated Copper Arsenate (CCA) Treated Wood as sold/shipped in its solid, treated wood product form does not present an inhalation, ingestion or contact hazard, nor would any of the following exposure data apply. However, operations such as sawing, drilling, sanding, burning, grinding or other similar processes may produce fumes and/or particulates. The following exposure limits are offered as reference, for an experienced industrial hygienist to review. US. OSHA Components

Type

Value

Form

Wood/Wood dust

PEL

US. OSHA Table Z-1 Limits for Air Contaminants (29 CFR 1910.1000) Components Trivalent Chromium (CAS 1308-38-9) ACGIH Components Wood/Wood dust U.S. NIOSH: Pocket Guide to Chemical Hazards Arsenic Pentoxide (CAS 1303-28-2) Copper Oxide (CAS 1317-39-1) Arsenic Pentoxide (CAS 1303-28-2) Wood/Wood dust Biological limit values ACGIH Biological Exposure Indices Components Arsenic Pentoxide (CAS 1303-28-2)

Type

5 mg/m3 15 mg/m3 Value

Respirable dust Total fraction

PEL Type TWA Type

0.5 mg/m3 Value 1 mg/m3 Value

Ceiling TWA TWA TWA

0.001 mg/m3 1 mg/m3 0.05 mg/m3 1 mg/m3

Dust and mist

Value 35 mg/l

Determinant Inorganic arsenic, plus methylated, metabolites as As

Specimen Urine

Form Inhalable fraction Form

Dust

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665

Agency., E. P. & Seahome/Housewaste/House/Woodpre.Htm. (n.d.) Copyright, 1996 by Purdue Research Foundation, West Lafayette, Indiana 47907. American Wood Preservers’ Association (AWPA) (1998) Book of Standards. P5–98 standard for Waterborne Preservatives. 8. ASTM (1995) Designation D 1625. Standard specification for chromated copper arsenate. In: 1995 Annual Book of ASTM Standards. Philadelphia, PA: American Society for Testing and Materials. ATSDR (1989) Arsenic Public Health Statement. ATSDR (1993) Toxicological Profile For Arsenic. Washington, DC. ATSDR (2002) Chromated Copper Arsenate (CCA), Agency for ToxicSubstances and Disease Registry. Atlanta: CDC, DHHS. ATSDR and Toxicological Profile For Arsenic. Atlanta, G. A. F. T. S. A. D. R (2007) US Public Health Service. Available From, as of Nov 20, 2014. https://www.Atsdr.Cdc.Gov/ Toxprofiles/Index.Asp. 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Cocker J, Morton J, Warren N, Wheeler JP, and Garrod AN (2006) Biomonitoring for chromium and arsenic in timber treatment plant workers exposed to CCA wood Preservatives. The Annals of Occupational Hygiene 50: 517–525. Cox C (1991) Chromated copper arsenate. Journal of Pesticide Reform 11: 2–6. Currance, P.L. Clements, B., Bronstein, A.C. (Eds) (2007) Emergency Care For Hazardous Materials Exposure. 3rd revised edition, Elsevier Mosby, St. Louis, MO, p. 370. Dartsch PC, Hildenbrand S, Kimmel R, and Schmahl FW (1998) Investigations on the nephrotoxicity and hepatotoxicity of trivalent and hexavalent chromium compounds. International Archives of Occupational and Environmental Health 71(Supplement): S40–S45. Fukuyama T, Ueda H, Hayashi K, Tajima Y, Shuto Y, Kosaka T, and Harada T (2008) Sensitizing potential of chromated copper arsenate in local lymph node assays differs with the solvent used. Journal of Immunotoxicology 5: 99–106. Gordon T, Spanier J, Butala JH, Li P, and Rossman TG (2002) In vitro bioavailability of heavy metals in pressure-treated wood dust. Toxicological Sciences 67: 32–37. Hingston JA, Collins CD, Murphy RJ, and Lester JN (2001) Leaching of chromated copper arsenate wood preservatives: A review. Environmental Pollution 111: 53–66. Hodson PV, Uwe B, and Harvey S (1979) Toxicity of Copper to Aquatic Biota. Katz SA and Salem H (1993) The toxicology of chromium with respect to its chemical speciation: A review. Journal of Applied Toxicology 13: 217–224. Katz SA and Salem H (2005) Chemistry and toxicology of building timbers pressure-treated with chromated copper arsenate: A review. Journal of Applied Toxicology 25: 1–7. Lew K, Acker JP, Gabos S, and Le XC (2010) Biomonitoring of arsenic in urine and saliva of children playing on playgrounds constructed from chromated copper arsenate-treated wood. Environmental Science & Technology 44: 3986–3991. 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Matos RC, Oliveira H, Fonseca H, Morais S, Sharma B, Santos C, and De Lourdes Pereira M (2020) Comparative Cr, As and CCA induced Cytostaticity in mice kidney: A contribution to assess CCA toxicity. Environmental Toxicology and Pharmacology 73(103): 297. Timothy F. Mcmahon, 2001. Hazard Identification and Toxicology Endpoint Selection for Inorganic Arsenic and Inorganic Chromium. EPA, U. (ed.) FIFRA Scientific Advisory Panel Background Document. Morais S, Fonseca H, Oliveira SMR, Oliveira H, Gupta VK, Sharma B, and De Lourdes Pereira M (2021) Environmental and health hazards of chromated copper arsenate-treated wood: A review. International Journal of Environmental Research and Public Health 18. National Research Council (US) Committee on Medical and Biological Effects of Environmental Pollutants. (1977) Arsenic Medical and Biologic Effects of Environmental Pollutants. Washington (DC): National Academies Press Nico PS, Ruby MV, Lowney YW, and Holm SE (2006) Chemical speciation and bioaccessibility of arsenic and chromium in chromated copper arsenate-treated wood and soils. Environmental Science & Technology 40: 402–408. Ohgami N, Yamanoshita O, Thang ND, Yajima I, Nakano C, Wenting W, Ohnuma S, and Kato M (2015) Carcinogenic risk of chromium, copper and arsenic in CCA-treated wood. Environmental Pollution 206: 456–460. Shalat SL, Solo-Gabriele HM, Fleming LE, Buckley BT, Black K, Jimenez M, Shibata T, Durbin M, Graygo J, Stephan W, and Van De Bogart G (2006) A pilot study of children’s exposure to CCA-treated wood from playground equipment. Science of the Total Environment 367: 80–88. Shamim AN, Mcmahon T, Chen J, Wormell L, and Hartman M (Feb 19, 2008) Environmental fate and transport assessment of CCA-C for reregistration eligibility decision (RED) process. In: Ii AD (ed.) Regulatory Management Branch. Washington, DC: United States Environmental Protection Agency. Sheet ABPF (2003) Chromated copper arsenate (CCA) treated wood. Pesticides and You: Beyond Pesticides/National Coalition Against the Misuse of Pesticides, vol. 23, 18–21. Siripitayakunlit U, Thonghong A, and Pradipasen M (2000) Growth of Children with Different Arsenic Accumulation, Thailand. University of Denver Poster, financed by the Thailand Health Research Institute. National Health Foundation. Solo-Gabriele HM and Townsend TG (1999) Disposal practices and management alternatives for Cca-treated wood waste. Waste Management & Research 17: 378–389. Song J, Dubey B, Jang YC, Townsend T, and Solo-Gabriele H (2006) Implication of chromium speciation on disposal of discarded CCA-treated wood. Journal of Hazardous Materials 128: 280–288. Stilwell DE (1998) Arsenic from CCA-treated wood can be reduced by coating. Frontiers of Plant Science 51: 6–8. Stilwell DE and Gorny KD (1997) Contamination of soil with copper, chromium, and arsenic under decks built from pressure treated wood. Bulletin of Environmental Contamination and Toxicology 58: 22–29. Takahashi N, Yoshida T, Kojima S, Yamaguchi S, Ohtsuka R, Takeda M, Kosaka T, and Harada T (2018) Pathological and clinical pathological changes induced by four-week, repeated-dose, oral administration of the wood preservative chromated copper arsenate in wistar rats. Toxicologic Pathology 46: 312–323. United Nations Environment Programme, World Health Organization and International Labour Organisation (1981) Arsenic - Environmental Health Criteria 18. https://wedocs.unep.org/ 20.500.11822/29304. USEPA/Office of Prevention, P (2008) Toxic Substances; Human Health Risk Assessment and Ecological Effects Assessment for the Reregistration Eligibility Decision Document of Inorganic Arsenicals and/or Chromium-Based Wood Preservatives. P. 22 EPA-HQ-OPP-2003-0250-0081. https://www.Regulations.Gov. Wang JS and Chiu K (2008) Extraction of chromated copper arsenate from wood wastes using green solvent supercritical carbon dioxide. Journal of Hazardous Materials 158: 384–391.

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Wang Z, Lin HP, Li Y, Tao H, Yang P, Xie J, Maddy D, Kondo K, and Yang C (2019) Chronic hexavalent chromium exposure induces cancer stem cell-like property and tumorigenesis by increasing c-Myc expression. Toxicological Sciences 172: 252–264. Wormell L, Chen J, Aviado D, Elkassabany N, and Cook N (2008) Occupational Exposure Chapter for Inorganic Arsenicals and Chromium-based Wood Preservatives in Support of the Reregistration Eligibility Decision (RED) Document for the Chromated Arsenicals (RED Case 0132). In: Pesticides, and Toxic Substances. Washington, DC: United States Environmental Protection Agency, O. O. P. Zhang W, Zhu Y, Gu R, Liang Z, Xu W, and Jat Baloch MY (2022) Health risk assessment during in situ remediation of Cr(VI)-contaminated groundwater by permeable reactive barriers: A field-scale study. In: International Journal of Environmental Health Research, 19. Zheng S, Jiang W, Rashid M, Cai Y, Dionysiou DD, and O’shea KE (2015) Selective reduction of Cr(Vi) in chromium, copper and arsenic (CCA) mixed waste streams using UV/TiO2 photocatalysis. Molecules 20: 2622–2635. Zhou Q and Xi S (2018) A review on arsenic carcinogenesis: Epidemiology, metabolism, genotoxicity and epigenetic changes. Regulatory Toxicology and Pharmacology 99: 78–88.

Relevant websites https://pubchem.ncbi.nlm.nih.gov/compound/Copper-Arsenate_-Chromated#section¼2D-Structure :2D structure of CCA. https://www.chemicalbook.com/ChemicalProductProperty_US_CB71325163.aspx :Structure of CCA. https://www.epa.gov/ingredients-used-pesticide-products/chromated-arsenicals-cca :Registration review of Chromated Arsenicals-Updated on Feb 4, 2022.

Cell cycle Aarthi Nivasini Mahesh, Karanpreet Singh Bhatia, and Shruti Bhatt, Department of Pharmacy, National University of Singapore, Singapore, Singapore © 2024 Elsevier Inc. All rights reserved. This is an update of N. Yang, A.M. Sheridan, Cell Cycle, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 753–758, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00273-6.

Introduction Cyclins and cyclin-dependent kinases Regulations of cyclins/cdk Retinoblastoma Isoforms of Rb protein Tumor suppressor function of Rb protein Checkpoints P53 ATM and ATR Role of P53 in apoptosis Clinical application of cell cycle modulatory drugs Inflammation Cancer treatment Flavopiridol Palbociclib (PD-0332991) Paclitaxel Docetaxel Vincristine sulfate liposome Brentuximab vedotin Conclusion References

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Abstract Cell cycle is a series of events that involves the duplication and separation of cellular contents in two newly formed identical daughter cells. This chapter describes the different phases of the cell cycle and various factors required for progression and regulation of cell cycle phases. Besides the mechanism, this chapter outlines the role of cell cycle during normal homeostasis and disease states, as well as outlines existing therapies that target cell cycle regulators for the treatment of inflammation and cancer. Furthermore, we discuss toxic compounds that considerably alter the cell cycle events. Overall, targeting of cell cycle events has shown potential therapeutic values.

Keywords Apoptosis; Cancer; Cardiovascular disease; Cdk inhibitors; Cdks; Cell cycle arrest; Checkpoints; Cyclins; DNA damage; E2F; p53; Rb

Key points

• • • •

Summary of key factors required for cell cycle progression at G1, S, G2 and M phase. Role of regulatory proteins including cyclins, cdks, INK4, Kip/Cip, p53 and Rb in cell cycle regulation. Discussion of existing therapy targeting cell cycle regulators for the treatment of inflammation and cancer. Outline of adverse effects of clinically approved agents on cell cycle phases.

Introduction The cell cycle is the orderly progression of cells through specific stages during which DNA gets replicated and distributed to two daughter cells, resulting in cell division. Precise regulation of the passage of the cells through this process is necessary to ensure the maintenance of DNA integrity through successive generations. Cell cycle regulation also ensures that cell proliferation occurs only

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under defined conditions in response to growth factors and in the presence of a suitable environment. Cell division and cell proliferation both are necessary for the optimal functioning of organs in an organism. Loss of cell cycle regulation is a characteristic of cancer. The factors that control the Cell cycle regulation are: 1. Mitogens control the rate of the cell cycle by downregulating the inhibitory barriers that otherwise block the cell cycle progression. 2. Growth factors control the growth of a cell in size and mass by increasing the rate of synthesis of proteins and macromolecules required for cell cycle. 3. Survival factors inhibit apoptosis and promotes cell survival. The cell cycle comprises of four stages, G1, S, G2 and M phases (Fig. 1). The G1, S and G2 phase is called the interphase and it usually makes the largest part of the cell cycle, about 90% of the cell’s lifetime followed by M phase. G1 (gap phase 1) is the stage immediately prior to S during which the cell increases in size, initiates transcription of genes and synthesis of protein responsible for cell cycle control such as Cdks and Cyclins. At this time, the cell does a series of checks before DNA synthesis. S (DNA synthesis phase) is the stage in which the entire genomic DNA is duplicated. G2 (gap phase 2) is the stage preceding M phase during which the cell prepares for cell division by checking size and error in the DNA duplication. M (Mitosis) is the stage in which the cell divides into two new daughter cells and transfers the copy of chromosomes to each daughter cell. Mitotic phase is the shortest phase of the cell cycle and comprises of 5 phases prophase, prometaphase, metaphase, anaphase and telophase and cytokinesis. Cell cycle is normally regulated by cell cycle checkpoints to ensure proper production of daughter cells. The three major cell cycle checkpoints are at the transitions from G1 to S phase, G2 to M phase, and metaphase to anaphase. The progression of cells through late G1/S checks the presence of growth factors, optimum cell size and possible DNA damage. A restriction point in late G1 marks the point at which cell cycle progression becomes growth factor independent. G2/M checks for improper DNA duplication and DNA damage. Metaphase/anaphase checkpoint is also called spindle apparatus checkpoint (SAC) that checks the proper attachment of chromosomes to the mitotic spindle. Cells that are actively proliferating progress from M phase back to G1 where preparations for DNA synthesis occurs immediately. Cells that are not actively proliferating are known as quiescent and remain in G0 phase. The entry of cells from G0 into the cell cycle is also a tightly regulated process and requires an extracellular stimulus or growth factor. Critical proteins involved in the regulation of G1/S and G2/M transition is described below.

Cyclins and cyclin-dependent kinases Numerous proteins have been identified that strictly regulate the passage of cells at G1/S and G2/M phase transitions. Conserved serine/threonine kinases, called cyclin-dependent kinases (CDKs), phosphorylate and activate specific regulatory proteins that drive

Fig. 1 Overview of the different phases of cell cycle and corresponding regulatory proteins. Quiescent cell remains in G0 phase and re-enter the cell cycle at G1, during which cell prepares for DNA synthesis. After passing the restriction point in late G1 and G1/S checkpoint, cell is committed to enter the S phase, during which DNA replication occurs. Cell in G2 phase prepares for mitosis (M phase) and this transition is tightly regulated at G2/M checkpoint. During M phase, spindle assembly checkpoint ensures proper assembly of the spindle fibers in metaphase to anaphase transition. Cell cycle progression is controlled by various positive and negative cell cycle regulatory proteins, including cyclins (A, B, D, E); cyclin-dependent kinases (cdk 1, 2, 4, 6); cdk inhibitors (INK4 and Kip/Cip); and retinoblastoma (Rb).

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cell cycle progression. The activity of cdks is regulated at three levels. First, cdks are activated by their interaction with proteins, called cyclins. Cyclins have very short half-lives usually less than 30–60 min. Whereas cdks are constitutively expressed throughout the cell cycle, the level of the cyclins varies throughout the entire process. Cyclin levels are controlled by both regulated synthesis and ubiquitin-mediated proteolysis. Specific cyclin-cdk complexes function at different cell cycle phases. Formation of the heterodimers cyclin D/cdk4, cyclin D/cdk6, and cyclin E/cdk2 is necessary for entry into and progression through G1. Expression of cyclin D family members is initiated by an extracellular signal or growth factor, due to which quiescent cells move from G0 to enter into G1. Cyclin D/cdk heterodimers phosphorylate and inactivate retinoblastoma protein (pRb), causing the release and activation of the E2F family of transcription factors. This family of transcription factors drives transcription of genes necessary for the G1/S transition, including cyclin E. Cyclin E/cdk2 also phosphorylate pRb but unlike cyclin D heterodimers, its activity is mitogen independent. Both cyclin E/cdk2 and cyclin A/cdk2 drive entry and progression through S phase via the phosphorylation of non-Rb proteins that initiate DNA synthesis. Cyclins A and B form complexes with cdk1 (also called cdc2) and are called the mitotic cyclins since these complexes regulate mitosis. Cyclin B/cdk1 control the G2/M transition. Cyclin B is synthesized as the cell progresses through G2. Upon binding of cyclin B to cdk1, the activated heterodimer phosphorylates proteins that are involved in mitosis (Ding et al., 2020; Zabihi et al., 2022).

Regulations of cyclins/cdk Activity of the cyclin/cdk complexes is also regulated by phosphorylation/dephosphorylation by cdk activating kinases (CAKs) and phosphatases. A third level of regulation is achieved by control of protein levels of cdk inhibitors. Cdk inhibitors are proteins that accumulate in response to multiple environmental stimuli, including DNA damage, hypoxia, cell–cell contact, and cytokines to inhibit the activity of cyclin/cdk heterodimers. The cdk inhibitors include two classes of proteins. The INK4 proteins, which include p16INK4a, p15INK4b, p18INK4c, and p19INK4d. They specifically inhibit the activity of cdk4 and cdk6 by competitive inhibition of cyclin D binding to the monomeric kinases. Mutations and deletions of the p16INK4a gene and inactivation by hypermethylation have been shown to play a role in tumorigenesis in many different types of tumors. The Kip/Cip proteins include three structurally related proteins, p21, p27, and p57. In contrast to the INK4 proteins, the Kip/Cip proteins inhibit most cyclin/cdk heterodimers. Specific Kip/Cip proteins are induced by upstream events. p21 is induced in response to DNA damage and specifically inhibits cyclin E/cdk2. Protein levels of p27 are highest in quiescent cells and induce G1 arrest in response to conditions that typically result in cell quiescence such as growth factor deprivation or contact inhibition. Both the INK4 and the Kip/Cip proteins inhibit the phosphorylation and inactivation of pRb. Recent genetic studies using knockout mouse models indicate that Cdk2, Cdk4, or Cdk6 singular knockout mouse embryos are viable while Cdk1 knockout mice are embryonically lethal. These suggests that Cdk4 and Cdk6 are not required for organogenesis and cell cycle except for some endocrine and hematopoietic cells. Cdk2 is not needed for the mitotic cell cycle. Overall, these studies suggest the compensatory role of various Cdks. Recent studies have also established O-GlcNAc cycling which is an addition and removal of O-GlcNAc as an important cell cycle regulator in mitosis. O-GlcNAcylation is an important post translational modification that adds b-N-acetylglucosamine to the mitochondria, nuclear and cytoplasmic proteins. Alterations in enzymes involved in O-GlcNAc directly affect the cell cycle by dysregulating the expression of cyclins (Zabihi et al., 2022).

Retinoblastoma The retinoblastoma gene (Rb) was the first tumor suppressor to be identified. Mutations in Rb gene were first shown to be causal in familial and sporadic retinoblastoma, a rare tumor of the eye, but have since been associated with many other tumors, including osteosarcoma, small cell lung cancer, prostate cancer and breast cancer. In addition, mutations in the upstream Rb signaling pathway that result in the functional inactivation of the Rb gene product are found in virtually all malignancies. Three Rb homologs are p110Rb (or Rb), p107Rb (or p107), and p130Rb (or p130). All Rb homologs are characterized by a ‘pocket’ domain, which is highly conserved, and necessary for pRb’s tumor suppressive function. All the Rb homologs bind viral oncoproteins as well as E2F family members. Binding of viral oncoproteins disrupts the pocket domain of pRb and impairs pRb’s tumor suppressor function. All pRb homologs cause G1 arrest. The primary role of pRb is the inhibition of transcription of genes that mediate passage across the G1/S transition.

Isoforms of Rb protein Rb exists in three different isoforms: (i) Un-phosphorylated Rb, (ii) Hypo-phosphorylated Rb, and (iii) Inactive hyper-phosphorylated Rb. The un-phosphorylated Rb regulates cell cycle at the Go Phase. Hypophosphorylated or monophosphorylated Rb is present throughout the early G1 phase in both normal and tumor cells, and hyper-phosphorylated in late G1, S, G2, and M phases. Cyclin D/cdk4/6 is a Rb mono-phosphorylating kinase which initiates phosphorylation in early G1 and cyclin E/cdk2 hyperphosphorylates Rb in late G1. Cyclin A/cdk2 and Cyclin B/cdk1 maintains an inactive hyperphosphorylated Rb throughout S, G2 and M phase. pRb may perform other roles in addition to regulation of G1/S including the regulation of apoptosis. A decrease in functional pRb results in the activation of p53-induced apoptosis, which appears to be mediated via the release of E2F1. Free E2F1 activates

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transcription of ARF (alternate reading frame of the p16INK4a locus), which inhibits mdm-2 ubiquitin ligase (mdm-2). Mdm-2 targets proteins for ubiquitin-mediated proteolysis. Since mdm-2 initiates degradation of p53, its inhibition results in an increase in p53 and a corresponding increase in apoptosis. Thus, a decrease in functional pRb could otherwise result in unchecked cell proliferation and trigger an apoptotic response. A decrease in functional pRb also creates selection pressure for p53 mutations, since only cells that have mutated & dysfunctional p53 survive. Not surprisingly, p53 mutations are often found to co-exist with Rb mutations in malignant tumors.

Tumor suppressor function of Rb protein The phosphorylated form of Rb (pRb) pRb binds to and inhibits the E2F family of transcription factors. The binding characteristics of the homologs vary slightly, while pRb binds preferentially to E2F1-4, p107 and p130 bind preferentially to E2F4 and E2F5. Phosphorylation of Rb regulates its interaction with E2F. The phosphorylation status of Rb fluctuates throughout the cell cycle. Hypophosphorylated Rb is active and binds to E2F family members thus sequestering E2F and inhibiting its transcriptional activity. Hyperphosphorylated Rb is inactive and releases E2F, which results in the transcription of genes that allow the cell to progress to S phase. Upon release from pRb, E2F binds to DP-1 or DP-2, and the resulting heterodimer activates genes involved in DNA replication. The mechanism by which pRb inhibits E2F transcriptional activity is still debated, but it may be via the recruitment of chromatin remodeling enzymes such as histone deacetylases (HDACs) that directly repress transcription by removing acetyl groups from chromatin, which causes the chromatin to be less accessible to transcription factors. pRb-bound HDAC may counteract activity of the E2F-bound acetyltransferase protein, p300/CBP, which transfers acetyl groups to chromatin and enhances transcriptional activity. In addition to inactivation of E2F via pRb that results in a decrease in transcription of E2F-responsive genes, the complex of pRb and E2F actively represses transcription, which may also be via the recruitment of HDACs to the promoter regions. The regulation of pRb activity is complex. There are 16 possible sites for cdk-mediated phosphorylation, and studies suggest that phosphorylation at each site regulates a distinct pRb function. pRb is phosphorylated by multiple cyclin/cdk complexes (Engeland, 2022).

Checkpoints Checkpoints are surveillance mechanisms comprising numerous genes that detect DNA damage and induce either cell cycle arrest or DNA repair mechanisms, or apoptosis. The data elucidating this surveillance network are incomplete but has been advanced significantly since the discovery of the mutation, associated with ataxia telangiectasia (AT). AT is a rare inherited condition that affects the nervous system, the immune system and other body systems. It is characterized by the presence of progressive ataxia (lack of coordination) due to a defect in the cerebellum. AT also increases susceptibility to cancer. Prior to the identification of AT mutation, it was long thought that the stimuli that induce DNA damage delay progression through the cell cycle. For years, this phenomenon was assumed to be the passive response of the cell as a direct result of the DNA damage itself. By contrast, cells that harbor the AT mutation demonstrate marked decrease in cell cycle arrest after DNA damaging radiation. These data suggested that an active system exists in normal cells that retard cell cycle progression in the presence of DNA damage. The checkpoint surveillance system comprises sensor proteins (that detect DNA damage and initiate a signaling cascade); transducers (modifying enzymes such as kinases that relay the signal to effector proteins); and effectors (downstream target proteins that cause cycle arrest). Of these, the least is known about sensor proteins, although several candidate genes have been suggested. The effector proteins include kinase inhibitors such as p21, or cyclin/cdk heterodimers that are either activated or inhibited to cause cell cycle arrest. Major transducer proteins include p53, ATM (AT-mutated protein kinase), and ATR (ATM and Rad3-related protein kinase) (Matthews et al., 2022).

P53 P53 activates the transcription of genes that cause cell cycle arrest at either G1/S or G2 phase. In addition, p53 activates genes that initiate DNA repair and cause apoptosis. The result of p53 activation is cell type-specific and depends on the type and severity of injury. P53-induced G1 cell cycle arrest is mediated via the induction of p21 and p16. p53 has a very short half-life and is generally undetectable in healthy cells. In the presence of DNA damage induced by either ultraviolet or g-irradiation, p53 is activated via posttranscriptional modifications, including phosphorylation and acetylation, that either enhances its stability or alter its affinity for protein binding. ATM and its related protein, ATR, phosphorylates p53, which decreases its binding to mdm-2. A decrease in the interaction between p53 and mdm-2 causes a decrease in ubiquitin mediated proteolysis of p53 and a resulting increase in p53 protein levels. As described previously, ARF also activates p53 via inactivation of mdm-2. In addition to phosphorylation, the acetylation status of p53 also determines its stability. P53 is acetylated and stabilized by p300/CBP, which increases apoptosis. The recently described NAD-dependent deacetylase protein SIRT1 removes acetyl groups from p53 and decreases apoptosis.

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ATM and ATR ATM and ATR are closely related phosphoinositide 3-kinases that are activated by DNA damage. Upon activation, ATM phosphorylates and activates Chk-2 and activated Chk-2 phosphorylats p53. ATR phosphorylates and activates Chk-1, which also phosphorylates p53. p53, ATM, and ATR also contribute to G2 arrest. Upon activation, cdk1 initiates mitosis. cdk1 is activated via its interaction with cyclin B and via dephosphorylation by cdc25C phosphatase. Upon phosphorylation by ATM and ATR, Chk-2 and Chk-1 phosphorylate and inhibit cdc25C, which prevents the activation of cdk1. P53 activates transcription of two genes that inhibit cdk1 activity, including GADD45 and 14-3-3s. GADD45 disrupts the cyclin B/cdk1 heterodimer. The protein product of 14-3-3s sequesters cdc25C, which prevents the dephosphorylation of cdk1.

Role of P53 in apoptosis p53 activates multiple genes that are involved in apoptosis, including genes that encode proteins that function via receptor mediated signaling and those which encode proteins that modulate downstream effectors. p53-activated IGF-BP3 inhibits binding of IGF-1 to the IGF-1 receptor, which can induce apoptosis. p53 activates transcription of the death receptor ligands fas/CD95 and the death receptor KILLER/DR5. p53 also induces the proapoptotic BCL-2 protein bax as well as other proteins that enhance cytochrome c release from mitochondria, including AIP1, PUMA, and Noxa. Apoptosis may also be induced via increase in oxidative stress generated by multiple p53-induced genes that are homologous to NADPH-quinone oxidoreductase. Importantly, no singular p53-activated gene product has been conclusively shown to initiate apoptosis. It appears that many p53-induced proapoptotic genes need to be activated concurrently for apoptosis to occur. It is not clear which variables determine whether p53 induces cell cycle arrest or apoptosis. The decision between p53-induced cell cycle arrest and apoptosis may depend on the protein levels of p53 and the cell type. Higher levels of p53 may induce apoptosis and lower levels cause cell cycle arrest. Certain cell types, such as T lymphocytes, are highly sensitive to apoptosis whereas fibroblasts are more likely to undergo cell cycle arrest. While p53 induces arrest or senescence in normal cells, p53 activation usually causes apoptosis in transformed cells. The enhanced sensitivity to p53-induced apoptosis in transformed cells may be related to the deregulation of E2F due to the inactivation of pRb. Cycle arrest induced by p21 may protect cell from apoptosis. Other factors that may predispose cells toward p53-induced apoptosis include alterations in the Bax/Bcl-2 ratio, concurrent absence of growth factors, and a greater intensity of stress. Post-translational modifications may also determine p53 promoter specificity, which may play a major role in determining whether p53 expression results in cell cycle arrest or apoptosis (Yang et al., 2017).

Clinical application of cell cycle modulatory drugs Inflammation Cell cycle plays an important role during normal tissue repair and inflammation. All tissues may be stratified by proliferative capability into three categories including labile, quiescent, or permanently non-dividing cells. Labile cells are continuously dividing and include surface epithelial cells such as stratified squamous epithelial cells of the skin and columnar epithelial cells of the gastrointestinal tract. Quiescent cells are non-dividing under normal circumstances but can be induced to re-enter cell cycle by exposure to growth factors. Quiescent cells include parenchymal cells of the liver, kidney, pancreas, and mesenchymal cells such as fibroblasts. The cytokine-induced re-entry of quiescent cells into G1 phase is an important step of the inflammatory response, which has been well characterized in the kidney. Glomerular mesangial cells proliferate in many models of glomerular disease, including lupus nephritis and diabetes. The proliferation of mesangial cells occurs in response to cytokines such as platelet-derived growth factor and basic fibroblast growth factor. Inhibition of mesangial cell proliferation may abrogate the glomerulosclerosis or the glomerular scarring that occurs because of inflammation. Nondividing cells do not have proliferation capacity, for example nerve cells and cardiomyocytes. In cardiovascular diseases, such as myocardial infarction, there is a significant loss of cardiomyocytes, however, due to the limited regenerative capacity of cardiomyocytes, the damaged cells are not replaced. Thus, there is an increasing interest in developing cell cycle modulators of cardiomyocytes or neurons that can initiate cell division Table 1.

Cancer treatment The deregulation of the cell cycle resulting in unchecked cell proliferation is a hallmark of cancer. Most human cancer cells have defects in restriction point control, checkpoints, DNA repair, or apoptosis. Defects of restriction point control allow for uncontrolled proliferation and results in the loss of terminal differentiation. Although cdks are seldom mutated in human cancers, many human tumors harbor mutations or epigenetic changes in upstream targets of cdks, including INK4, Cip/Kip, cyclin D, cyclin E, and downstream targets of cdks, mainly Rb. Targeting cdks in cancer has shown to restore cell cycle checkpoints and induce slow growth and apoptosis. Some of the CDK inhibitors are listed in Table 2.

Summary of some toxicants that are known to influence specific cell cycle events.

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Cell cycle stage

Specific mechanism

References

Acetaminophen Alcohol Antidiabetic drugs Ciglitazone Insulin Metformin BCNU Benzidine Cannabinoid

G1/S border G2/M transition

G1/S arrest by inhibiting DNA synthesis G2/M arrest by activating CHK-2

Wiger et al. (1997) Clemens et al. (2011)

G2,M Phase G1, S phase G0, G1 phase G2/M G2/M transition phase G1 phase

G2/M arrest by increasing p53, p27 and decreasing cyclin B1 Stimulates cell proliferation by increasing cyclin D/E Blocks the cell cycle in G0–G1 phases by decreasing cyclin D, Cdk4/6. G2/M arrestby increasing the cyclinB1/p-Cdc2 (Tyr15). G2/M arrest by upregulation of the p53. G1 phase arrest by upregulation of p53 and p27/KIP1 and down-regulation of cyclins D1, D2, E, cdk-2, -4, and -6.

Plissonnier et al. (2011) Chappell et al. (2001) Kato et al. (2012) Yan et al. (2005) Ching Chen et al. (2011) Sarfaraz et al. (2006)

Chemotherapy agents Cisplatin Dexamethasone Etoposide Hydroxyurea Paclitaxel Chloramphenicol

All phases G1 phase S, G2 phase G1, S border M phase G1/S transition phase

As an alkylating agent, it causes cell arrest at various phases due to dosage and cell types. It induces G1 arrest by inhibiting cyclin D, E2F, and increasing p21. As a specific topoisomerase II inhibitor, it arrests cells in late S and G2 phase As a specific inhibitor of DNA synthesis, it blocks cells at G1/S border. It stabilizes microtubules and causes M phase arrest. As an antimicrobial agent chloramphenicol, inhibits the cell cycle progression by decreasing the Cyclin D1 and E2F-1. Chloropicrin is a toxic irritant to respiratory system. It causes cell cycle arrest by upregulating the phospho-Erk1/2, p53, p21 and p27. As an antimalarial drug Chloroquine inhibits cell cycle progress at G2/M transition phase by decreasing the phospho- ERK1/2. Chlorpromazine exhibit anticancer activity and it inhibits cell cycle at G2/M phase by decreasing the cyclin D1, cyclin A, and cyclin B1. It inhibits cell cycle by down-regulating cyclin B, cyclin E, and p21. It inhibits cell cycle at S phase by down-regulating cyclin B1 and phosphorylated CDK1. It blocks the cell cycle at G1 and S phase by down-regulating cyclin D1 and cyclin E. It induces cell cycle arrest at G1 phase by down-regulating Cyclin D1.

Basu et al. (2010) Greenberg et al. (2002) Fearnhead et al. (1994) Kim et al. (1967) Carlier et al. (1983) Kang et al. (2005)

Toxic metals induce an S-phase-specific cell cycle block by their selective interaction with DNA metabolism. It induces cell cycle arrest at G1 phase by down-regulating cyclin D1, CDK4 and CDK6 and upregulates p21 and p27. As an antidepressant drug, it arrests cells at G0/G1 phase by accumulating p21 and p27. As an anti-inflammatory drug, it causes cells to accumulate in the G0/G1 phase by increasing p53 and p21. It arrests cells at G1/S transition by up-regulating P53.

Costa et al. (1982)

Chloropicrin Chloroquine

G1/G0 or G2/M transition phases G2/M transition phase

Chlorpromazine

G2/M transition phase

Ciprofloxacin Cresol Cycloheximide Diazoxide Metals Water insoluable (As, Ni) Water soluble (Cd2+, Hg2+,Co2+, Cu2+, Ni2+, Zn2+, Pb2+) NT1014, a novel biguanide

S/G2-M transition phase S phase G1 and S phases G1 phase

G1 Phase

Prozac (or fluoxetine) Sulindac

G0, G1 phase G0, G1 phase

Vit D

G1/S transition phase

Go, G1 phase S phase

Pesonen et al. (2014) Zhao et al. (2008) Shin et al. (2010) Aranha et al. (2000) Zhu et al. (2012) Liu et al. (2010) Ding et al. (2009)

NT1014, a novel biguanide, inhibits ovarian cancer growth in vitro and in vivo Krishnan et al. (2008) Jung et al. (2005) Tabasi et al. (2015)

Cell cycle

Compound

Cell cycle Table 2

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Cell cycle checkpoint inhibitors in cancer therapy (Whittaker et al., 2017).

Inhibitor

Developmental status

Tumor type

Palbociclib (IBRANCE) Ribociclib (LEE011) Paclitaxel (Taxol) Nab-paclitaxel (Abraxane)

FDA approved FDA approved FDA approved FDA approved

Docetaxel (Taxotere)

FDA approved

Vincristine sulfate liposome (Marqibo) Brentuximab vedotin (Adcetris)

FDA approved FDA approved

Flavopiridol (Alvocidib)

Orphan designation

AZD-1775 AT9283 Ispinesib

Investigational drug Investigational drug Investigational drug

HR-positive, HER2-negative advanced or metastatic breast cancer HR-positive, HER2-negative advanced or metastatic breast cancer Ovarian cancer, breast cancer and non-small cell lung cancer (NSCLC) Metastatic breast cancer NSCLC Late-stage pancreatic cancer Metastatic breast cancer, NSCLC, metastatic prostate cancer, gastric adenocarcinoma head and neck cancer Adult Philadelphia chromosome-negative (Ph-) acute lymphoblastic leukemia Hodgkin lymphoma Systemic anaplastic large cell lymphoma B-cell chronic lymphocytic leukemia (B-CLL) Prolymphocytic leukemia arising from CLL Acute myeloid leukemia Hematological malignancies and solid tumors Acute myeloid leukemia Hematological malignancies and solid tumors

Flavopiridol

Flavopiridol (5,7-dihydroxy-8-(4-N-methyl-2-hydroxypyridyl)-60 -chloroflavone) is the first pan CDK inhibitor that inhibits Cdk 2, 4, 6, and 7. It is a flavonoid derivative extracted from an indigenous plant of India, Dysoxylum binectariferum. Flavopiridol binds to the ATP binding domain of the CDK to impair the cell cycle regulation, leading to cell cycle arrest and caspase-dependent cell death in hematological malignancies and solid tumors. Additionally, it decreases the transcription of RNA polymerase II by inhibiting the phosphorylation activity of CDK9.

Palbociclib (PD-0332991) Palbociclib is an oral, highly selective inhibitor of CDK4 and CDK6. It is a pyridopyrimidine derivative that binds to the ATP binding site of the CDK4/6. Palbociclib inhibits the binding of CDK4/6-Cyclin D, blocking the Rb phosphorylation and E2F1 release. Thus, preventing cell proliferation and cell cycle arrest at the G1/S phase. In 2015, FDA approved Palbociclib as an endocrinal-based combination therapy with letrozole for postmenopausal women with estrogen receptor, and progesterone receptor-positive and human epidermal growth factor receptor 2 negative breast cancer. Preclinical studies have also shown anti-tumor activity against multiple myeloma and lung cancer.

Paclitaxel Paclitaxel, also known as taxol, is a natural diterpene alkaloid. It is an FDA-approved drug used to treat ovarian cancer, breast cancer, and non-small cell lung cancer (NSCLC). Paclitaxel binds to the microtubules and stabilizes polymerization of the microtubules by promoting the alpha and beta subunits of the tubulin (building blocks of microtubules). Thus, due to the stability of microtubules, the critical concentration of the tubulin reduces; therefore, the cell cycle is halted at the G2/M phase leading to apoptosis. In lower doses, paclitaxel can also be used in skin disorders, renal and hepatic fibrosis, inflammation, axon regeneration, limb salvage, and coronary artery restenosis. Nab-Paclitaxel is a solvent-free, albumin-bound nanoparticle form of paclitaxel. It binds to the gp60, albumin receptor of the cell resulting in enhanced drug delivery to the tumor site. It is an FDA-approved drug to treat metastatic breast cancer, NSCLC, and late-stage pancreatic cancer.

Docetaxel Docetaxel is a semisynthetic taxane used to treat metastatic breast cancer, NSCLC, metastatic prostate cancer, gastric adenocarcinoma, and head and neck cancer. It inhibits the microtubule depolymerization and halts the cell cycle at the G2/M phase. It blocks and inhibits BCL-2 and BCL-XL (anti-apoptotic) gene expression by BCL-2 phosphorylation. Both mechanisms lead to the prevention of cell proliferation and apoptosis.

Vincristine sulfate liposome Vinca alkaloids vincristine (VCR) is used as a chemotherapeutic agent since the 1960s. It binds and inhibits the equilibrium of the tubulin polymerization leading to G2/M phase arrest. In combination with other chemotherapeutic agents, VCR is effective in acute lymphocytic leukemia, Hodgkin’s and non-Hodgkin’s lymphoma, rhabdomyosarcoma, and neuroblastoma. However, VCR has a short plasma half-life and high neurotoxicity, limiting its anticancer activity. Thus, a liposomal formulation of VCR was formulated called vincristine sulfate liposome injection (VSLI) that had a higher retention time in the plasma. In 2012, vincristine sulfate

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liposome injection (VSLI) was approved for adult patients with Philadelphia chromosome-negative (Ph-) Acute lymphoblastic Leukemia (ALL) as second line or greater relapse phase.

Brentuximab vedotin Brentuximab vedotin is an antibody-drug conjugate that combines mouse-human chimeric IgG1 anti-CD30 mAb with a drug monomethyl auristatin A (MMAE) via a protease-sensitive dipeptide linker. Brentuximab vedotin binds to CD30 expressing tumor cells and is internalized via endocytosis. The MMAE drug gets released upon exposure to intracellular proteolytic enzymes, binds to tubulin, and inhibits tubulin polymerization, leading to G2/M arrest and apoptosis. Brentuximab vedotin is FDA approved for the treatment of Hodgkin lymphoma and systemic anaplastic large cell lymphoma (Klein et al., 2018; Łukasik et al., 2021).

Conclusion Cell cycle regulation plays important role during normal tissue homeostasis. Defect in cell cycle regulation resulting in uncontrolled proliferation or evasion of apoptosis are considered as major hallmarks of cancer development. Cell cycle regulation involves numerous signaling pathways giving rise to different cell fates such as proliferation, quiescence, arrest, or apoptosis depending on the stimuli, context, upstream singling, and cell type. It has been established that both carcinogenic and non-carcinogenic toxicants can considerably alter cell cycle specific events. Table 1 shows the list of agents that can influence cell cycle specific events. Many drug or chemical-induced alterations are reversible, and many are irreversible; it depends on whether the cell cycle regulatory elements are altered/influenced by parent compounds, their metabolites, and/or their biological reactive intermediates. While enormous progress has been made in discovering signaling pathways, our understanding of cell cycle regulation remains incomplete. Further studies may allow better understanding of diseases that originate from deregulated pathways.

References Aranha O, et al. (2000) Clinical Cancer Research 6(3): 891–900. Basu A, et al. (2010) Journal of Nucleic Acids 2010: 201367. Carlier MF, et al. (1983) Biochemistry 22: 4814. Chappell J, et al. (2001) The Journal of Biological Chemistry 276(38): 023. Ching Chen S, et al. (2011) Environmental and Molecular Mutagenesis 52(8): 664–672. Clemens DL, et al. (2011) Alcohol 45: 785. Costa M, et al. (1982) Research Communications in Chemical Pathology and Pharmacology 38: 405. Ding J, et al. (2009) Lung 187(1): 61–67. Ding L, Cao J, Lin W, Chen H, Xiong X, Ao H, Yu M, Lin J, and Cui Q (2020) The roles of cyclin-dependent kinases in cell-cycle progression and therapeutic strategies in human breast cancer. International Journal of Molecular Sciences 21(6): 1960. Engeland K (2022) Cell cycle regulation: p53-p21-RB signaling. Cell Death and Differentiation 29(5): 946–960. Fearnhead HO, et al. (1994) Biochemical Pharmacology 48: 1073. Greenberg AK, et al. (2002) American Journal of Respiratory Cell and Molecular Biology 27: 320. Jung B, et al. (2005) Cancer Letters 219: 15. Kang KY, et al. (2005) Journal of Microbiology and Biotechnology 15(5): 913–918. Kato K, et al. (2012) Molecular Cancer Therapeutics 11: 549. Kim JH, et al. (1967) Cancer Research 27: 1301. Klein ME, Kovatcheva M, Davis LE, Tap WD, and Koff A (2018) CDK4/6 inhibitors: the mechanism of action may not be as simple as once thought. Cancer Cell 34(1): 9–20. Krishnan A, et al. (2008) Biochemical Pharmacology 75: 1924. Liu X, et al. (2010) BMC Cancer 10(1): 1–8. Łukasik P, Baranowska-Bosiacka I, Kulczycka K, and Gutowska I (2021) Inhibitors of cyclin-dependent kinases: Types and their mechanism of action. International Journal of Molecular Sciences 22(6): 2806. Matthews HK, Bertoli C, and de Bruin RA (2022) Cell cycle control in cancer. Nature Reviews Molecular Cell Biology 23(1): 74–88. Pesonen M, et al. (2014) Toxicology Letters 226(2): 236–244. Plissonnier ML, et al. (2011) PLoS One, e28354. Sarfaraz S, et al. (2006) Journal of Biological Chemistry 281(51): 39480–39491. Shin SY, et al. (2010) Experimental & Molecular Medicine 42(5): 395–405. Tabasi N, et al. (2015) Iranian Journal of Basic Medical Sciences 18(11): 1107–1111. Whittaker SR, Mallinger A, Workman P, and Clarke PA (2017) Inhibitors of cyclin-dependent kinases as cancer therapeutics. Pharmacology & Therapeutics 173: 83–105. Wiger R, et al. (1997) Pharmacology & Toxicology 81: 285. Yan L, et al. (2005) Oncogene 24(13): 2175–2183. Yang HW, Chung M, Kudo T, and Meyer T (2017) Competing memories of mitogen and p53 signalling control cell-cycle entry. Nature 549(7672): 404–408. Zabihi M, Lotfi R, Yousefi AM, and Bashash D (2022) Cyclins and cyclin-dependent kinases: from biology to tumorigenesis and therapeutic opportunities. Journal of Cancer Research and Clinical Oncology 1–22. Zhao YL, et al. (2008) Cellular Physiology and Biochemistry 22(5–6): 431–440. Zhu JZ, et al. (2012) Nephrology, Dialysis, Transplantation 27(12): 4323–4330.

Cell phones Mahshid Ataei and Mohammad Abdollahi, Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of S. Saeidnia, M. Abdollahi, Cell Phones, Editor (s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 759–760, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01061-7.

Introduction Immune system effects Cytotoxicity and genotoxicity Reproductive effects Brain and neurological effects Pregnancy-related exposure risk Pregnancy and dental amalgam mercury exposure Effects of prolonged exposure Prevention methods Cell phones as electronic waste (E-waste) Conclusion References Further reading

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Abstract Radiofrequency is non-ionizing electromagnetic radiation typically emitted by cell phones. While there may once have been questions about whether cell phone usage could be associated with cancer and other health risks to humans, the scientific consensus as of 2023 is that there is no credible evidence to implicate cell phones in adverse human health effects. Neither ongoing epidemiological nor in vivo studies, such as those conducted by the US FDA and the National Cancer Institute over, in some cases, decades, have not shown any significant cause for concern for cellphone use. However, the IARC considered cell phones a possible carcinogen to humans. Radiation from cell phones could target several organs and tissues like CNS and the reproductive systems. Some studies did not report significant harmful results. Moreover, distance and time of exposure are two critical factors that should be considered. Monitoring continues, including for possible consequences of 5G (5th generation), but to date, cell phones and other wireless devices have generally proven their safety to humans based on their intended and recommended use. On the other hand, electronic waste, to including cell phones, has become a burgeoning problem that impacts the environment and health (especially children) and must be controlled. Waste materials from discarded cell phones could reach water and result in ecotoxicity. This chapter discusses the concerns about possible toxic and adverse effects in both experimental animals and humans, focusing on radiation and e-waste.

Keywords Batteries; Brain tumor in animals; Electronic devices; Environmental toxicant; E-Waste; Microwave; Radiation; Recycling; Reproductive system

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Cell phones and other wireless devices have generally proven their safety to humans based on their intended and recommended use. Both experimental toxicology and human epidemiological studies and results are summarized in this chapter. Based on NTP’s 2 year rats and mice study results from 2018, rats and mice were exposed to 2G and 3G cell phone frequencies from about 700–2700 MHz (MHz). There was clear evidence of an association with tumors (malignant schwannomas) in the hearts of male mice exposed to RFR 900 MHz. There was evidence of an association with brain tumors (malignant gliomas) in the male rats. “Some evidence of an association with tumors in the adrenal glands of male rats. The tumors were benign, malignant, or complex combined pheochromocytoma.”

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“It was unclear if tumors observed in the studies were caused by exposure to RFR in female rats (900 MHz) and male and female mice (1900 MHz).” Cell phone radiation and toxicity may target several organs and tissues, especially the brain, nervous system, reproductive organs, and possibly genetic materials (DNA and RNA) in experimental animals and some human users Short-term microwave radiation exposure can stimulate the immune response, but long-term exposure inhibits the activity of the immune systems in experimental animals. Long-term radiofrequency exposure can induce oxygen species, increase the formation of reactive oxygen species, and may lead to DNA damage in experimental animals. Electromagnetic frequency exposure can affect both the male and female reproductive systems in experimental animals and humans. Experimental toxicology evidence suggests an increase in testicular proteins in adult rats exposed to radio frequencies, possibly increasing cancer risk and reproductive effects. Cell phone radiation may cause adverse effects on the brain and nervous systems in animals and some human users. As EMF exposure can lead to embryonic neurological impairments; it is suggested that pregnant women limit their cell phone use. E-waste is a critical issue that increases yearly as the number of electronic device users grows and causes many health and environmental problems. One should focus on preventative methods and strategies to reduce, manage and/or eliminate potential risks from RF radiation sources.

Introduction Cell phones emit radiofrequency (RF) radiation. Reviewing the evidence showed that glioma and acoustic neuroma were the only types of cancer reported by heavy users of cell phones (30 min a day for more than 10 years). No significant result was declared about other types of cancer. Therefore, in 2011, the World Health Organization (WHO) and the International Agency for Research on Cancer (IARC) recommended that electromagnetic radiation from cell phones and other wireless devices may be classified as a ‘possible human carcinogen’ 2B.1 Based on IARC’s classification, “this category is used for agents for which there is limited evidence of carcinogenicity in humans and less than sufficient evidence of carcinogenicity in experimental animals. It may also be used when there is inadequate evidence of carcinogenicity in humans but sufficient evidence of carcinogenicity in experimental animals. In some instances, an agent for which there is insufficient evidence of carcinogenicity in humans and less than sufficient evidence of carcinogenicity in experimental animals, together with supporting evidence from mechanistic and other relevant data, may be placed in this group.” An agent may be classified in this category based on solid evidence from mechanistic and other relevant data. However, a review of published literature between 2008 and 2018 by the United States Food and Drug Administration (USFDA) concluded no risk for tumor formation after RFR exposure (US Food and Drug Administration, 2020). In 2022, a new study conducted by IARC in partnership with researchers from various Nordic countries provided important information on temporal trends of glioma incidence rates among men between 1979 and 2016 (Deltour et al., 2022). IARC summarized the study and reported that the “absence of an observable impact on the glioma incidence rates provides evidence against any significant contribution of mobile phone use to the risk of glioma.2” Furthermore, the study reported some uncertainties for a latency period greater than 20 years and risk less than an 8% increase. Several studies have evaluated the safety of RF-EMF; only two adverse events, including tissue heating and nerve stimulation like tingling, were reported, not cancer formation. These side effects depend on the time and distance cell phones are used (Russell, 2018). Cell phones are globally responsible for ubiquitous and quickly growing exposure as they have two-way microwave radios that emit low levels of electromagnetic radiation. In addition, children are increasingly using cell phones since ‘Family Plans’ encourage parents to buy cell phones for their children. Since children and adolescents are probably more sensitive than adults to the adverse effects of cell phone exposure, the risk of brain cancer did not increase among them (Aydin et al., 2011). This study also reported that increased brain tumor risk was more related to time than the amount used. Aydin et al. (2011) reported that for a sub-group of the total study participants, brain tumor risk was related to the “elapsed time” since the start of their cell phone subscription” but not linked to the “amount of use.” Furthermore, the authors reported that there is increased risk of tumors for brain areas “receiving the highest amount of exposure.” The authors concluded that there was no exposure-response relationship and no causal association between exposure and risk of brain tumors. Cell phone exposure increases the risk of kidney dysfunction in rats (Deniz et al., 2017; Borzoueisileh et al., 2020) and changes in salivary flow rate and enzymes in some cell phone users (Shivashankara et al., 2015; Mishra et al., 2017).

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https://www.iarc.who.int/wp-content/uploads/2018/07/pr208_E.pdf (accessed Dec 02, 2022).

https://www.iarc.who.int/news-events/time-trends-in-mobile-phone-use-and-glioma-incidence-among-males-in-the-nordic-countries-1979-2016/ (accessed Dec 02, 2022).

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Immune system effects The fast and increasing application of cell phones in recent years has made concerns about the possible immune effects of RF/ microwave frequency (MW) radiation. The impacts of weak RF/MW fields, including cell phone radiation, on various immune functions have been discussed. Much evidence reveals alterations in the number and (or) the activity of immunocompetent cells may occur. For instance, several lymphocyte functions might be enhanced or weakened within a single experiment based on exposure to similar intensities of MW radiation. Also, some studies suggested that RF exposure can act as a tumor promoter by inhibiting anti-tumor immune responses (De Vocht, 2011). For example, a study reported a higher risk of myeloid than lymphoma. The study explains this result by suggesting that innate immunity (myeloid cell-related) can involve RF exposure (Cooke et al., 2010; Kaufman et al., 2009). However, in a study on rats after 1 year of exposure to RF radiation, tumors were not significant between exposed and control animal groups (Jin et al., 2012). Furthermore, in vitro studies showed that human skin fibroblast exposure to RF slightly increased cellular ROS levels. It is important to remember that skin is the first line of body defense and can easily be targeted by RF exposure (Szilágyi et al., 2020). Generally, short-term exposure to low MW radiation can temporarily stimulate particular humoral and cellular immune responses, while prolonged irradiation inhibits the same functions (Abdollahi and Saeidnia, 2014).

Cytotoxicity and genotoxicity The IARC has classified mobile phone electromagnetic fields (MP EMF) as “possibly carcinogenic to humans, for which there is limited evidence of carcinogenicity in humans and less than sufficient evidence of carcinogenicity in experimental animals (Nersesyan et al., 2016). In a study on the Allium cepa, the cytotoxicity and genotoxicity of plutonium-239 alpha particles and GSM 900 modulated cell phone (model Sony Ericsson K550i) radiation were compared. The authors stated that cell phone radiation had a time-dependent enhancement of the mitotic index and its frequency, chromosome abnormalities, and micronucleus frequency (Abdollahi and Saeidnia, 2014). Furthermore, electromagnetic radiation (EMR) enhances reactive oxygen species (ROS) formation. It promotes apoptosis (Hou et al., 2015) and oxidative stress (OS) by reducing antioxidant parameters leading to DNA strand breaks and damage, which have been assessed in rat livers (Alkis et al., 2021). Also, another study revealed the RF effects on buccal mucosal cells, such as the formation of micronuclei and broken eggs, which are considered biomarkers in genotoxicity (Revanth et al., 2020). Such results should inform us of the possible effects of long-term exposure to cell phones. Also, a review article published by the National Toxicology Program in 2018 analyzed the evidence of rats and mice exposed to RF (900 MHz) for 2 years by designing a standard scale. This study concluded that heart tumors in male rats correlate with RF exposure. Furthermore, there was some evidence of brain and adrenal gland tumors in male rats after 2-year exposure to RF. However, results showed that the tumor incidence in female and male rats and female mice exposed to RF was unclear (National Toxicology Program, 2018).

Reproductive effects A systematic review and meta-analysis of in vivo and in vitro studies suggest that cell phones were associated with the quality of semen by reducing sperm motility (but not sperm counts, as its results were more equivocal), viability, and morphology. Therefore, pooled results of in vivo and in vitro studies suggest that cell phone exposure negatively affects sperm quality (Adams et al., 2014). Although there are consistent results, further studies are necessary to show the possible association between cell phone radiation and male reproductive system impacts. To address the concern that carrying a cell phone influences the reproductive system (like the testes), one group of researchers conducted a study to investigate the effects of a 1.95 GHz electromagnetic field on testicular function in male rats. Their findings indicated that the number of sperm in the testis and epididymis did not decrease in the electromagnetic field exposed groups. Further, no abnormalities were observed for sperm motility or morphology, and the histological appearance of seminiferous tubules, including the stage of the spermatogenic cycle, was normal (Imai et al., 2011). In studies reported by a recent systematic review, total numbers of primary spermatocytes and spermatids, Leydig cells in Swiss albino mice, serum testosterone levels, and wet weight of testes were significantly decreased in the rat group exposed to EMF. The RFR effect on spermatogenesis and lowering of sperm count is a crucial issue in humans and rodents (Maluin et al., 2021). Furthermore, talking while charging the device for an hour or more daily can be a risk factor for abnormal sperm concentration. Also, keeping a cell phone less than 50 cm from the groin area can increase abnormal sperm concentrations. However, the findings were reported as insignificant. Multivariate analysis showed that cell phone usage (while charging) and smoking-related exposures were potential risk factors for abnormal sperm concentrations in men. The authors recommended that large-scale studies be carried out to evaluate further cellphone use exposure and sperm quality (Zilberlicht et al., 2015). Additionally, long-term exposure to the 4th generation mobile communication technology (4G) smartphone radiofrequency electromagnetic radiation (SRF-EMR) diminished male fertility in the rat model (Yu et al., 2020). A review article by Okechukwu et al. concluded that animal and human EM exposure from cell phones had reduced structural anomalies, motility, and increased oxidative stress in spermatozoa. These adverse effects may be related to the duration of cellphones usage (Okechukwu, 2020).

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Moreover, EMF exposure in a study on rats revealed an increased risk of testicular cancer. Most often, men put their cell phones in pockets near their gonads, where exposure can increase. Also, another study revealed morphological and functional impairments in the testis and ovary following EMF exposures that increased ROS rates and decreased antioxidants in both animal and human studies (Altun et al., 2018). Santini et al. (2018) concluded that mitochondria play an essential role as the source of ROS in both male and female reproductive systems of rats under EMF exposure. Moreover, in the female rat model, EMF exposure caused endometrial hyperplasia (Fadiloglu et al., 2019). More specific studies are needed to evaluate these effects in humans.

Brain and neurological effects The severity of possible effects associated with cell phone radiation exposure will depend on the intensity and duration of exposure. Some studies did not report any adverse effects (de Pomerai et al., 2016; Petitdant et al., 2016; Nakatani-Enomoto et al., 2020) based on the short-term and low-intensity nature of RF exposures. In other studies, RF radiation significantly reduced the antioxidant parameters (glutathione, superoxide dismutase, and glutathione peroxidase) (Hussein et al., 2016) and induced cellular stress (Miah and Kamat, 2017) in different parts of the rat’s brain. Also, degenerative changes (Hussein et al., 2016), like autophagy which inhibits apoptosis (Kim et al., 2018), were observed in the hippocampus pyramidal cells, dark cells, and cerebellar Purkinje cells of mice. In addition, significant DNA fragmentation and over-expression of the cyclooxygenase-2 apoptotic gene were detected in rat hippocampus cells. Therefore, apoptosis and oxidative stress significantly increased in brain tissues. To decline these adverse events, the study suggests shortening the length of phone calls and keeping cell phones away from the body (Hussein et al., 2016). A correlation study reinforces the statement that continuous exposure to cell phones could elevate the risk of MS disease (Khaki-Khatibi et al., 2019). Additionally, another animal study found that radiation exposure prolonged particular auditory brainstem response (ABR) components (Kaprana et al., 2021). The results for brain cancer are controversial. Some studies illustrated no correlation between cancer and EMF or RF exposure (Yoon et al., 2015; Miah and Kamat, 2017; Ouadah et al., 2018). A case-control study of five northern European countries suggested that there is no significant risk of acoustic neuroma in the first decade of cell phone use (Schoemaker et al., 2005). As acoustic neuroma is a slow-growing tumor, longer-term use should be evaluated in future studies (US Food and Drug Administration, 2020). In contrast, other studies showed a strong link between cell phone exposure and adult acoustic neuroma (Vienne-Jumeau et al., 2019). Acoustic neuroma, also known as “vestibular schwannoma” is a non-cancerous brain tumor that grows in the ear and affects hearing and balance (US Food and Drug Administration, 2020). In addition, a review article demonstrated that longtime exposure over 10 years to low-intensity EMR could increase the risk of brain cancers. Another review article showed that 10-year EMR exposure did not increase the risk of brain and head tumors (Sri, 2015). Thermal damage is another effect of long-term cell phone use in which brain glucose can burn. One study showed that more than 50 min of cell phone use would increase the risk of dementia and other thermal damage shields (London et al., 1991). Impaired cognition, like memory problems, is the subsequent adverse effect of exposure to cell phones (Barthélémy et al., 2016; Foerster et al., 2018; Hong et al., 2020). However, another review showed that more than 86% of studies found no relation between cell phone exposure and cognitive problems (Ishihara et al., 2020). Also, memory problems and Blood-Brain Barrier (BBB) permeability damage are observed in the male rat model exposed to a 900 MHz EMF (Tang et al., 2015). Headache is one of the most common complaints of cell phone users (Cho et al., 2016; Durusoy et al., 2017; Zheng et al., 2015), which can be more frequent in migraine patients (Demir and Sümer, 2019). In contrast, other studies found no relation between Global System for mobile communications (GSM) and headaches. In children, a greater risk for fatigue is significantly associated with the years and daily duration of cell phone usage. A study reported limitations like misclassification of the self-reported cell phone usage field (Zheng et al., 2015). Furthermore, insomnia resulting from using cell phones before sleep is another issue affecting learning ability (Arora et al., 2014; Zarghami et al., 2015; Zheng et al., 2015). A study on rats also explained circadian rhythmicity impairments, which may happen after RF exposure by decreasing the daily antioxidant levels (Cao et al., 2015). Furthermore, cell phone dependency was correlated with poor subjective sleep quality and latency. Lower grades were reported for students having cellphone dependency (owners>1y, used it >5 h/day) (Ibrahim et al., 2018). Another experimental animal study in the lumbar region spinal cords asserted that continuous exposure to EMF could result in impairments at both morphological in gray matter (higher apoptotic index of glia cells and neurons) and biochemical levels (Malondialdehyde (MDA)) increased. In contrast, Superoxide Dismutase (SOD) and Reduced glutathione (GSH) levels decreased significantly in Sprague Dawley rats (Kerimo glu et al., 2016).

Pregnancy-related exposure risk Pregnancy and dental amalgam mercury exposure Two Mortazavi et al. studies revealed that mercury could be released from dental amalgam restorations after exposure to electromagnetic fields (Mortazavi and Mortazavi, 2015; Mortazavi et al., 2016). As mercury can pass through cord blood (Mortazavi and Mortazavi, 2015), there can be an increased risk for developmental disorders such as autism spectrum disorders (ASD), attention-deficit hyperactivity disorder (ADHD), and other neurological problems (Mortazavi et al., 2016). Therefore, it is

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suggested that pregnant women with dental amalgam fillings, including cell phones, should limit their EMF exposure (Mortazavi and Mortazavi, 2015). During pregnancy, exposure to environmental and maternal RF for fetuses can increase the risk of neurobiological impairments (Petitdant et al., 2018), like a decrease in the Purkinje cells’ excitability (Haghani et al., 2020). However, in an experimental animal study, RF exposure did not harm embryo development (Safian et al., 2016).

Effects of prolonged exposure Several papers conclude that human health risks exist after prolonged and intermittent cell phone exposure. For instance, in Taiwan, a 10-year observational study revealed that the intensive rate of cell phone use is not relative to the incidence rate or the mortality of malignant brain tumors in that country. Also, there was no significant association between the morbidity/mortality of malignant brain tumors and cell phone applications (Hsu et al., 2013). Moreover, a large prospective study reported that cell phone usage might not be related to the enhanced incidence of glioma, meningioma, or non-centrally nervous system cancers (Benson et al., 2013). Various research groups have explored an enhanced risk of some low-incidence brain cancers among users exposed for a long time and known as heavy users. There is no etiological link between cell phone use and cancer initiation. A 14-year follow-up study on 776156 women did not show a significant increase in brain tumors and cell phone use (Joachim et al., 2022). Children may be at a greater risk because of being in earlier stages of neural development. The challenging issue is that datasets related to children subpopulations have not been incorporated in many investigations until very recently. Further studies, documentation, and evidence analyses are essential to statistically define the relationship between cell phone use and brain cancer in different age groups. Taken together, it is safer to use precautions and encourage users to decrease their exposure to cell phone radiation fields (Abdollahi and Saeidnia, 2014).

Prevention methods Use of headsets, Bluetooth, or cell phones in speaker mode, reduction in length, and several calls and messages are recommended. Keeping your cell phone away from the body while using a wired earpiece or speaker phone (also not placing it in the front pocket while it is switched on) lowers the amount of radiation absorbed. Furthermore, text messaging, rather than talking, also reduces radiation exposure. Children, adolescents, and pregnant women should minimize cell phone use. A child’s brain can absorb as much radiation as an adult brain (Sri, 2015). Another preventive method for children is allowing cell phones only in flight mode and deactivating Wi-Fi access points when not in use (Hedendahl et al., 2017). Cell phones should not be used when the signal is weak, like in a moving vehicle or an elevator, as the phone increases its signal strength to compensate (Sri, 2015). Administration of antioxidants, thymoquinone (Yahyazadeh and Altunkaynak, 2019), and luteolin (Yahyazadeh and Altunkaynak, 2020) showed decreased spinal cord tissue damage in the rat model. Also, melatonin and omega-3 can protect cells against neuronal damage in the hippocampus (Erdem Koç et al., 2016). EGb761, a free-radical scavenger, decreased the toxic effects in testicular tissue, restoring normal spermatogenesis and hormone levels (Gevrek et al., 2017). Melatonin reduces oxidative damage to the testicular tissue (Shokri et al., 2020). Moreover, a study on zebrafish embryos explained that electromagnetic shielding fabric, a non-woven textile, is suitable for keeping individuals safe from EM radiations by blocking electromagnetic waves. Oxidant-antioxidant system parameters were evaluated. tp53 expression was impaired, and casp3, responsible for apoptosis, decreased (Üstünda g et al., 2020). Overall, reducing the distance from cell phones and length of usage to reduce RF absorption is recommended. Several studies showed that administering antioxidants would reduce EM oxidative damage to different body parts. A recent study reported that a particular electromagnetic shielding fabric, by its antioxidant action, decreased oxidative stress and its consequences.

Cell phones as electronic waste (E-waste) E-waste is one of the fastest-growing ecotoxicological problems globally because many electronics contain toxic metals and toxic organics. Such dangerous wastes pose serious health risks, particularly to pregnant women and children (Abdollahi and Saeidnia, 2014). Between 2002 and 2013, in a case study by Chen et al. (2018), 36 waste mobile phones (WMPs) were analyzed for metals concentration. WMPs should be considered hazardous based on elevated lead, copper, and chromium levels (Chen et al., 2018). A recent study illustrated that the metals in newer devices had been decreased over time, complying with the US non-hazardous waste definition (Intrakamhaeng et al., 2019). Copper (Chen et al., 2018) and nickel (Singh et al., 2019) showed the most significant potential for ecotoxicity risks, and chromium (Chen et al., 2018) posed significant risks related to cancer and non-cancer diseases. Additionally, Chen et al. (2018) demonstrated that WMP toxicity increased with technology innovation. Moreover, the WHO reported that even small amounts of lead, cadmium, and mercury (found in old phones) could generate irreversible neurological damage and affect a child’s development. Electronic waste is quickly flooding Asian countries, especially China, which inevitably can lead to environmental toxicological concerns on a massive scale if the e-wastes are not handled properly. The critical question is,

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to what extent can metal and other toxicants leach from cell phone wastes or their parts into the water? Also, which class of toxicants (metals or hydrophobic organics) is responsible for toxicity? Also, toxicological assessment with C18 and ethylenediaminetetraacetic acid (EDTA) addition reported that metals were responsible for the observed toxicity from leachates of cell phones. The findings indicated that electronic waste could leach toxic components even in short-term leaching with pure water. Bisphenol A (BPA) (Win-Shwe et al., 2019) and its derivate tetrabromobisphenol A (TBBPA) (Kim et al., 2017) are used in commercial and household products, such as cell phones, computers, and televisions as significant constituents. Exposure to a high dose of BPA in the presence of allergens can cause memory impairment (Win-Shwe et al., 2019). Lithium-based cell phone batteries can lead to further metal depletion and cause remarkable environmental and health impacts (Abdollahi and Saeidnia, 2014). Lithium is toxic to CNS as it can interfere with other drugs and cause irreversible hand tremors and gastrointestinal, endocrine, and reproductive systems effects by passing through the placenta and accelerating the incidence of Ebstein’s anomaly in babies. Also, Lithium exposure may lead to gastrointestinal, cardiovascular, neurological, endocrine, and renal effects (Hedya et al., 2022). While skin exposure to LiOH was deemed safe in humans (Park et al., 2018); however, exposure to electronic devices, notably cell phones, may also contribute to additional exposure to an organophosphorus ester (OPE) (Yang et al., 2019). In addition, lithium can also be incorporated into the food chain from soils via plant uptake and to humans. A 2020 study showed that toxic substances from cell phones are below the limit values of substances regulated in the Restriction of Hazardous Substances (RoHS) Directive in China and Europe (Singh et al., 2020).

Conclusion The use of cell phones, especially for a long time and with high frequency, might have many adverse health effects on humans and experimental animals. Based on available evidence, these adverse effects may include possible debilitation of the immune system, cytotoxicity and genotoxicity, different central and peripheral nervous system disorders, reproductive system impairments, and effects on the fetus during pregnancy. Many studies were not definitive and conclusive. However, one must be aware of increasing cell phone use for various needs and increasing body burden and risk of radiation exposure. Also, in recent years, some studies measured E-wastes, which can harm the environment if chemical contaminants are released into the ecosystem resulting in exposure and possible health effects in biota. Therefore, using preventive methods will help us further to reduce radiation exposure.

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Further reading Andeobu L, Wibowo S, and Grandhi S (2021) A systematic review of E-waste generation and environmental management of Asia Pacific countries. International Journal of Environmental Research and Public Health 18(17): 9051. https://doi.org/10.3390/ijerph18179051. PMID: 34501640. PMCID: PMC8430537. Choi Y-J, Moskowitz JM, Myung S-K, Lee Y-R, and Hong Y-C (2020) Cellular phone use and risk of tumors: Systematic review and meta-analysis. International Journal of Environmental Research and Public Health 17(21): 8079. https://doi.org/10.3390/ijerph17218079. Davis DL, Kesari S, Soskolne CL, et al. (2013) Swedish review strengthens grounds for concluding that radiation from cellular and cordless phones is a probable human carcinogen. Pathophysiology 20: 123–129. Hardell L, Carlberg M, and Hansson MK (2011) Pooled analysis of case-control studies on malignant brain tumors and the use of mobile and cordless phones including living and deceased subjects. International Journal of Oncology 38: 1465–1474. Herrala M, et al. (2018) Assessment of genotoxicity and genomic instability in rat primary astrocytes exposed to 872 MHz radiofrequency radiation and chemicals. International Journal of Radiation Biology 94(10): 883–889. https://doi.org/10.1080/09553002.2018.1450534. Hsu MH, Syed-Abdul S, Scholl J, Jian WS, Lee P, Iqbal U, and Li YC (2013) The incidence rate and mortality of malignant brain tumors after 10 years of intensive cell phone use in Taiwan. European Journal of Cancer Prevention 22(6): 596–598. https://doi.org/10.1097/CEJ.0b013e328360f456. PMID: 23591455. Huang MF, Chang YP, Lu WH, and Yen CF (2022) Problematic smartphone use and its associations with sexual minority stressors, gender nonconformity, and mental health problems among young adult lesbian, gay, and bisexual individuals in Taiwan. International Journal of Environmental Research and Public Health 19(9): 5780. https://doi.org/10.3390/ ijerph19095780. PMID: 35565175. PMCID: PMC9105429. Iakovidis S, Apostolidis C, Manassas A, and Samaras T (2022) Electromagnetic fields exposure assessment in europe utilizing publicly available data. Sensors 22(21): 8481. Jagetia GC (2022) Genotoxic effects of electromagnetic field radiations from mobile phones. Environmental Research 212: 113321. https://doi.org/10.1016/j.envres.2022.113321. Kesari KK, Siddiqui MH, Meena R, et al. (2013) Cell phone radiation exposure on brain and associated biological systems. Indian Journal of Experimental Biology 51: 187–200. Moradi M, et al. (2016) Effect of ultra high frequency mobile phone radiation on human health. Electronic Physician 8(5): 2452. Panagopoulos DJ (2019) Comparing DNA damage induced by mobile telephony and other types of man-made electromagnetic fields. Mutation Research 781: 53–62. https://doi.org/ 10.1016/j.mrrev.2019.03.003.

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Pesnya DS and Romanovsky AV (2012) Comparison of cytotoxic and genotoxic effects of plutonium-239 alpha particles and mobile phone GSM 900 radiation in the Allium cepa test. Mutation Research 750: 27–33. Schüz J, Pirie K, Reeves GK, Floud S, Beral V, and Million Women Study Collaborators (2022) Cellular telephone use and the risk of brain tumors: Update of the UK million women study. Journal of the National Cancer Institute 114(5): 704–711. https://doi.org/10.1093/jnci/djac042. PMID: 35350069. PMCID: PMC9086806. Sepehrimanesh M, et al. (2017) Proteomic analysis of continuous 900-MHz radiofrequency electromagnetic field exposure in testicular tissue: A rat model of human cell phone exposure. Environmental Science and Pollution Research 24(15): 13666–13,673. https://doi.org/10.1007/s11356-017-8882-z. Szmigielski S (2013) Reaction of the immune system to low-level RF/MW exposures. The Science of the Total Environment 454–455: 393–400. Tyagi A, Prasad AK, and Bhatia D (2021) Effects of excessive use of mobile phone technology in India on human health during COVID-19 lockdown. Technology in Society 67: 101762. https://doi.org/10.1016/j.techsoc.101762. PMID: 34566205. Epub 2021 Sep 22. PMCID: PMC8456111. Vaverka F, Smetana M, Gombarska D, and Psenakova Z (2023) Investigation of microwave electromagnetic fields in open and shielded areas and their possible effects on biological structure. Sensors 23(4): 2351. Yahyazadeh A, Altunkaynak BZ, and Kaplan S (2020) Biochemical, immunohistochemical and morphometrical investigation of the effect of thymoquinone on the rat testis following exposure to a 900-MHz electromagnetic field. Acta Histochemica 122(1): 151467. https://doi.org/10.1016/j.acthis.2019.151467. Yilmaz A, et al. (2017) Lasting hepatotoxic effects of prenatal mobile phone exposure. Journal of Maternal-Fetal and Neonatal Medicine 30(11): 1355–1359. https://doi.org/ 10.1080/14767058.2016.1214124.

Relevant websites https://seawave-project.eu/home-3/, https://www.iarc.who.int/news-events/iarc-to-coordinate-production-of-a-risk-assessment-on-5g-exposures-as-part-of-the-eufunded-seawave-project/ :5G risk assessment project—Scientific-Based Exposure and Risk Assessment of Radiofrequency and Millimetre-Wave Systems (SEAWave). https://www.canada.ca/en/health-canada/services/health-risks-safety/radiation/everyday-things-emit-radiation/cellphones-towers.html :5G technology, cell phones, cell phone towers and antennas https://www.hopkinsmedicine.org/health/conditions-and-diseases/brain-tumor/vestibular-schwannoma :Acoustic Neuroma https://www.fcc.gov/consumers/guides/wireless-devices-and-health-concerns :Wireless devices and Health Concerns—Factsheet from Federal Communications Commission https://ntp.niehs.nih.gov/whatwestudy/topics/cellphones/index.html :Cell Phone Radio Frequency Radiation resources, National Toxicology Program https://www.fda.gov/radiation-emitting-products/cell-phones/scientific-evidence-cell-phone-safety :Scientific Evidence for Cell Phone Safety https://www.iarc.who.int/wp-content/uploads/2018/07/pr208_E.pdf :IARC Classifies Radiofrequency Electromagnetic Fields as Possibly Carcinogenic to Humans https://www.iarc.who.int/news-events/time-trends-in-mobile-phone-useand-glioma-incidence-among-males-in-the-nordic-countries-1979-2016/ :Time trends in mobile phone use and glioma incidence among males in the Nordic countries, 1979–2016 https://www.cancer.gov/about-cancer/causes-prevention/risk/radiation/cell-phones-fact-sheet :Cell Phones and Cancer Risk https://www.eurekalert.org/news-releases/967282 :5.3 billion mobile phones will become waste in 2022; a small fraction will be properly disposed of (News release as of October 13, 2022) https://www.fda.gov/radiation-emitting-products/cell-phones/children-and-teens-and-cell-phones :Children and Teens and Cell Phones—Resources from US FDA https://www.fda.gov/radiation-emitting-products/cell-phones/potential-cell-phone-interferencepacemakers-and-other-medical-devices :Potential Cell Phone Interference with Pacemakers and Other Medical Devices https://www.who.int/publications/i/item/9789240023901, https://www.who.int/news/item/15-06-2021-soaring-e-waste-affects-the-health-of-millions-of-children-whowarns :WHO Report on E-Waste “Children and Digital Dumpsites” (2021)

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Cell proliferation Iva Srdanovica, Ningning Yangb, and Sidhartha D Raya, aDepartment of Pharmaceutical and Biomedical Sciences, Touro University College of Pharmacy, New York, NY, United States; bLake Erie College of Osteopathic Medicine, Bradenton, FL, United States © 2024 Elsevier Inc. All rights reserved. This is an update of N. Yang, S.D. Ray, K. Krafts, Cell Proliferation, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 761–765, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00274-8.

Introduction Division cycle of cells Role of tight junction proteins in cell proliferation Genetic control of cell structure and function during and after embryonic development Terminal differentiation and cell division Role of growth factors and cytokines Cell senescence and reluctance to divide Cell proliferation as a compensatory response to toxic tissue injury Stem cells and terminally differentiated cells Cell proliferation and cancer Cell proliferation, cardiovascular disease and drug-eluting stents with cell-proliferation inhibiting agents Importance of understanding the mechanisms in control of cell proliferation Conclusion References Further reading

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Abstract Cell cycle and cell proliferation mastermind tissue repair and renovation after a toxic injury. Multicellular organism’s proper structure and function depends on the minute-to-minute balance between the cell cycle and cell proliferation given the fact that billions of cells die each day in human body. Underneath these complex processes, cell regeneration and repair are the key for life’s sustainability. Humans have approximately 30 trillion cells total, out of which 50 billion die every day and 330 billion are produced each day, although majority of the new cells are blood cells (Fischetti and Christiansen, 2021). To ensure organism’s proper growth and survival, cell cycle and cell proliferation always work in concert and are controlled by various factors (genetic, environmental, physiological) which play critical roles from early on during embryonic development to fully-formed adult organisms and beyond. Therefore, different cells proliferate at different times and at different rates depending on the cell type and the changing needs of the organism. Eukaryotic cells undergo the process of division by progressing through a highly regulated process known as the cell cycle divided into four distinct phases: G1 phase, S phase (synthesis), G2 phase (collectively known as interphase) and M phase (mitosis and cytokinesis). Cell division is tightly regulated by specific cyclins, cyclin-dependent kinases (CDKs) and other components of the core cell cycle machinery. In order to complete the cycle of cell growth and division, cells require highly specific growth factors and cytokines for the activation of the cycle and cell growth, as is the case with tissue injury and repair, as well as arrest of the cell cycle should something go wrong, as is the case with cancer.

Keywords Anti-proliferative agents, Cell cycle; Cell senescence; Cyclins, Cyclin-dependent kinases (CDKs), Mitosis, Cytokines; DNA mutations; Drug-eluting stents; Embryonic development; G1 phase, S phase, G0 phase, G2 phase, M phase, Tight junction proteins, Cell division; Gastrulation; Growth factors; Mutagens cancer; Neurulation; Organogenesis; Proto-oncogenes; Reendothelization; Restenosis; Stem-cells; Thrombosis

Key points

• • • • •

Cell cycle and cell proliferation both are tightly regulated complex processes which work hand in hand to control the development and survival of multicellular organisms. Most drugs, chemicals and biological toxins directly or indirectly influence cell proliferation. There are four distinct phases of the cell cycle: G1 phase, S phase (synthesis), G2 phase (collectively known as interphase) and M phase (mitosis and cytokinesis), which significantly influence cell proliferation. Different cells in an adult organism divide at different pace, different times and at different rates which are influenced by regulatory homeostatic mechanisms coordinating and the endocrine status. Growth factors, hormones and cytokines and several other physiological elements are involved in the regulation of cell proliferation and cell cycle. These are coordinated by cyclins and cyclin-dependent kinases.

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00216-5

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Cell proliferation

Cell proliferation automatically takes place in normal tissues upon chemical or physical injury to replace the damaged cells with healthy cells to restore normal tissue homeostasis. Stem cells are required wherever there is a recurring need to replace non-dividing, terminally differentiated cells to mature into specialized cells that will have the ability to carry out a specific function. Cells with mutation and/or DNA damage when enter into cell cycle can give rise uncontrolled cell proliferation, aka as cancer. However, well-orchestrated cell cycle has appropriate mechanisms to scan for such mistakes (such as damaged DNA) and disallow defective cells to enter into cell division and eventually cell proliferation. Interestingly, drugs used in cardiovascular drug-eluting stents and cancer treatments work by arresting the cell cycle at a specific checkpoint to prevent the cells from further replication.

Introduction Unicellular organisms like yeasts, bacteria, or protozoa have a strong selective pressure to grow and divide as rapidly as possible. The rate of cell division in these cases is limited only by the rate at which nutrients can be taken from the medium and converted into cellular materials. Multicellular organisms, in contrast, are made up of many different types of cells performing a variety of functions. The primary drive is the survival of the organism as a whole rather than the survival or proliferation of a single individual type of cell. Therefore, different cells proliferate at different times, depending on the changing needs of the organism (Fischetti and Christiansen, 2021; Yang et al., 2014). Some tissues contain cells that, once terminally differentiated, are incapable of reentering the cell cycle. These tissues are called non-dividing tissues; an example is neural tissue. Other tissues, such as liver tissue, contain cells that normally reside outside the cell cycle, but which can be stimulated to proliferate when necessary. These tissues are called dividing tissues. A third type of tissue, termed continuously dividing tissue, is continuously being replaced due to frequent cell sloughing or cell death (Krafts, 2010). Skin and bone marrow are examples of this type of cell. Replacement in these tissues is accomplished through native stem cell populations (Blanpain and Fuchs, 2006; Weissman, 2000). For multicellular organisms to survive, some cells must refrain from dividing even when nutrients are plentiful. In tissues capable of cell proliferation (stable and continuously dividing tissues), when the need arises for new cells, as in the case of tissue injury, cells are replaced either by cell division or by replenishment from stem cell reserves. A clear understanding of the dynamics of the cell cycle machinery in stem cell compartments will likely have important implications for regenerative medicine and therapeutics.

Division cycle of cells Adult multicellular animals must produce tons of new cells in order to replace dead cells. Cells undertake the process of division by progressing through a highly regulated process known as the cell cycle (Fig. 1), the end product being a duplication of the contents of the mother cell into two daughter cells. In an adult animal, most cells are stable cells, and reside in the G0 (gap) phase of cell cycle. When division is necessary, cells that are capable of doing so enter the G1 phase of the cell cycle. In most cells the DNA in the nucleus is replicated during only a limited portion of the cell cycle called the S (synthesis) phase (Yang et al., 2014; Rhind and Russell, 2012; Carter et al., 2020). The central machinery that propels cell cycle in the nucleus are a series of proteins called cyclins (11 cyclins are known todate: A, B1, B2, C, D1, D2, D3, E, F, G, and H) that bind, activate, and provide substrate specificity to their associated catalytic partners, aka the CDKs (11 CDKs have been found todate). Upon appropriate signaling, cell cycle progress via four phases: gap 1 (G1), DNA synthesis (S), gap 2 (G2) and mitosis (M). Depending on the mitogenic environment, cells traversing G1 phase either activate a program that will result in cell division, or they enter a quiescent G0 state. When cells encounter growth-promoting factors, it results in upregulation of the D-type cyclins (D1, D2 and D3), which activate the cyclin-dependent kinase 4 (CDK4) and CDK6. However, classical cell cycle pathway models cyclin D-CDK4/6 complex together with E-type cyclins (E1 and E2), but their associated kinases (mostly CDK2, but also CDK1 and CDK3) phosphorylate and functionally inactivate the retinoblastoma protein RB1, and pRB1-related RBL1 and RBL2 proteins. This leads to the activation or de-repression of E2F transcription factors, which then transactivate genes required for the entry and progression of cells into S phase. Although this model has merits, it has been queried by the observation that, RB1 exists in a mono-phosphorylated state throughout most of G1 phase and becomes fully phosphorylated by cyclin E-CDK2 at the end of G1 phase. After the S phase, the cells enter a second gap phase called the G2 phase. Finally, in the M (mitosis) phase, the contents of the nucleus condense to form visible chromosomes, which through an elaborately orchestrated series of movements are pulled apart into two equal sets. The cell itself then splits into two daughter cells. Upon loss of tissues due to injury, or due to normal cell sloughing, the speed of the cell division is accelerated in tissue- or organ-specific fashion so that the lost tissues can be replaced promptly to restore tissue function. New cell or tissue replacement is mainly governed by repair and renovation processes governed by the tissue type and physiological state of the organism. The phases of cell division and the transitions between the phases are orchestrated by an intricate series of signaling mechanisms. When the lost tissue is replaced, the cells return to the normal resting state, thereby reestablishing the cellular, organ, and tissue homeostatic mechanisms (Liu, 2019).

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Interphase

S Growth and DNA Synthesis

G1 checkpoint

Each of the 46 chromosomes is duplicated

G1

G2

Growth Cellular contents excluding the chromosomes are duplicated

Growth and final preparaƟons for division

M

The cell double checks the duplicated chromosomes for errors, making any needed repairs

Mitosis

G2 checkpoint

Fig. 1 In adult organisms normally cells are in resting phase (G0) of the cell division cycle. Upon appropriate stimulus the cells enter the division cycle, which is characterized by G1, S, G2, and M phases. After division, the daughter cells (D) may either reenter the division cycle or enter the resting phase, depending on the stimulus (Yang et al., 2014).

Role of tight junction proteins in cell proliferation Tight junction (TJ) proteins form a continuous intercellular network creating a barrier with selective regulation of water, ion, and solutes across endothelial, epithelial, and glial tissues. Intercellular communication and dependency are extended by Claudin family (provides barrier properties), MARVEL family (participates in barrier regulation), and JAM molecules (regulate tight junctional organization and diapedesis). In addition, the membrane-associated proteins such as MAGUK family members form the scaffold linking the transmembrane proteins to both cells signaling molecules and the cytoskeleton. Several investigations on TJ proteins made efforts to understand cell-cell adhesion and tissue barrier properties. However, investigations disclosed that, similar to adherens junction proteins, TJ proteins contribute to the control of cell proliferation. It is clear that TJ proteins dispense specific roles in the control of epithelial and endothelial cell proliferation. TJ proteins can potentially act as a pool of important cell cycle modulators, by binding and regulating their nuclear access, while in some cases, TJs proteins found inside cellular organelles, regulate transcription and proliferation. In summary, TJ proteins play a role in controlling cell proliferation and differentiation necessary for forming and maintaining a tissue barrier (Díaz-Coránguez et al., 2019).

Genetic control of cell structure and function during and after embryonic development Multicellular animals are clones of cells descended from a single original cell, the fertilized egg. The cells in the body, as a rule, are genetically alike. However, they are phenotypically different; some are muscle cells, some are neurons, and yet others are hepatocytes, and so on. The different cell types are arranged into precisely organized tissues and organs, and the entire structure has a well-defined shape. All of these features are determined by the DNA sequence of the genome, which is reproduced in every cell. Each cell must act according to the same genetic instructions, but it must interpret them with due regard to time and circumstance so as to play a proper part in multicellular organization. The development of vertebrates can be divided into three phases. In the first phase, the fertilized egg divides into many smaller cells that become organized into epithelium. Following a complex series of gastrulation and neurulation movements, a rudimentary gut cavity and neural tube are formed. In the second or organogenesis phase, the various organs, such as limbs, eyes, and heart, are formed. In the third phase, the generated structures grow to their adult size. These phases are not sharply distinct but overlap considerably in time.

688

Cell proliferation

Terminal differentiation and cell division After embryonic development, cells in the normal adult human body divide at very different rates. Some, such as neurons and skeletal muscle cells, do not divide at all; others, such as liver cells, normally divide once every year or two; and certain epithelial cells in the gut divide more than twice a day so as to provide constant renewal of the gut lining. Most cells in the vertebrates fall somewhere between these extremes: they are able to divide, but normally do so infrequently. Almost all the variation in division rate lies in the time cells spend between mitosis and the S phase; slowly dividing cells remain arrested after mitosis for weeks or even years. By contrast, the time taken for a cell to progress from the beginning of S phase through mitosis is brief (typically 12–24 h in mammals) and remark- ably constant, irrespective of the interval from one division to the next. The time cells spend in a non-proliferative state (G0 in the cell cycle) varies according to both the cell type and the circumstances under which division is stimulated. Hepatocytes, for example, exist mostly in a resting state unless liver damage provokes proliferation. In contrast, uterine lining cells enter the cell cycle for a few days each month. A fraction of all hematopoietic precursors are always dividing to compensate for normal cell loss; this fraction increases after an episode of blood loss. Delicately adjusted and highly specific controls govern the proliferation of each class of cells in the body in each situation.

Role of growth factors and cytokines When put into an artificial culture medium completely devoid of serum, vertebrate cells normally do not pass through the G1/S restriction point, even though all the requisite nutrients are present in the medium. Rather, they halt their progress through the cell cycle. In order to complete the cycle of cell growth and division, cells require highly specific growth factors and cytokines, usually present in very small concentrations (10-9–10-11 mol-1) in the serum. Different cells require different combinations of growth factors and cytokines. Some directly stimulate cell division and are called complete mitogens. Others control cell division by directly inhibiting cell cycle progression; these are called growth inhibitors. Yet others cause cell cycle progression in an indirect way and are called growth triggers. Table 1 provides examples of growth factors and cytokines involved in cell division along with their particular functions. Growth factors and cytokines often interact with cell-surface receptors in order to carry out their particular functions. For example, epidermal growth factor (EGF) binds to a receptor tyrosine kinase (RTK) (a cell-surface molecule present on many different kinds of cells). This triggers phosphorylation of tyro- sine residues and dimerization of the receptors. An adaptor protein called Grb2 (growth factor receptor-bound protein 2) is then recruited to promote the formation of Ras-GTP. Active Ras-GTP binds to and activates a protein kinase termed Raf. Raf further phosphorylates and activates MEK (MAP kinase kinase). The downstream target is MAP kinase (MAPK). Active MAPK phosphorylates various proteins, including transcription factors that regulate the expression of cell cycle proteins to induce cell proliferation.

Table 1

Example of growth factors, cytokines and medications known to regulate cell proliferation.

Platelet–derived growth factor (PDGF) Epidermal growth factor (EGF) Insulin like growth factors I and II (IGF-I and II) Fibroblast growth factor (FGF) Interlukin-2 (IL-2) Transforming growth factor b (TGT-b) Interleukin-1 (IL-1) Hepatocyte proliferation inhibitor Nerve growth factor (NGF) Hematopoietic cell growth factors (IL-3, GM-CSF, M-CSF, G-CSF, and erythropoietin) Angiotensin converting enzyme inhibitors (ACEi) Antioxidants Statins Angiotensin receptor blockers (ACBs) Renin inhibitors, beta blockers and estrogens Nintedanib (OfevW) and Pirfenidone (EsbrietW) Pentoxifylline Published in Elsevier EOT3, 2014.

Stimulates proliferation of connective tissue cells and neuroglial cells Stimulates proliferation of many cell types Work with PDGF and EGF to stimulate cell proliferation Stimulates cell proliferation of many cell types including fibroblasts, endothelial cells, and myoblasts Stimulates proliferation of T lymphocytes Inhibits cell cycle progression of different cell types Inhibits proliferation of hepatocytes and other cells types Inhibits hepatocyte proliferation Promotes axon growth and survival of sympathetic and some sensory and CNS neurons Promote division of different blood cells and various other types of cells Used to treat endothelial dysfunction and vascular dysfunction (mainly cardiac and pulmonary) Upregulate the eNOS pathway leading to an increase in plasma NO availability and vasodilation Interfere with fibroblast proliferation, migration and differentiation Inhibits hepatic stellate cell proliferation in treatment of liver fibrosis

Cell proliferation

689

Cell senescence and reluctance to divide Most normal mammalian cells show a striking inability to proliferate indefinitely. Fibroblasts taken from a normal human fetus, for example, undergo approximately 50 population doublings when cultured in a standard growth medium. Toward the end of this time, proliferation slows down and after spending some time in a quiescent state, the cells die. Similar cells taken from a 40-year-old person stop dividing after approximately 40 doublings, while cells from an 80-year-old stop after approximately 30 doublings. Fibroblasts from animals with shorter life spans stop after a smaller number of division cycles in culture. Because of the correspondence with aging of the body as a whole, this phenomenon is called cell senescence. According to one theory, cell senescence is the result of a catastrophic accumulation of self-propagating errors in a cell’s biosynthetic machinery. An alternative theory suggests that cell senescence is the result of a mechanism that has evolved to protect us from cancer by limiting the growth of tumors (Alberts et al., 2007). It has been re- ported that telomeres play an essential role in chromosome capping affecting the cell proliferation. Telomere DNA undergoes progressive shortening of 50 and 200 bp per round of DNA replication. And then, telomere dysfunction leads to disruption of the telomere structure, resulting in end-to-end chromosome fusions and genomic instability. It can activate DNA damage- induced pathways that trigger cell cycle arrest or apoptosis.

Cell proliferation as a compensatory response to toxic tissue injury Humans are exposed to numerous toxic insults directly or indirectly every day. Fortunately, the body has several defense mechanisms to combat toxicants. Some toxicants are prevented from entering the body by virtue of their particle size. Many that enter the body are detoxified by robust antioxidants (such as glutathione, Vit E, Vit C, tocopherol etc.) (Ray et al., 2004; Bulku et al., 2012; Stohs, 2020). Toxicants that do enter the body are metabolized or conjugated in an attempt to safely carry out their detoxification and excretion. When these first lines of defense crumble, toxic substances may cause severe cell injury or even cell death (apoptosis, necrosis, apocrosis, autophagy etc.). At this point, the tissue may respond by stimulating its healthy cells to divide, replace injured cells, and restore normal tissue architecture and function as quickly as it can. The ability of the tissue to undergo repair depends on the type of tissue damaged and the extent of the damage. Damage occurring in tissues that are unable to proliferate (for example, cardiac muscle) will not result in replacement of parenchymal cells (cardiac myocyte), and a scar will replace the dead cells. Likewise, damage that disrupts the supporting structure of the tissue—the basement membrane scaffolding upon which the cells reside—will likewise involve scarring rather than complete resolution to the normal, pre-damaged state. The process of tissue repair stops at a precise, preordained point. For example, liver regeneration ends when the functional mass of the liver is restored. Epithelial cell proliferation subsequent to renal cell injury was described by Lee et al. (2021). At low to moderate doses of a particular toxicant, the process functions well, and repair is usually adequate. At high doses of a toxicant, however, the ability of the cells to progress through the cell cycle is inhibited, leading to two consequences. First, dead cells are not replaced, which may lead to organ failure and death. Second, in the absence of compensatory cell division, which normally serves to contain the toxic injury, tissue injury can progress in an unrestrained manner. The ability of cells to enter and progress through the cell cycle following toxic injury decreases with age, a finding which explains, in part, why an 80-year-old person may be more susceptible to the same dose of a toxicant as a 40-year-old. Table 2 shows a list of drugs and chemicals that affect cell proliferation in a variety of model systems.

Stem cells and terminally differentiated cells Many of the tissues in the body undergoing constant renewal, such as skin and the lining of the intestine, accomplish this task by means of a small population of tissue stem cells. The defining properties of a stem cell are (1) the ability to divide virtually without limit throughout the lifetime of the organism and (2) the ability to divide symmetrically (leading to two terminally differentiated cells) or asymmetrically (giving rise to one stem cell and one terminally differentiated cell) (Shahriyari and Komarova, 2013; Morrison and Kimble, 2006). Stem cells are required wherever there is a recurring need to replace non-dividing, terminally differentiated cells. Some terminally differentiated cells, such as mature mammalian red blood cells and the cells in the outermost layer of the skin, lack a cell nucleus and are therefore unable to divide. Others contain cytoplasmic structures (such as the myofibrils of striated muscle cells) that hinder cell duplication. And in some terminally differentiated cells, the chemistry of differentiation may simply be incompatible with cell division (Yang et al., 2014). The job of the stem cell is not to carry out the function of the differentiated cell, but rather to produce the cells that will carry out those functions. Stem cells that give rise to only one type of differentiated cell are called unipotent; these are capable of differentiating along only one lineage. Also, adult stem cells in many differentiated undamaged tissues are typically unipotent and give rise to just one cell type under normal conditions. Additionally, a small number of cell types are called oligopotent. These stem cells have the ability to differentiate into just a few types of cells. A lymphoid stem cell is an example of an oligopotent stem cell. These stem cells cannot develop into any type of blood cell as bone marrow stem cells can. They give rise to only blood cells of the lymphatic system, such as T cells. Cells that give rise to many cell types are called pluripotent or totipotent. Yet, there is another type of stem cell that is called multipotent. These cells are progenitor cells that have the genetic potential to differentiate into multiple, but limited cell types (Yang et al., 2014; Chen et al., 2021).

690

Cell proliferation

Table 2

Summary of some natural and manmade toxicants that are reported to influence cell proliferation.

Compound Natural food toxicants Ochratoxin A Citrinin Okadaic acid Glucosinolates Psoralen Pyrrolizidine alkaloid Environmental toxicants Nonmetals (As, S) Metals (Cd2þ, Hg2þ, Pb2þ) 1,3-Butadiene Polycyclic aromatic hydrocarbons (PAHs) Dichlorodiphenyl-trichloroethane (DDT) Polychlorinated biphenyls (PCBs) Lithium Dimethylnitrosamine Doxorubicin caffeine TCDD

Origin

Specific mechanism

Source reference

Fungi Fungi Algae Plants Plants Plants

Induces cell proliferation by activating Cyclin-D and Cox-2 Inhibits cell proliferation by increasing p53 and p21 Inhibits cell proliferation by inducing c-Myc Inhibits cell proliferation by G2/M arrest Inhibits cell proliferation by BMP signaling Inhibits cell proliferation by activating p38

Kumar et al. (2013) Chang et al. (2011) Zhang et al. (2007) Liang et al. (2008) Tang et al. (2011) Ji et al. (2002)

Soil, air Water

Wu et al. (2006) Costa et al. (1982)

Rubber, plastics Tobacco smoke

Inhibit cell proliferation by DNA damage Inhibit cell proliferation by their interaction with DNA metabolism Increases cell proliferation by DNA modification Increases cell proliferation by increasing ER.

Pesticides

Increases cell proliferation by chromosomal alterations

Uppala et al. (2005)

Pesticides

Induces cell proliferation of collecting ducts in kidneys

Nephrogenic diabetes insipidus Food and environment Anticancer drug Environmental toxin

Induces cell proliferation in the liver

Chaudhuri et al. (2010) Gao et al. (2013)

Inhibits cell proliferation in the liver Increases in hematopoietic cell proliferation

Melnick et al. (2001) Plísková et al. (2005)

Syed et al. (2012) Motegi et al. (2013) NTP (2010)

This table was published in EOT3, 2014.

Cell proliferation and cancer Cells within a tissue exert an inhibitory effect on each other’s growth. This restraining force is called social control of cell division, and it is mediated by a set of genes called social control genes. The process is called ‘contact inhibition’ in cell culture systems. This property is lost in cancer cells. A cell that acquires a DNA mutation that disrupts this social restraint will divide without regard to the needs of the organism as a whole, and its progeny may become tumor cells. Approximately 1016 cell divisions take place in a human body in the course of a lifetime. Even in an environment that is free of mutagens, mutations occur spontaneously at an estimated rate of about 10−6 mutations per gene per cell division, a value set by fundamental limitations on the accuracy of DNA replication and repair. Thus, in an average person’s lifetime, every single gene is likely to have undergone mutations on about 1010 separate occasions. Some mutated genes—if not repaired by the cell’s DNA repair mechanisms but somehow get into cell division, may adversely affect the host. Consequently, the affected cell, now lacking the normal ‘brakes’ on cell growth, may relentlessly continue to progress through the cell cycle eventually ending in uncontrolled cell proliferation. To transform into a malignant cell, however, a cell must acquire not just one but a number of DNA mutations to escape the multiple controls and scanning processes (p53 checkpoint control is one example) during cell cycle. Further mutations endow the cell with the capacity for invasion and metastasis. Statistically, it is estimated that somewhere between three and seven independent random events, each of low probability, are typically required to turn a normal cell into a cancer cell; the smaller numbers apply to leukemia and the larger numbers to carcinomas (Sun et al., 2021; Matthews, 2021). Proto-oncogenes are normal genes encoding proteins involved in cell proliferation. Like any gene, a proto-oncogene may undergo mutation. When a mutation in a proto-oncogene confers a gain of function, the new mutant gene (now called an oncogene) will dramatically stimulate cell division. Tumor suppressor genes, in contrast, are normal genes that encode proteins that inhibit cell proliferation. If a tumor suppressor gene is mutated in such a way as to inactivate the gene, this removal of inhibition may make the cell grow continuously. Mutations may be spontaneous, or they may result from exposure to chemical carcinogens or radiation. Mutations in tumor suppressor genes (p53) generally need to occur on both alleles for a tumor to arise, whereas most proto-oncogenes need only one mutated allele (one oncogene) to contribute to malignancy (Yang et al., 2014; Sun et al., 2021; Engeland, 2022).

Cell proliferation, cardiovascular disease and drug-eluting stents with cell-proliferation inhibiting agents Drug-eluting stent (DES) deployment in patients suffering from cardiovascular disease leads to local arterial or peripheral tissue injury. As a result, the tissue responds by stimulating its healthy cells to divide and restore tissue structure and function. The injury

Cell proliferation

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induces smooth muscle cell (SMC) and endothelial cell (EC) proliferation conducive to cell regeneration and vascular repair. In order to prevent in-stent restenosis, DES devices contain cell proliferation inhibiting agents that prevent SMC proliferation and neointimal hyperplasia (Htay and Liu, 2005). However, drugs eluted from DES also inhibit EC proliferation and migration, blocking re-endothelization at the site of injury (Joner et al., 2006; Liuzzo et al., 2005; Lüscher et al., 2007). Healthy vascular endothelial cells normally provide anti-coagulant properties, fluid and nutrient trafficking, the regulation and repair of vascular homeostasis, and the control of vascular tone (Gori, 2018). However, endothelium that has regenerated after stent implantation is lacking in its integrity and function, with poorly formed cell junctions, reduced expression of antithrombotic molecules, and decreased nitric oxide production (Otsuka et al., 2012). In addition to inhibiting SMC proliferation and migration, anti-proliferative agents used in DES also inhibit EC proliferation and migration, delaying proper endothelium regeneration (Liuzzo et al., 2005; Lüscher et al., 2007) (Table 3). Delayed wound healing characterized by poor re-endothelization has been associated with stent thrombosis (Finn et al., 2007; Joner et al., 2006; Räber et al., 2011). Stent thrombosis is a serious and life-threatening adverse event in patients treated with DES suffering from cardiovascular disease. High and extreme drug doses released from DES and overwhelming tissue receptors can cause tissue toxicity, augmented fibrin deposition, intra-intimal hemorrhages, mural thrombus, medial necrosis, and excessive arterial expansion. All of the mentioned have been associated with stent thrombosis and exacerbated neointimal tissue (Lysitsas et al., 2007; Nakazawa et al., 2008) As a cell proliferation inducer, basic fibroblast growth factor (bFGF) has been studied in vitro as a potential sent coating to enhance rapid re-endothelization without excessive SMC proliferation (Kitamura et al., 2014). Some of the more common cell proliferation inhibiting agents used in DES include paclitaxel and the -limus family of drugs. Paclitaxel was used in 1st generation DES. It is no longer used as a cell proliferation inhibiting agent in DES due to its cytotoxicity at higher concentrations, narrow therapeutic window, high lipophilicity, and low transmural diffusivity resulting in long tissue-retention times, (Puranik et al., 2013; Ishida and Tanaka, 1982; Naito et al., 2000; Levin et al., 2004), conducive to delayed would healing, inhibited re-endothelization, and stent thrombosis (Finn et al., 2005; Liuzzo et al., 2005). The lipophilic nature and low transmural diffusivity of paclitaxel make it a better candidate for drug-coated balloon devices (DCB). Paclitaxel stabilizes the microtubule making them dysfunctional and inhibiting SMC proliferation and migration by causing cell cycle arrest in the G2/M phase (Puranik et al., 2013; Auchampach et al., 2022). Sirolimus, everolimus, and zotarolimus are used as cell proliferation inhibitors and immunosuppressants in 2nd generation DES. The -limus drugs are an improvement over paclitaxel in being cytostatic, having better transmural diffusivity, and a wider therapeutic window (Levin et al., 2004). Sirolimus and its derivatives cross cell membranes, bind to the FKBP12 binding protein, which subsequently binds to mammalian TOR receptor (m-TOR), blocking cell cycle mainly of the smooth cell between G1 and S phases to inhibit SMC proliferation. Paclitaxel and the -limus drugs are potent cell-proliferation inhibitors used in cancer treatment, stents for cardiovascular disease and organ-transplantation therapy. Sirolimus was not genotoxic in the in vitro bacterial reverse mutation assay, the Chinese hamster ovary cell chromosomal aberration assay, and the mouse lymphoma cell forward mutation assay (RAPAMUNE®FDA LABEL, 2011). Everolimus was not genotoxic in the in vitro assays, such as Ames mutation test in Salmonella, Mutation test in L5178Y mouse lymphoma cells, and chromosome aberration assay in V79 Chinese hamster cells. (AFINITOR/AFINITOR DISPERZ® FDA LABEL, 2007).

Importance of understanding the mechanisms in control of cell proliferation An understanding of the mechanisms in control of cell proliferation is critical in the development of new tissue restoration therapies. Current clinical treatments for patients with drug overdoses or chemical poisoning are aimed primarily at preventing additional injury, either by blocking further formation of toxic metabolites or by increasing clearance of the toxin from the body. While these strategies are useful, the survival of the tissue—and sometimes the patient—is heavily dependent on tissue repair, the success of which is in turn contingent on the ability of cells to proliferate. In cases of toxic exposure in which tissue repair is delayed, either due to the massivity of the exposure or to a delay in treatment, organ loss and even death may occur because the damage compromises the regenerating ability of the cells, thereby paving the way for unrestrained progression of injury. If cellular regeneration after massive tissue damage could be actively stimulated, it might be possible to prevent organ loss and death. Animal experiments provide concrete examples of how modification of tissue repair can directly influence survival. Animals given ordinarily lethal doses of toxins are able to survive—even when there is massive liver injury—when tissue repair in the liver is stimulated. Conversely, animals receiving otherwise nonlethal doses of toxins develop liver failure and die if cell division (and therefore tissue repair) is blocked by antimitotic agents. Perhaps carefully induced suppression of pathways involved in cell death and stimulation of pathways involved in cell division could stop the progression of toxic injury and restore organ structure and function. With the advent of gene therapy, specific genes could, 1 day, be delivered directly to the damaged organ to induce expression/suppression of the appropriate factors needed for recovery. Although this may require time, with technological advances this route of therapy appears to be doable (Table 3).

Table 3

Systemic and local tissue toxicity of cell proliferation-inhibiting agents.

USE Paclitaxel CAS ID 33069-62-4 C47H51NO14 Anticancer agent: Breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer, HIV-related Kaposi sarcoma Systemic toxicity TAXOLW ABRAXANEW

MOA

Toxicity in animals

Toxicity in humans

References

Reproductive toxicity-testicular atrophy & degeneration in mice; Fetal Toxicity and Reproductive Toxicity/ Impaired Fertility in rats; Fetal Toxicity in rabbits; Inhibits endothelial cell proliferation, contributing to delayed wound healing and inhibited re-endothelization of coronary and peripheral tissue upon stent implantation, which have been associated with stent thrombosis High and extremely high doses overwhelm tissue receptors; can cause augmented fibrin deposition, intra-intimal hemorrhages, mural thrombus, medial necrosis and excessive arterial expansion, all associated with stent thrombosis and exacerbated neointimal tissue

Myelosuppression (neutropenia); UT infections; Sepsis, Pneumonia; Hypersensitivity reactions; Cardiovascular: Hyper- & hypotension, bradycardia; Peripheral neuropathy; Musculoskeletal and connective tissue toxicity (myalgia & arthralgia); GI and alopecia; Pulmonary fibrosis

Herdeg et al. (2000)

Cytotoxic Agent

Neurotoxicity in mice

NCI Thesaurus

Vincristine arrests tumor cell in metaphase by binding irreversibly to microtubules and spindle proteins in S phase of the cell cycle and interfering with the formation of the mitotic spindle

Reproductive organ toxicity- testicular degeneration and atrophy, and epididymal aspermia; Neurotoxicity; Carcinogenesis Genotoxicity; Impaired fertility in rats

Neurotoxicity SIADH Cutaneous toxicity (alopecia) GI enterotoxicity Hepatic toxicity Hematologic toxicity: myelosuppression may occur

Cytotoxic agent

Neurotoxicity Reproductive toxicity in mice

Hematologic: myelosuppression, primarily neutropenia

NCI Thesaurus

Thymus, testes, bone marrow toxicity Mutagenesis Cardiovascular (myocardial damage) as in necroptosis hepatic toxicity in rats

GI enterotoxicity

Cytotoxic agent Inhibits cell proliferation in the late G2 phase (G2/M) by stabilizing current microtubules (promotes the assembly of microtubules and inhibits their disassembly) Inhibits smooth muscle cell proliferation and neointimal hyperplasia as an anti-proliferative agent in drug-eluting stents (DES) and drug-coated balloons (DCB)

Cardiovascular Devices: 1. Drug Eluting Stents Used as an anti-proliferative agent in 1st generation DES 2. Drug Coated Balloon Used as an anti-proliferative agent [Local tissue toxicity] Vincristine CAS ID 57-22-7 C46H56N4O10 Anticancer agent: Hodgkin’s disease; lymphocytic leukemia, chronic myelogenous leukemia; Wilms tumor; sarcoma; multiple myeloma; breast cancer, cervical cancer, lung cancer and ovarian cancer ONCOVINW MARQIBOW (VinCRIStine Sulfate) CASS ID 2068-78-2 C46H56N4O10•H2SO4 Vinblastine CAS ID 865-21-4 C46H58N4O9 Anticancer agent: Hodgkin’s disease, lymphoma, cancer of breast, bladder and testis; Kaposi sarcoma VELBANW

Vinblastine arrest tumor cells in the M phase of the cell cycle and disrupts the mitotic spindle assembly by binding to tubulin and inhibiting microtubule formation

Abal et al. (2003) Li et al. (2010) Lysitsas et al. (2007) Nakazawa et al. (2008) Granada et al. (2014)

Neurotoxicity SIADH (13)

Liu et al. (2018) MarqiboW [FDA LABEL]

BC Cancer Drug Manual© (2015) Zimmermann et al. (1991) Zhou et al. (2021)

Methotrexate CAS ID 59-05-2 C20H22N8O5 Anticancer agent: Cancers that begin in the tissues that form around a fertilized egg in the uterus, breast cancer, lung cancer, certain cancers of the head and neck, certain types of lymphoma, and leukemia XATMEPW

Cytotoxic agent Methotrexate (MTX) inhibits the enzyme dihydrofolate reductase, resulting in inhibition of purine nucleotide and thymidylate synthesis which leads to inhibition of DNA and RNA syntheses. MTX reversibly inhibits the cell cycle in the late G1 phase and may cause cytotoxicity of cells in the S phase

Chronic toxicity: Hepatic toxicity-subacute (28 days) Reproductive Toxicity Nephrotoxic-subacute (28 days) Respiratory toxicity Cardiovascular toxicity Embryo-Fetal toxicity Reproductive organ toxicity

Immunosuppressive action: Severe psoriasis and severe active rheumatoid arthritis (RA) OTREXUPW TREXALLW RASUVOW

Fluorouracil CAS ID 51-21-8 C4H3FN2O2

Acute toxicity: GI toxicity in rats-oxidative toxicity and intestinal inflammation

Anticancer drug: Colorectal cancer, metastatic breast cancer XELODAW

NCI Thesaurus

Neurologic-acute (arachnoiditis and headache) and chronic (dementia, motor deficits, seizures, coma) Cardiovascular toxicity; Cutaneous toxicity

Abdel-Daim et al. (2017) Hyoun et al. (2012)

Chronic toxicity: Myelosuppression and GI toxicity (prolonged exposure at high doses) Embryo-fetal toxicity Reproductive organ toxicity

Felemban et al. (2020)

Famurewaa et al. (2018)

Patel et al. (2014) RasuvoW [FDA LABEL] https://www. accessdata.fda.gov/ drugsatfda_docs/ label/2014/ 205776s000lbl.pdf

Cytotoxic Agent Arrests cell cycle in the G1 phase and induces apoptosis

Embryo-Fetal toxicity Reproductive organ toxicity Cardiotoxicity

Anticancer agent: GI adenocarcinoma, carcinoma of lung, breast, ovary, prostate, ovary, bladder, head and neck cancers ADRUCILW

Capecitabine CAS ID 154361-50-9 C15H22FN3O6

Pulmonary toxicity—acute or chronic interstitial pneumonitis Renal toxicity (acute renal failure) Hepatotoxicity: fibrosis and cirrhosis

The active moiety of capecitabine, fluorouracil, arrests cell cycle in the G1/S phase and induces apoptosis

Embryo-Fetal toxicity: embryolethality and teratogenicity in mice and embryolethality in monkeys when drug administered during organogenesis

Acute toxicity: Coma with hyperammonemia, lactic acid elevations and respiratory alkalosis (hepatoxicity) Chronic toxicity: Myelosuppression Cutaneous toxicity GI enterotoxicity Cardiotoxicity Hepatoxicity Neurotoxicity (acute and chronic) Acute toxicity: Acute renal failure due to dehydration Chronic toxicity: GI enterotoxicity Myelosuppression Cutaneous toxicity Neurotoxicity Hepatotoxicity Coagulopathy Hyperbilirubinemia

Kwok et al. (2017) Filgueiras et al. (2013) FLUOROURACIL injection, for intravenous use [FDA LABEL] NIDDK (2012a)

Filgueiras et al. (2013) BC Cancer Drug Manual© (2001) Kwok et al. (2017)

(Continued )

Table 3

(Continued)

USE

MOA

Toxicity in animals

Toxicity in humans

References

Mercaptopurine CAS ID 50-44-2 C5H4N4S

Mercaptopurine inhibit DNA synthesis. Incorporation of Mercaptopurine into DNA as deoxythioguanosine, that causes disruption of DNA replication

Mutagenic Carcinogenic - chromosomal abbreviation in animals - dominant-lethal mutations in mice ! reproductive organ toxicity and embryofetal toxicity in mice

NCI Thesaurus

Hepatoxicity (chronic)

Mutagenic - chromosomal abbreviation - carcinogenic potential Myelosuppression ! immunosuppression Myelosuppression is linked to higher levels of methyl-mercaptopurine (product of mercaptopurine metabolism)

Myelosuppression-linked to higher levels of methyl-mercaptopurine (product of mercaptopurine metabolism)

Renal toxicity Hepatoxicity (chronic, rarely acute) Embryo-Fetal toxicity

Chronic: Embryo-Fetal Toxicity (mutagenic) Hepatoxicity Myelosuppression- likely

Myelosuppression-chronic - manifests as anemia, leukopenia, thrombocytopenia, or any combination of these ! immunosuppression - myelosuppression

NCI Thesaurus

Hepatoxicity-chronic

Oancea et al. (2012)

Embryo-Fetal Toxicity Potentially carcinogenic

Henry et al. (1973)

Anticancer agent Acute lymphocytic leukemia, acute promyelocytic leukemia, chronic myeloid leukemia Immunosuppressant Crohn’s disease Ulcerative Colitis PurinetholW PurixanW Thioguanine CAS ID 154-42-7 C5H5N5S

Cytotoxic Agent Causes cell death in the S phase specific

Anticancer agent: Acute nonlymphocytic leukemia, chronic myelogenous leukemia TABLOIDW

Cytarabine CAS ID 147-94-4 C9H13N3O5 Anticancer agent Acute lymphomatic leukemia, Acute myeloid leukemia, chronic myelogenous leukemia, meningeal leukemia lymphoma (non-Hodgkin’s)

Cytotoxic Agent When converted to active cytarabine triphosphate, this agent kills cells undergoing DNA synthesis at S-phase.

Embryo-Fetal Toxicity (Both acute and chronic)

It blocks the progression of cells from the G1 phase to the S-phase under certain conditions. Cytarabine also has antiviral and immunosuppressant properties

Myelosuppression (acute and chronic) - anemia, leukopenia (acute), Thrombocytopenia (chronic), megaloblastosis and reduced reticulocytes ! immunosuppression (chronic) Hepatoxicity (chronic) Cutaneous toxicity (acute-skin reactions observed from 2 to 8 days) Neurologic toxicity (CNS) ! (5–50% increased incidence with high-dose)

Cytosar-U DepoCytW

PURINETHOLW (mercaptopurine) 50-mg Scored Tablets [FDA LABEL] NIDDK (2012b)

TABLOIDW brand Thioguanine 40-mg Scored Tablets [FDA LABEL]

Kwok et al. (2017) Cytarabine Injection For Intravenous, Intrathecal and Subcutaneous Use Only [FDA LABEL] DepoCytW (cytarabine liposome injection) for Intrathecal Use Only 50 mg vial [FDA LABEL] Kwok et al. (2017)

Embryo-Fetal toxicity

Hydroxyurea

Antimetabolite Cytotoxic agent

Embryo-Fetal toxicity (embryotoxic and teratogenic toxicities)-chronic

CNS, GI and pulmonary toxicity can be severe and at times fatal at high experimental injection doses CNS toxicity induced by high experimental doses Myelosuppression, most commonly and first to occur is leukopenia; thrombocytopenia and

NCI Thesaurus

CAS ID 127-07-1 CH4N2O2 Anticancer agent: Resistant chronic myeloid leukemia; Locally advanced squamous cell carcinomas of the head and neck (excluding the lip) in combination with chemoradiation. Sickle cell Anemia

Hydroxyurea selectively inhibits ribonucleoside diphosphate reductase, thereby preventing cells from leaving the G1/S phase of the cell cycle. This agent also maintains cells in the radiation-sensitive G1 phase and interferes with DNA repair.

Reproductive organ toxicity-males Carcinogenic-chronic; higher doses increased the incidence of mammary tumors in rats surviving to 18 months compared to control

It is a cell-cycle specific for the S phase, causing cell arrest at G1 to S

HYDREAW DROXIAW SIKLOSW

Topotecan

Cytotoxic Agent

CAS ID 123948-87-8 C23H23N3O5

Topotecan arrests the cell cycle in the S phase

Embryo-Fetal Toxicity: embryolethality, fetotoxicity, and teratogenicity in rats and rabbits when administered during organogenesis-chronic

Anticancer agent: Metastatic carcinoma of ovary as a single agent Small cell lung cancer in patients with platinum-sensitive disease Indicated for the treatment of patients with Stage IV-B, recurrent, or persistent cervical cancer not amenable to curative treatment, in combination with cisplatin HYCAMTINW Teniposide CAS ID 29767-20-2 C32H32O13S Acute lymphomatic leukemia, lymphoma, carcinoma of lung and breast

anemia occur less often and are rarely seen without a preceding leukopenia-chronic Carcinogenic (secondary leukemia and skin cancer)-chronic

HYDREA/DROXIA (hydroxyurea) capsules, for oral use Initial. [FDA LABEL]

Cutaneous toxicity acute and chronic (acute with higher doses) Cutaneous vasculitis Mucocutaneous toxicity-chronic Acute mucocutaneous toxicity caused by overdose Macrocytosis Neurotoxicity-rare side effect Pancreatitis, hepatotoxicity, and peripheral neuropathy Hematologic toxicity ! Myelosuppression: neutropenia, anemia, thrombocytopenia, and febrile neutropenia. Chronic Acute—high-risk patients are more likely to incur higher-grade acute hematologic toxicities during the standard regimen GI toxicity (acute and chronic) Late neurotoxicity Extravasation and tissue injury-adverse reaction

NCI Thesaurus HYCAMTINW (topotecan) for injection, for intravenous use Initial US. [FDA LABEL]

Interstitial Lung Disease as an adverse reaction

Cytotoxic Agent A phase-specific drug that acts in the late S or early G2 phase of the cell cycle, and subsequently preventing cells from entering mitosis.

Embryo-Fetal Toxicity: teratogenic and embryotoxic in laboratory animals Reproductive organ toxicity-male Chromosome aberrations in vivo in the embryonic tissue of pregnant Swiss albino mice

Elevated hepatic enzymes have been reported with intravenous topotecan overdose; skin toxicity with overdoses Hematologic toxicity (chronic) ! myelosuppression Neutropenia (primarily); Leukopenia; Thrombocytopenia Anemia Alopecia likely

VUMONW (teniposide injection) [FDA LABEL] Hartmann and Lipp (2006)

(Continued )

Table 3

(Continued)

USE

MOA

VUMONW

Toxicity in animals

Toxicity in humans

Dose-related increase in sister chromatid exchanges in Chinese hamster ovary cells

Neurotoxicity (including severe cases of neuropathy) when given with vincristine sulfate

References

GI toxicity (chronic) Potential carcinogen-chronic

Bleomycin (Sulfate) Bleomycin A2 Bleomycin

Cytotoxic Agent Causes cell cycle arrest in G2 phase and in mitosis

Carcinogenic in animals Mutagenic both in vitro and in vivo Teratogenic in rats Abortifacient in rabbits

CAS ID 11056-06-7 C55H84N17O21S3 +

OVERDOSE: Acute CNS depression, hypotension, and metabolic acidosis in patients who were receiving higher than recommended doses Pulmonary toxicity (most frequently presented as pneumonitis occasionally progressing to pulmonary fibrosis)—acute and chronic Pulmonary toxicity- Acute stage diffuse interstitial fibrosis that resembles the Hamman-Rich syndrome.

Anticancer drug: Testicular cancer, Hodgkin’s disease Lymphoma, Sarcoma Squamous cell carcinoma of head and neck, GI tumors

GI enterotoxicity (acute and chronic emetogenic potential)-anorexia and vomiting most common

Sclerosing agent: Malignant pleural effusion and prevention of recurrent pleural effusions

Renal toxicity or hepatic toxicity have been reported

BLENOXANEW (Bleomycine sulfate for injection) Everolimus CAS ID 159351-69-6 C53H83NO14 Anticancer agent: Kidney, breast, pancreas, lung, stomach/ intestinal cancers Certain genetic disorder (tuberous sclerosis complex) to treat certain types of benign tumors in the brain or kidney AfinitorW Afinitor DisperzW ZortressW Systemic toxicity Cardiovascular Devices: 1. Drug Eluting Stents - Everolimus as an antiproliferative agent in 2nd generation DES

BLENOXANEW (bleomycin sulfate for injection, USP) [FDA LABEL] BC Cancer Drug Manual (1994) Thomson (2000)

Cutaneous toxicity-chronic

Cytostatic agent Arrests cell cycle in G1 phase Inhibits SMC proliferation, but not migration which may account for better clinical outcomes as compared to other anti-proliferative agents used in DES Endothelial cell activation is attenuated by everolimus via transcriptional and post-transcriptional regulatory mechanisms following stent implantation ! associated with lower rates of in-stent restenosis as compared to bare metal stents Associated with tissue factor increase related to stent thrombosis

Embryo-fetal toxicity Reproductive organ toxicity

Hematologic toxicity anemia, thrombocytopenia, neutropenia, leukopenia, lymphopenia-chronic

AFINITOR/AFINITOR DISPERZW FDA LABEL

Renal toxicity (acute and chronic) Acute renal failure

Xu and Tian (2014)

Delayed wound healing Cardiotoxicity

Karvelasa et al. (2018) Fejas et al. (2018) Camici et al. (2010)

[Decrease local tissue toxicity following DES implantation as compared to paclitaxel and sirolimus] Sirolimus Rapamycin CAS ID 53123-88-9 C51H79NO13 Immunosuppressant antibiotic: Renal transplantation Lymphangioleiomyomatosis

Generally cytostatic agent

Embryo-fetal toxicity in rats, but not in rabbits Reproductive organ toxicity-chronic

Blocks cell cycle between G1 and S phases to inhibit cell proliferation

Carcinogenic-chronic

RAPAMUNEW Systemic toxicity Cardiovascular Devices: 1. Drug Eluting Stents - Sirolimus as an antiproliferative agent in 1st generation DES 2. Drug Coated Balloon - Sirolimus as an anti-proliferative agent

Inhibits smooth muscle cell proliferation and neointimal hyperplasia as an anti-proliferative agent in drug-eluting stents (DES) and drug-coated balloons (DCB) Antimigratory and antiproliferative effect on SMC ! arrests cell cycle without causing cell death

Edema, vasculitis, myocarditis, scar, diffuse and focal inflammation and focal degeneration ! sirolimus inhibits endothelial cell function contributing to delayed wound healing and stent thrombosis

Hematologic toxicity ! Myelosuppression (anemia, leukopenia, thrombocytopenia) Increased susceptibility to infections and possible development of lymphoma and other malignancies

Li et al. (2014) Puranik et al. (2013) Clever et al. (2016)

Chronic: Interference with normal wound healing due to their anti-proliferative effects

RAPAMUNEW FDA LABEL.

Renal toxicity when co administration with cyclosporine-chronic

Webster et al. (2006)

Hepatoxicity-chronic exposure at 3rd week

NIDDK (2012c) Liu et al. (2010)

Local tissue toxicity Other DES Drugs -Limus Family (Immunosuppresants) Zotarolimus CAS ID 221877-54-9 C52H79N5O12 Associated with tissue factor increase related to stent thrombosis Biolimus A9 CAS ID 851536-75-9 C55H87NO14 Novolimus CAS ID 151519-50-5 C50H77NO13 Ridaforolimus CAS ID 572924-54-0 C53H84NO14P

USE/MAO

Local tissue toxicity in animal models

Local tissue toxicity in humans

References

Cardiovascular Devices:

Local tissue toxicity associated with stent thrombosis

Histological studies studying local tissue toxicity have been most frequently conducted in rabbits and rats

Puranik et al. (2013)

Drug Eluting Stents: antiproliferative agents in 2nd generation DES Cytostatic agents Blocks cell cycle between G1 and S phases to inhibit cell proliferation Inhibit smooth muscle cell proliferation and prevent neointimal hyperplasia after DES implantation

Lee et al. (2018) Camici et al. (2010) Lysitsas et al. (2007) Nakazawa et al. (2008)

698

Cell proliferation

Conclusion Cell proliferation and factors regulating checkpoints in the cell cycle determine a multicellular organism’s development and survival. Cell growth and the cell cycle are necessary for embryonic development, tissue repair and regulation of cell division in case of tumorigenesis. Different types of cells proliferate at different times and different rates during an organism’s lifetime depending on the cell type and the specific stimuli to chemical or physical tissue injury. In case of gene mutations-if not repaired by the cell’s DNA repair mechanisms, can lead to uncontrolled cell proliferation. Although every cell with damaged DNA may not succeed to transform into a malignant cell, chances of cell proliferation increase with each such mutation. As indicated in the text, cells require a number of DNA mutations to escape the multiple controls and scanning processes during cell division to form cancerous cell. Mutations occur spontaneously per gene per cell division even in an environment that is free of mutagens. Drugs used in cardiovascular stents and cancer treatments work by arresting the cell cycle at a specific checkpoint to prevent the cells from further replication. A vast amount of literature has accumulated how many of the antiproliferative agents are used in stent therapy in multiple target organs (Lee et al., 2019; Huang et al., 2020; Torii et al., 2020).

See also: Cell cycle

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(2020) Highly bioavailable forms of curcumin and promising avenues for curcumin-based research and application: A review. Molecules 25(6): 1397. https://pubmed. ncbi.nlm.nih.gov/32204372/. Sun Y, et al. (2021) The influence of cell cycle regulation on chemotherapy. International Journal of Molecular Sciences 22(13): 6923. https://doi.org/10.3390/ijms22136923. Syed I, et al. (2012) Molecular and Cellular Biochemistry 365(1–2): 351–361. Tang DZ, et al. (2011) Biochemical and Biophysical Research Communications 405: 256. Thomson PDR (2000) Bleomycin. USP DI. Volume 1. Drug Information for the Health Care Professional, 20th edn. Torii S, et al. (2020) Drug-eluting coronary stents: Insights from preclinical and pathology studies. Nature Reviews. Cardiology 17(1): 37–51. https://pubmed.ncbi.nlm.nih.gov/ 31346257/. Uppala PT, et al. (2005) Environmental and Molecular Mutagenesis 46: 43. Webster AC, et al. (2006) Transplantation 81(9): 1234–1248. Weissman IL (2000) Stem cells: Units of development, units of regeneration, and units in evolution. Cell 100(1): 157–168. https://doi.org/10.1016/s0092-8674(00)81692-x. Wu JZ, et al. (2006) European Journal of Pharmaceutical Sciences 29: 35. Xu J and Tian D (2014) Current Medical Research and Opinion 30(1): 67–74. https://doi.org/10.1185/03007995.2013.844116. Yang N, Ray SD, and Krafts K (2014) Cell Proliferation. Encyclopedia of Toxicology, 3rd edn. Academic Press 761–765. https://doi.org/10.1016/B978-0-12-386,454-3.00274-8. Zhang L, et al. (2007) Cancer Research 67: 10198. Zhou, et al. (2021) Molecular and Cellular Biochemistry 476(2): 1233–1243. Zimmermann JL, Todd G, and Tamura RN (1991) Fundamental and Applied Toxicology 17(3): 482–493.

Further reading Wollin L, Wex E, Pautsch A, et al. (2015) Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis. The European Respiratory Journal 45(5): 1434–1445.

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Centipedes Timothy J Wiegand, University of Rochester Medical Center and Strong Memorial Hospital, Rochester, NY, USA © 2024 Elsevier Inc. All rights reserved. This is an update of H.H. Lin, T.J. Wiegand, Centipedes, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 766–767, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00707-7.

Background Exposure routes and pathways Mechanism of toxicity Acute and short-term toxicity (or exposure) Animal Human Chronic toxicity or exposure Animal Human Clinical management Miscellaneous Millipedes References Further reading

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Abstract Centipedes are nocturnal multi-segmented elongated arthropods known for the distinct feature of having a pair of legs for each body segment except for the last. The first pair of legs on the cranial segment is in actuality modified into a pincer-like apparatus, known as forcipules, used to inject venom into prey. The last segment contains a pair of filamentous caudal appendages that despite popular belief play no role in envenomation. There are over 3000 species, varying in size from 1 to 30 cm with body segments numbering from 15 to more than 100. Centipedes are divided into four orders; Geophiulomorpha (soil centipedes), Lithobiomorpha (rock or garden centipedes), Scolopendromorpha (tropical or giant centipedes), and Scutigeromorpha (house or feather centipedes) of which only two, Scolopendromorpha and Scutigeromorpha, are of medical significance. Because the majority of centipede envenomations result in minor symptoms alone, treatment consists mainly of symptomatic alleviation. Centipedes are most commonly found in tropical and subtropical regions but can be found worldwide on all six inhabited continents. The Scutigera coleoptrata is a centipede about 25 mm in length with long thin legs that is commonly found in United States households. Lithobius spp. are ground dwelling species about 30–50 mm in length commonly found in eastern United States gardens. Scolopendra spp., the largest and most dangerous centipedes worldwide are attributed with the majority of symptomatic envenomations and are associated with at least two known fatalities. Scolopendra spp. are found throughout Asia, Africa, Australia, and the Americas including the southern United States.

Keywords Arthropoda; Bites; Centipedes; Chilopoda; Envenomation; Geophiulomorpha; Lithobiomorpha; Scolopendromorpha; Scutigeromorpha

Key points

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There are over 3000 species of centipede, varying in size from 1 to 30 cm with body segments numbering from 15 to more than 100. The majority of centipede envenomations result in minor symptoms alone, with treatment consisting mainly of symptomatic alleviation. Scolopendra spp., found throughout Asia, Africa, Australia, and the Americas, are the largest and most dangerous centipedes worldwide. The majority of symptomatic envenomations, and at least two known fatalities, are associated with this species.

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Background Centipedes are nocturnal multi-segmented elongated arthropods known for the distinct feature of having a pair of legs for each body segment except for the last. The first pair of legs on the cranial segment is in actuality modified into a pincer-like apparatus, known as forcipules, used to inject venom into prey. The last segment contains a pair of filamentous caudal appendages that despite popular belief play no role in envenomation. There are over 3000 species, varying in size from 1 to 30 cm with body segments numbering from 15 to more than 100. Centipedes are divided into four orders; Geophiulomorpha (soil centipedes), Lithobiomorpha (rock or garden centipedes), Scolopendromorpha (tropical or giant centipedes), and Scutigeromorpha (house or feather centipedes) of which only two, Scolopendromorpha and Scutigeromorpha, are of medical significance. Because the majority of centipede envenomations result in minor symptoms alone, treatment consists mainly of symptomatic alleviation. Centipedes are most commonly found in tropical and subtropical regions but can be found worldwide on all six inhabited continents.

Exposure routes and pathways A venom gland is found at the base of the forcipules which act as fangs. Upon penetration of human skin by the forcipules, venom passes through the ducts of the forcipules and is injected into the bite site, causing local envenomation. Distal extremities such as hands and feet are common sites of envenomation. There has been one case report in the literature of an accidental ingestion resulting in symptomatic presentation.

Mechanism of toxicity Much is still unknown regarding the components that make up centipede venom but considerable work has been done surrounding the venom of Scolopendra spp. Centipede venom is a complex mixture of lipid-toxin, similar to scorpion venom, thus facilitating local cellular penetration and absorption (Cherniack, 2011). Known components include polysaccharides, lipoproteins, various enzymes including proteinases and esterases, amino acid naphthylamidase, alkaline phosphatase, 5-hydroxytryptamine, histamine, and a cardiotoxic agent known as Toxin-S. Toxin-S is a large heat-labile acid that has been shown to increase capillary permeability, induce vasoconstriction and act as a cardiodepressant. In addition, there is an identified presence of a smooth muscle contractile agent that has muscarinic activity as well. It is thought that the digestive enzymes and Toxin-S serve as a primary predatory function of the venom whereas pain mediators such as 5-hydroxytryptamine and histamine serve as a secondary defensive function of the venom.

Acute and short-term toxicity (or exposure) Animal Acute toxicity of centipede extract has been demonstrated in animal models. One study demonstrated cardiac arrest in a toad shortly after being injected with an extract made from Scolopendra morsitans. The effect was prevented by giving atropine. Though the underlying mechanism is not clearly understood, it is thought to be associated with the muscarinic activity agent present in centipede venom.

Human The majority of acute centipede envenomations produce minor localized symptoms that are self limiting and generally resolve spontaneously within 48 h (Bush et al., 2001). Localized symptoms around envenomation sites include burning pain, edema, erythema, bullae formation, ecchymosis, paresthesia, delayed itchiness, lymphangitis, and lymphadenopathy. Occasionally the bite wound is complicated by necrosis and gangrene but this is thought to be secondary to wound infection and not a property of envenomation (Fung et al., 2011). A minority of acute centipede envenomations produce minor systemic symptoms including chest pain, palpitations, headache, dizziness, and vomiting. It is thought that the severity of symptoms correlates with the size of the centipede. There have been several case reports of acute centipede envenomations resulting in acute myocardial infarction or acute coronary ischemia. In one case, a previously healthy 22-year-old male with no cardiac risk factors developed severe retrosternal chest pain radiating to his left arm, diaphoresis, nausea, and vomiting 2 h after being bitten by a Scolopendra sp. 14 h after envenomation, the patient presented to the emergency department where electrocardiogram, echocardiogram, and cardiac marker levels revealed an anterior wall myocardial infarction. An emergent coronary angiography revealed entirely normal coronary arteries. Three days after admission all abnormal findings with the electrocardiogram, echocardiogram, and cardiac marker levels had resolved (Senthilkumaran et al., 2011). Another case report described a 20-year-old male who presented to the emergency department

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with severe chest pain and electrocardiogram findings suggestive of an inferolateral myocardial infarction approximately 24 h after being bitten by a suspected Scolopendra sp. (Yildiz et al., 2006). The exact pathophysiology has not yet been identified but the suspected mechanism is thought to be an acute release of inflammatory mediators causing increased capillary permeability, hypotension, and coronary artery spasm which may result in an acute myocardial infarction. Rare but severe presentations of centipede envenomations have been documented in the literature. In one case, a 46-year-old female in the southwestern United States was bitten on the right foot by Scolopendra heros. She went on to develop necrosis of the peroneal muscles, rhabdomyolysis, acute renal failure, and massive edema of the leg, requiring fasciotomy to relieve increased compartmental pressures within the leg (Logan and Ogden, 1985). Another case documented eosinophilic cellulitis or Wells’ syndrome associated with the bite of an unidentified centipede (Friedman et al., 1998). An Israeli female bitten on the neck was unable to turn her head, most likely secondary to muscle spasm. A 54-Year-old male developed Acute Disseminated Encephalomyelitis after a bite on the foot from scolopendra subspinipes mutilans, a common centipede species found in China (Xiaoying et al., 2013). There have been no reported deaths associated with centipede envenomation within the United States. In the early 20th century, there was a reported case of a 7-year-old female in the Philippines who died after being bitten on the head by a Scolpendra subspinipes (Pineda, 1923). In the one reported case of symptomatic ingestion, a six-month-old Australian infant accidentally ingested a centipede identified as Scutigera morpha and proceeded to develop pallor, hypotonia, vomiting, and lethargy. The symptoms were thought to be secondary to the systemic absorption of venom. The child fully recovered after approximately 48 h (Barnett, 1991). A prospective study of centipede exposures identified three cases of ingestions that resulted in no adverse effects or symptoms (Balit et al., 2004).

Chronic toxicity or exposure Animal Chronic exposure of centipede extract has been demonstrated as an antihyperlipidemic agent in animal models. One study isolated the substance centipede acidic protein (CAP) from an alcohol extract comprised of powdered dried Scolopendra subspinipes mutilans and injected it into rodent models. Rodents that received CAP were shown to have lower levels of triglycerides, cholesterol, and LDL when compared to control models injected with saline.

Human In one death associated with Scolopendra spp. envenomation a 21-year-old Thai female who had been previously exposed to repeated envenomations. The 9 months prior her death, she had been envenomated on three separate occasions, each resulting in a local urticarial reaction. Upon her fourth envenomation, she presented with chest discomfort, hypotension, tachypnea, and a generalized urticarial rash. Despite aggressive medical intervention, she decompensated into respiratory failure and died from cardiovascular collapse and acute respiratory distress syndrome. The mechanism was thought to be secondary to an allergic reaction. It is theorized that there is similarity between a centipede venom allergy and a hymenoptera venom allergy suggesting that chronic or repeated envenomations in an individual with a centipede venom allergy may result in life-threatening allergic reactions that may not have been seen with prior envenomations.

Clinical management As the majority of centipede envenomations produce minor and self limiting symptoms, the mainstay of treatment is supportive care and symptomatic relief. Appropriate wound care and cleaning should be performed along with the administration of tetanus prophylaxis. The use of antibiotics should be reserved for recognized wound infections. The use of ice packs, hot water immersion, and analgesics have all been shown to alleviate pain from centipede envenomations (Chaou et al., 2009). The mechanism of analgesia produced from hot water immersion is not yet identified but it is thought to be secondary to the denaturing of the heat-labile toxin. Severe pain may also be treated with injection of local anesthetic around the envenomation site.

Miscellaneous Millipedes Like centipedes, millipedes are also multi-segmented elongated arthropods. Millipedes differ from centipedes in that they do not possess venom injecting apparatuses and the majority of their body segments have two pairs of legs per segment instead of one. Though millipedes do not bite, they can secrete and spray noxious chemicals that upon exposure can act as skin and eye irritants. Like centipedes, millipedes are also commonly found in the southern United States.

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References Balit CR, Harvey MS, Waldock JM, and Isbister GK (2004) Prospective study of centipede bites in Australia. Journal of Toxicology. Clinical Toxicology 42(1): 41–48. PMID: 15083935 https://doi.org/10.1081/clt-120028743. Barnett PL (1991) Centipede ingestion by a six-month-old infant: Toxic side effects. Pediatric Emergency Care 7(4): 229–230. https://doi.org/10.1097/00006565-19910800000009. PMID: 1758778. Bush SP, King BO, Norris RL, and Stockwell SA (2001) Centipede envenomation. Wilderness & Environmental Medicine 12(2): 93–99. https://doi.org/10.1580/1080-6032(2001)012 [0093:ce]2.0.co;2. PMID: 11434497. Chaou CH, Chen CK, Chen JC, Chiu TF, and Lin CC (2009) Comparisons of ice packs, hot water immersion, and analgesia injection for the treatment of centipede envenomations in Taiwan. Clinical Toxicology 47(7): 659–662. https://doi.org/10.1080/15563650802084821. PMID: 19640231. Cherniack EP (2011) Bugs as drugs, part two: Worms, leeches, scorpions, snails, ticks, centipedes, and spiders. Alternative Medicine Review 16(1): 50–58. PMID: 21438646. Friedman IS, Phelps RG, Baral J, and Sapadin AN (1998) Wells’ syndrome triggered by centipede bite. Int. The Journal of Dermatology 37(8): 602–605. https://doi.org/10.1046/ j.1365-4362.1998.00570.x. PMID: 9732008. Fung HT, Lam SK, and Wong OF (2011) Centipede bite victims: A review of patients presenting to two emergency departments in Hong Kong. Hong Kong Medical Journal 17(5): 381–385. PMID: 21979475. Logan JL and Ogden DA (1985) Rhabdomyolysis and acute renal failure following the bite of the giant desert centipede Scolopendra heros. The Western Journal of Medicine 142(4): 549–550. PMID: 4013269. Pineda EV (1923) A fatal case of centipede bite. Journal of the Medical Association 3: 59–61. Senthilkumaran S, Meenakshisundaram R, Michaels AD, Suresh P, and Thirumalaikolundusubramanian P. (2011) Acute ST-segment elevation myocardial infarction from a centipede bite. Journal of Cardiovascular Disease Research 2(4): 244–246. https://doi.org/10.4103/0975-3583.89811. PMID: 22135485. Xiaoying Y, Quan D, Ying C, Zhiying F, and Yansheng L (2013) Acute disseminated encephalomyelitis following biting by a scolopendra subspinipes mutilans. Clinical Toxicology 51(6): 519–520. https://doi.org/10.3109/15563650.2013.804929. Yildiz A, Biçeroglu S, Yakut N, Bilir C, Akdemir R, and Akilli A (2006) Acute myocardial infarction in a young man caused by centipede sting. Emergency Medicine Journal 23(4): e30. https://doi.org/10.1136/emj.2005.030007. PMID: 16549562.

Further reading Lersloompleephunt N, Eakthunyasakul S, Sittipunt C, et al. (2003) Severe hypotension and adult respiratory distress syndrome (ARDS) following centipede bite. In: Proceedings of the 5th Asia-Pacific Congress on Animal, Plant and Microbial Toxins. Thailand: International Society on Toxinology. Haddad V, Haddad de Amorim PC, Rassi da Cruz C, and Amaral A (2022) Centipede envenomation (Chilopoda): Case report. Revista da Sociedade Brasileira de Medicina Tropical 55. https://doi.org/10.1590/0037-8682-0601-2021.

Relevant websites http://emedicine.medscape.com/article/769448-overview :Medscape eMedicine-Information regarding clinical presentation and management of centipede envenomations. http://soilbugs.massey.ac.nz/chilopoda.php :Massey University-Images and information regarding centipedes. http://www.ipm.ucdavis.edu/PMG/PESTNOTES/pn7472.html :University of California Davis-Images and information regarding centipedes and millipedes. http://www.caes.uga.edu/publications/pubDetail.cfm?pk_id¼6198 :The University of Georgia College of Agricultural and Environmental Sciences website related to centipede and millipedes. https://www.ncbi.nlm.nih.gov/books/NBK542312/ :StatPearls-Centipede envenomations National Library of Medicine.

Cephalosporins Shayne C Gad, Gad Consulting Services, Raleigh, NC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.C. Gad, Cephalosporins, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 768–770, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00825-3.

Chemical profile Background Uses/occurrence Exposure Toxicokinetics (ADME) Mechanism of toxicity In vitro toxicity data Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines Other PubChem URL References

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Abstract Originally isolated from microorganisms. Routes of exposure to cephalosporins are commonly oral, intravenous, or intramuscular. Cephalosporins are generally well absorbed following administration with oral bioavailability being greater than 75%. Hematologic when given in higher doses, cephalosporins bind to cell membrane proteins and act as haptens. High-dose treatments can induce a hemolytic anemia. They can also prolong bleeding times by reducing platelet adhesion and activation. Cephalosporins (a class of b-lactam antibiotics) induce their antimicrobial effect by inhibiting the integration of bacterial peptidoglycan. There are substantial differences among the cephalosporins in their spectra of activity as well as levels of activity against susceptible bacteria. Later-generation cephalosporins also are used in the later stages of livestock raising for adding weight.

Keywords Anaphylaxis; Cefepidime; Cephalosporium acremonium; Enzymuria; Proteinuria; b-Lactam antibiotics

Chemical profile

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Chemical/Pharmaceutical/Other Class: b-Lactam antibiotics (as are penicillins) Name: Cephalosporins Synonyms: Example Compounds: cephalosporin C, Cephalosporanic acid, Cefaclor, Cefadroxil, Cefamandole, Cefazolin, Cefepidime, Cefoperazone, Cefotaxime, Cefoxitin, Ceftriaxone, Cephalexin, Cephalothin, Cephradine, Cephaprin, Cefmetazole, Cefonicid, Ceforanide, Cefotetan, Cefprozil, Loracarbef, Cefperazone, Cefpodoxime, Cefixime, Ceftazidime, Ceftizoxime, Moxalactarn CAS Number: Example Compounds CAS Numbers: 61-24-5 Molecular Formula Examples: C16H21N3O8S, C10H11NO5S, C15H21N3O7S Chemical Structure: Examples

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Background The cephalosporins have sustained their position as a very significant class of antibiotics worldwide for many years, comprising over one-half of the available b-lactam antibiotics. Initially, cephalosporin compounds produced by the fungal organism Cephalosporium acremonium were isolated in the early 1940s from fungus in sewage seawater in Cagliari, Sardinia, after it was observed that a natural pattern of periodic clearing of microbes was taking place from a local harbor area. Filtrates from C. acremonium cultures were found to have antimicrobial activity against infections in animals and humans, including the injection of filtrates into ‘boils’ and other cutaneous infections. The initial work later expanded to result in the discovery of cephalosporin C, the structural nucleus for cephalosporin compound development over the next four decades. Ongoing research and development have led to several additional cephalosporin antibiotics that have been released since the 1960s, with 24 unique, yet structurally similar compounds currently available for clinical use in the United States. The first three generations of cephalosporin antibiotics include both parenterally (i.e., intravenously or injection) and orally administered agents. The fourth-generation compound, cefepidime, is available for parenteral administration (Walsh, 2003).

Uses/occurrence Cephalosporins (a class of b-lactam antibiotics) induce their antimicrobial effect by inhibiting the integration of bacterial peptidoglycan. Individual peptidoglycan units are synthesized in the cytoplasm of the bacterial cell and are transported across the cytoplasmic membrane where they are inserted by peptidase enzymes into a cross-linked lattice that forms the structural support of the bacterial cell wall. The peptidase enzymes present in the outer cytoplasmic membrane are referred to as penicillin-binding proteins and represent the target sites for antibacterial action of cephalosporins and other b-lactam antibiotics. Cephalosporins are active in vitro against many gram-positive aerobic bacteria and some gram-negative aerobic bacteria. There are substantial differences among the cephalosporins in their spectra of activity as well as levels of activity against susceptible bacteria. Later-generation cephalosporins also are used in the later stages of livestock husbandry for adding weight.

Exposure The routes of exposure to cephalosporins are commonly oral, intravenous, or intramuscular. Accidental ingestion of oral dosage forms by children is the most common poisoning exposure. There are some workers who may be exposed to cephalosporins, primarily those involved in the production of drug forms.

Toxicokinetics (ADME) Cephalosporins are generally well absorbed following administration, with bioavailability being greater than 75%. Many of these compounds are not stable in the acid environment of the stomach; therefore, only a limited number are useful for oral administration. The distribution is limited to the extracellular fluid space, with volumes of distribution for most ranging from 0.25 to 0.5 l kg−1. Protein binding is primarily to albumin. Most cephalosporins are widely distributed to tissues and fluids, including pleural fluid, synovial fluid, and bone. Some of the third-generation compounds have good distribution to the cerebrospinal fluid. The metabolites possess antibacterial activity. The cephalosporins and their metabolites are rapidly excreted by the kidneys by glomerular filtration and/or tubular secretion. Serum half-lives of these compounds range from 0.4 to 10.9 h. Patients with immature renal systems or with renal compromise are at risk for toxicity due to decreased elimination. Cefamandole, cefmetazole, cefmenoxime, cefoperazone, and moxalactam have been associated with coagulopathies due to inhibition of platelet aggregation and prolongation of bleeding time (Walsh, 2003; Ballantyne et al., 2009; Karalis et al., 2003).

Mechanism of toxicity Hematologic when given in higher doses, cephalosporins bind to cell membrane proteins and act as haptens. High-dose treatments can induce a hemolytic anemia. They can also prolong bleeding times by reducing platelet adhesion and activation. Many cephalosporins are substrates for the organic anion transport system in the proximal tubules and can accumulate in the kidney, competing for and inhibiting the transport system, leading to renal necrosis. Nephrotoxicity, with potency ranging from mild to high (cephaloridine), can cause renal tubule injury. There is great variability in the potential for renal toxicity among different members of the family of compounds. Renal toxic members deplete glutathione levels in the renal cortex. There is evidence that this nephrotoxicity is due to action of the drugs on mitochondria (Walsh, 2003).

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In vitro toxicity data Antibiotic, cytotoxic to bacterial cells (Dancer, 2001).

Chronic toxicity Animal The extent of renal accumulation and effect is species dependent (rabbit > guinea pig > rat). There is also great variability in toxicity between different compounds.

Human Like penicillins, cephalosporins are a relatively nontoxic group of antibiotics. The primary adverse effect reported is hypersensitivity, a rare event. Cross-allergenicity and sensitization with penicillins may occur and is of concern, though allergic responses to cephalosporins are less likely than to penicillins. Toxicity is unlikely in children less than 6 years of age who acutely ingest less than 250 mg kg−1. Nephrotoxicity is a possible, but rare, occurrence with acute ingestion. Coagulopathies have been reported following chronic intravenous use of certain cephalosporins. At higher concentrations, cephalosporins cause renal tubular injury, characterized by decreased glomerular filtration rate, glucosuria, enzymuria, and proteinuria (Ballantyne et al., 2009).

Immunotoxicity Lymphocyte response may be inhibited by some of the cephalosporin class. There are also effects on interleukin-2 function and production. Additional testing will be required to make a positive determination on the immunotoxic affects of cephalosporins, and there will be variability, possibly wide, among the members of the class. Hypersensitivity does occur but is less common than with penicillin. Cross reacting does occur (Dancer, 2001).

Reproductive and developmental toxicity Cefotan: Has adverse effects on the testes of prepubertal rats. Subcutaneous administration of 500 mg kg−1 day−1 (8–16 times the usual adult human dose) on days 6–35 of life (thought to be developmentally analogous to late childhood and prepuberty in humans) resulted in reduced testicular weight and seminiferous tubule degeneration in 10 of 10 animals. Affected cells included spermatogonia and spermatocytes; Sertoli and Leydig cells were unaffected. Incidence of severity of lesions was dose dependent; at 120 mg kg−1 day−1 (B2–4 times the usual human dose) only 1 of 10 treated animals was affected, and the degree of degeneration was mild. Similar lesions were observed in experiments of comparable design with other methylthiotetrazole-containing antibiotics, and impaired fertility has been reported, particularly at high dose levels. No testicular effects were observed in 7-week-old rats treated with up to 1000 mg kg−1 day−1 subcutaneously for 5 weeks, or in infant dogs (3 weeks old) that received up to 300 mg kg−1 day−1 intravenously for 5 weeks. Pregnancy category B: Reproduction studies have been performed in rats and monkeys at doses up to 20 times the human dose and have revealed no evidence of impaired fertility or harm to the fetus due to cefotetan.

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Cefixime: In rats, fertility and reproductive performance were not affected by cefixime at doses up to 125 times the adult therapeutic dose. Pregnancy category B: Reproduction studies have been performed in mice and rats at doses up to 400 times the human dose and have revealed no evidence of harm to the fetus due to cefixine. Cefoperazone: Produced no impairment of fertility and had no effects on general reproductive performance or fetal development when administered subcutaneously at daily doses up to 500–1000 mg kg−1 prior to and during mating, and to pregnant female rats during gestation. These doses are 10–20 times the estimated usual single clinical dose. Cefoperazone had adverse effects on the testes of prepubertal rats at all doses tested. Subcutaneous administration of 1000 mg kg−1 day−1 (16 times the average adult human dose) resulted in reduced testicular weight, arrested spermatogenesis, reduced germinal cell population, and vacuolation of Sertoli cell cytoplasm. The severity of lesions was dose dependent in the 100–1000 mg kg−1 day−1 range; the low dose caused a minor decrease in spermatocytes. This effect has not been observed in adult rats. Historically, the lesions were reversible at all but the highest dosage levels. However, these studies did not evaluate subsequent development of reproductive function in the rats. Pregnancy category B: Reproduction studies have been performed in mice, rats, and monkeys at doses up to 10 times the human dose and have revealed no evidence of impaired fertility or harm to the fetus due to cefoperazone.

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Genotoxicity Cefixime: SUPRAX did not cause point mutations in bacteria or mammalian cells, DNA damage, or chromosome damage in vitro and did not exhibit clastogenic potential in vivo in the mouse micronucleus test. Cefoperazone: The maximum duration of cefoperazone animal toxicity studies is 6 months. In none of the in vivo or in vitro genetic toxicology studies did cefoperazone show any mutagenic potential at either the chromosomal or subchromosomal level (Ballantyne et al., 2009).

Carcinogenicity Studies have yet to be undertaken for the carcinogenicity of cephalosporins in animals or humans. Cephalosporins are not listed by regulatory agencies.

Organ toxicity Renal Tubular toxicity.

Interactions Drugs for acid reflux and live typhoid vaccine.

Clinical management If a toxic or unknown amount of cephalosporin has been ingested, gastric decontamination and the administration of activated charcoal are usually all that is needed. In the symptomatic patient, evaluation of renal function and electrolytes may be necessary. Chronic exposure usually requires discontinuation of the drug and supportive care. Anaphylaxis should be treated with epinephrine and/or diphenhydramine (Del Rosso, 2003; Greenberg et al., 2003).

Environmental fate and behavior The environmental behavior of most of the cephalosporin compounds is quite similar. In the atmosphere, most are expected to exist solely in the particulate phase and are removed via wet or dry deposition. In aquatic environments, due to their anionic nature, they are not expected to volatize, and they adsorb slowly. Cephalosporins are highly mobile in soils, with low adsorption. Data suggest that even with water treatment, some of these compounds will persist. They may be susceptible to photolyzation.

Ecotoxicology Cephalosporins have a low bioconcentration factor and are not expected to bioconcentrate or bioaccumulate to any serious degree. They are not expected to significantly impact species outside of the bactericidal activity they are in use for.

Exposure standards and guidelines Exposure guidelines have yet to be set by regulatory agencies.

Other When heated to decomposition, many of the cephalosporins will emit significant toxic fumes.

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PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/65536

See also: Hemocompatibility; Kidney

References Ballantyne B, Marrs T, and Syversen T (2009) General and Applied Toxicology, 3rd edn, New York: Macmillan References Ltd pp. 1138–1140. 686–687. Dancer SJ (2001) The problem with cephalosporins. The Journal of Antimicrobial Chemotherapy 48: 463–478. Del Rosso JQ (2003) Cephalosporins in dermatology. Clinics in Dermatology 21: 24–32. Greenberg ML, Hendrickson RG, and Muller AA (2003) Occupational exposure to cephalosporins leading to clostridium difficile infection. Journal of Toxicology. Clinical Toxicology 41: 205–206. Karalis V, Tsantili-Kakoulidou A, and Macheras P (2003) Quantitative structure–pharmacokinetic relationships for disposition parameters of cephalosporins. European Journal of Pharmaceutical Sciences 20: 115–123. Walsh C (2003) Antibiotics. Washington DC: ASM Press.

Relevant websites https://comptox.epa.gov/ :United States Environmental Protection Agency. http://medical-dictionary.thefreedictionary.com :Search for `Cephalosporins’. http://www.drugs.com :Search for `Cephalosporins’. http://accessmedicine.com/ :Search for `Cephalosporins’.

Cerium Shayne C Gad, Gad Consulting Services, Raleigh, NC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.C. Gad, Cerium, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 773–775, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00826-5.

Chemical profile Background Uses/occurrence Exposure Toxicokinetics (ADME) Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines PubChem URL References Further reading

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Abstract Cerium (7440-45-1) is a lanthanide (rare earth metal) and it is relatively abundant in the earth’s crust, (among the lanthanides, the most abundant). It is one of the 78 common elements in the earth’s crust, and ranks 25th in occurrence at an average distribution of 20–60 ppm. Inhalation, dermal, and oral are the possible exposure routes. Cerium is poorly absorbed by the intestine. Cerium resembles aluminum in its biologic and chemical properties. Cerium can increase blood coagulation rate and produce adverse gastrointestinal effects. Inhalation can lead to polycythemia. Toxicity to aquatic species is primarily dependent upon the particle size.

Keywords Granulomatosis; Polycythemia

Chemical profile

• • • • •

Name: Cerium Synonyms: Ce, cerio, Cerium; EINECS 231–154-9; UNII-30K4522N6T CAS Number: 7440-45-1 Molecular Formula: Ce3+, Ce4+ Chemical Structure:

Ce

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Background Cerium is a rare earth metal and the most abundant member of the lanthanide series discovered by Jons J. Berzelius and W. von Hisinger in 1803 in Sweden. Berzelius and Hisinger discovered the new element in a rare reddish-brown mineral now known as cerite, a cerium-lanthanide silicate. Although they could not isolate the pure metal, they found that cerium had two oxidation states: trivalent state (Ce3+, cerous, usually orange-red) and the tetravalent state (Ce4+, ceric, usually colorless). Cerium is the only material known to have a solid-state critical point (Wells and Wells, 2012).

Uses/occurrence Cerium is used in metallurgy as a stabilizer in alloys and in welding electrodes; in glass as a polishing agent, decolorizer, and to render glass opaque to near-ultraviolet radiation. It is also used in ceramics and as a catalyst. Cerium is used as a component of some diesel fuel additives, and may be added to residual fuel oils to improve combustion. Cerium is found in portable rechargeable batteries.

Exposure Inhalation, dermal, and oral are the possible exposure routes. Exposure to commercially used cerium compounds is most likely through exposure to cerium oxide.

Toxicokinetics (ADME) Cerium is poorly absorbed by the intestine following oral exposure in animals, except for water-soluble cerium compounds and cerium oxide. As poorly soluble particles, cerium oxide my dissolve slowly from the lung into systemic circulation and is observed in the liver, skeleton, and tracheobronchial lymph nodes. Cerium has also been observed to be localized in the cell, particularly in the lysosomes, where it is concentrated and precipitated in an insoluble form in association with phosphorus. As an element, cerium is neither created nor destroyed within the body. The particular cerium compounds, such as cerium chloride and cerium oxide, may be altered as a result of various chemical reactions within the body, particularly dissolution. Following inhalation exposure, the initial rapid elimination of insoluble cerium from the body is due primarily to transport up to the respiratory tract via the mucociliary escalator and eventual swallowing of the material. Initial short-term clearance rates range from 35 to 95% of initial cerium body burden, depending on the species tested and length of clearance time investigated. Elimination of orally administered soluble cerium has been shown to be age dependent in animals. The cerium may remain in the intestinal cells, may not be available systemically, and may eventually be eliminated in the feces. Cerium is capable of crossing the placenta and entering the fetal circulation in mice, but the amounts found in the uterus and placenta were generally less than 5% of the maternal body burden and decreased rapidly with increased time after exposure (Wells and Wells, 2012).

Mechanism of toxicity Cerium resembles aluminum in its biologic and chemical properties. Cerium and cerium compounds have low to moderate toxicity unless the associated anions are toxic. Intratracheally administered nanoparticles tend to accumulate in liver and cause damage there (Nalabotu et al., 2010).

In vitro toxicity data Particle shape influences cytotoxicity. Causes oxidative stress in cells.

Acute and short-term toxicity Animal The LD50 values reported in rats ranged from 4 to 50 mg kg−1 for cerium nitrate with female rats being more sensitive than males. After peritoneal injection, the LD50 of cerium nitrate was 470 mg kg−1 in female mice and 290 mg kg−1 in female rats; the LD50 of cerium chloride was 353 mg kg−1 in mice and 103 mg kg−1 in guinea pigs. The oral toxicity of cerium nitrate was much lower (LD50 of 4200 mg kg−1 in female rats and 1178 mg kg−1 in female mice) than after intravenous or intraperitoneal administration. The LD50 of ingested cerium oxide could not be determined in rats when delivered at a dose of 1000 or 5000 mg kg−1. An LD50 of 622 mg kg−1 has been reported for cerium oxide ingested by mice. The LC50 after inhalation of cerium oxide in rats was greater than 50mgm−3. The primary targets after inhalation of cerium are the lung and the associated lymph nodes; other organs could be affected via clearance through the blood. Studies of cerium injected systemically have shown that, once in the circulation, cerium

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can cause liver toxicity with a no observed-adverse-effect level of 1 mg kg−1 after a single intravenous injection and a lowest-observed adverse-effect level (LOAEL) of 2 mg kg−1 for effects on liver detoxifying enzymes. Effects on other organs where cerium can accumulate (such as spleen, bones, and kidney) have not been studied. A single-dose study on the effects of in utero intravenous administration reported reduced weight in newborn mouse pups, with an LOAEL of 80 mg kg−1. Cerium has been found to depress certain behaviors in mice administered this chemical, and cerium administered to pregnant mice on day 7 or 12 of gestation or 2 days postpartum caused significant decreases in open field activity of offspring. Fetal growth was impaired, as evidenced by weight decreases of 7–19%. The potential carcinogenicity of cerium-containing particles has not been studied in conventional rodent bioassays; in vivo mutagenicity studies have been negative.

Human Cerium can increase blood coagulation rate and produce gastrointestinal effects. Inhalation can lead to polycythemia.

Chronic toxicity Animal An animal inhalation study involved exposure of rats to cerium oxide particles substantially larger than those in diesel emission. The exposure concentrations ranged between 5 and 500 mgm−3 for 13 weeks. Effects observed included lung discoloration, enlargement of lymph nodes, and increased lung and spleen weight at all concentrations (Arvela et al., 1991).

Human Case reports of workers occupationally exposed to rare earth metals (including cerium) describe a condition termed rare earth pneumoconiosis with pathologic features including interstitial fibrosis, granulomatosis, and bilateral nodular chest X-ray infiltrates. Although the disease sometimes is associated with accumulation of cerium in particles, the role of cerium in this complex disease is unclear relative to other metals or gases to which workers may also have been exposed.

Immunotoxicity No relevant human or animal data are available regarding immunotoxicity in cerium or cerium compounds.

Reproductive and developmental toxicity A cross-fostering design was employed to separate effects of prenatal and postnatal exposure following a single subcutaneous dose of cerium citrate (80 mg kg−1) in pregnant female mice. Analyses revealed that neonatal weight was reduced both in offspring exposed to cerium in utero and in the offspring of mothers receiving cerium during lactation. Cerium also appeared to affect maternal- offspring interaction: pups exposed prenatally to cerium were retrieved in less time than control pups.

Genotoxicity In vitro mutagenicity studies have been negative. Other cerium compounds, such as cerium chloride observed no induction DNA damage in two strains of B. subtilis by using rec-assay, but cerium nitrate was reported to induce chromosomal breaks and reduce the mitotic index in rat bone marrow in vivo, and cerium sulfate was reported to cause differential destaining of chromosomal segments in plants (Wells and Wells, 2012). No information has been located regarding genotoxic effects of cerium and cerium compounds in humans.

Carcinogenicity Data regarding the carcinogenicity of cerium compounds in humans or experimental animals are unavailable. In accordance with U.S. EPA, cerium and cerium compounds are classified as “inadequate information to assess the carcinogenic potential” in humans.

Organ toxicity Pulmonary, red blood cells, lymph nodes, and liver.

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Clinical management Methylene blue is used to treat oxidative damage induced methemoglobinemia.

Environmental fate and behavior Although cerium is a rare earth element, it is relatively abundant in the earth’s crust. It makes up about 0.0046% of the Earth’s crust by weight and ranks 25th in occurrence at an average distribution of 20–60 ppm. Cerium is a malleable, soft, ductile, iron-gray metal, slightly harder than lead. It is very reactive and readily tarnishes in the air. Cerium oxidizes slowly in cold water and rapidly in hot water. It dissolves in acids. Cerium can burn when heated or scratched with a knife. Cerium is not expected to exist in elemental form in the environment since it is a reactive metal. Cerium is dumped in the environment in many different places, mainly by petrol-producing industries. It can also enter the environment when household equipment is thrown away. Cerium compounds exist solely in particulate form if release into air and not expected to volatilize. Water-soluble cerium compounds usually have a pKa of 8.5 which indicates that the hydrated Ce3+ ion will remain in solution at environmental pHs 4–9. The ion is expected to hydrolyze and polymerize at environmental pH and may precipitate out of solution. Thus, cerium will gradually accumulate in soils and water which eventually lead to increasing concentrations in humans, animals and soil particles (Biswas et al., 2011).

Ecotoxicology Toxicity to aquatic species is dependent upon the particle size (Biswas et al., 2011).

Exposure standards and guidelines No standards have been recommended for elemental cerium, cerium compounds, or any other lanthanides because either suitable data for setting a standard, such as inhalation studies or studies on these compounds are lacking. However, because of the accumulating evidence of induction of fibrosis with the lanthanides and their expanding use, the exposure should probably be limited to 1 mg m−3.

PubChem URL Cerium | Ce - PubChem (nih.gov).

References Arvela P, Kraul H, Stenback F, and Pelkonen O (1991) The cerium-induced liver injury and oxidative drug metabolism in DBA/2 and C57BL/6 mice. Toxicology 69: 1–9. Biswas A, Gaiser BK, Jepson MA, Lead JR, Rosenkranz P, and Stone V (2011) Effects of silver and Cerium dioxide micro- and nano-sized particles on Daphni. Journal of Environmental Monitoring 5: 1227–1235. Nalabotu SK, Kolli MB, Triest WE, Ma JY, and Manne NDPK (2010) Intratracheal instillation of cerium oxide nanoparticles induces hepatic toxicity. Nanomedicine 6: 2327–2335. Wells HW and Wells VI (2012) Cerium. In: Bingham E, Cohrssen B, and Powell CH (eds.) Patty’s Toxicology, 6th edn. New York: Wiley-Interscience Publication.

Further reading Berry JP, Meignan M, Escaig F, and Galle P (1988) Inhaled soluble aerosols insolubilised by lysosomes of alveolar cells. Application to some toxic compounds; electron microprobe and ion microprobe studies. Toxicology 14: 127–139.

Relevant websites http://www.healtheffects.org :Evaluation of Human Health Risk from Cerium Added to Diesel Fuel. Health Effects Institute Communication 9 (August 2001). http://www.chemicool.com/elements/cerium.html :“Cerium.” Chemicool Periodic Table. Chemicool.com (June 2011).

Cesium Shayne C Gad, Gad Consulting Services, Raleigh, NC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.C. Gad, T. Pham, Cesium, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 776–778, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00827-7.

Chemical profile Background Uses/occurrence Exposure Toxicokinetics (ADME) Mechanism of toxicity Acute and short-term toxicity (e.g., animal, human; oral, inhalation, dermal) Animal Human Chronic toxicity (e.g., animal, human; oral, inhalation, dermal) Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines Other PubChem URL References

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Abstract Cesium (CAS 7440-46-2) was discovered in 1860 by Robert Bunsen and Gustav Kirchoff. It is used in drilling fluid, in manufacturing of special artificial glasses, in photovoltaic cells, in vacuum tubes and most accurate atomic clocks. Inhalation and ingestion are the routes of exposure. Cesium displaces potassium. It is neither genotoxic or carcinogenic. Cesium has been reported to cause hyperirritability and muscle spasms. Prussian blue is administered by a duodenal tube to act as a chelating agent.

Keywords Cesium; Gustav Kirchoff; Nuclear accidents; Prussian blue; Radiocesium; Robert Bunsen

Chemical profile

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Name: Cesium Synonyms: Cesium CAS Number: 7440-46-2 Molecular Formula: Cs+ Chemical Structure:

Cs ^ Background Cesium was discovered in 1860 by Robert Bunsen and Gustav Kirchoff. It is used in the most accurate atomic clocks. Cesium melts at 28.41  C (just below body temperature) and occurs in Earth’s crust at 2.6 ppm. Cesium is the rarest of the naturally occurring alkali metals as the isotope 133Cs. Its compounds are correspondingly rare. Granites contain about 1 ppm cesium and sedimentary rocks

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contain approximately 4 ppm cesium. The most common commercial source of cesium is pollucite, which contains between 5% and 32% cesium oxide. Radioactive forms of cesium (134Cs and 137Cs) can also be found in the environment. They are produced during nuclear fission, and are used in cancer treatment (ATSDR, 2011).

Uses/occurrence Cesium is used in drilling fluids, making of special optical glasses, photovoltaic cells, vacuum tubes, scintillation counters, and atomic clocks (Bingham et al., 2012).

Exposure Occupational exposure to cesium compounds occurs primarily through inhalation and dermal contact at workplaces in which pollucite is mined or cesium compounds are manufactured or used. General population exposure to cesium occurs by ingestion of food and drinking water, inhalation of ambient air, and dermal contact with cesium compounds in soil. Current exposure of the general population of the United States to radioactive cesium-134 and cesium-137 is expected to be low because atmospheric testing of nuclear weapons has been discontinued for many years.

Toxicokinetics (ADME) Stable and radioactive cesium can be taken into the body by ingestion of food, water, or inhalation. Once absorbed, cesium behaves in a manner similar to potassium and distributes uniformly throughout the body as cations, becoming incorporated into intracellular fluids. Gastrointestinal absorption from food or water is the principal source of internally deposited cesium in the general population. Essentially all cesium that is ingested is absorbed into the bloodstream through the intestines. Cesium tends to concentrate in muscles because of their relatively large mass. Like potassium, cesium is excreted from the body fairly quickly. In an adult, 10% is excreted with a biological half-life of 2 days, and the rest leaves the body with a biological half-life of 110 days. Clearance from the body is somewhat quicker for children and adolescents. This means that if someone is exposed to radioactive cesium and the sources of exposure are removed, much of the cesium will readily clear the body along the normal pathways for potassium excretion within several months. Urinary excretion is the major route of elimination of cesium. The metabolism and tissue distribution of cesium-137 were studied in rats injected intraperitoneally and sacrificed 1–300 days post-injection. In a chronic study, rats were administered cesium-137 in their drinking water daily. In the acute study, with the exception of the brain, muscle, and total animal, all tissues showed retention curves resolvable into three exponential components with half-lives of 1.5–2, 5–8, and 15–17 days. Retention in muscles was resolvable into a two-exponential function with half-lives of 8 and 16 days. In the chronic study, the highest equilibrium cesium-137 concentrations, 10% of the average daily intake per gram, occurred in the muscle. The authors concluded that the muscle should be considered as the formal critical organ for cesium-137 (Ballou and Thompson, 1958; Bingham et al., 2012).

Mechanism of toxicity Stable cesium was shown to affect various central nervous system functions, mainly involving displacing potassium, with which it competes for transport through the potassium channel, and it can also activate sodium pump and subsequent transport into the cell across membranes. Thus, this resulted in potassium deficiency. Radioactive isotopes of cesium, such as 134Cs and 137Cs, are a greater health concern than stable cesium. These radioactive isotopes of cesium are formed during nuclear fission. Both 134Cs and 137Cs emit beta and gamma radiations. Beta radiation travels short distances and can penetrate the skin and superficial body tissues, whereas gamma radiation can travel great distances and penetrate the entire body. Both beta and gamma radiations may induce tissue damage and disruption of cellular function (Nordberg et al., 2007).

Acute and short-term toxicity (e.g., animal, human; oral, inhalation, dermal) Animal Animal studies indicated that stable cesium and its salts have relatively low toxicity, with acute oral LD50 values for rats and mice in the range 800–2000 mg kg−1, with the exception of cesium hydroxide, which is more toxic than cesium iodide or cesium chloride and has an intraperitoneal LD50 of 89 mg kg−1. Stable cesium is found to be a primary skin and eye irritant in rabbits and cutaneous sensitizer in pigs. Cesium can induce cardiac arrhythmias (Cochran et al., 1950; Bingham et al., 2012; Johnson et al., 1975).

Human Cesium has been reported to cause hyperirritability and muscle spasms. The symptoms of cesium toxicity include fatigue, muscle weakness, palpitations, and cardiac arrhythmia. Human data for cesium hydroxide are unavailable.

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Chronic toxicity (e.g., animal, human; oral, inhalation, dermal ) At very high exposure doses to radionuclides of 134Cs and 137Cs, the beta and gamma radiations can cause adverse effects, such as radiation syndrome (vomiting, nausea, and diarrhea) erythema, ulceration, tissue necrosis, neurological signs, chromosomal abnormalities, compromised immune function, and death (ATSDR, 2011; Bingham et al., 2012).

Immunotoxicity No data has been located regarding immunological effects in animals or humans following acute- or chronic-duration oral exposure to stable cesium.

Reproductive and developmental toxicity Exposure to radioisotopes of cesium may result in reduced fertility in males, as evidence by reduced concentration of spermatozoa in men who were exposed externally and internally to 137CsCl for about 1 month before testing. Reduced fertility, including sterility, was also reported in male mice exposed to gamma radiation from 137Cs. Developmental effects such as reduced postnatal body weight, impaired motor activity, morphological changes in the brain, reduced head size, and retarded odontogenesis and palatal closure had been reported in rats that were exposed to radioactive cesium sources (137Cs) in utero (ATSDR, 2011; Bingham et al., 2012).

Genotoxicity Chromosome damage has been induced by cesium chloride in the bone marrow cells of mice given a single oral dose and human blood cells in culture. The chloride was not mutagenic in an Ames bacterial test, but caused DNA damage in bacteria, as did carbonate, sulfate, and nitrate.

Carcinogenicity Animal studies indicate an increased risk of cancer following external and internal exposure to relatively high doses of radiation from 137Cs sources, but not from the nonradioactive element itself. An increased lifetime incidence of mammary tumors was noted in female rats that were acutely exposed to whole-body radiation. Intravenous injection of 137CsCl in dogs resulted in long-term increased risk of all cancers combined in males and females. However, studies of increased cancer risk specifically associated with exposure to humans to radioactive cesium isotopes were not found (Bingham et al., 2012).

Organ toxicity Cardiovascular and Gastrointestinal.

Clinical management Prussian blue is administered by a duodenal tube to act as a chelating agent, which binds to cesium chemically and reduces the biological half-life to 30 days (Dart, 2004).

Environmental fate and behavior Appearance: silvery white, soft, ductile metal; liquid (slightly above room temperature). Boiling point: 685  C. Solubility: Soluble in liquid ammonia. Naturally occurring cesium can enter the environment mostly from the erosion and weathering of rocks and minerals. The production and use of cesium compounds may also result in their release to the environment through various waste streams. However, there are relatively few commercial uses for cesium compounds, such as cesium radioactive isotopes (134Cs and 137Cs), and they have been released into the environment by human activities such as the atmospheric testing of nuclear weapons

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(1945–80) and leakages at nuclear power plants. Cesium compounds can travel long distances in the air before being brought back to the earth by rainfall and gravitational settling. If released to water, cesium compounds are deposited on land and water via wet and dry deposition. These deposited-cesium particles may be resuspended into the atmosphere from soil and dust. If released to soil, cesium compounds have low mobility and do not migrate below 40 cm in depth. The majority of cesium ions are retained in the upper 20 cm of the soil surface. Clay and zeolite minerals strongly bind cesium cations irreversibly. Soils rich in organic matter also adsorb cesium ions. However, cesium compounds are readily exchangeable and highly available for plant uptake in these soils. If released into water, cesium compounds are very water soluble and exist primarily as cesium cations. Because most cesium compounds are ionic, they will not volatilize from water surfaces. Most cesium compounds released to water adsorb to suspended solids in the water column and ultimately they are deposited in sediments. Cesium compounds bioconcentrate and have been shown to bioaccumulate in both terrestrial and aquatic food chains. The half-life of 134Cs is 2 years and that of 137Cs is 30 years (ATSDR, 2011).

Ecotoxicology Toxic effects to aquatic organisms by pH shift. Natural soil cesium concentrations are generally low but are nontoxic to plants. Consequently, cesium is not readily available for uptake by vegetation through their roots. However, radiocesium can enter plants upon falling onto the surface of leaves (Bingham et al., 2012).

Exposure standards and guidelines The National Institute for Occupational Safety and Health (NIOSH) recommended a limit of 2 mg m−3 of cesium hydroxide as an average for a 10-h workday, 40-h workweek. The EPA has established a maximum contaminant level of 4 mg per year for beta particles and photon radioactivity for synthetic radionuclides (including radioactive cesium) (ATSDR, 2011).

Other o Radioactive cesium is much more dangerous due to radioactivity and not the cesium itself. o Radioactive decay is a way of decreasing the amount of 134Cs and 137Cs in the environment.

PubChem URL Cesium | Cs - PubChem (https://pubchem.ncbi.nlm.nih.gov/compound/5354618).

See also: Lithium; Metals; Potassium; Sodium

References ATSDR (2011) Toxic Substance Portal, Cesium. Atlanta, GA: Agency for Toxic Substances & Disease Registry (ATSDR). Ballou JE and Thompson RC (1958) The metabolism of cesium-137 in the rat: Comparison of acute and chronic administration experiments. Health Physics 1: 85–89. Bingham E, Cohressen B, and Powell CH (2012) Patty’s Toxicology, 6th edn. Wiley Interscience: New York. Cochran KW, Doull J, Mazur M, and DuBois KP (1950) Acute toxicity of zirconium, columbium, strontium, lanthanum, cesium, tantalum and yttrium. Archives of Industrial Hygiene and Occupational Medicine 1: 637–650. Dart RC (2004) Medical Toxicology, 3rd edn. Philadelphia PA: Lippincott, Williams, and Wilkens 247–270. Johnson GT, Lewis TR, and Wagner WD (1975) Acute toxicity of cesium and rubidium compounds. Toxicology and Applied Pharmacology 32: 239–245. Nordberg GF, Fowler BA, Nordberg M, and Friberg LT (2007) Handbook on the Toxicology of Metals, 3rd edn. Academic Press.

Relevant website http://www.epa.gov :Cesium. US Environmental Protection Agency.

Charcoal Sara Mostafaloua and Perham Mohammadib, aDepartment of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran; bDepartment of Physiology and Pharmacology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran. © 2024 Elsevier Inc. All rights reserved. This is an update of M. Abdollahi, A. Hosseini, Charcoal, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 779–781, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00685-0.

Chemical profile Introduction Background Uses Exposure and exposure monitoring Routes and pathways Human exposure Toxicokinetics Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Toxicogenomics Clinical management Environmental fate and behavior Routes and pathways relevant physico-chemicals properties Partition behavior in water, sediment and soil Environmental persistency Bioaccumulation and biomagnifications Ecotoxicology Exposure standards and guidelines PubChem URL Conclusion References

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Abstract Charcoal is an odorless, tasteless, fine black powder obtained by removing water and other volatile constituents from animal and vegetation substances. It has a range of applications, but as a gastrointestinal decontaminant, charcoal is used to treat people who have ingested toxic substances. Charcoal is neither absorbed in the gastrointestinal (GI) tract nor metabolized. Due to its large surface area, it absorbs chemicals in the GI tract, trapping and carrying them out of the body without allowing them to be absorbed into the blood. Acute exposure to charcoal is irritating to the skin, eyes, and GI and respiratory tracts.

Keywords Activated charcoal; Carbon; Charcoal; Gut decontamination; Poisoning management

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Charcoal is a GI decontaminant agent. Charcoal is neither absorbed in the GI tract nor metabolized. Charcoal can irritate skin, eyes, and GI and respiratory tracts in acute exposure.

Chemical profile

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Name: Charcoal Chemical Abstracts Service Registry Number: 16291-96-6, Other registry number: 1333-85-3. Synonyms: Acticarbone, Activated carbon, Activated charcoal, Adsorba, Adsorbit, Anthrasorb, Black pearls, Carbolac, Carbomet, Carbon, CARBON BLACK, CHARCOAL, Charcoal activated, DIAMOND, Graphite, Tinolite Molecular Formula: Unspecified Chemical Structure:

Introduction Activated charcoal is originally the carbon that has been treated to create small, low-volume pores in order to increase the surface area available for adsorption.

Background The first use of charcoal comes from the black pigment used in European cave paintings around 32,000 years ago. It is possible that the earliest use of charcoal as a fuel in the smelting of copper began over 7000 years ago. The first definite evidence of human involvement with charcoal as a fuel goes back to 5500 years ago in the Middle East and Southern Europe, when the Egyptians, who were expert metal workers, discovered the smelting of iron using charcoal (WebExhibits, 2022).

Uses Charcoal has been used since the earliest times for several purposes, including medicine and art, but by far its most important use has been as a metallurgical, cooking, industrial, and automotive fuel. Charcoal is used as a conventional fuel where an intense heat is wanted. Charcoal was also used historically as a source of carbon black in chemical reactions by grinding it up. In this form charcoal was a constituent of formulas for mixtures such as gunpowder and was important to early chemists. Due to its high surface area, charcoal can be used as a catalyst, a filter, or an adsorbent. Other uses of charcoal include in the decolorizing of sugar, water and air purification, waste treatment, solvent recovery, removal of jet fumes from airports, removal of sulfur dioxide from stack gases and ‘clean’ rooms, deodorant, catalyst in natural-gas purification, brewing, chromium electroplating, and air conditioning (ESHA, 2022; PubChem, 2004).

Exposure and exposure monitoring Routes and pathways Occupational exposure to charcoal may occur through inhalation and dermal contact with this compound at workplaces where it is produced or used. The general population may be exposed to charcoal via dermal contact with consumer products containing carbon black (Bautista et al., 2009; ESHA, 2022).

Human exposure Charcoal can be absorbed into the body by inhalation. In addition to inhalation, exposure can be occurred by skin and/or eye contact (ESHA, 2022; PubChem, 2004).

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Toxicokinetics Activated charcoal is neither absorbed in the gastrointestinal (GI) tract nor metabolized and excreted in the feces. The adsorptive capacity of activated charcoal may be decreased by concurrent use of isoosmolar electrolyte solution and polyethylene glycol for whole-bowel irrigation. The adsorptive efficacy of activated charcoal may also be decreased by the emesis induced by ipecac syrup. In general, activated charcoal can reduce the absorption of and therapeutic response to other orally administered drugs; therefore, medications other than those used for GI decontamination or antidotes for ingested toxins should not be taken orally within at least 2 h of administration of activated charcoal. When concomitant drug therapy is needed, drugs can be given parenterally if activated charcoal has been administered (PubChem, 2004).

Mechanism of toxicity Due to its large surface area, charcoal exerts its effects by absorbing a wide variety of drugs and chemicals. After the toxic substance attaches to the surface of the charcoal and because charcoal is not absorbed, it stays inside the GI tract, being eliminated in the feces along with the charcoal. In single-dose therapy, activated charcoal adsorbs some of the toxic substance ingested, and thus inhibits GI absorption which prevents or reduces toxicity. When used repeatedly, as in multiple-dose therapy, activated charcoal also creates and maintains a concentration gradient across the wall of the GI tract that facilitates passive diffusion of the toxic substance from the blood stream into the GI tract lumen, where it is adsorbed onto the charcoal and thus prevented from reabsorption. In this process, activated charcoal also interrupts the enterohepatic or enteroenteric cycle or recirculation and increases the rate of elimination of the toxic substance from the body. Use of multiple-dose charcoal in this manner is also called GI (gut) dialysis (PubChem, 2004; Skov et al., 2021).

In vitro toxicity data There is no available data on toxicity of charcoal in vitro.

Acute and short-term toxicity Animal Special remarks on toxicity of charcoal to animals are not available. The lethal doses of charcoal in animal models including dog, mouse and rat by intraperitoneal, subcutaneous, and oral administration have been estimated to be higher than 5 g/kg body weight. The LD50 of charcoal in rat through oral administration has been estimated to be higher than 10,000 mg/kg body weight. The LD50 of charcoal in mouse through intravenous administration has been reported 440 mg/kg body weight (PubChem, 2004).

Human Acute exposure to charcoal irritates skin, eyes, GI track, and the respiratory tract. Redness, swelling, and pain may occur with dermal exposure. Stinging pain, watering of eyes, inflammation of eyelids, and conjunctivitis may happen with occular exposure. Aspiration pneumonitis, decreased GI transit time of ingested substances, vomiting, GI obstruction, constipation, pulmonary fibrosis and subsequent emphysema, intestinal perforation, charcoal deposits in the esophageal and gastric mucosa, and ulceration of rectal mucosa are possible if charcoal is ingested. Cough, tachypnea, wheezing, rapid irregular breathing, headache, fatigue, mental confusion, nausea, and vomiting may occur after inhalation. Burning charcoal in a fireplace or in a grill may be hazardous as carbon monoxide (CO) is produced. CO is a tasteless, odorless, invisible gas that is toxic when inhaled in significant amounts. Symptoms of CO poisoning include headaches, confusion, dizziness, nausea, and, at high concentrations, loss of consciousness and death (PubChem, 2004).

Chronic toxicity Animal Special remarks on charcoal toxicity to animals are not available. There are some reports on the biochemical changes by inducing inflammation and oxidative stress in animal studies evaluating carbon derivatives toxicities (PubChem, 2004).

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Human Chronic exposure to charcoal may damage mucous membranes and lungs. Chronic skin exposure can result in dryness, rashes, and clogging of hair follicles, rendering them black. Chronic inhalation can cause accumulation of carbon particles in the lungs. Chronic exposure to coal causes a pneumoconiosis called coal workers pneumoconiosis or black lung disease. No evidence has been found for the equivalent with occupational exposure to charcoal (PubChem, 2004).

Immunotoxicity There is not enough evidence on immunotoxicity of charcoal in animals or human.

Reproductive and developmental toxicity There is no evidence that charcoal causes reproductive or developmental problems.

Genotoxicity No genotoxic effect was confirmed for charcoal.

Carcinogenicity Charcoal is not listed as a potential carcinogen by any agency. But tumorigenic effects in lung, thorax, and respiratory system by inhalational exposure of rat to carbon derivatives have been reported. According to the American Conference of Governmental Industrial Hygienists (ACGIH), charcoal is classified in the A4 group (Not classifiable as a human carcinogen). The International Agency for Research on Cancer (IARC) reported that there is inadequate evidence in humans for the carcinogenicity of carbon black while there is sufficient evidence in experimental animals for the carcinogenicity of carbon black. According to IARC classification, carbon black has been listed in the Group 2B (possibly carcinogenic to humans) (PubChem, 2004).

Organ toxicity inhalational exposure to carbon can irritate respiratory system leading to the symptoms including cough, dyspnea (breathing difficulty), black sputum, decreased pulmonary function, lung fibrosis. Skin and eye contact can also cause irritation and redness (ESHA, 2022; PubChem, 2004).

Interactions The distribution of carbon black throughout the lung tissue can provide a greater surface area for desorption and resorption of the carcinogen benzopyrene. Additive as well as synergistic interaction effects of 14 nm or 95 nm ultrafine carbon black particles and staphylococcal lipoteichoic acid (LTA) have been indicated in producing early pulmonary inflammatory responses in male BALB/c mice. In a study, intratracheal instillation of ozone and carbon black, alone and/or in combination, to the rats showed that these two agents have counteractive effects. Ozone was shown to stimulate phagocytotic and chemotactic activity of alveolar macrophages whereas carbon black caused retardation of these activities. In clinical research, it has been shown that concurrent use of polyethylene glycol and isoosmolar electrolyte solution (PEG-ELS) for whole-bowel irrigation can decrease adsorptive capacity of activated charcoal. Concurrent use of activated charcoal and ipecac syrup in the management of oral poisoning can reduce efficacy of both agents. Activated charcoal has been reported to adsorb ipecac alkaloids. So, it is recommended that activated charcoal should be administered after vomiting has ceased. In general, activated charcoal can decrease the absorption of and therapeutic response to other orally administered drugs. Medications other than those used for GI decontamination or antidotes for ingested toxins should not be taken orally within 2 h of administration of activated charcoal; if necessary, concomitant drug therapy can be given parenterally (PubChem, 2004).

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Toxicogenomics Based on the literature, charcoal or generally carbon, has been shown to interact with various genes and enzymes in the living organisms. The most reported genes and enzymes having interaction with charcoal include slc28a3, gpkow, ribulose-bisphosphate carboxylase, peroxidase, axin2, cellulase, and glucose oxidase (CTD, 2022).

Clinical management In oral exposure, inducing emesis is not indicated due to the irritant nature of charcoal, in particular if aspiration occurs. Dilution is recommended with 120–240 ml of water or milk, not exceeding 120 ml in children. In inhalation exposure, move the patient to fresh air and monitor for respiratory distress. If cough or difficulty in breathing develops, the patient should be evaluated for respiratory tract irritation, bronchitis, or pneumonitis. As the patient is being evaluated assist ventilation and administer oxygen as required. Bronchospasm can be treated with inhaled b2 agonists and oral or parenteral corticosteroids. In ocular exposure to charcoal, wash the exposed eyes with copious amounts of room-temperature water for at least 15 min and if pain, irritation, swelling, lacrimation, or photophobia persists, the patient should be seen in a health care facility (ESHA, 2022).

Environmental fate and behavior Routes and pathways relevant physico-chemicals properties Charcoal is an odorless, tasteless, fine black powder or black porous solid. It is typically encountered as course granules or powder. It is insoluble in water and also in organic solvents. Other physical properties include specific gravity: 0.08 to 0.5; heat of combustion: 14100 Btu/lb. ¼ 7830 cal g−1 ¼ 3.28  10 + 5 J kg−1; boiling point: 4200  C (PubChem, 2004).

Partition behavior in water, sediment and soil The presence of charcoal in a compound elevates the Koc value, resulting in reduction of mobility of compounds through the sediment and soil (PubChem, 2004).

Environmental persistency Charcoal is stable under ambient environmental conditions (PubChem, 2004).

Bioaccumulation and biomagnifications Hazardous short-term degradation products of charcoal are not likely. Charcoal and its products of degradation are not toxic. Special remarks on the products of biodegradation are not available (PubChem, 2004).

Ecotoxicology Charcoal in its original state is not harmful to the environment. No specific information is available on the effect of charcoal on animals or plants in the environment. No specific information is available on the effect of charcoal on aquatic life (PubChem, 2004).

Exposure standards and guidelines Drug products containing charcoal are offered over the counter for certain uses for instance in GI tract. Charcoal (activated) is included in antidiarrheal and digestive aid drug products. The US Consumer Product Safety Commission regulations require that two highly visible warning labels are included at the top of every bag of charcoal briquettes that identify the hazard of carbon monoxide poisoning. The US Occupational Safety and Health Administration: Hazardous by definition of Hazard Communication Standard (29 CFR 1910.1200). This product is on the European Inventory of Existing Commercial Chemical Substances. This product is not classified according to the EU regulations. Not applicable (OSHA, 2020).

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/5462310

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Conclusion Charcoal is a GI decontaminant agent which is neither absorbed in the GI tract nor metabolized. In acute exposure through inhalation, dermal or eye contact, charcoal can irritate respiratory tracts, skin, and eyes leading to cough, dyspnea, and redness. There are no strong evidences on reproductive and developmental toxicity, as well as genotoxicity, carcinogenicity and immunotoxicity of charcoal.

References Bautista LE, Correa A, Baumgartner J, Breysse P, and Matanoski GM (2009) Indoor charcoal smoke and acute respiratory infections in young children in the dominican republic. American Journal of Epidemiology 169: 572–580. https://doi.org/10.1093/aje/kwn372 %J American Journal of Epidemiology. CTD (2022) Charcoal. Comparative Toxicogenomics Database. http://ctdbase.org/detail.go?type¼chem&acc¼D002606. 2022. ESHA (2022) Carbon Black. European Chemical Agency. https://echa.europa.eu/registration-dossier/-/registered-dossier/16056/9. 2022. OSHA (2020) Carbon Black. Occupational Safety & Health Administration. https://www.osha.gov/chemicaldata/236. PubChem (2004) PubChem Compound Summary for CID 5462310, Carbon. National Center for Biotechnology Information. https://pubchem.ncbi.nlm.nih.gov/compound/Carbon. 2021. Skov K, Graudal NA, and Jürgens G (2021) The effect of activated charcoal on drug exposure following intravenous administration: A meta-analysis. Basic & Clinical Pharmacology & Toxicology 128: 568–578. https://doi.org/10.1111/bcpt.13553. WebExhibits How Carbon Black is Made. webexhibits.org. http://www.webexhibits.org/pigments/indiv/recipe/charcoal.html. 2022

Chemical hazard communication and safety data sheets María J Ramos-Peralonso*, Occupational Risk Prevention and in Social Communication and Health, Madrid, Spain © 2024 Elsevier Inc. All rights reserved. This is an update of M.J. Ramos-Peralonso, Chemical Hazard Communication and Safety Data Sheets, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 787–792, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00460-7.

Introduction A globally harmonized classification and labeling system (GHS) Classification of chemical substances and mixtures Labeling of hazardous chemical substances and mixtures Safety data sheets The GHS implementation The EU extended SDSs with exposure scenarios Chemical hazard communication for substances in articles Conclusion References

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Abstract Communicating the hazards of chemical substances to workers, consumers and emergency services is essential for ensuring a safe use of hazardous chemicals. As the communication covers both industrial/professional users and consumers, different strategies are needed. The classification and labeling of those chemicals considered as hazardous is the first and more important hazard communication tool. This classification and labeling is applied to substances marketed on its own or as mixtures and covers all users including consumers. The United Nations Organization has harmonized the classification and labeling worldwide through the globally harmonized system. The safety data sheets (SDSs), previously named as material SDSs in the United States and other jurisdictions, expand the information provided to industrial and professional users. The communication of the hazards related to substances in articles has received less attention; however, in the European, Union, the REACH Regulation has created new hazard communication obligations for those substances identified as of very high concern and included in the ‘candidate list’ for authorization.

Keywords Candidate list; Classification and labeling; Globally harmonized system; Hazard communication; Hazardous substances; REACH; Safety data sheets; Substances of very high concern

Key points

• • • •

The communication of chemical hazards is based on a classification and labeling system. Different jurisdictions and sector developed their own systems; for facilitating the communication in a globalized environment, the UN developed a Globally Harmonized System, also applicable to transport. Pictograms and other mandatory elements in the label of recipients containing hazardous substances and mixtures constitute the first communication tool. For professional users including workers the communication is complemented with safety data sheets.

Introduction The use of chemical products is a widespread practice not only at industrial and professional levels but also in the domestic environment. Alongside the benefits of these products, many chemicals have also the potential to cause adverse effects on human health and the environment. This potential is based on the intrinsic properties of the chemical, which can be used for setting specific concerns and information to be communicated to the users. The properties include some physical characteristics, such as flammability or corrosion, health concerns related to its toxicity to humans, and environmental concerns related to its ecotoxicity for environmental organisms and other environmental concerns. The identification of the specific hazards of each chemical substance is essential for ensuring a safe management during manufacturing, transport, use, and disposal. The identification of the relevant hazards of each marketed chemical product should be communicated through the supply chain to ensure that all users, including consumers, are aware of these potential hazards and take the appropriate precautions and implement protective measures.


Former Director and CEO of Green Planet Environmental Consulting.

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Chemicals are used worldwide in the workplace and by consumers; and the hazard communication strategy must consider the needs and possibilities of the different target audiences. For workers, communication on chemical hazards should be included in the general safety and health training, which requires dedicated professionals and comprehensive material (Sinyai et al., 2018; Scott et al., 2019). For consumers, the available tools are information campaigns and dissemination activities, such as the Infocards developed by the European Chemicals Agency (ECHA, 2018). In addition, there are specific communication needs during transport and in the case of emergencies. Employers and workers manufacturing and using the chemicals need to know the specific hazards of the chemicals they are or may be exposed to in the workplace, the protective measures required to avoid the adverse effects that might be caused by those chemicals, the proper storage and disposal conditions, and the mitigation measures to be adopted in case of unintended spills or accidental exposure. Labeling is the primary source of information but it is combined with other elements such as the safety data sheet (SDS). Comprehension can be enhanced by training when provided. In principle, consumers have similar needs but the label is, in most cases, the sole source of information. The hazard pictograms are far from being self-explanatory (European Commission, 2011) and awareness-raising activities are essential (ECHA, 2012). In addition, the hazard pictograms and phrases are integrated in the over label, and while simplification is essential (Geuens et al., 2021) it is not sufficient and should be complemented to maximize comprehension. There are two different approaches for the hazard communication to consumers, one focus on effectiveness and is based on the likelihood of injury, the other focuses on the ‘right to know and to take educated decisions’ and is based on the communication of hazards independently of the likelihood for those hazards to result in adverse effects. It should be noted that even well-informed consumers have misconceptions and difficulties for a proper understanding of chemical hazards (Hartmann and Klaschka, 2017). The transport of dangerous chemical products requires communication on safe practices to different types of dedicated workers, from drivers to those handling directly the products. The main elements are the conditions for proper handling, e.g., during storage, loading, and unloading, as well as in the case of accident. Similarly, emergency responders have specific hazard communication needs in order to facilitate immediate and adequate responses. The needs for firefighters, those involved in the contention, cleaning, and risk assessment, or the medical personnel treating the victims are very different and should be considered.

A globally harmonized classification and labeling system (GHS) The chemical hazard communication is mostly based on the classification and labeling system, which supports additional downstream implementation tools. Chemicals are classified as hazardous following expert assessments of their dangerous properties; and, following the regulatory implementation of the proposed classification, must be labeled when distributed to users and marketed. The classification system not only identifies that the chemical is hazardous, but also informs on which particular hazards are relevant for each chemical substance or mixture, as well as its potency. During the twentieth century, different sectors and jurisdictions developed their own classification and labeling systems, creating confusion and reclassification/relabeling obligations when hazardous chemicals were transported among countries/regions. The harmonization of the classification and labeling systems of chemicals started, in fact, in relation to the need for safe international transport of dangerous goods. The United Nations Economic and Social Council’s Committee of Experts on the Transport of Dangerous Goods (UNCEDTG) started a process to harmonize physical hazards and acute toxicity in the transport sector. However, transport requirements in countries were often not harmonized with those of other sectors in that country and particularly in relation with the workplace or consumer sectors. Chapter 19 of Agenda 21, adopted at the United Nations Conference on Environment and Development (UN, 1992), provided the international mandate to develop a single, globally harmonized system (GHS) to address classification of chemicals, labels, and SDSs. The work was coordinated and managed under the auspices of the Interorganization Program for the Sound Management of Chemicals (IOMC) Coordinating Group for the Harmonization of Chemical Classification Systems (CG/HCCS). It required a long-term commitment from the three technical focal points:

• • •

The International Labour Organization (ILO) for the hazard communication; The Organization for Economic Cooperation and Development (OECD) for the classification of health and environmental hazards; and The United Nations Sub-Committee of Experts on the Transport of Dangerous Goods (UNSCETDG) and ILO for the physical hazards.

The examination of existing systems at that moment clarified that although many countries had some requirements; four ‘major’ existing systems need to be considered: 1. 2. 3. 4.

Requirements of systems in the United States for the workplace, consumers, and pesticides. Requirements of Canada for the workplace, consumers, and pesticides. European Union directives for classification and labeling of substances and preparations. The UN recommendations on the transport of dangerous goods.

These systems were the basis for developing the GHS of classification and labeling of chemicals.

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The first version of the GHS was adopted in December 2002 by the Sub-Committee on the GHS (SCEGHS), and endorsed by the Committee on the Transport of Dangerous Goods and the GHS of Classification and Labeling of Chemicals. The first version of the GHS was published in 2003 (UN, 2003). The system has been amended biannually. The first revised edition of the GHS (GHS Rev. 1, published in 2005) included various revised provisions concerning classification and labeling, new provisions for aspiration hazards, and new guidance on the use of precautionary statements and pictograms and on the preparation of SDSs. The second revised edition of the GHS (GHS Rev. 2, published in 2007) included new and revised provisions concerning the classification and labeling of explosives, respiratory and skin sensitizers, toxic by inhalation gases and gas mixtures, additional guidance on the interpretation of the building block approach and on the evaluation of the carcinogenic potential of chemicals, and the codification of hazard and precautionary statements. The third revised edition of the GHS (GHS Rev. 3, published in 2009) included new provisions for the allocation of hazard statements and for the labeling of small packaging, two new subcategories for respiratory and skin sensitization, the revision of the classification criteria for long-term hazards (chronic toxicity) to the aquatic environment, and a new hazard class for substances and mixtures hazardous to the ozone layer. The fourth revised edition of the GHS (GHS Rev. 4, published in 2011) included new hazard categories for chemically unstable gases and nonflammable aerosols, further rationalization of precautionary statements, and further clarifications of some of the criteria to avoid differences in their interpretation. The fifth revised edition of the GHS (GHS rev.5, published in 2013) included new test methods for oxidizing solids and further clarifications on different criteria and the information to be included in the SDS as well as a new codification system for the pictrograms. The sixth revised edition of the GHS (GHS rev.6, published in 2015) included new hazard categories and classes for physical hazards and further clarifications on different criteria and the information to be included in the SDS. The seventh revised edition of the GHS (GHS rev.7, published in 2017) included revised criteria, clarifications and additional guidance for transport and labeling. The eight edition of the GHS (GHS rev.8, published in 2019) included new classification criteria, hazard communication elements, decision logics and guidance for chemicals under pressure; new provisions for the use of in vitro/ex vivo data and non-test methods to assess skin corrosion and skin irritation; additional clarifications and examples, as well as guidance on the identification of dust explosion hazards and the need for risk assessment, prevention, mitigation, and hazard communication. The most recent (UN, 2021), is the ninth edition of the GHS (GHS rev.9, published in 2021), which included the revision of chapter 2.1 (explosives), the revision of decision logics and classification and labeling summary tables in Annex 1, further rationalization of precautionary statements and the updating of references to OECD test guidelines for the testing of chemicals in annexes 9 and 10. This revised version contains four parts and 10 annexes (11 are listed but one is empty):

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Part 1: Introduction Part 2: Physical hazards Part 3: Health hazards Part 4: Environmental hazards Annex 1: Classification and labeling summary tables Annex 2: (reserved) Annex 3: Codification of hazard statements, codification and use of precautionary statements, codification of hazard pictograms, and examples of precautionary pictograms. Annex 4: Guidance on the preparation of SDSs. Annex 5: Consumer product labeling based on the likelihood of injury. Annex 6: Comprehensibility testing methodology. Annex 7: Examples of arrangements of the GHS label elements. Annex 8: An example of classification in the GHSs. Annex 9: Guidance on hazards to the aquatic environment. Annex 10: Guidance on transformation/dissolution of metals and metal compounds in aqueous media. Annex 11: Guidance on other hazards not resulting in classification.

The GHS describes the classification criteria and the hazard communication elements by type of hazard and covers the classification and labeling of chemical substances in its own and of mixtures. It covers 17 physical hazard classes, 10 health hazard classes, and 2 environmental hazard classes. It also includes details on the labeling and guidance on other hazard communication elements such as the SDS.

Classification of chemical substances and mixtures Classification is the process for identifying the physical, health, and environmental hazards of a substance or a mixture and it is done by experts which compare the intrinsic properties of the substance, measured through standardized assays or coming from other information sources, with defined criteria in order to propose the classification of the substance or mixture. It follows three

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consecutive steps: (1) the identification and examination of relevant data regarding the potential hazards of the substance or mixture; (2) the comparison of the data with the classification criteria; and (3) the decision on whether the substance or mixture shall be classified as hazardous in relation to each of the hazard classes, and the degree of hazard, where appropriate. The classification system is based on a set of hazard classes which describe the nature of the physical, health, or environmental hazard, and a set of categories, within each class, which describe the severity of the harm. These categories may represent the potency with quantitative criteria for the allocation of the chemical within the category, e.g., in the case of acute toxicity or aquatic toxicity, or the extend/type of evidence, e.g., for carcinogens or chemicals toxic to reproduction. For example, acute toxicity is based on the adverse effects occurring after the administration of a single dose (or multiple doses within 24 h for oral and dermal routes and 4 h for inhalation) and is mostly based on the oral/dermal LD50 and inhalation LC50 observed in animals under experimental standardized conditions. This hazard class contains five categories (Category 1–5) for each exposure route defined by fixed ranges in the oral/dermal LD50 and inhalation LC50. Consequently, chemicals with similar acute toxicity potencies are classified within the same category. Regarding carcinogenicity, the criteria are based on the strength and type of the evidence and distinguish three different types: chemicals known to have carcinogenic potential for humans, chemicals presumed to have carcinogenic potential for humans (mostly based on evidence on animals), and suspected human carcinogens. The potency or dose at which the effects have been observed is not part of the criteria and the classification does not provide information on the level of expected human cancer risk associated to a particular level of exposure. For the environment, the criteria cover aquatic organisms and the ozone layer. Initila criteria were refined allowing the use of chronic aquatic toxicity (González-Doncel et al., 2006), while proposals for covering terrestrial organisms (Tarazona and Vega, 2002) have not been implemented yet. Mixtures can be classified similarly to substances, i.e. by comparing directly their intrinsic properties measured through standardized methods with the classification criteria, or by applying a set of equations based on the classification of their components. It should be noted that in some occasions each system provides different classification results (Kurth et al., 2019). The classification in a particular hazard class and category implies the need to communicate this information through the labeling and other hazard communication elements such as the SDS.

Labeling of hazardous chemical substances and mixtures As described above, each hazard class and category has a set of associated labeling requirements. The GHS labeling approach is based on four complementary label elements: the symbol, the signal word, the hazard statements, and the precautionary statements. The nine GHS hazard symbols are basically similar to those used in the UN transport system, except one specific for certain health hazards but are presented in a specific graphic composition (shape, border, background pattern, and color), the pictogram, which is GHS specific: a square set at a point with a black symbol on a white background with a red frame (see Fig. 1). The signal words are words used to indicate the relative level of severity of the hazard and to alert the reader to a potential hazard on the label. The word ‘danger’ is used for the more severe hazard categories and the word ‘warning’ is used for intermediate categories, while no signal word is allocated to the less severe categories. The hazard statements are phrases assigned to each hazard class and category describing the nature of the hazards and its degree when appropriate. Each hazard statement is associated to a code, which starts with the letter ‘H,’ the number 2, 3, or 4 for physical, health, and environmental hazards, respectively, and two additional digits with sequential numbering. The precautionary statements are phrases (and/or pictograms) that describe the measures recommended to minimize or prevent adverse effects resulting from the exposure to a hazardous product or its improper storage or handling. Each precautionary statement is associated to a code which starts with the letter ‘P,’ the number 1, 2, 3, 4, or 5 for general, prevention, response, storage, and disposal statements, respectively, and two additional digits with sequential numbering. Annex 3 of the GHS includes two sections, Sections 2 and 3, detailing the link between the classification of the substance and the precautionary statements that should be considered in the label. The label should also include a product identifier and information on the supplier. A particularly relevant annex regarding communication is Annex 6, which describe the methodology for assessing the comprehensively of the labels and SDSs by the target population. Includes 11 modules with general recommendations, questionaries, exercises and simulations in order to check that the proposed labels and SDSs are understood by workers and citizens.

Safety data sheets SDSs, previously named as material SDSs in the United States and other jurisdictions, are the complementary hazard communication tool for users and professional at the workplace. The SDS provides employers and workers, including those involved in transport and emergency personnel, with procedures for handling, storing and disposing the substance or mixture in a safe manner, advice on safety precautions, and to react properly in the case of accidental spills, mishandling, or emergencies. The SDS is in fact the main tool for transferring detailed information on hazardous substances, as such or in mixtures, to workers and professional users through the supply chain. It is product specific and according to the GHS should be provided at least for all substances and mixtures that meet one or more of the classification criteria, as well as for mixtures which contain ingredients that meet the criteria for carcinogenic, toxic to reproduction or specific target organ toxicity in concentrations exceeding the selected

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Fig. 1 GHS pictograms.

cutoff criteria. The competent authorities may also choose to require SDSs for mixtures not meeting the criteria for classification as hazardous but which contain hazardous substances in certain concentrations. Annex 4 of the GHS provides guidance for drafting SDS, while Annex 6 provides a tool for checking the comprehensibility of the text by the targeted population. The information in the SDS has been harmonized and should be presented using 16 specific headings: 1. Identification • GHS product identifier. • Other means of identification. • Recommended use of the chemical and restrictions on use. • Supplier’s details (including name, address, phone number, etc.) • Emergency phone number. 2. Hazard(s) identification • GHS classification of the substance/mixture and any national or regional information. • GHS label elements, including precautionary statements. (Hazard symbols may be provided as a graphical reproduction of the symbols in black and white or the name of the symbol, e.g., flame, skull, and crossbones.) • Other hazards which do not result in classification (e.g., dust explosion hazard) or are not covered by the GHS. 3. Composition/information on ingredients (Note: For information on ingredients, the competent authority rules for CBI take priority over the rules for product identification) • For substances o Chemical identity. o Common name, synonyms, etc. o CAS number, EC number, etc. o Impurities and stabilizing additives which are themselves classified and which contribute to the classification of the substance. • For mixtures o The chemical identity and concentration or concentration ranges of all ingredients which are hazardous within the meaning of the GHS and are present above their cutoff levels.

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4. First aid measures • Description of necessary measures, subdivided according to the different routes of exposure, i.e., inhalation, skin and eye contact, and ingestion. • Most important symptoms/effects, acute, and delayed. • Indication of immediate medical attention and special treatment needed, if necessary. 5. Firefighting measures • Suitable (and unsuitable) extinguishing media. • Specific hazards arising from the chemical (e.g., nature of any hazardous combustion products). • Special protective equipment and precautions for firefighters. 6. Accidental release measures • Personal precautions, protective equipment, and emergency procedures. • Environmental precautions. • Methods and materials for containment and cleaning up. 7. Handling and storage • Precautions for safe handling. • Conditions for safe storage, including any incompatibilities. 8. Exposure controls/personal protection • Control parameters, e.g., occupational exposure limit values or biological limit values. • Appropriate engineering controls. • Individual protection measures, such as personal protective equipment (PPE). 9. Physical and chemical properties • Basic physical and chemical properties specified by Chapter 1.45 Table 1.5.2., such as physical state, color, odor, pH, melting point/freezing point, initial boiling point and boiling range, flash point, vapor pressure, solubility(ies), partition coefficient: n-octanol/water, etc. • Data relevant with regard to physical hazard classes, such as sensitivity to shock for explosives, critical temperature for gases under pressure, etc. • Further safety characteristics, such as mechanical sensitivity, self-accelerating polymerization temperature, formation of explosible dust/air mixtures, etc. 10. Stability and reactivity • Reactivity. • Chemical stability. • Possibility of hazardous reactions. • Conditions to avoid (e.g., static discharge, shock, or vibration). • Incompatible materials. • Hazardous decomposition products. 11. Toxicological information: Concise but complete and comprehensible description of the various toxicological (health) effects and the available data used to identify those effects, including: • Information on the likely routes of exposure (inhalation, ingestion, and skin and eye contact); • Symptoms related to the physical, chemical, and toxicological characteristics; • Delayed and immediate effects and also chronic effects from short- and long-term exposure. • Numerical measures of toxicity (such as acute toxicity estimates). • Interactive effects. • Where specific chemical data are not available, data on the chemical class of statement indicating which information is not available. • For mixtures, details on the classification and information available. • Other information on adverse health effects even if not required by the GHS classification system. 12. Ecological information • Toxicity (aquatic and terrestrial, where available). • Persistence and degradability. • Bioaccumulative potential. • Mobility in soil. • Other adverse effects. 13. Disposal considerations • Description of waste residues and information on their safe handling and methods of disposal, including the disposal of any contaminated packaging. 14. Transport information • UN number. • UN proper shipping name. • Transport hazard class(es). • Packing group, if applicable. • Environmental hazards (e.g., marine pollutant (yes/no)).

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• •

Special precautions for user Transport in bulk according to IMO instruments 15. Regulatory information: • Safety, health, and environmental regulations specific for the product in question. 16. Other information

The GHS implementation New Zealand was the first country in implementing the GHS. Since then, the GHS has been already implemented, at least partially, in over 80 countries worldwide. In order to allow flexibility, the GHS has been developed for allowing an implementation as “building blocks,” thus each jurisdiction can decide the relevant elements to be implemented at national level. Detailed information on the level of implementation is provide by UNECE. In addition to national provisions, the implementation has been also facilitated through international regional agreements. In the European Union (EU) and European Economic Area (EEA), the GHS was implemented through Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008, the CLP Regulation, complementing the REACH Regulation. The European Chemicals Agency provides guidance and has created tools for disseminating the information; including the largest and most extensive inventory on substances classified according to the GHS, created as part of the requirements of the CLP Regulation. The inventory includes the self-classification by manufacturers and importers of all substances marketed, as such or in mixtures, in the EU and contains about 6 million notifications for more than 120.000 substances. In addition to the EU, other regions have developed international agreements for implementing the GHS. In de ANDEAN Community is implemented for transport and for pesticides, in MERCOSUR is implemented for transport, and is expected implementation in the Eurasian Economic Union during 2022.

The EU extended SDSs with exposure scenarios The SDS information acts as a reference source for the management of hazardous chemicals in the workplace. The SDS is product related and, usually, is not able to provide specific information that is relevant for any given workplace where the product may finally be used. Instead, the information is designed to enable employers to develop active programs for worker protection measures specific to individual workplaces and to consider any measures, which may be necessary to protect the environment. However, the EU chemicals legislation implemented through the REACH and CLP Regulations has gone a step further, considering that the SDS should also provide specific information relevant to the assessment of each use. The UE legislation requires extended SDSs indicating the uses that are covered by the provider of the substance (and the uses advised against and the reasons for this advice). In addition, the exposure scenarios for each relevant use should be attached. These exposure scenarios present clear indications regarding the operational conditions for proper handling of the substance, the risk reduction measures that should be applied to reduce the exposure and risks, and the resulting exposure level, covering the workplace, the environment, and consumers when relevant, if possible in quantitative terms. Updated guidance is available from the European Chemicals Agency (ECHA, 2020). This EU approach combines the standard approach of SDS as a hazard (not risk) communication tool, with additional information on use patterns and expected exposure. Consequently, it offers new opportunities for the employers, facilitating their obligation to ensure a safe use of hazardous chemicals at the workplace, including environmental emissions. European downstream users are expected to implement the operational conditions and risk management measures indicated in the extended SDS for each described use pattern, as part of their obligations to ensure a safe handling and use of hazardous products. If a downstream user is unable to implement these conditions, must conduct its own safety assessment, which in certain cases should be notified to ECHA. The use patterns, and the associated exposure assessment tools (Schlueter and Tischer, 2020), are based on a use descriptor system, which consist of five separate descriptor lists which, in combination with each other, form a brief description of use. The five descriptors are the following: 1. The sector of use category (SU) describes in which sector of the economy the substance is used. This includes mixing or repacking of substances at formulator’s level as well as industrial, professional, and consumer end-uses. 2. The chemical product category (PC) describes in which types of chemical products (¼substances as such or in mixtures) the substance is finally contained when it is supplied to end-uses (by industrial, professional, or consumer users). 3. The process category (PROC) describes the application techniques or process types defined from the occupational perspective. 4. The environmental release category (ERC) describes the broad conditions of use from the environmental perspective. 5. The article category (AC) describes the type of article into which the substance has eventually been processed. This also includes mixtures in their dried or cured form (e.g., dried printing ink in newspapers; dried coatings on various surfaces). In addition to their description function, these use descriptors define the name of the exposure scenario and support the identification of the suitable exposure estimation. In fact, some descriptors are directly associated to one of the available Tier 1

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exposure estimation tools (Schlueter and Tischer, 2020) developed for the implementation of REACH, allowing an initial release/ exposure estimation based on default values, which can be used for demonstrating safe use or as the starting point for the exposure refinement if needed. As a complement, the EU REACH Regulation has extended the need to provide the SDS to other chemicals not covered by the GHS criteria. These include the PBT and vPvB substances. In addition, an SDS should be provided for mixtures that contains at least one substance on the candidate list of substances of very high concern (SVHC) and the individual concentration of this substance in the mixture is  0.1% (w/w) for nongaseous mixtures if the substance is persistent, bioaccumulative, and toxic (PBT) or very persistent and very bioaccumulative (vPvB).

Chemical hazard communication for substances in articles The communication of the hazards related to substances in articles has received less attention. The classification and labeling approach is restricted to ‘chemical products’: substances in its own or as mixtures. The hazards, of chemical or other nature, in articles intended for industrial, professional, or consumer use are communicated as elements in the label or just as indications in the instruction pamphlets that are distributed with the articles. With few exceptions, these obligations have not been harmonized and defined labeling requirements for covering specifically chemical risks in articles have not been developed yet. A typical exception is the classification of explosive articles in the EU, as the CLP Regulation applies the GHS principles to explosive articles. Nevertheless, the need to communicate the chemical hazards of articles to users, including consumers, is receiving significant attention. In the European Union, the REACH Regulation has created new hazard communication obligations for those substances identified as of very high concern and included in the ‘candidate list’ of SVHC. EU or EEA suppliers of articles which contain substances on the candidate list in a concentration above 0.1% (w/w) have to provide sufficient information to allow safe use of the article to their customers. The obligation is also applied to consumers’ articles if requested by the consumer. This information must contain as a minimum the name of the substance, and as a complement, ECHA published in the web page the notifications received from producers and importers of articles regarding the presence of SVHC in their articles. However, additional efforts are still needed in order to fully implement the societal concerns for access to the information and the ‘right to know’ in a way that could really allow educated decisions by each consumer regarding personal assessments of the risk and benefits related to the presence of hazardous chemicals in consumer articles. In addition, this information is essential for proper management during the end-of-service step of the article, supporting recycling and circular economy. Under the 2018 revision of the EU Waste Framework Directive, new obligations for communication were introduced, and since January 2021, companies supplying articles that contain more than 0.1% weight by weight of substances of very high concern, SVHCs, have to notify this information to ECHA. The notifications are stored in the Substances of Concern in Products (SCIP) database, which ensures that the information is publicly available throughout the whole lifecycle of products and materials, including the waste stage. In February 2022, 1 year after the SCIP entered into force, the SCIP database included 7 million searchable article notifications, from nearly 7000 companies across the EU; mostly from Germany, Italy and France. The database is public and the access is free of charge. Searching is possible by article name, brand, product category, type of material, chemical name or SCIP number. According to ECHA, the information is expected to help consumers to make more informed and sustainable purchasing choices. In addition, waste operators can use the data to improve their current re-use and recycling practices. The data also increases knowledge about harmful chemicals in supply chains and can help drive the phasing out of these harmful chemicals.

Conclusion Communicating the hazards of chemicals to professional users and consumers is an essential element for handling chemical risks. This requires to establish first an strategy for deciding which chemicals should be considered hazardous, a classification strategy, and then to implement a system for communicating this information to users through a labeling system that could be understood by non-experts. The initial efforts by several sectors and jurisdictions have been harmonized by the UN in a Globally Harmonized System (GHS) for the Classification and Labeling of chemical substances, implemented already in many jurisdictions around the world. The label is the main communication element, and the only available for consumers, while the communication to professional users is complemented by detailed information provided in the Safety Data Sheet (SDS). The system covers substances marketed as such and as mixtures. In the EU additional provisions have been implemented for hazardous substances in articles.

References ECHA (2012) Communication on the safe use of chemicals: Study on the communication of information to the general public (ECHA-12-A-01-EN). https://echa.europa.eu/documents/ 10162/17203/clp_study_en.pdf. ECHA (2018) What is an Infocard? Helsinki: European Chemicals Agency, 33pp. https://doi.org/10.2823/54931. ECHA (2020) Guidance on the compilation of safety data sheets. ECHA-20-H-25-EN. https://doi.org/10.2823/77155.

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European Commission (2011) Special Eurobarometer 360. Consumer understanding of labels and the safe use of chemicals. https://www.reach.gov.it/sites/default/files/allegati/ eurobarometer%20special_360_en.pdf. Geuens M, Byrne D, Boeije G, Peeters V, and Vandecasteele B (2021) Investigating the effectiveness of simplified labels for safe use communication: The case of household detergents. International Journal of Consumer Studies 45: 1410–1424. https://doi.org/10.1111/ijcs.12662. González-Doncel M, Ortiz J, Izquierdo JJ, Martín B, Sánchez P, and Tarazona JV (2006) Statistical evaluation of chronic toxicity data on aquatic organisms for the hazard identification: The chemicals toxicity distribution approach. Chemosphere 63(5): 835–844. https://doi.org/10.1016/j.chemosphere.2005.07.060. Hartmann S and Klaschka U (2017) Interested consumers’ awareness of harmful chemicals in everyday products. Environmental Sciences Europe 29: 29. https://doi.org/10.1186/ s12302-017-0127-8. Kurth D, Wend K, Adler-Flindt S, and Martin S (2019) A comparative assessment of the CLP calculation method and in vivo testing for the classification of plant protection products. Regulatory Toxicology and Pharmacology 101: 79–90. https://doi.org/10.1016/j.yrtph.2018.11.012. Schlueter U and Tischer M (2020) Validity of Tier 1 modelling tools and impacts on exposure assessments within reach registrations—ETEAM Project, Validation Studies and Consequences. International Journal of Environmental Research and Public Health 17: 4589. https://doi.org/10.3390/ijerph17124589. Scott JG, Shore E, Brown C, Harris C, and Rosen MA (2019) Highlights from occupational safety and health continuing education needs assessment. American Journal of Industrial Medicine 62: 901–907. https://doi.org/10.1002/ajim.23014. Sinyai C, MacArthur B, and Roccotagliata T (2018) Evaluating the readability and suitability of construction occupational safety and health materials designed for workers. American Journal of Industrial Medicine 61: 842–848. https://doi.org/10.1002/ajim.22901. UN (1992) United Nations Conference on Environment & Development AGENDA 21. Rio de Janerio, Brazil, 3 to 14 June 1992. https://sustainabledevelopment.un.org/content/ documents/Agenda21.pdf. Tarazona JV and Vega MM (2002) Hazard and risk assessment of chemicals for terrestrial ecosystems. Toxicology 181–182: 187–191. https://doi.org/10.1016/s0300-483x(02) 00279-2. UN (2003) Globally Harmonized System of Classification and Labelling of Chemicals (GHS), 1st edn. https://unece.org/ghs-1st-edition-2003. UN (2021) Globally Harmonized System of Classification and Labelling of Chemicals (GHS), 9th edn. https://unece.org/sites/default/files/2021-09/GHS_Rev9E_0.pdf.

Relevant websites https://echa.europa.eu/scip-database :ECHA: SCIP database. https://ec.europa.eu/environment/chemicals/index_en.htm :European Commission. DG Environment Chemicals home page. https://ec.europa.eu/environment/strategy/chemicals-strategy_en :European Commission Chemicals strategy. https://unece.org/about-ghs :UNECE. GHS. https://www.osha.gov/hazcom :US OSHA Hazard communication.

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Chemical safety assessment and reporting tool (Chesar), REACH María J Ramos-Peralonso⁎, Occupational Risk Prevention and in Social Communication and Health, Madrid, Spain © 2024 Elsevier Inc. All rights reserved. This is an update of M.J. Ramos-Peralonso, Chemical Safety Assessment and Reporting Tool (Chesar), REACH, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 797–800, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00548-0.

Introduction Chesar workflows Use maps Exposure assessments in Chesar Human exposure assessment Environmental exposure assessment Conclusion References

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Abstract The CHEmical Safety Assessment and Reporting tool (Chesar) is an application developed by the European Chemicals Agency (ECHA) to help companies carry out their chemical safety assessments (CSAs) under the European REACH Regulation and to prepare their chemical safety reports (CSRs) that must be included in the REACH registration dossiers and the exposure scenarios (ES) that also should be added to the extended safety data sheets (SDSs) for communication in the supply chain. Chesar enables REACH registrants to carry out their safety assessments in a structured, harmonized, and efficient way. This includes the importing of substance-related data directly from IUCLID, describing the uses of the substance, identifying risk management measures if needed, carrying out exposure estimates, and demonstrating control of risks. Based on this, Chesar automatically generates the CSR and ES for communication in an electronic exchange format and as a text document. It also facilitates the reuse (or update) of assessment elements generated in a single Chesar instance or imported from external sources.

Keywords Chemical safety assessment; Chesar; Exposure assessment; IUCLID; REACH; Risk characterization

Key points

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In the EU, REACH Regulation includes a process for the identification of certain chemicals as ‘Substances of Very High Concern (SVHC). The identification focuses on the following hazards: carcinogenicity, mutagenicity, toxic to reproduction, persistent– bioaccumulative–toxic and very persistent and very bioaccumulative, or substances of equivalent concern, including endocrine disruptors. A specific identification process, which includes a broad consultation, has been established for this identification. Following the identification, the substances are included in the Candidate List to be further consider for requiring an Authorization process. The inclusion in the list triggers specific obligations regarding communication in the supply change, to consumers, and to ECHA.

Introduction REACH (Registration, Evaluation, Authorization, (and Restriction), of Chemicals) is the European Union (EU) regulation adopted to improve the protection of human health and the environment from the risks that can be posed by chemicals, while enhancing the competitiveness of the EU chemicals industry. REACH Regulation: Regulation (EC) No 1907/2006 of The European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending ⁎

Former Director and CEO of Green Planet Environmental Consulting.

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Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. The Regulation created the European Chemicals Agency (ECHA), responsible for setting guidance and provide support to authorities and industry for implementing the legislation. REACH also promotes alternative methods for the hazard assessment of substances in order to reduce the number of tests on animals (ECHA, 2016, 2020). The implementation of REACH is a combination of challenges and opportunities (Tarazona, 2013), and includes the incorporation of scientific progress as well as the development of practical tools for facilitating the implementation by registrants and downstream users. The CHEmical Safety Assessment and Reporting tool (Chesar) (ECHA, 2021a) is an application developed by the European Chemicals Agency (ECHA) to help companies carry out their chemical safety assessments (CSAs) under the European REACH Regulation. Chesar provides support to registrants for preparing their chemical safety reports (CSRs) in a structured, harmonized, transparent and efficient way. In order to facilitate the communication process in the supply change, Chesar generates CSRs and exposure scenarios as text documents. In the EU, CSRs must be included in the REACH registration dossiers; and the exposure scenarios (ES) should also be added to the extended safety data sheets (SDSs) and communicated through the supply chain. The CSR is a regulatory requisite that must be included in the registration dossiers for chemicals registered under the REACH Regulation and manufactured or imported above 10 tons per year. Basically, Chesar is an IT tool that implements the process for developing a CSA described in the ECHA “Guidance on Information Requirements and Chemical Safety Assessment.” This is a very complex guidance provided through a set of documents. Currently consist of six Concise Guidance documents, and eight In Depth Guidance documents. This guidance is frequently updated, and consequently the IT tool Chesar needs also updates. By March 2022, Chesar has experienced three main revisions, and the most recent version is Chesar 3.7.1, published in 2021. Chesar is directly connected to IUCLID, the electronic database system for storing and presenting the information on the substance identity, its uses, and the hazard information (physical–chemical properties, ecotoxicology, and toxicology end-points). Chesar was initially developed as a “plug-in” for IUCLID5, and as evolved with the evolution of IUCLID. When a new Chesar version is released, full compatibility is ensured only with the newest IUCLID version available at that time. Chesar 3 is compatible with IUCLID6, and specifically Chesar 3.7 is compatible with IUCLID 6.5 and 6.6. Chesar provides a structured workflow for carrying out a standard safety assessment for the different uses of a substance, but provides enough flexibility to accommodate specific situations. The tool also helps to structure the information needed for the exposure assessment and risk characterization, facilitating the generation of a transparent CSR. Chesar allows both quantitative and qualitative risk assessments. The quantitative assessments are based on predicted no-effect levels for human health and for the environment. Under REACH, the predicted no-effect levels for human health assessment are named DNELs, Derived No-Effect Levels, and those for environmental protection, PNECs, Predicted No-Effect Concentrations. Regarding the quantitative exposure assessment, Chesar has combined three complementary generic exposure assessment models, the ECETOC TRA models for workers and for consumers and the EUSES 2.1.2 fate model for predicting the environmental concentrations. Chesar can also be used in assessments based on other exposure estimation tools or measured data; however, no automated calculation of exposure estimates can be performed in these cases, and the exposure data should be introduced manually. Qualitative risk characterization approaches are used when hazards are identified for a substance but the information does not permit setting predicted no-effect levels. In these cases Chesar supports ES building with qualitative risk characterization. The main aim of Chesar is to increase the efficiency when conducting CSAs under REACH and to provide consistency between CSAs and the information communicated to downstream users, with the automated generation of CSRs and ESs. Chesar facilitates the reuse (or update) of assessment elements generated in Chesar or imported from external sources, and has considered developments by industry, such as generic exposure scenarios (GESs), specific environmental release categories (SPERCs), Sector-specific Worker Exposure Descriptions (SWEDs), Specific Consumer Exposure Determinants (SCEDs). It also enables the use of the phrase catalog and the information exchange format for ESs that have been developed under Cefic’s ESCom project (exposure scenario for communication phrase catalogue and XML).

Chesar workflows Chesar is divided into seven major groups of functionalities called Boxes (ECHA, 2021a). All Boxes are connected and contribute to the generation of the CSR and/or the ES for the extended SDS:

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Box 1: Substances. It is used for importing the hazard information from the IUCLIC dossier. This includes the conclusions from the hazard assessment, directly determining the scope of exposure assessment and the type of required risk characterization (qualitative or quantitative). Chesar is used once the hazard assessment according to Annex I of REACH has been finalized, and consequently all the information related to those substances’ intrinsic properties (single end-point summaries and overall toxicological and ecotoxicological summaries) needed for the exposure assessment and risk characterization is available in IUCLID. This information is imported into Chesar with the Box 1 functionalities, which also allow import and export of full CSAs, e.g., for exchange with other assessors. Information on the substance’s intrinsic properties can also be entered directly in Chesar, but this information cannot be exported to IUCLID and Chesar cannot generate a full CSR for these substances. Box 2: Uses. Here the uses of the substance are described in a structured way to ensure consistency between use description, the exposure assessments, and the exposure scenario building. The information is presented using Chesar’s life-cycle tree structure, reporting the relevant uses of the substance, considering human health and environmental aspects as well as the

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tonnage breakdown to the different uses. Chesar’s life cycle trees may have a maximum of eight cycle stages or use types: Manufacture; Formulation or repacking; Use (of substances as such or in mixture) at industrial sites; Widespread use (of substance as such or in mixture) by professional workers; Consumer use (of substance as such or in mixture); Service life (consumer); Service life (industrial sites); Service life (professional workers). For each use one environmental contributing scenario is automatically created. Other contributing scenarios for human health and for the environment can be created in addition. The labels (names), the appropriate set of use descriptors according to REACH, and further specifications are included here to be used later on for setting a default conservative exposure assessment. This Chesar element also allows import or export of CSA building blocks (life-cycle tree or parts of it with corresponding exposure assessment). Box 3: Exposure assessment. One or more quantitative exposure assessments for each contributing scenario are conducted here. Chesar allows the selection of different exposure assessment methods, while the route/types of effects (for humans) and environmental compartments to be covered have been determined when importing the hazard conclusions from IUCLID in Box 1. Depending on the substance properties and the uses, the plugged in exposure estimation tools could be sufficient or not to demonstrate that the expected exposure is lower than the no-effect thresholds (the DNELs for human effects and the PNECs in the case of environmental assessments). Additional assessment methods may be needed in some cases, for example when the uses or the specific use conditions are not covered by the plugged-in tools included in Chesar, or when even after considering all refinement options, Chesar predicts an unacceptable level of risk (exposure exceeding the derived no-effect levels). In such situations other exposure assessment methods or measured data should be included manually to demonstrate a safe use. Alternatively, additional risk management options or significant changes in the operational conditions may be considered. Chesar supports the systematic and transparent manual reporting of such assessments. Box 4: CSR. In this part the final exposure scenarios are built by consolidating the assessments carried for the contributing scenarios conducted in Box 3. At this stage hazards without DNELs or PNECs are also taken into account, and appropriate conditions of use are added, if needed to reach a sufficient level of exposure minimization for these hazards. Chesar offers quantitative and qualitative approaches as appropriate for completing the risk characterization. The justifications and final assessment can be clarified by entering explanations on single exposure scenarios or on the overall assessment approach. These explanations will be transferred automatically to the Chemical Safety Report, generating sections 9 and 10 of the CSR as well as a full CSR (part A and B), integrating the information from IUCLID. In Box 4 it is also possible to report risk management measures that are applicable to all uses, and do not result from the exposure assessment carried out for each specific use. Box 5: ES for communication. It covers the establishment of the exposure scenarios for communicating the information to downstream users. These scenarios, SDS ES, are meant to transport selected parts of the information documented in the Chemical Safety Report to the users, covering the full life cycle or selected uses. The exposure scenarios describe how the substance can be safely used. This includes operational conditions and risk management measures. The scenarios are identified by a short title, which should enable the user of the substance to identify whether the exposure scenario contains information relevant to him. The short titles of the exposure scenarios for a substance shall be consistent with the brief general description of uses in IUCLID. The information should allow downstream users to establish whether or not they work within the conditions for which a safe use has been demonstrated by the registrant in the CSA. The SDS ES can be exported in ESCom XML format so that the information can be further processed in the recipient ITsystems, and in rtf format allowing translation in any EU language if ESCom standard phrases have been used systematically (the translation ESCom phrase catalogue should be uploaded in the library). Box 6: Library. Box 6 includes the functionalities of the Chesar library. The library enables creation, storage, import, and export of objects that the assessor may need in his Chemical Safety Assessment work process. In Chesar 3 the following library objects can be managed: Condition of use templates, Specific Environmental Release Categories (SPERCs), Specific Consumer Exposure Determinants (SCEDs), Sector-specific Worker Exposure Description (SWEDs) and standard phrases. SPERCs, SCEDs and SWEDs correspond to sets of information describing specific conditions of use (and the corresponding release estimates to water, air, soil and waste for SPERCs) developed by industry sectors as part of their use maps. These library objects are meant to be used across various assessments, contributing to the harmonization and efficiency of the assessments conducted under REACH. CEFIC has developed a phrase catalogues with standard phrases respecting the Chesar format through the Cefic’s ESCom project. The library should be regularly updated for maximizing the capacities of Chesar. Box 7: Users. Box 7 covers those aspects related to the user and role management. For example users can use this section for assigning a legal entity to objects created in the library, identifying the author (and therefore the source) of the object.

Use maps In order to facilitate the harmonization and exchange of information within the supply chain on the use of chemicals, ECHA in cooperation with industry associations have developed the concept of “use maps” (ECHA, 2021b). Use maps are typically generated by organizations that represent downstream users. They collect information on the uses and the conditions of use of chemicals in their sector in a harmonized and structured way; and create use maps by making use of the templates available at the ECHA web page. Following the submission, the information is made publicly available by ECHA and the users to be downloaded by the users and included in the Chesar Library. Four different templates are available:

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Use maps—overview of common uses in a sector. SWEDs–sector-specific workers exposure descriptions. SPERCs—specific environmental release categories. SCEDs—specific consumer exposure determinants.

Registrants can use available use the maps developed by the downstream user sectors to prepare their chemical risk assessments, and to communicate the information to the users. The following associations have already implemented this possibility, and offer use maps, SWEDs, SPERCs or SCEDs, covering different sectors:

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AISE: Soaps, Detergents and Maintenance products CEPE: Pigments, solvents, binders, additives, . . . Concawe: Fuels Cosmetics Europe: Cosmetics and personal care products ECPA: Plant protection products EFCC: Construction Chemicals ESIG: End-products containing solvents EuPC: Plastics Additives FEICA: Adhesives, sealants Fertilizers Europe: Fertilizers I&P Europe/I&P Europe Imaging and printing products: Imaging and printing products IFRA: Fragrance compounds

Exposure assessments in Chesar The exposure assessment under REACH is required for hazardous substances (those classified according to the CLP/GHS criteria) and for PBT/vPvB substances. The exposure assessment should present a quantitative or qualitative estimate of the dose/concentration of the substance to which humans and the environment are or may be exposed. The exposure assessment is expected to be an interactive process, but the reporting process for REACH registration is limited to the last step, once the use is considered safe by the registrant, and should include the description of use conditions proposed for ensuring a safe use. Chesar implements these fundamental principles, but may be also used for exchanging preliminary results among registrants and within the supply change. The use of tiered and targeted approaches for information acquisition improves not only the efficiency but also the confidence in the outputs (Money, 2018). Exposure assessment under REACH includes two elements. The first element is the characterization/definition of the operational conditions and risk management measures for the identified uses; based on this first step, the exposure to humans and to the environment occurring under these conditions should be predicted as a second element of the exposure assessment. The exposure assessment results are used later on during the risk characterization process. Quantitative risk assessments require quantitative exposure estimates, and those are generated in Chesar Box 3. The identification of operational conditions and risk management measures driving this exposure estimation is an essential element for these quantitative exposure assessments. Based on Box 3 results, the final exposure scenarios are built in Box 4, considering also the integration of conditions and measures needed to control risks from hazards for which a qualitative exposure assessment is considered.

Human exposure assessment The human exposure assessment estimates the expected exposure for workers and consumers and the indirect exposure via the environment of the general population. For workers, the ECETOC TRA workers v3 method has been plug-in in Chesar. Exposure assessment based on measured data are also possible. The ECETOC TRA workers plugged-in tool covers three routes of exposure: inhalation acute exposure, inhalation long-term exposure, and dermal long-term exposure. In all three cases, local and/or systemic effects can be assessed. In addition, Chesar allows the use of external exposure estimation tools, which results are later manually incorporated to be reported in the CSA. Sector-specific worker exposure descriptions (SWEDs) can be used as input in order to carry out the assessment based on realistic and representative conditions of use. In order to run the ECETOC TRA tool, information on the substance properties (e.g., molecular weight, physical state of the substance, vapor pressure if liquid) is needed for estimating the exposure concentrations. The assumptions are defined by the use conditions. The exposure estimation based on measured data requires the manual introduction for each route of all exposure estimation data (exposure value, units, and explanations on the source and representativity of those values). In addition, the conditions of use associated to the measured data set should be introduced. If the exposure estimates based on measured data will be used as key

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information for the risk characterization (not just as supportive information), determinant types for the use conditions should be created in the library under Box 6; to ensure that the related conditions of use are part of the ES. Chesar allows the incorporation of exposure estimates obtained using external exposure estimation tools. Some developers provide outputs in Chesar compatible forms. In other cases, all exposure estimations data (exposure values, units, and explanations on the exposure value if relevant) have to be manually entered for each route. In addition, relevant determinants reporting the conditions of use should be added and transferred to the ES. Standard sets of determinants corresponding to the input parameters of the tool can be used. Similar exposure assessment methods are considered for consumers estimations. The ECETOC TRA Consumers exposure estimation tool (v 3.1) is built-in to Chesar 3. For the implementation in Chesar, the tool has been aligned with the principles laid down in the update of Guidance R15, and there are some deviations compared to the ECETOC implementation of the TRA. The tool can provide estimates for long-term (repeated) inhalation, dermal and oral exposure. Specific Consumer Exposure Determinants (SCEDs) can be used as input where no product specific inputs are available in the TRA, or where an assessor wishes to overwrite the build-in defaults. The indirect human exposure via the environment is part of the environmental exposure assessment described below.

Environmental exposure assessment The environmental protection targets considered in Chesar are aquatic and sediment dwelling organisms (freshwater and marine), predators in the aquatic food chain (freshwater and marine), sewage treatment plant functioning, agricultural soil organisms, predators in the terrestrial food chain, and air. In addition, exposure estimates are generated for intakes by humans via the environment, estimating the concentration of substance in air, drinking water, and different food items (e.g., root crops, dairy products, meat, and fish). Chesar allows two complementary assessment types. Measured data are preferred when there is good quality information on the release and/or environmental exposure levels for one or more environmental protection targets. Model estimation, using generic or specific tools can be used when good quality measurements are not available. The exposure assessment starts with the quantification of the release rate. Chesar offers four possibilities for setting the release rates to each environmental compartment:

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Default release estimations based on the Environmental Release Categories (ERC-based release estimations) for any of the release route: water, air, and soil. This is the release method selected by default and corresponds to a worst case estimate of release under the assumption that no specific risk management measures have been put in place. Targeted estimations based on Specific Environmental Release Category (SPERC-based release estimations) describing the expected release under specific conditions of use and the corresponding release estimates to water, air, soil, and waste, covering all release routes. Ad hoc release factors for water, air, and/or soil (reported as the percentage of the daily tonnage released to each compartment). An explanation and justification giving details on the source and quality of these release factors must be provided. Measured release for water and air for the local release rate (kg/day) for each release route. In addition to the release rate, an explanation and justification giving details on the source for these measured release values should be provided.

Based on the outcome of these release estimations, Chesar estimates the exposure for the relevant compartments according to the EUSES 2.1.2 fate model. The EUSES 2.1 fate model uses a set of default parameters and assumptions, which can be modified and adapted to more realistic conditions. The exposure assessment for uses at industrial sites and wide dispersal uses differ from each other, and these conditions are systematically differentiated in Chesar. Uses at industrial sites are assessed for one generic representative site per use. It is assumed that a certain tonnage is used at this one single generic site and that a certain fraction of the use tonnage at this site is released (depending on the conditions of use). As explained above, the release factor depends on the operational conditions and risk management assumed for the generic site. As there may be very large and very small industrial sites for one use, two contributing scenarios (or even the definition of two different uses) may be needed to reflect the difference in conditions and tonnage. By default it is assumed that the discharge from the site is treated in a municipal sewage treatment plant (STP) and the STP sludge is applied to agricultural soil. In addition, it is assumed that the tonnage released from the site is diluted in the sewage system and further by an additional dilution factor when the sewage is discharged into the receiving river water. The Chesar plug-in tool has incorporated the default values and assumptions used in the EUSES fate model, these assumptions can be changed in Chesar if needed. For wide dispersal uses it is assumed that the market tonnage is evenly distributed in space and time. The assessment is carried out for a standard town with 10,000 inhabitants and a corresponding use tonnage (fraction of the market tonnage). Thus wide dispersal uses are those uses that correspond to consumer activities, services in a municipality or housing. Depending on the conditions of use, a certain fraction of this tonnage is assumed to be released to the sewage system. It is also assumed that the tonnage released from the 10,000 inhabitants is diluted within the 2000 m3 of wastewater generated by this population (average of 200 L of wastewater per person and day) and then by a generic additional factor of 10 in the receiving river water. By default it is

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assumed that the STP-sludge is applied to agricultural soil. Usually, the assessor will define one contributing scenario per use and will not overwrite the default assumptions on the local conditions for a substance marketed across Europe. The releases from all uses into the municipal sewage system are aggregated to derive an exposure estimate. The Predicted Environmental Concentrations (PECs) for the generic site or generic town are estimated taking into account the so-called “local concentrations” (Clocal) and “regional concentrations.” The regional concentration results from all the uses of a substance and is calculated by EUSES in the background. The regional concentration is added to the local concentration for deriving the local PEC, in order to take into account that a single site or a single town do not discharge into a virgin environment but just add to emissions released by other site or towns. Measured regional concentrations for one or several compartments can also be reported in Chesar. When measured concentrations are reported they are automatically used as regional concentrations (overwriting the EUSES estimates), and thus also impact on the local PECs for each contributing scenario. Consequently, only very reliable regional concentrations should be reported. For substances considered to be PBT or vPvB, only local concentrations for water are provided. This is due to the large uncertainties of the regional estimations that should predict the fate, distribution, and long-term effects of such substances in the environment, in particular regarding accumulation in the food chain. However, measured data may be provided. Detailed explanations of environmental exposure assessment are available in the Guidance on Information Requirements and Chemical Safety Assessment Chapter R16: Environmental Exposure estimation and in the EUSES user manual and supplementary information provided by ECHA.

Conclusion The European Chemicals Agency (ECHA) has developed several tools to help companies to fulfil their obligations under the EU REACH Regulation. One of these tools is Chesar, an application connected to IUCLID and that conducts chemical safety assessments (CSAs) and prepares the chemical safety report (CSRs) to be included in the REACH registration dossiers. Chesar also prepares the exposure scenarios (ES) to be attached to the extended safety data sheets (SDSs). The Chesar reports describe the uses of the substance, identify risk management measures, and conduct the exposure estimations.

References ECHA (2016) Practical Guide: How to use alternatives to animal testing to fulfil the information requirements for REACH registration. ECHA-16-B-25-EN https://doi.org/ 10.2823/194297https://echa.europa.eu/documents/10162/17250/practical_guide_how_to_use_alternatives_en.pdf/148b30c7-c186-463c-a898-522a888a4404. ECHA (2020) The use of alternatives to testing on animals for the REACH Regulation Fourth report (2020) under Article 117(3) of the REACH Regulation. ECHA-20-R-08-EN https://doi. org/10.2823/092305https://echa.europa.eu/documents/10162/0/alternatives_test_animals_2020_en.pdf/db66b8a3-00af-6856-ef96-5ccc5ae11026. ECHA (2021a) Chesar 3 use manual. ECHA-20-H-26-EN https://doi.org/10.2823/638991 Available at: https://chesar.echa.europa.eu/documents/736332/8711025/Chesar_3-6_ user_man_en.pdf/65edfa9e-57b8-f334-07f7-afb9841e8099?t¼1610721136527. ECHA (2021b) Chesar 3 for sector associations: Creating use maps—Including SPERCs, SWEDs, and SCEDs. ECHA-20-H-27-EN https://doi.org/10.2823/163707 Available at: https://chesar.echa.europa.eu/documents/736332/11080857/chesar_manual_sector_associations_3_en.pdf/ee7daedd-1316-133e-bf45-d8e195f918dd? t¼1637235644866. Money C (2018) Improving the relevance and efficiency of human exposure assessments within the process of regulatory risk assessment. Environmental Science: Processes & Impacts 2018(20): 12–19. https://doi.org/10.1039/C7EM00434F. Tarazona JV (2013) Use of new scientific developments in regulatory risk assessments: Challenges and opportunities. Integrated Environmental Assessment and Management 9: e85–e91. https://doi.org/10.1002/ieam.1445.

Relevant websites https://chesar.echa.europa.eu/ :Chesar https://ec.europa.eu/environment/chemicals/index_en.htm :European Commission. DG Environment Chemicals home page https://ec.europa.eu/environment/strategy/chemicals-strategy_en :European Commission. Chemicals strategy https://echa.europa.eu/csr-es-roadmap/use-maps/use-maps-library :European Chemicals Agency: use maps

Chemical specific adjustment factor: A shift from default/refined toward hybrid uncertainty Seyed Mojtaba Daghighia,∗, Maryam Baeeria, and Hamid Rashidi Nodehb,∗, aPharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran, Iran; bFood Technology and Agricultural Products Research Center, Standard Research Institute (SRI), Karaj, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of M.A. Rezvanfar, Chemical-Specific Adjustment Factor (CSAF), Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 810–813, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00594-7.

Introduction Refined uncertainty factor Hybrid uncertainty analysis Conclusion References

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Abstract For a decade, the chemical-specific adjustment factor has been developed based on the chemical-specific data obtained from the risk assessment of chemicals toxicity. The traditional methodology of evaluating chemical toxicity relied on animal experiments and default uncertainty analysis with particular attention to the interspecies and interindividual differences. The current knowledge about the determination of chemical-specific adjustment factor (CSAF) concerning the uncertainty for chemicals and biological risk assessment has been reviewed. Due to the lack of knowledge in hazard and exposure uncertainty analysis, a refinement process is needed to reduce the default uncertainty to a minimum level. Refinement processes are developing based on a combination of the current CSAF (common structural characteristics, common mode of action, and physiologically based toxicokinetic) and new data (in vitro, in silico, in vivo, clinical, and epidemiological data), resulting in more precise uncertainty analysis and an increased tier of initial data from a lower to a higher level. Thus, the influence of default, refined, and, a combination of both, hybrid uncertainty analysis on the development of CSAF methodology is updated regarding the present state of knowledge.

Keywords CSAFs; Default uncertainty; Hybrid uncertainty; Interspecies; Mode of action; Refined uncertainty; Refinement; Risk assessment; Toxicodynamic; Toxicokinetic

Key points

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Chemical-specific adjustment factor (CSAF) has been determined based on default uncertainty for chemicals and biological risk assessment. A refinement process in hazard and exposure uncertainty analysis is required to reduce the default uncertainty to a minimum level. Although the current CSAF is developing based on the default uncertainty and refined uncertainty, a combination of hybrid uncertainty analysis results in a more precise uncertainty analysis.

Introduction In order to quantify the health risk assessment for decision-making in risk management, the US environmental protection agency (EPA) regulated the exposure levels for each chemical in terms of reference doses and reference concentrations (Gentry et al., 2002). Although linear uncertainty is traditionally used for the risk assessment, nonlinear approaches, including chemicals mode of action, dose-response and uncertainty analysis, have been considered according to the latest EPA guideline (Liao et al., 2007; Bhat et al., 2017). Similarly, the world health organization (WHO) and the international program on chemical society (IPCS) provided guidance based on CSAFs for a better understanding of the default pharmacodynamics and pharmacokinetic uncertainty factors (IPCS, 2005; WHO, 2005). The use of CSAFs appears to be attainable when chemical information on interspecies and human ∗

Equally as the corresponding author.

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interindividual (human to human) variations are accessible for the mode of action in toxicokinetic or toxicodynamic uncertainty (Leusch et al., 2020; Rietjens et al., 2021). Therefore, CSAFs first create a method by incorporating quantitative data on interspecies differences and human variability in toxicokinetic and toxicodynamic data, then it provides broader approaches in the more precise and personalized risk assessment procedures (Meek et al., 2002; WHO, 2005). In practice, toxicokinetic data must be derived from human volunteers for a quantitative comparison between animals and humans (Rezvanfar, 2014). Analogously, toxicodynamic components in CSAFs are related to the ratios of a concentration that induce the toxic effect. This can also be attributed to a measurable related response from in vitro data of animal tissues or a representative sample of healthy individuals (interspecies differences). New broader approaches can be developed based on the combined use of refined in vitro and in silico neurotoxicology. Although these models cannot cover all aspects of in vivo models, these alternative models can potentially facilitate the development of CSAFs toxicodynamic of interspecies and interindividual differences (Kasteel and Westerink, 2021). The use of uncertainty factors (UFs) in establishing exposure limits extends back at least as far as 1954 when Lehman and Fitzhugh (1954) proposed a 100-fold factor (which they referred to as a “margin of safety”) for extrapolating from animal toxicity data to safe levels of human exposure to food additives and pesticide residues. Therefore, this 100-fold default uncertainty factor has been used in human risk assessment so far (Kasteel et al., 2020). After introducing CSAFs in 2005, the default uncertainty factor is expanded to an interspecies and interindividual variable with different subdivided factors. CSAFs can be replaced with default sub-factors by modifying the relevant default interspecies uncertainty and the default interindividual uncertainty factor of 10 (Fig. 1). The subdivided factors provided a different uncertainty cut point which “4” reflects the interspecies differences in kinetic (AKUF), “2.5” reflects the interspecies differences in dynamic (ADUF), and “3.16” reflects both interindividual differences in kinetic (HKUF) and dynamic (HDUF) (Rietjens et al., 2021). Hence, uncertainty factors are often associated with lower confidence in risk assessment and provide a greater uncertainty than CSAFs (Kasteel and Westerink, 2021). In addition to the dose-response and mode of action, the uncertainty analysis also plays a crucial role in the modern hazard/ toxicity assessment processes. Therefore, the purpose of uncertainty analysis, via specifying all uncertainties, is to increase the transparency of risk assessments and supply full information for the decision-making process (Fraize-Frontier and Roth, 2020). Due to the broad application of CSFAs in the risk assessment, CSAFs with a minor change in methodology is used to set other uncertain parameters of in vivo concentration as well as a complete sequence of in vitro target concentrations for synthetic chemical such as perfluorooctanoic acid (Loizou et al., 2021). On the road of shifting from default to refined uncertainty, the in vitro Ellman method has been used for acetylcholinesterase (AChE) toxicity to assess the relevance of toxicodynamic’s default uncertainty in human variability interspecies differences. In this study, Ellman method has been employed to measure the AChE inhibition after exposure of human blood and Electric eel enzyme of healthy individuals to different chemical compounds such as diazinon, rivastigmine, chlorpyrifos and etc. (Kasteel et al., 2020). Ellman’s method gained a factor of 10 for each interindividual differences and 3.16 for kinetic and dynamic uncertainty. The data of this experiment suggest that the toxicodynamic default uncertainty factor of AChE (3.16) may be in an overestimated range of human variability for the toxicity of this chemical. This information implies that the toxicodynamic associated adjustment factors successfully support in vivo and in vitro kinetic and dynamic data extrapolating to improve the CSFA. Because the in vitro data can refine the default uncertainty factor based on reduction, refinement, and replacement of animal testing, the Ellman in vitro data can be supportively used to improve chemical risk assessment and derivation of toxicodynamic (Kasteel et al., 2020). Similarly, the risk assessment of exposure to biologicals or chemicals such as isopropanol (Gentry et al., 2002), acetone (Gentry et al., 2003), chloroform (Liao et al., 2007), and chromium (Fallahzadeh et al., 2018) have been quantitatively studied based on uncertainty parameters.

Fig. 1 Diagram of default uncertainty factor (UF) and subdivided factors abased on CSAFs concept. From Rietjens IMCM et al. (2021) A chemical-specific adjustment factor for human interindividual differences in kinetics for glutamates (E620-625). Food and Chemical Toxicology. Elsevier, 147: 111910 on the basis of Creative Commons CC-BY license.

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Refined uncertainty factor Traditional chemical risk assessment methods rely on animal experiments and default uncertainty analysis to consider interspecies and interindividual differences. Due to the lack of enough knowledge after the hazard uncertainty and exposure uncertainty stage, also to get more precise uncertainty, refinement is needed to reduce the uncertainty (Kasteel and Westerink, 2021). Hence, to personalize the risk assessment and hazard risk analysis with more precise uncertainty, default uncertainty analysis must be refined and replaced by common CSAFs (Bhat et al., 2017; Kasteel and Westerink, 2021). Refinements of uncertainty are well depicted based on chemical and biological knowledge that the data sets can be utilized to optimize (data calibration) and verify the risk assessment model between the experimental and simulated data. The refinement is defined as a refined mode of action developed physiologically based on toxicokinetic new data, etc. (Bhat et al., 2017). The refinement for exposure assessment is described as refined scenarios, refined parameters, refined method, and new data. Fig. 2 depicts the main purpose of refinement, hazard assessment, and exposure assessment, increasing the initial tier from lower to higher.

Hybrid uncertainty analysis Uncertainties are involved in every stage of the risk assessment process, and an uncertainty analysis has to be an excellent built-in strategy in hazard characterization. It is crucial to describe the uncertainties concerned in assessing the exposure of multi chemicals/ species. It will be more significant than in single chemical/species (More et al., 2019). Therefore, there is a substantial relationship between hazard/toxicity characterization of risk and acceptable level of uncertainty (Bhat et al., 2017). The uncertainty analysis in hazard assessment leads to the risk assessment in available conditional data, risk management, comparison across hazard data set, estimation of the exposure concentration, methodologies for hazard characterization, and toxicity factor sets (Rezvanfar, 2014; Bhat et al., 2017). The different equipment and techniques that are relevant to describe the uncertainty analysis are human health (masking human fitness), animal health (food additives), food contaminants (in situ producing or external), and ecological areas (impact of binary combination interactions on hazard characterization in bees) (More et al., 2019). Single or unimodal population distribution and bimodal population distribution represented the human validity model based on CSAFs derived on pharmacokinetics and physiology, biochemistry, and chemical structure parameters. It has been shown that the population distribution uncertainty is applicable to measure the whole population analyzed active entity and calculated CSAFs based on differences between the central values of the main groups and given percentiles (95th, 97.5th, and 99th) (Rezvanfar, 2014). Uncertainty analysis is thus imperative to investigate the level of self-belief in the chemical effects (ANSES, 2016). Step-wise mythology (Fig. 3) is recommended to calculate the uncertainty analysis in chemical risk assessment as represented in the scheme (Fraize-Frontier and Roth, 2020).

Fig. 2 A schematic diagram indicating the relationship between consideration of uncertainty relevant to tiered assessment and problem formulation. From Bhat VS et al. (2017) Evolution of chemical-specific adjustment factors (CSAF) based on recent international experience; increasing utility and facilitating regulatory acceptance. Critical Reviews in Toxicology. Taylor & Francis, 47(9): 733–753, based on Creative Commons Attribution License.

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Fig. 3 Representation of step approaches describing the uncertainty analysis in risk assessment.

In practice, the carrying of hybrid uncertainty method for the risk assessment deals with the combination of random sample distribution with fuzzy interval based on uncertainties co-exist (epistemic uncertainty) and variability distribution (aleatory) (Dutta and Ali, 2012; Dutta, 2017). Hence, by applying the hybrid uncertainty analysis, large-scale sustainability risk can be measured by comparing risk standard value and risk change value (Tang et al., 2018). However, in recent years, hybrid uncertainty analysis has been further developed and used for chemical risk assessment (Fraize-Frontier and Roth, 2020), including qualitative non-probabilistic, approximate probabilistic and full probabilistic models (Karuna and Manohar, 2017; Rohmer, 2018).

Conclusion Uncertainty analysis in hazard evaluations is the essential fundamental step. This step is accountable for making risk management decisions that influence the hazard administration. On the other hand, currently and for most chemicals, critical appraisal of hazardous risks is performed habitually without analyzing uncertainty and comparing instability examination. Suppose there is a disappointment to analyze uncertainty in hazard evaluation methodically. In that case, the chances of assessors and directors, who may be incapable of entirely judging the processes and the conclusions, are not high. To improve the actual hazard appraisal, it is inevitable to bargain with unavoidable instabilities through the number of possible ways to decrease to a reasonably expected level. Therefore, vulnerability must be characterized and measured with default/refined uncertainty to account for interspecies and interindividual differences. The integration of adverse outcome pathways and physiologically based kinetic models provide a robust quantitative understanding of a chemical’s toxicodynamic. This combined use of integrated approaches will aid in structuring hybrid uncertainty leading to increased suitability of alternative methods for regulatory purposes.

References ANSES (2016) Traitement de l’incertitude dans le processus d’évaluation des risques sanitaires des substances chimiques - réfexion sur la caractérisation et la prise en compte de l’incertitude en évaluation des risques sanitaires au sein de l’anses. Maisons-Alfort: ANSES. Bhat VS, et al. (2017) Evolution of chemical-specific adjustment factors (CSAF) based on recent international experience; increasing utility and facilitating regulatory acceptance. Critical Reviews in Toxicology 47(9): 733–753. Taylor & Francis. Dutta P (2017) Modeling of variability and uncertainty in human health risk assessment. MethodsX 4: 76–85. Elsevier. Dutta P and Ali T (2012) A hybrid method to deal with aleatory and epistemic uncertainty in risk assessment. International Journal of Computer Applications 42(11): 37–44. Fallahzadeh RA, et al. (2018) Spatial distribution variation and probabilistic risk assessment of exposure to chromium in ground water supplies; a case study in the east of Iran. Food and Chemical Toxicology 115: 260–266. Elsevier. Fraize-Frontier S and Roth C (2020) Uncertainty analysis in chemical risk assessment. In: Risk Assessment Methods for Biological and Chemical Hazards in Food, pp. 457–482. CRC Press. Gentry PR, et al. (2002) Application of a physiologically based pharmacokinetic model for isopropanol in the derivation of a reference dose and reference concentration. Regulatory Toxicology and Pharmacology 36(1): 51–68. Elsevier.

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Gentry PR, et al. (2003) Application of a physiologically based pharmacokinetic model for reference dose and reference concentration estimation for acetone. Journal of Toxicology and Environmental Health Part A 66(23): 2209–2225. Taylor & Francis. IPCS, W (2005) Chemical-specific adjustment factors for interspecies differences and human variability: Guidance document for use of data in dose/concentration–response assessment harmonization project document No. 2. World Health Organization Geneva. Karuna K and Manohar CS (2017) Inverse problems in structural safety analysis with combined probabilistic and non-probabilistic uncertainty models. Engineering Structures 150: 166–175. Elsevier. Kasteel EEJ and Westerink RHS (2021) Refining in vitro and in silico neurotoxicity approaches by accounting for interspecies and interindividual differences in toxicodynamics. Expert Opinion on Drug Metabolism & Toxicology 1–11. Taylor & Francis. Kasteel EEJ, et al. (2020) Acetylcholinesterase inhibition in electric eel and human donor blood: an in vitro approach to investigate interspecies differences and human variability in toxicodynamics. Archives of toxicology 94(12): 4055–4065. Springer. Lehman AJ and Fitzhugh OG (1954) 100-fold margin of safety. Quarterly Bulletin - Association of Food & Drug Officials of the United States 18: 33–35. Leusch FDL, et al. (2020) Deriving safe short-term chemical exposure values (STEV) for drinking water. Regulatory Toxicology and Pharmacology 110: 104545. Elsevier. Liao KH, et al. (2007) Bayesian estimation of pharmacokinetic and pharmacodynamic parameters in a mode-of-action-based cancer risk assessment for chloroform. Risk Analysis: An International Journal 27(6): 1535–1551. Wiley Online Library. Loizou G, et al. (2021) Derivation of a human in vivo benchmark dose for perfluorooctanoic acid from toxcast in vitro concentration–Response data using a computational workflow for probabilistic quantitative in vitro to in vivo extrapolation. Frontiers in Pharmacology 12: Frontiers Media SA. Meek ME, et al. (2002) Guidelines for application of chemical-specific adjustment factors in dose/concentration–response assessment. Toxicology 181: 115–120. Elsevier. More SJ, et al. (2019) Guidance on harmonised methodologies for human health, animal health and ecological risk assessment of combined exposure to multiple chemicals. EFSA Journal 17(3). Wiley Online Library. Rezvanfar MA (2014) Chemical-Specific Adjustment Factor (CSAF). Elsevier. Rietjens IMCM, et al. (2021) A chemical-specific adjustment factor for human interindividual differences in kinetics for glutamates (E620-625). Food and Chemical Toxicology 147: 111910. Elsevier. Rohmer J (2018) HYRISK: An R package for hybrid uncertainty analysis using probability, imprecise probability and possibility distributions. HYRISK. EarthArXiv. https://doi.org/ 10.31223/OSF.IO/J67CY. Tang W, Li Z, and Tu Y (2018) Sustainability risk evaluation for large-scale hydropower projects with hybrid uncertainty. Sustainability 10(1): 138. Multidisciplinary Digital Publishing Institute. WHO (2005) Chemical-specific adjustment factors for interspecies differences and human variability: Guidance document for use of data in dose/concentration-response assessment. In: Chemical-Specific Adjustment Factors for Interspecies Differences and Human Variability: Guidance Document for Use of Data in Dose/Concentration-Response Assessment, 96.

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Chemical toxicity of per- and poly-fluorinated alkyl substances (PFAS) Noah Peter Christian, Leidos, Incorporated, San Diego, CA, United States © 2024 Elsevier Inc. All rights reserved.

Introduction PFAS as a chemical class PFAS “Heavy Hitters” related to toxicity, supplanting PFOS and PFOA, and research depth and breadth Hexafluoropropylene oxide (HFPO) Name and background Background Uses/occurrence Exposure Mechanism of toxicity Environmental fate and behavior Toxicity PubChem URLs PFDA Name and background Toxicity PubChem URL PFBA Name and background Toxicity PubChem URL PFBS Name and background Toxicity PubChem URL PFHxA Name and background Toxicity PubChem URL Conclusion/summary/outlook References Further reading

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Abstract Per- and poly- fluorinated alkyl substances (PFAS) include a large number of chemicals that have the general characteristic of a carbon to fluorine bond. The draft EPA method 1633 for analysis of PFAS targets 40 analytes. However, over 14,735 substances are listed as PFAS as per EPA’s CompTox Chemical dashboard PFAS database. The Organization for Economic Cooperation and Development (OECD) PFAS definition is a saturated carbon-fluorine (C-F) alkyl chain (CF2), which encompasses over 6 million compounds. As of writing of this chapter, PubChem Classification browser contains more than 21 million PFAS and Fluorinated compounds There are over 400,000 known organofluorine compounds. Specific toxicities of key PFAS (other than perfluorooctane sulfonic acid; PFOS, and perfluorooctanoic acid; PFOA, both of which are discussed in other chapter entries) are summarized across the range of human and animal studies, including perfluorodecanoic acid (PFDA) as the next most studied compound behind PFOS and PFOA, Perfluorobutanesulfonic acid (PFBS), Perfluorohexanoic acid (PFHxS), and Hexafluoropropylene oxide dimer acid (HFPO-DA), the last of which is often referred to as GenX chemicals. Much attention is being paid by independent investigators and the media for this class of compounds, because of the pervasiveness, longevity, and profound toxicological effects of these compounds at very low levels.

Keywords Fluorinated substances; GenX; HFPO; HFPO-DA; Human studies; Mutagenicity; PFAS; PFBS; PFDA; PFHxA; PFHxS; Toxicity

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Perfluoro compounds have all hydrogens in carbon-hydrogen (CdH) bonds replaced with fluorine. According to the EPA, over 14,735 substances are listed as PFAS (as of August 2022) in their COMPTOX PFAS database. Recent California state legislation (California AB-1817, 2022; California AB-2247, 2022) has defined any compound with a carbon-fluorine bond to be defined as PFAS, leading to hundreds of thousands of potential PFAS, including non-alkyl fluorinated substances. Average blood PFOS and PFOA levels have been steadily dropping in humans over the past 20 years as these compounds are being phased out. Despite this, a large number of compounds categorized as PFAS are still in production and low levels still lead to high toxicity in the blood. In this chapter, HFPO, PFBA, PFDA, PFBS and PFHxA are highlighted. PFOA and PFOS each has been addressed under separate chapters. In addition, PFAS as a chemical class is briefly summarized.

Introduction Per- and Poly- fluoroalkylated substances (PFAS) represent a class of compounds that are used in a variety of applications from firefighting foam to omniphobic surface treatments in consumer goods, including cosmetics, home products, carpets, and clothing. These materials have high stability under ambient conditions and have been called “forever chemicals” due to their long lifetimes and high pervasiveness. There has been a general search for lower toxicity replacements to perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) compounds in applications where the properties of those compounds are of benefit. Many of these substitutes can have higher toxicity as described in this chapter and more significant environmental pervasiveness due to their high solubility. Exposure can follow a number of routes, from chemical dumping to leaching from landfills to use of PFAS by individuals. For example, PFAS have been observed in human serum concentrations as high as 423 ng/mL due to the use of Scotchgard on carpets (Beesoon et al., 2012).

PFAS as a chemical class The PFAS category represents a broad range of chemicals. The current EPA CompTox Chemical Dashboard database (updated August 2022) lists over 14,735 unique chemicals in this category, (U.S. EPA COMPTOX Dashboard, 2022) and additional side- and degradation- products are expected to further expand this list. The Government of Canada intends to address PFAS as a chemical class because replacements are still associated with environmental and/or human health effects (Canada Gazette, 2021). California has recently introduced legislation (California AB-2247, 2022; California AB-1817, 2022) that state that any compound with a carbon-fluorine bond is PFAS and will be regulated against inclusion in cosmetics, personal care products, and clothing. Legislation in California and New York (New York Department of Environmental Conservation (DEC), 2022) will bring the total number of unique PFAS to over 400,000 compounds (Schofield, 1999). These recent regulations have further driven PFAS compounds to be broadly defined, and the toxicology and environmental fate of many different compounds overlap in significant ways (Anderson et al., 2022; Konkel, 2021; Balan et al., 2021; OECD, 2021). PFAS in general, due to the diversity of materials and categories (fluorotelomers, fluorosulfonates, fluorinated acids, fluoroethers, etc.), can be generalized as a class of compounds with carbon-fluorine bonds (Kwiatkowski et al., 2020). Many of these concepts are based on the persistence of these materials and the longevity of these materials in the environment (Cousins et al., 2020; Cousins et al., 2022). This is a direct consequence of the high bond strength of carbon with fluorine, and the difficulty of biological enzymes and chemical methods to degrade these materials in the environment. USEPA Draft methods for PFAS analysis (U.S. EPA Press Office, 2021a) target 40 compounds, yet it is understood that tens of thousands of compounds exist with CdF bonds. Studying the toxicity of all of these materials (beyond high-throughput in vitro multiplexed assays) is not practical, and animal toxicity needs to be assessed through analogy, metabolic pathways, or direct study of specific compounds. Methods for high-throughput screening of environmental samples to detect organofluorine (CdF bonded) materials to enable rapid assessment of the extent of pollution will be increasingly important to detect these contaminants. Targeted analysis of the 40 compounds in draft method EPA 1633, for example, (U.S. EPA, 2022b) will miss the many thousands of compounds with (a) high environmental persistence (b) potential low biological turnover via low solubility or high partitioning to lipid/aqueous interfaces (and hence high biological persistence) and (c) high potential biotoxicity.

PFAS “Heavy Hitters” related to toxicity, supplanting PFOS and PFOA, and research depth and breadth A database of PFAS is maintained at the EPA CompTox database under a PFAS master list category (U.S. EPA COMPTOX Dashboard, 2022). Some primary PFAS are covered in this chapter, using the following criteria: (1) A high incidence of literature studies as

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reported by CompTox, (2) Billing as a direct replacement and/or safer alternative to PFOS or PFOA, and (3) Presence of an EPA summary toxicological assessment report and/or EPA discussion of regulatory levels for a material. The toxicities of these replacements are, by nature, not as thoroughly studied, though generally believed to be of lower toxicity compared to PFOS and PFOA. To confirm these suppositions, these compounds are being studied in the literature using a variety of in vivo and in vitro models; experimental toxicology (mouse, rats, monkey, rabbits, etc.) and epidemiological investigations. The Environmental Protection Agency (EPA) has been consolidating toxicity information and related databases via published literature and reporting findings through peer-reviewed publications and summaries. To date, based on systematic reviews protocol, the US EPA has published toxicological assessments for PFBS, PFHxA, PFHxS, PFNA, and PFDA (US EPA, 2021b). Furthermore, US EPA has published reference doses (RfDs) for various PFAS to compare toxicity in humans. These values are defined by the EPA as “An estimate (with uncertainty spanning perhaps an order of magnitude) of a daily oral exposure to the human population (including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a lifetime”(U.S. EPA, 2002; US EPA, 2023a). As a baseline, the other chapter entries for PFOS and PFOA have an RfD of 1.8
10−6 mg/kg/day and 0.00002 mg/kg/day respectively (Pachkowski et al., 2019; U.S. EPA, 2016a; US EPA, 2016b). Significant reductions to PFOS and PFOA RfDs on the order of three orders of magnitude are currently under peer review. The EPA is proposing a reference dose (RfD) of 1.5
10−9 mg/kg-day for PFOA and an RfD of 7.9
10−9 mg/kg-day for PFOS (US EPA, 2021c). At the time of writing this chapter, US EPA released a proposed national primary drinking water regulation (NPDWR) for PFOA and PFOS, and four other PFAS and mixtures based on the latest scientific and/or toxicological updates. In their proposed rule, US EPA also presented updated draft noncancer toxicity values that are different from those used to calculate the 2022 interim Health Advisories (HAs). The proposed Maximum Contaminant Level Goals (MCLGs) of zero including supporting technical information and the draft 2023 toxicity values for PFOA and PFOS are under review and public comment period. The 2022 interim HAs will remain available as the agency finalizes a national primary drinking water regulation for those contaminants. For additional information, readers can refer to https://www.epa.gov/sdwa/drinking-water-health-advisories-pfoa-and-pfos#: (website last updated, March 29, 2023).

Hexafluoropropylene oxide (HFPO) Name and background HFPO and derivatives HFPO dimer acid (HFPO-DA), as well as the ammonium salt form of HFPO-DA (HFPO-DA ammonium salt), represent the largest contributors in the GenX (a DuPont Chemours tradename) chemical product, a processing aid technology introduced as a replacement to PFOA for fluoropolymer manufacture. GenX chemicals have been discharged into the Cape Fear River for several decades as a by-product of manufacturing (US EPA, 2021d; North Carolina Department of Environmental Quality (NCDEQ), 2018). Synonyms [Chemsrc]: Refer to https://www.chemsrc.com/en/searchResult/HFPO/ for additional information. HFPO Synonyms: perfluoropropylene oxide; hexafluoro-1,2-epoxypropane7; epoxy-1,1,2,3,3,3-hexafluoro-. HFPO-DA Synonyms: Perfluoro(2-methyl-3-oxa hexanoic) acid; 2-(Heptafluoropropoxy)tetrafluoropropionic acid; (heptafluoropropoxy)perfluoropropionic acid. HFPO-DA Ammonium Salt Synonyms: Perfluoro(2-methyl-3-oxa hexanoic) acid ammonium salt; GenX; Ammonium 2,3,3,3-tetrafluoro-2-(perfluoropropoxy)propanoate. CAS Numbers [Chemsrc]: HFPO: 428-59-1 HFPO-DA: 13252-13-6 HFPO-DA Ammonium Salt: 62037-80-3 Molecular Formulae: HFPO: C3F6O HFPO-DA: C6F12O2 HFPO-DA Ammonium Salt: C6H4F11NO3 Chemical Structures [PubChem]: HFPO

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HFPO-DA

HFPO-DA Ammonium Salt

Background HFPO is a key intermediate in the synthesis of organofluorine compounds. The chemistry of HFPO has been studied across polymerization and intermediate formation reactions (Tarrant et al., 1971; Millauer et al., 1985).

Uses/occurrence HFPO is a fluorointermediate that can be used for the synthesis of fluoromonomers and fluoropolymers and to add fluorine functionality to a variety of precursors. The perfluorinated Krytox™ lubricant has HFPO as the monomeric unit. HFPO is supplied as a liquified, nonflammable pressurized gas (Chemours, 2018). HFPO-DA is the dimeric form of HFPO. It is the common form of GenX in the environment, though more specifically the GenX tradename registered by DuPont Chemours refers to the ammoniated salt form of HFPO-DA. HFPO-DA ammonium salt is most commonly termed GenX. The tradename is now used to address the general class of compounds associated with this structure which vary in chain length. Many additional longer chain components are still associated with GenX toxicity as well as environmental contamination (US EPA, 2021d; NCBI PubChem, 2023a, 2023b).

Exposure Exposure to HFPO is most commonly a result of the widespread use of this compound, long environmental persistence, and disposal of bulk materials from global suppliers. The long persistence of these compounds leads to exposure from contaminated drinking water, food sources, consumer products, and dermal absorption.

Mechanism of toxicity HFPO can rearrange to toxic HFA in the presence of Lewis acids. This can occur in shipping containers, storage vessels, or other process equipment. Because higher temperatures cause faster rearrangement, it is highly recommended to maintain HFPO below 25  C (77  F), unless required for process purposes (i.e., purification or chemistry) (Chemours, 2018; US EPA, 2021c; NCBI PubChem, 2023a, 2023b). For additional mechanistic insights, readers can refer to U.S. EPA Press Office (2021b) report and NCBI PubChem (2023a, 2023b) information resources. Acute, short-term, subchronic, chronic, reproductive, and developmental studies are available in rats and mice.

Environmental fate and behavior HFPO-DA is a liquid and the ammoniated form is a solid. Both are infinitely soluble in water. Both can volatilize in air and migrate to soil particles. They are stable against environmental degradation via photolysis, hydrolysis, and biodegradation.

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The US EPA reference dose for GenX is 3 ng/kg-day (U.S. EPA, 2021a). Environmental contamination of the Cape Fear River Basin in North Carolina and wells in the region exceed 0.140 mg/L as a result of industrial release. A reference dose for HFPO-DA was determined to be 0.0001 mg/kg/day (North Carolina Department of Environmental Quality (NCDEQ), 2018; U.S. EPA, 2021d).

Toxicity HFPO-DA is rapidly absorbed from the GI tract by male and female rats. A higher absorption was observed in males vs. females. Invitro human and rat dermal uptake studies demonstrated dermal absorption, though lower than well-absorbed dermal materials. Dermal absorption is a significant potential source of chemical uptake for PFAS (Ragnarsdóttir et al., 2022). HFPO-DA acute oral toxicity median lethal doses (LD50) are 1730 mg/kg for male rats and 1750 mg/kg for female rats. Clinical signs include altered posture, lethargy, discoloration of lungs, skin, stomach, lymph nodes, liver, and esophagus, and enlarged liver and hepatocytes. HFPO-DA can be readily transferred to a fetus as demonstrated in multiple studies. In a chronic toxicity/carcinogenicity study of 7-week-old rats HFPA-DA ammonium salt was administered at varied rates of 0, 0.1, 1, 50, and 500 mg/kg/day for males and females. Numerous animals of all dose groups were found dead over the course of the study. Males most commonly died from pituitary tumors and the females most commonly died from pituitary and mammary tumors. High dosed females group showed papillary necrosis and kidney inflammation as well as alopecia (US EPA, 2021d).

PubChem URLs HFPO: https://pubchem.ncbi.nlm.nih.gov/compound/9883 HFPO-DA: https://pubchem.ncbi.nlm.nih.gov/compound/114481 HFPO-DA Ammonium Salt: https://pubchem.ncbi.nlm.nih.gov/compound/51342034

PFDA Name and background Perfluorodecanoic acid (PFDA) is a fluorosurfactant and a perfluoroalkyl acid (PFAA). PFAAs have been frequently detected in both the environment and in plants, fish, and animals. It is a breakdown product of stain- and grease-proof coatings on food packaging, furniture (e.g., couches), and carpets. Like many PFAAs, it is persistent and bioaccumulative. PFAAs are thought to be endocrine disruptors. Many are known toxicants and carcinogens. PFAAs have been used in the manufacture of such prominent consumer goods as Teflon and Gore-Tex (NCBI PubChem, 2023a, 2023b). At the time of writing of this chapter, US EPA released a draft PFDA toxicological assessment (April 2023) for public comment. Readers can refer to https://cfpub.epa.gov/ncea/iris_drafts/recordisplay. cfm?deid¼354408 for additional information. Synonyms [Chemsrc]: Nonadecafluorocapric acid; DECANOIC ACID, NONADECAFLUORO; Nonadecafluorodecanoic acid; perfluoro1-decanoic acid; EINECS 206-400-3; perflurodecanoic acid; Perfluorodecanoic acid; MFCD00004175; perfluoro-n-decanoic acid; PFDA; Nonadecafluoro-n-decanoic acid; Ndfda; 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-nonadecafluorodecanoic acid; Decanoic acid, 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-nonadecafluoro-; Perfluorocapric acid CAS Number [Chemsrc]: 335-76-2 Molecular Formula [PubChem]: C10HF19O2 Chemical Structure [PubChem PFDA]:

Toxicity According to the CompTox database number of entries, after PFOA and PFOS, perfluorodecanoic acid (PFDA) is the most studied compound related to human toxicity. PFDA has relatively high toxicity and promotes tumor growth (Dong et al., 2017). Acute toxicity determined by LD50 is 57 mg/kg in rat (oral) and 150 mg/kg in mouse and guinea pig (intraperitoneal). Toxicity has been studied in mouse liver using microarray analyses. PFDA greatly affected the immune system. Expression levels of IL-1beta and IL-18 were decreased after exposure to PFDA. Mechanistically, it appears to inhibit caspase-1 activation and decrease the mRNA levels of NLRP1, NLRP3, and NLRC4. Further studies indicated that cIAP2 and its binding proteins were also reduced. These results point to a suppression of inflammasome response (Zhou et al., 2017). PFDA has toxicity similar to dioxin, but its effect on the body is not through a single target or pathway. In other studies, cIAP2 was found to be upregulated in order to suppress gastric cell senescence (Zhang et al., 2019). PFDA toxicity in mouse studies has been shown to differ from 2,3,7,8-tetrachlorodibenzo-p-dioxin (Brewster and Birnbaum, 1989). PFDA has been found to induce meiotic defects and deterioration of mouse oocytes in vitro (Li et al., 2022).

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Chemical toxicity of per- and poly-fluorinated alkyl substances (PFAS)

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/9555

PFBA Name and background Heptafluorobutyric acid, more commonly known as perfluorobutanoic acid (PFBA), is a fluorosurfactant and a breakdown product of other PFAS that are used in stain-resistant fabrics, paper food packaging, and carpets; it is also used for manufacturing photographic film, and it is used as a substitute for longer chain perfluoroalkyl carboxylic acids (PFCAs) in consumer products. PFBA has been found to accumulate in agricultural crops and has been detected in household dust, soils, food products, and surface, ground, and drinking water (US EPA 2022c). Synonyms [Chemsrc]: butyric acid, heptafluoro-; Butanoic acid, heptafluoro-; Perfluorobutanoic acid; CAS Number [PubChem]: 375-22-4 Molecular Formula [PubChem]: C10HF19O2

Chemical Structure [PubChem]:

Toxicity PFBA exposure effects include liver weight changes, morphological changes in the liver and thyroid, decreased thyroxine (T4), and decreased red blood cell count, hematocrit, and hemoglobin levels. Other effects include decreased cholesterol and delayed eye response. PFBA has been tested for endocrine, developmental, and neurotoxicity effects (Minnesota Department of Health, 2018). Endocrine effects include decreased T4 levels, altered follicular epithelium of the thyroid, and increased thyroid weight. Developmental delays were observed in offspring of mice exposed during pregnancy at a twofold higher than human equivalent dose. Delayed bilateral pupillary reflex was observed in males exposed to a 10-fold higher dose over the benchmark dose lower confidence limit (BMDL). Histopathology of neural tissues did not reveal any treatment-related abnormalities. The EPA reference dose for PFBA is 1 mg/kg-day (US EPA 2022c).

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/9777

PFBS Name and background Nonafluoro-1-butanesulfonic acid, more commonly known as perfluorobutanesulfonic acid (PFBS), is a fluorosurfactant. PFBS is a member of a larger group of per- and poly-fluoroalkyl substances (PFAS) (U.S. EPA, 2021d). Synonyms [Chemsrc]: Nonafluorobutane-1-sulfonic acid; 1-Perfluorobutanesulfonic acid; 1,1,2,2,3,3,4,4,4-nonafluorobutane1-sulfonic acid. CAS Number [PubChem]: 375-73-5 Molecular Formula [PubChem]: C4HF9O3S

Chemical Structure [PubChem]:

Toxicity The US EPA reference dose for PFBS is 300 ng/kg-day. PFBS is found to primarily be accumulated in intracellular regions and shows half lives in human sera of 1000 h. Animal studies showed exposure of PFBS at levels as low as 50 mg/kg/day in rats lead to

Chemical toxicity of per- and poly-fluorinated alkyl substances (PFAS)

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significant decreases in tetraiodothyronine (T4), triiodothyronine (T3), and increase in thyroid stimulating hormone (TSH). No significant changes to thyroid weight or histopathology were shown. Some changes in reproductive hormonal levels are seen at levels higher than 200 mg/kg/day in animal studies. No human studies have been done related to growth or early development in children. No discernable change in absolute and relative kidney weights in rats exposed for up to 90 days at doses up to 600 mg/kg-day and in two generations at 1000 mg/kg-day. Renal changes were detected in animal studies at levels down to 100 mg/kg-day. Hepatic changes were detected in animal studies at levels up to 200 mg/kg-day. No human studies have been made relative to renal or hepatic studies. A birth cohort study revealed changes in adiposity with exposure-response relation in females only up to 5 years in age. There was a statistically significant positive association of asthma and PFBS exposure. No significant genotoxicity or mutagenicity has been observed in studies (U.S. EPA, 2021d).

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/67815

PFHxA Name and background Perfluorohexanoic acid (PFHxA) is a fluorosurfactant. Synonyms [Chemsrc]: Perfluorohexanoic acid (PFHXA); Undecafluoro-1-hexanoic acid; perfluoro-1-hexanoic acid CAS Number [PubChem]: 307-24-4 Molecular Formula [PubChem]: C6HF13O3S

Chemical Structure [PubChem]:

Toxicity The US EPA released the final toxicological review of PFHxA and related salts in April 2023. This assessment addresses the potential cancer and noncancer human health effects from exposure to perfluorohexanoic acid and related salts. The recommended chronic Reference Dose (RfD) is 5
10−4 mg/kg-day or 500 ng/kg-day. The chronic RfD is based on a decreased postnatal (PND 0) body weight in F1 Sprague-Dawley male and female rats (US EPA, 2023b). For additional information, readers can refer to the full toxicological review, summary toxicity values and supplemental information available at: https://cfpub.epa.gov/ncea/iris_drafts/ recordisplay.cfm?deid¼357314#. In a previously published study from 2019, a chronic human health RfD of 0.25 mg/kg/day was derived by Luz et al. (2019), based on the benchmark dose modelling of renal papillary necrosis in chronically exposed female rats (cited by Anderson et al., 2019). The Michigan Department of Environment, Great Lakes, and Energy (EGLE) completed the process of promulgating rules and established maximum contaminant levels (MCLs) for seven PFAS compounds under the authority of the Safe Drinking Water Act. The drinking water standards for PFHxA is 400 parts-per-billion. Readers can refer to the Michigan PFAS Action Response Team portal (https://www.michigan.gov/pfasresponse/drinking-water/mcl) for more information. A review of literature related to PFAS toxicity listed cardiovascular, renal, immunological, and reproductive effects from exposure to PFHxA in human epidemiological studies. Studies in laboratory animals displayed endpoint effects across the vast majority of tissues, organs, and systems (Agency for Toxic Substances and Disease Registry, 2021).

PubChem URL https://pubchem.ncbi.nlm.nih.gov/compound/67542

Conclusion/summary/outlook PFAS as a class chemical group cover a broad range of compounds and it is likely that while some exhibit relatively low toxicity, others may have high toxicity, mutagenicity, and other deleterious health effects. These compounds, by virtue of their longevity and unique properties, will continue to be challenging from an environmental and health perspective. Detailed toxicity studies have

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been carried out for some of the compounds in this chemical class. This is primarily driven by motivation to replace PFOS and PFOA compounds with safer alternatives. However, in some cases the material toxicity is higher, though animal studies show they are not as highly retained in the body. Given the recent trend of the EPA and other regulatory agencies to update toxicity values and regulatory limits of these compounds to address their persistence and complex toxicology, these compounds will continue to garner attention from a health and safety standpoint. Higher throughput analysis methods for chemical surveillance as well as higher throughput toxicological screening will be required for these compounds due to the sheer number of compounds currently being considered under the PFAS chemical class.

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U.S. EPA (2016a) Health Effects Support Document for Perfluorooctanoic Acid (PFOA). Available at: https://www.epa.gov/sites/default/files/2016a-05/documents/pfoa_hesd_final_ 508.pdf (Accessed on 1 March, 2023). U.S. EPA (2016b) Drinking Water Health Advisory for Perfluorooctanoic Acid (PFOA). Available at: https://www.epa.gov/sites/default/files/2016b-05/documents/pfoa_health_ advisory_final-plain.pdf (Accessed on 1 March, 2023). U.S. EPA (2021a) Systematic Review Protocol for the PFBA, PFHxA, PFHxS, PFNA, and PFDA (anionic and acid forms) IRIS Assessments, CASRN 335-76-2 (PFDA), CASRN 375-95-1 (PFNA), CASRN 307-24-4 (PFHxA), CASRN 355-46-4 (PFHxS), CASRN 375-22-4 (PFBA) Supplemental Information―Appendix A. U.S. EPA (2021b) Human Health Toxicity Values for Hexafluoropropylene Oxide (HFPO) Dimer Acid and Its Ammonium Salt (CASRN 13252-13-6 and CASRN 62037-80-3) Also Known As “GenX Chemicals”. Available at: https://www.epa.gov/system/files/documents/2021a-10/genx-chemicals-toxicity-assessment_tech-edited_oct-21-508.pdf (Accessed on 1 March, 2023). U.S. EPA (2021c) Fact Sheet: Human Health Toxicity Assessment for GenX Chemicals. Available at: https://www.epa.gov/system/files/documents/2021b-10/genx-final-toxassessment-general_factsheet-2021.pdf (Accessed on 1 March, 2023). U.S. EPA (2021d) Human Health Toxicity Values for Perfluorobutane Sulfonic Acid and Related Compound Potassium Perfluorobutane Sulfonate, EPA/600/R-20/345F, April 2021. Available at: https://cfpub.epa.gov/ncea/risk/recordisplay.cfm?deid¼350888 (Accessed on 1 March, 2023). U.S. EPA (2022a) IRIS Toxicological Review of Perfluorobutanoic Acid (PFBA) and Related Salts (Final Report, 2022). U.S. Environmental Protection Agency: Washington, DC. EPA/635/ R-22/277F. U.S. EPA (2022b) Clean Water Act Analytical Methods for Per- and Polyfluorinated Alkyl Substances (PFAS). Available at: https://www.epa.gov/cwa-methods/cwa-analytical-methodsand-polyfluorinated-alkyl-substances-pfas (Accessed on 1 March, 2023). U.S. EPA (2023a) Basic Information about the Integrated Risk Information System, IRIS Toxicity Values Reference Dose (RfD). Available at: https://www.epa.gov/iris/basic-informationabout-integrated-risk-information-system. (Accessed on 1 March, 2023). U.S. EPA (2023b) Toxicological Review of Perfluorohexanoic Acid (PFHxA) and Related Salts (Final Report, 2023). U.S. Environmental Protection Agency: Washington, DC. EPA/635/R23/027F. Available at: https://cfpub.epa.gov/ncea/iris_drafts/recordisplay.cfm?deid¼357314 (Accessed on 10 April, 2023). U.S. EPA COMPTOX Dashboard (2022) PFAS Master List of PFAS Substances. Available at: https://comptox.epa.gov/dashboard/chemical-lists/PFASMASTER (Accessed on 1 March, 2023). U.S. EPA Press Office (2021a) EPA Announces First Validated Laboratory Method to Test for PFAS in Wastewater, Surface Water, Groundwater, Soils. Available at: https://www.epa. gov/newsreleases/epa-announces-first-validated-laboratory-method-test-pfas-wastewater-surface-water (Accessed on 1 March, 2023). U.S. EPA Press Office (2021b) EPA Advances Science to Protect the Public From PFOA and PFOS in Drinking Water. Available at: https://www.epa.gov/newsreleases/epa-advancesscience-protect-public-pfoa-and-pfos-drinking-water (Accessed on 1 March, 2023). Zhang Z, Song N, Peng Y, Fan Z, Han M, Zhao M, Dong T, and Liu S (2019) Environmental pollutant perfluorodecanoic acid upregulates cIAP2 to suppress gastric cell senescence. Oncology Reports 41(2): 981–988. https://doi.org/10.3892/or.2018.6856. Zhou X, Dong T, Fan Z, Peng Y, Zhou R, Wang X, Song N, Han M, Fan B, Jihui J, and Liu S (2017) Perfluorodecanoic acid stimulates NLRP3 inflammasome assembly in gastric cells. Scientific Reports 7: 45468. https://doi.org/10.1038/srep45468.

Further reading Bil W, Ehrlich V, Chen G, Vandebriel R, Zeilmaker M, Luijten M, Uhl M, Marx-Stoelting P, Halldorsson TI, and Bokkers B (2023) Internal relative potency factors based on immunotoxicity for the risk assessment of mixtures of per-and polyfluoroalkyl substances (PFAS) in human biomonitoring. Environment International 171: 107727. Buck RC, Franklin J, Berger U, Conder JM, Cousins IT, de Voogt P, Jensen AA, Kannan K, Mabury SA, and van Leeuwen SPJ (2011) Perfluoroalkyl and polyfluoroalkyl substances in the environment: Terminology, classification, and origins. Integrated Environmental Assessment and Management 7(4): 513–541. https://doi.org/10.1002/ieam.258. Carlson LM, Angrish M, Shirke AV, Radke EG, Schulz B, Kraft A, Judson R, Patlewicz G, Blain R, Lin C, and Vetter N (2022) Systematic evidence map for over one hundred and fifty per-and polyfluoroalkyl substances (PFAS). Environmental Health Perspectives 130(5): 056001. Carstens KE, Freudenrich T, Wallace K, Choo S, Carpenter A, Smeltz M, Clifton MS, Henderson WM, Richard AM, Patlewicz G, and Wetmore BA (2023) Evaluation of Per-and polyfluoroalkyl substances (PFAS) in vitro toxicity testing for developmental neurotoxicity. Chemical Research in Toxicology. CFPUB (2019) Initial Draft Posted November 2019 Update posted July 2020 (in response to public comments). https://cfpub.epa.gov/ncea/iris_drafts/recordisplay.cfm? deid¼345065; https://ordspub.epa.gov/ords/eims/eimscomm.getfile?p_download_id¼542033 (Update posted January 2021). East A, Dawson DE, Brady S, Vallero DA, and Tornero-Velez R (2023) A scoping assessment of implemented toxicokinetic models of per-and polyfluoro-alkyl substances, with a focus on one-compartment models. Toxics 11(2): 163. Hawkey AB, Mead M, Natarajan S, Gondal A, Jarrett O, and Levin ED (2023) Embryonic exposure to PFAS causes long-term, compound-specific behavioral alterations in zebrafish. Neurotoxicology and Teratology: 107165. Joerss H and Menger F (2023) The complex ‘PFAS world’-how recent discoveries and novel screening tools reinforce existing concerns. Current Opinion in Green and Sustainable. Chemistry: 100775. https://doi.org/10.1016/j.cogsc.2023.100775. Pelch KE, Reade A, Kwiatkowski CF, Merced-Nieves FM, Cavalier H, Schultz K, Wolffe T, and Varshavsky J (2022) The PFAS-tox database: A systematic evidence map of health studies on 29 per- and polyfluoroalkyl substances. Environment International 167: 107408. https://doi.org/10.1016/j.envint.2022.107408. PMID: 35908389. Rice PA, Cooper J, Koh-Fallet SE, and Kabadi SV (2021) Comparative analysis of the physicochemical, toxicokinetic, and toxicological properties of ether-PFAS. Toxicology and Applied Pharmacology 422: 115531. https://doi.org/10.1016/j.taap.2021.115531. https://www.sciencedirect.com/science/article/pii/S0041008X21001381. Solan ME, Koperski CP, Senthilkumar S, and Lavado R (2023) Short-chain per-and polyfluoralkyl substances (PFAS) effects on oxidative stress biomarkers in human liver, kidney, muscle, and microglia cell lines. Environmental Research 223: 115424. U.S. EPA (2023) IRIS Toxicological Review of Perfluorodecanoic Acid (PFDA) and Related Salts (Public Comment and External Review Draft). Washington, DC: U.S. Environmental Protection Agency. EPA/635/R-23/056a. Available at: https://cfpub.epa.gov/ncea/iris_drafts/recordisplay.cfm?deid¼354408 (Accessed on 10th April, 2023). Yao, et al. (2022) Non-target discovery of emerging PFAS homologues in Dagang Oilfield: Multimedia distribution and profiles in crude oil. Journal of Hazardous Materials 437: 129300. https://doi.org/10.1016/j.jhazmat.2022.129300.

Relevant websites https://www.atsdr.cdc.gov/toxprofiles/tp200.pdf :Agency for Toxic Substances and Disease Registry. https://www.cdc.gov/niosh/topics/pfas/default.html :Per- and polyfluoroalkyl substances (PFAS). https://www.chemsrc.com/en :Chemical abstracts number lookup. https://pubchem.ncbi.nlm.nih.gov/classification/#hid¼120 :NCBI PubChem. https://www.oecd.org/chemicalsafety/portal-perfluorinated-chemicals/; https://www.oecd.org/chemicalsafety/portal-perfluorinated-chemicals/aboutpfass/; https://www.oecd.org/ chemicalsafety/portal-perfluorinated-chemicals/countryinformation/ :OECD Portal on Per and Poly Fluorinated Chemicals.

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https://www.oecd.org/chemicalsafety/portal-perfluorinated-chemicals/synthesis-report-on-understanding-side-chain-fluorinated-polymers-and-their-life-cycle.pdf :OECD. https://www.ewg.org/interactive-maps/pfas_contamination/ :Oil and Gas PFAS Mapping Effort. https://www.natlawreview.com/article/pfas-risk-assessment-pfhxa-epa-impact-business#::text :PFAS Risk Assessment for PFHxA by EPA – Impact on Business. https://psr.org/wp-content/uploads/2022/09/fracking-with-forever-chemicals-in-ohio.pdf; https://psr.org/wp-content/uploads/2021/07/fracking-with-forever-chemicals.pdf :PFAS and Fracking Reports from “Physicians for Social Responsibility (PSR)” organization. https://ntp.niehs.nih.gov/whatwestudy/topics/pfas/index.html :Per- and Polyfluoroalkyl Substances (PFAS). https://ehp.niehs.nih.gov/curated-collections/pfas-2022 :PFAS Research Papers Collection from Environmental Health Perspective Journal. https://ehp.niehs.nih.gov/curated-collections/PFAS :PFAS. https://tools.niehs.nih.gov/pfas/index.cfm :NIEHS-supported Publications on Per- and Polyfluoroalkyl Substances (PFAS). https://pfastoxdatabase.org/ :PFAS Toxicological Database. https://comptox.epa.gov/dashboard/chemical-lists/PFASSTRUCTV5 :US EPA: PFAS structures in DSSTox. https://www.epa.gov/pfas/basic-information-pfas :US EPA Basic information on PFAS. https://www.pfonline.com/news/epa-releases-draft-health-based-levels-for-pfas-in-drinking-water :US EPA Releases Draft Health-Based Levels for PFAS in Drinking Water. https://comptox.epa.gov/dashboard/chemical-lists/pfasmaster :US EPA Comptox Chemicals Dashboard: Master List of PFAS Substances. https://www.epa.gov/chemical-research/human-health-toxicity-assessments-genx-chemicals :US EPA Toxicity of GenX Chemicals. https://hawc.epa.gov/assessment/100000037/ :US EPA Health Assessment Workspace Collaborative (HAWC) PFBS. https://hawc.epa.gov/assessment/100500070/ :PFHxA.

Chemical warfare Steven A Burr, Peninsula Medical School, University of Plymouth, Plymouth, United Kingdom. © 2024 Elsevier Inc. All rights reserved. This is an update of S.A. Burr, Chemical Warfare, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 814–816, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01003-4.

Prohibition Historical use of chemical agents Exposure and toxic effects Personal protective equipment Decontamination References Further reading

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Abstract Chemical warfare has a long history, using a wide variety of agents. It is now accepted as prohibited by most nations. Accurate information to compare effects, onset and potency by different routes in humans can be collated with difficulty but often has unverifiable military provenance. Recent work focuses on countermeasures such as personal protective equipment and decontamination.

Keywords Anthrax; Arsenical vomiting agents; Bio warfare and terrorism; Blister agents/vesicants; Botulinum toxin; Chemical warfare delivery systems; Chlorine; Cyclosarin; G-series nerve agents; Lewisite; Nerve agents; Nitrogen mustards; Sarin; Soman; Tabun; V-series nerve agents; VX

Key points

• • • • •

Previously employed by nations, but now coveted by extreme terrorist organizations. Fortunately expertise in both the chemistry of production and the engineering of delivery systems are required for application. This area includes the most potently harmful chemicals to humans, many being deliberately developed. The purpose of these chemicals is to either incapacitate or kill people as quickly as possible, or to deter and deny occupation of a space. Emphasis is now on developing protective measures.

Prohibition There have been several international agreements attempting to limit and ultimately abolish chemical warfare. The Brussels Declaration in 1874 (unratified) forbade parties ‘to employ poison or poisoned arms.’ The Hague Conference signed in 1899 (entered into force in 1900 and updated in 1907) forbade the “use of projectiles the object of which is the diffusion of asphyxiating or deleterious gases.” The Geneva Protocol signed in 1925 (entered into force in 1928) forbade “the use in war of asphyxiating, poisonous or other gases, and of all analogous liquids, materials or devices.” Most recently the Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and their Destruction of 1993 (entered into force in 1997) is administered by the Organisation for the Prohibition of Chemical Weapons (OPCW). This Chemical Weapons Convention has been ratified by all member states of the United Nations with the following exceptions: Israel has signed but not ratified; and Egypt, North Korea and South Sudan have neither signed nor acceded (as of April 2021). For Schedule 1 chemicals with no known general purpose use other than as a weapon (e.g. tabun, sarin, soman, cyclosarin, VX, lewisite, nitrogen mustard, sulfur mustard, ricin, saxitoxin, and the precursors chlorosarin, and chlorosoman), no more than 100 g per year can be produced for use in research. For Schedule 2 chemicals with established small-scale alternative applications (e.g. the lewisite precursor arsenic trichloride, which is also used in ceramic manufacture, the mustard gas precursor thiodiglycol, which is also used as a solvent for inks, and the sarin precursor dimethyl-methylphosphonate, which is also used as a flame retardant), facilities producing more than 1 kg, 100 kg, or 1 ton per year (depending on the specific chemical) must be declared and inspected. For Schedule 3 chemicals (e.g. phosgene and

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00560-1

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hydrogen cyanide) with established large-scale alternative applications, facilities producing more than 30 tons per year must be declared and inspected (Marrs et al., 2007). Many consider chemical weapons emotive, while others consider that the injuries inflicted are no more immoral than those inflicted by explosive agents. Furthermore, chemical weapons could be considered less environmentally destructive and hence preferable to nuclear weapons. Indeed, prohibition of chemical weapons means that the only remaining proportional response to an attack with a weapon of mass destruction would be to counterattack using nuclear weapons.

Historical use of chemical agents The use of poison-coated arrowheads and spearheads are referred to in some of the earliest historical records of warfare, which themselves are predated by references in mythological accounts. The poisoning of water supplies during sieges was first reported in the 6th century BCE (when the Assyrians used ergot and Greeks used hellebore). The first recorded device for delivery is possibly the poisonous (probably arsenic and croton) smoke bomb used by the Chinese Sung dynasty in 1161 CE. Many more devices were developed over the centuries culminating in a wide range of modern military delivery systems. It is estimated that 125,000 tons of chemical weapons (in descending order of quantity employed: chlorine, phosgene, diphosgene, chloropicrin, sulfur mustard, and cyanides) were used on the battlefield by both sides during WWI (from 1915 to 1918), causing more than 1 million casualties. As Germany sought to increase food production in the face of shortages following the Treaty of Versailles, research into organophosphates for new pesticides led to the discovery of the G-series nerve agents. More agents have been developed since then and been actively used on a comparatively small scale by several nations (Marrs et al., 2007).

Exposure and toxic effects Exposure is normally intended to be through breathing or skin contact, but can be through soil, dust, water and food ingestion, or via wounds. The predominant adverse effect of an agent is usually on the nervous system (increasing secretions and causing paralysis), skin (causing irritation, blisters, and burns) or lungs (causing oedema; see Table 1). Onset of effects is generally quicker following inhalation than percutaneous exposure. However, having effective respiratory and dermal absorption dictates that both breathing and skin countermeasures are required for protection. Protective countermeasures may be circumvented by a combination of agents to compromise the integrity of protective equipment (e.g., addition of an emetic such as diphenyl chloroarsine (DA) to act as a ‘mask breaker,’ forcing the removal of breathing apparatus in order to vomit, or a corrosive agent such as phosgene oxime (CX) to penetrate barriers), or by achieving surprise and rapidly causing debilitating effects (e.g., hydrogen cyanide). thus preventing the effective use of countermeasures. High potency reduces the quantity needed to be delivered for coverage of any given target area, but is often offset by the higher expense of a more complex manufacturing process (e.g., VX; Marrs et al., 2007; Kuca and Pohanka, 2010).

Personal protective equipment Major incident planning (for public health protection and counterterrorism) by local government emergency services includes provision for personal protective equipment. Emergency civilian personnel (i.e., police, fire and ambulance services) wear hazardous materials (‘hazmat’) suits for first response recovery and decontamination tasks (e.g., following spillages). Different degrees of protection for breathing and skin are available based on knowledge of the type of agent, concentration, and associated risk. Suit levels in the United States are designated A–D: Level A provides most protection, B provides more respiration protection than C, and D provides the least protection of all (inversely proportional to impingement of dexterity and comfort). Suit types in Europe are designated 1–6: Type 1 is gas tight (equivalent to A in the United States), 2 protects against gases and liquids but is not gas tight (B in the United States), 3 protects against liquids rather than gases and is liquid tight, 4 is spray tight (C in the United States), 5 protects most of the body against liquids (D in the United States), and 6 protects parts of the body against liquids. Individuals may use a self-contained breathing apparatus, air-purifying respirator, air-purifying escape respirator, self-contained escape respirator, or powered air-purifying respirator. It is also possible to provide collective protection using shelters that can be fixed, mobile, or improvised. Military personnel wear equivalent nuclear biological chemical/chemical biological radiological nuclear suits that are designed for uses extending to several days, and protection may be augmented by the use of prophylactic medication.

Decontamination Dissipation of non-persistent gases (e.g., chlorine, phosgene, sarin) may be facilitated by exhaust fans in enclosed areas. Neutralization of persistent liquids can be achieved chemically using liquid sodium dichlorocyanate (Fischor), or by absorption using powdered aluminum silicate (Fullers’ earth). Contaminated items need to be collected and contained for disposal, and protective equipment and people should be washed with hot water and soap (or 10% sodium carbonate or 5% household bleach) by blotting (rather than wiping) and rinsing. Pulmonary resuscitation and emetics are not normally recommended due to the increased risk of

Table 1

Comparison of the effects of scheduled chemical warfare agents.

OPCW schedule

1

2

3

Name

Code

Tabun ¼ O-ethyl N, N-dimethyl phosphoramidocyanidate

GA

Sarin ¼ O-isopropyl methylphosphonofluoridate Soman ¼ O-pinacolyl methylphosphonofluoridate O-ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate. Sulfur mustards: bis(2-chloroethyl) sulfide

GB GD VX H (HD when distilled)

Primary effect(s)

Paralysis (nerve)

Blister (vesicant)

Onset

Potency Inhalation LCt50 mg min–1 m–3

Dermal LD50 mg kg–1

100–200

14–57

4–24 h

70–100 70 39–70 300–1500

28 5 0.142 20–100

10–20 s

1500

37

Via flechettes, iv ¼ 0.57 mg kg–1 Via flechettes, iv ¼ 5 mg kg–1 Irritant, poorly absorbed Poorly absorbed

Seconds (fatal 1–10 m after inhaling, 1–2 h after dermal)

2-Chloroethylchloromethylsulphide Bis(2-chloroethylthio)methane Sesquimustard ¼ 1,2-bis (2-chloroethylthio)ethane 1,3-Bis(2-chloroethylthio)-n-propane 1,4-Bis(2-chloroethylthio)-n-butane 1,5-Bis(2-chloroethylthio)-n-pentane Bis(2-chloroethylthiomethyl)ether O-mustard ¼ bis(2-chloroethylthioethyl)ether Lewisite 1 ¼ 2-chlorovinyldichloroarsine Lewisite 2 ¼ bis(2-chlorovinyl)chloroarsine Lewisite 3 ¼ tris(2-chlorovinyl)arsine Nitrogen mustard 1 ¼ bis(2-chloroethyl) ethylamine Nitrogen mustard 2 ¼ bis(2-chloroethyl) methylamine Nitrogen mustard 3 ¼ tris(2-chloroethyl)amine Saxitoxin

T L1 L2 L3 HN1 HN2 HN3 TZ

Paralysis (nerve)

Minutes (fatal after 15 m)

5

Ricin

W

Gut lining (cytotoxic)

3–24 h

40

1,1,3,3,3-Pentafluoro-2-(trifluoromethyl)-1-propene

PFIB

Choking (pulmonary)

5 m–4 h

320

3-Quinuclidinyl benzilate

BZ

0.5–20 h

3800–200 000 (ICt50 ¼ 110–112)

Phosgene ¼ carbonyl dichloride

CG

Mental confusion (incapacitating), but effects are unpredictable Choking (pulmonary)

20 m–24 h

120–3200

Cyanogen chloride

CK

Asphyxiation (metabolic)

11 000

Hydrogen cyanide

AC

Asphyxiation (metabolic)

15 s (fatal after 30 s–30 m)

Chloropicrin ¼ trichloronitromethane

PS

Choking (pulmonary)

HK Q

1–12 h

3–30 s

1000–5000, (NB detoxified at 17 mg kg–1 min–1) 2000

Potent irritant, poorly absorbed 100 100 Potent irritant, poorly absorbed

Where: s ¼ seconds, m ¼ minutes, and h ¼ hours. NB. VG (Amiton ¼ O,O-diethyl S-[2-(diethylamino)ettiyl] phosphorothiolate) has similar properties to VX although with one-tenth the potency and hence is classified as schedule 2. Source: Encyclopedia of Toxicology (2014), Vol. 1 doi: 10.1016/B978-0-12-386454-3.01003-4.

760

Chemical warfare

contaminating additional people. Subsequent to decontamination, individuals should not be confined in small spaces with poor ventilation, or wrapped so as to contain any residual agent next to the body. Medical treatment will depend on the specific agent and development of systemic signs and symptoms (Rodgers and Condurache, 2010; Smirnov et al., 2013; Moshiri et al., 2012).

See also: Arsenical vomiting agents; Blister agents; BZ (3-quinuclidinyl benzilate) a psychotomimetic agent; Chemical warfare delivery systems; Cyclosarin (GF); G-series nerve agents; Nerve agents; Lewisite—A toxic warfare agent; Nerve agents; Nitrogen mustards; Sarin; Soman; Tabun; Vseries nerve agents other than VX; VX

References Kuca K and Pohanka M (2010) Chemical warfare agents. EXS 100: 543–558. Marrs TC, Maynard RL, and Sidell FR (2007) Chemical Warfare Agents: Toxicology and Treatment. Chichester, UK: John Wiley & Sons, Ltd. Moshiri M, Darchini-Maragheh E, and Balali-Mood M (2012) Advances in toxicology and medical treatment of chemical warfare nerve agents. Daru 20(1): 81. Rodgers GC Jr. and Condurache CT (2010) Antidotes and treatments for chemical warfare/terrorism agents: An evidence-based review. Clinical Pharmacology and Therapeutics 88(3): 318–327. Smirnov I, Belogurov A, Friboulet A, Masson P, Gabibov A, and Renard P-Y (2013) Strategies for the selection of catalytic antibodies against organophosphorus nerve agents. Chemico-Biological Interactions 203: 196–201.

Further reading Abo-zeid Y and Williams GR (2020) The potential anti-infective applications of metal oxide nanoparticles: A systematic review. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology 12(2): e1592. Bolt HM and Hengstler JG (2022) Recent research on Novichok. Archives of Toxicology 1–4. Bigley AN and Raushel FM (2019) The evolution of phosphotriesterase for decontamination and detoxification of organophosphorus chemical warfare agents. Chemico-Biological Interactions 308: 80–88. Costanzi S and Koblentz GD (2019) Controlling novichoks after salisbury: Revising the chemical weapons convention schedules. The Nonproliferation Review 26(5–6): 599–612. Costanzi S, Slavick CK, Hutcheson BO, Koblentz GD, and Cupitt RT (2020) Lists of chemical warfare agents and precursors from international nonproliferation frameworks: Structural annotation and chemical fingerprint analysis. Journal of Chemical Information and Modelling 60(10): 4804–4816. Fan S, Zhang G, Dennison GH, FitzGerald N, Burn PL, Gentle IR, and Shaw PE (2020) Challenges in fluorescence detection of chemical warfare agent vapors using solid-state films. Advanced Materials 32(18): 1905785. Giannakoudakis DA, Pearsall F, Florent M, Lombardi J, O’Brien S, and Bandosz TJ (2018) Barium titanate perovskite nanoparticles as a photo-reactive medium for chemical warfare agent detoxification. Journal of Colloid and Interface Science 531: 233–244. Haslam JD, Russell P, Hill S, Emmett SR, and Blain PG (2021) Chemical, biological, radiological, and nuclear mass casualty medicine: A review of lessons from the Salisbury and Amesbury Novichok nerve agent incidents. British Journal of Anaesthesia. Jacquet P, Rémy B, Bross RP, van Grol M, Gaucher F, Chabrière E, et al. (2021) Enzymatic decontamination of G-type, V-type and Novichok nerve agents. International Journal of Molecular Sciences 22(15): 8152. Jeong K and Choi J (2019) Theoretical study on the toxicity of ‘Novichok’ agent candidates. Royal Society Open Science 6(8): 190,414. Jeong WH, Lee JY, Lim KC, and Kim HS (2021) Identification and study of biomarkers from Novichok-inhibited butyrylcholinesterase in human plasma. Molecules 26(13): 3810. Jeong K, Lee JY, Woo S, Kim D, Jeon Y, Ryu TI, et al. (2022) Vapor pressure and toxicity prediction for Novichok agent candidates using machine learning model: preparation for unascertained nerve agents after chemical weapons convention schedule 1 update. Chemical Research in Toxicology. Kloske M and Witkiewicz Z (2019) Novichoks: The A group of organophosphorus chemical warfare agents. Chemosphere 221: 672–682. Lukey BJ, Romano JA Jr., and Salem H (eds.) (2019) Chemical Warfare Agents: Biomedical and Psychological Effects, Medical Countermeasures, and Emergency Response. CRC Press. Nepovimova E and Kuca K (2018) Chemical warfare agent NOVICHOK-mini-review of available data. Food and Chemical Toxicology 121: 343–350. Picard B, Chataigner I, Maddaluno J, and Legros J (2019) Introduction to chemical warfare agents, relevant simulants and modern neutralization methods. Organic & Biomolecular Chemistry 17(27): 6528–6537. Ploskonka AM and DeCoste JB (2019) Insight into organophosphate chemical warfare agent simulant hydrolysis in metal-organic frameworks. Journal of Hazardous Materials 375: 191–197. Rahmania TA, Wardhani BWK, Renesteen E, and Harahap Y (2021) Chemical properties, biological activities and poisoning treatment of novichok: A review. Pharmaceutical Sciences and Research 8(2): 2. Vanninen P, Östin A, Bełdowski J, Pedersen EA, Söderström M, Szubska M, et al. (2020) Exposure status of sea-dumped chemical warfare agents in the Baltic Sea. Marine Environmental Research 161(105): 112.

Relevant websites http://www.opcw.org :Organisation for the Prohibition of Chemical Weapons, Search for Convention. http://toxnet.nlm.nih.gov :TOXNET, Toxicology Data Network, National Library of Medicine, Search for Chemical Weapon. https://www.ncbi.nlm.nih.gov/books/NBK441997/ :Statpearls educational resources, Toxicology, V-Series Nerve Agents. https://costanziresearch.com/cw-nonproliferation/cw-control-lists/ :Chemical Warfare Agent Control Lists – Curated Tables. “A part of this work has been done within the scope of the Stimson Center’s Cheminformatics project, a collaborative project involving the Stimson Center’s Partnership in Proliferation Prevention’s Program and the Costanzi Research Group at American University. We gratefully acknowledge financial support from Global Affairs Canada.” https://www.epa.gov/emergency-response-research/chemical-fate-transport :USEPA Research reports on several Chemical Warfare Agent Fate and Transport. https://www.atsdr.cdc.gov/toxprofiledocs/index.html :Agency for Toxic Substances and Disease Registry (ATSDR, Centre for Disease Control, USA) – Toxicological Profiles. https://www.atsdr.cdc.gov/ToxProfiles/tp49.pdf :Sulphur Mustard, Toxicological Profile (2003).

Chemical warfare agents and delivery systems Alicia P DeFalco, South College School of Pharmacy, Knoxville, TN, United States © 2024 Elsevier Inc. All rights reserved.

Introduction Types of agents Nuclear and radiological Biological Chemical Vesicants Nerve agents Blood agents Choking agents Incapacitating agents Irritants/riot control agents Delivery methods Exposure Acute and short-term toxicity Vesicants Nerve agents Blood agents Choking agents Incapacitating agents Irritants/riot control agents Chronic toxicity Vesicants Nerve agents Blood agents Choking agents Incapacitating agents Irritants/riot control agents Clinical management Chemical properties/environmental fate Conclusion References Further reading

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Abstract The aim of this chapter is to discuss warfare agents and their delivery systems, with a focus on chemical warfare agents, and the toxicology associated with these agents. Chemical warfare agents are further classified as vesicants, nerve agents, blood agents, choking agents, incapacitating agents, and irritants/riot control agents. Exposure to these substances may occur by inhalation, dermal absorption, oral ingestion, and/or injection. The management of toxicity prioritizes early decontamination to reduce sequelae and further contamination.

Keywords Blister agents; Blood agents; Chemical warfare; Choking agents; Decontamination; Incapacitating agents; Nerve agents; Nuclear warfare; Riot control agents; Vesicants

Key points

• •

Warfare agents may be categorized as biological, nuclear, radiological, or chemical. Chemical warfare agents are further categorized as vesicants, nerve agents, blood agents, choking agents, incapacitating agents, and irritants/riot control agents. The four major properties that contribute to exposure injury are toxicity, latency, persistency, and transmissibility.

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Dissemination of these substances may occur through explosives, spray tanks, and aerosolization which may lead to direct contact and/or inhalation of the agents. Treatment is mainly focused on decontamination and limiting further spread and contamination. In addition to early decontamination, antidotes are available for nerve agents and cyanide, but these must be administered early to be effective.

Introduction Warfare agents may take on a number of forms and delivery systems. They include substances from nuclear fission or fusion, radioactive material, biological agents that may induce disease, and toxic chemicals (International Committee of the Red Cross, 2014). Toxicity may occur via radiation, inhalation, dermal contact, or ingestion. Chemical warfare agents were first developed during World War I when chlorine was released from large cylinders, and eventually, mustard gas was introduced. By the end of World War II, a new type of warfare agent known as, “nerve gases” were introduced. Warfare agents can be broken down into chemical, biological, radiological and nuclear agents. There are four key components which contribute to their ability to inflict injury: toxicity, latency, persistency, and transmissibility. Toxicity is the propensity to cause harm or death, and latency is the amount of time between exposure and first signs of illness. These two attributes are important to consider in treating patients exposed to chemical warfare agents. Persistency is the ability of an agent to induce harm for a prolonged period of time, and transmissibility is the capacity to contaminate additional victims. Persistency and transmissibility are important when responding to and containing exposure events (International Committee of the Red Cross, 2014). There are a variety of agents that fall under the warfare agent umbrella. Nuclear and radiological may emit radiation such as alpha radiation, beta radiation, gamma radiation and x-rays. Biological agents are microorganisms that induce infection, toxicity, and/or allergic reactions. Chemical agents are toxic chemical substances that have been developed under military guidance and include blister agents, nerve agents, blood agents, choking agents, incapacitating agents, and irritants (International Committee of the Red Cross, 2014). A summary of the types of agents belonging to each category may be found in Table 1.

Table 1

Types of warfare agents.

Types of warfare agents Category

Examples

Biological

Anthrax Botulinum toxin Staphylococcal enterotoxin B Alpha radiation Beta radiation Gamma radiation X-rays Sulfur mustard (HD) Nitrogen mustard (HN) Phosgene oxime (CX) Lewisite (L) Tabun (GA) Sarin (GB) Soman (GD) Methylphosphonothioic acid (VX) Arsine (SA) Cyanogen chloride (CK) Hydrogen cyanide (AC) Potassium cyanide Sodium cyanide White phosphorus Phosgene (CG) Diphosgene (DP) Chlorine Chloropicrin (PS) Fentanyl 3-Quinuclidinyl benzilate (BZ) Chloroacetophenone (CN) O-chlorobenzylmalonitrile

Nuclear and Radiological

Chemical

Vesicants

Nerve Agents

Blood Agents

Choking Agents

Incapacitating Agents Irritants/Riot Control Agents

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Dissemination of these substances may occur through explosives, spray tanks, and aerosolization which may lead to direct contact and/or inhalation of the agents. Treatment is mainly focused on decontamination and limiting further spread and contamination.

Types of agents Nuclear and radiological Nuclear agents are radioactive materials produced from nuclear fission or fusion. They may be released by nuclear weapons or nuclear power plants. Radiological agents may be released from mineral processing industries, industrial use, medical therapy, or may naturally occur in the environment. There are different types of ionizing radiation emitted by radioactive materials: alpha radiation, beta radiation, gamma radiation, and x-rays. Alpha radiation is considered short range and has low penetration capacity. The particles of protons and neutrons that are emitted are not able to penetrate skin but are able to be inhaled or ingested. Beta radiation has increased ability to penetrate the skin, reaching the top layer of the skin, and has the ability to travel several feet through the air. The particles that are emitted consist of negatively charged electrons. Gamma radiation and x-rays emit electromagnetic radiation that is highly penetrating, capable of reaching several centimeters into human tissue. Risks of exposure to nuclear and radiological agents range from increased cancer risk to acute, life-threatening injuries. The extent of complication depends on the type of exposure, duration of time, and dose (International Committee of the Red Cross, 2014).

Biological Biological agents are microorganisms that induce infection, toxicity, and/or allergic reactions. They may exist in the form of viruses, bacteria, or fungi. They are transmitted through direct contact with the substance, may be airborne, or can be transmitted by an arthropod vector. Examples include anthrax, botulinum toxin, staphylococcal enterotoxin B. They possess long latency, and the onset is typically delayed for days after exposure. The severity of injury varies depending upon type of agent, route and level of exposure, individual variability, and access to medical treatment (International Committee of the Red Cross, 2014).

Chemical While some chemical agents may be used for agricultural or industrial purposes, the focus of this chapter will be on those developed under military guidance to be used as weaponry. Categories of chemical warfare agents include vesicants, nerve agents, blood agents, choking agents, incapacitating agents, and irritants.

Vesicants Vesicants or blister agents are known to induce burns and blisters on the skin, mucous membranes, and may cause damage to the respiratory tract. Sulfur mustard is the most well-known of this category. Mustard works as an alkylating agent that generates a reactive sulfonium ion which alkylates sulfhydryl and amino groups. It also serves as a direct inhibitor of glycolysis which results in necrosis from adenosine triphosphate (ATP) depletion (Suchard, 2019). These agents exhibit low rates of fatalities, however, have the capacity to inflict painful burns and blisters at low doses.

Nerve agents Nerve agents inhibit acetylcholinesterase causing an excess of acetylcholine which results in cholinergic effects such as defecation, diaphoresis, urination, miosis, bradycardia, bronchorrhea, emesis, lacrimation, and salivation as well as respiratory paralysis and flaccidity. These are considered to be the most toxic chemical warfare agents. Examples include sarin (GB), tabun (GA), soman (GD), VX, cyclosarin (GF), and Russian VX (VR) (Suchard, 2019). Unless the cholinesterase is reactivated by targeted therapy with oximes, its binding to the enzyme is irreversible. Erythrocyte acetylcholinesterase activity recovers at approximately 1% per day. Plasma butyrylcholinesterase recovers more quickly and is a better guide to recovery of tissue enzyme activity (Romano and Newmark, 2022).

Blood agents Blood agents such as cyanide inhibit the transfer of oxygen within the blood. The last step of mitochondrial electron transport chain is damaged which halts cellular energy production. Tissues effected by cyanide cannot utilize oxygen from the blood and the cells die of hypoxia (Romano and Newmark, 2022).

Choking agents Choking agents, also referred to as pulmonary asphyxiants, cause damage to the lungs and delayed acute respiratory distress syndrome (ARDS) due to increased alveolar-capillary membrane permeability. Examples of choking agents include chlorine, phosgene, and diphosgene. Phosgene exerts its toxic effects by undergoing hydrolysis in the lungs to hydrochloric acid and forming diamides what crosslink cell components (Suchard, 2019).

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Incapacitating agents Incapacitating agents such as fentanyl and 3-quinuclidinyl benzilate (BZ) cause changes in mental status such as hallucinations and loss of consciousness and may result in respiratory failure (Suchard, 2019). BZ is now considered obsolete, however, it was known for having a long onset and duration of action of up to 48 h, causing confusion and impairing the patient’s ability to act decisively.

Irritants/riot control agents Irritants such as O-chlorobenzylmalonitrile were developed in the 1960s, are expected to be nonlethal, and are primarily used as riot gas to control large crowds. They are also referred to as riot control agents. These agents irritate the eyes, skin, and respiratory tract, but possess low toxicity (Suchard, 2019).

Delivery methods Chemical warfare agents may be disseminated using a variety of delivery methods. Some of the formats include bombs, submunitions, projectiles, warheads, and spray tanks. Explosive bombs usually take the form of central bursters which expel the product laterally. Agents detonated in this fashion may be diminished by incineration or by being forced into the ground. Some products are flammable which may result in the cloud of particles being ignited. Aerodynamic technology allows for non-explosive dispersion but requires that altitude be controlled, and that weather conditions, wind direction and velocity must be known. Thermal dissemination uses pyrotechnics to aerosolize agents. This produces fine, inhalable clouds of incapacitants. Many of the complex agent molecules are sensitive to high temperatures causing them to deteriorate if exposure is prolonged.

Exposure Chemical, biological, radiological, and nuclear (CBRN) agents may be difficult to detect. Many are odorless and/or colorless, and radioactive agents emit radiation that cannot be sensed. Products that take the form of liquid, vapor, smoke, dust, or aerosols may be inhaled, absorbed topically, ingested, and/or injected. Transmission may occur by person-to-person contact or contact with an arthropod vector (International Committee of the Red Cross, 2014).

Acute and short-term toxicity Vesicants Vesicants commonly impact the eyes, skin, and respiratory tract. Exposure manifests as blisters and lesions. While complications increase the risk of infection and are painful for the victim, there is a low mortality rate associated with vesicant exposure. The extent of injury is dependent upon the dose and form of the agent (Suchard, 2019). Timing of symptom onset depends on severity of exposure, ambient temperature, humidity, and type of skin exposed (Romano and Newmark, 2022). Erythema, vesicles, bullae, and necrosis typically ensue over 4–12 h. Vapor exposure can cause first- or second-degree burns while liquid exposure may be more detrimental, causing full-thickness burns. Ocular exposure may result in pain, miosis, photophobia, lacrimation, blurred vision, blepharospasm, and corneal damage. Inhalation may cause hoarseness, cough, sore throat, and chest pressure (Suchard, 2019).

Nerve agents Toxic effects develop within seconds to minutes with vapor exposure, however, liquid exposure may present in a delayed fashion. Some toxic effects mainly involving the central nervous system may persist for weeks. Persistence of toxicity and symptoms is dependent upon agent form. The most common acute symptomatic presentation includes defecation, diaphoresis, urination, miosis, bradycardia, bronchorrhea, emesis, lacrimation, and salivation. In severe toxicity, victims may experience loss of consciousness, seizures, muscle fasciculations, flaccid paralysis, and/or apnea. The following LD50 values have been documented in the literature: tabun aerosol: 400 mg-min/ m3, VX aerosol: 10 mg-min m3 -1, sarin dermal: 1700 mg, VX dermal: 6–10 mg. Data suggests that Harber’s rule does not apply in all exposures based on rat studies as rats exposed to sarin vapor survived higher concentrations than predicted at the shortest and longest exposure times (Suchard, 2019).

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Blood agents Blood agents rapidly impact victims and may result in death as quickly as 8 min after exposure. As early as 15 s after inhalation, patients may experience hyperpnea immediately followed by convulsions. Two to three minutes after exposure, respirations may stop leading to cardiac arrest. Skin may appear cherry-red or cyanotic (Romano and Newmark, 2022). The documented LD50 of aerosolized hydrogen cyanide is 2500–5000 mg-min/ m3 (Suchard, 2019).

Choking agents Choking agents produce eye and skin irritation, dyspnea, cough, sore throat, and chest tightness (Romano and Newmark, 2022). In some cases, it has been reported that smoking tobacco produces an unpleasant taste and some case reports have noted an outflow of pink foam from the mouths of those exposed. Respiratory distress may occur hours after exposure, and exercise may induce acute respiratory distress syndrome (Suchard, 2019).

Incapacitating agents Incapacitating agents primarily impact the central nervous system and manifest as altered level of consciousness, confusion, lack of coordination, and hallucinations (Romano and Newmark, 2022).

Irritants/riot control agents Irritants manifest toxic effects within seconds to minutes. Symptoms of exposure include burning eyes, lacrimation, burning or itching skin, and coughing (Suchard, 2019).

Chronic toxicity Vesicants Chronic toxic effects that may result from vesicant exposure include pulmonary carcinoma, chronic bronchitis, emphysema, scarring, pigmentation changes, chronic neuropathic pain, and pruritis (Suchard, 2019).

Nerve agents Long-term consequences associated with exposure to nerve agents may include peripheral neuropathy, intermediate syndrome, or posttraumatic stress disorder (Suchard, 2019).

Blood agents There is limited data available to support long-term complications associated with blood agent exposure as these agents carry a high mortality risk with acute exposure.

Choking agents Chronic toxicity is rare with choking agents, however, in some cases, delayed pulmonary edema may occur (Romano and Newmark, 2022).

Incapacitating agents Most victims exposed to incapacitating agents will recover after the acute effects.

Irritants/riot control agents Most victims exposed to irritants/riot control agents will recover after the acute effects.

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Clinical management Steps for managing those exposed to chemical warfare agents is dependent on the type of agent and exposure. Treatment mainly consists of decontamination and supportive care. Victims should promptly be removed from the area of exposure. Decontamination is key to prevent further absorption and spread of toxic substances. First responders and medical professionals caring for victims must don appropriate personal protective equipment (PPE). For those who experience dermal, hair, or clothing exposures, clothing must be removed and sealed in a hazardous waste bag, exposed areas of the skin should be scraped, wiped, and washed with disposable cloths, and for those who have contaminated hair, effort should be made to cleanse the hair first, keeping the body shielded to avoid additional exposure to the body upon washing (International Committee of the Red Cross, 2014). For mass exposures, patients should be triaged into groups able to decontaminate themselves and groups requiring assistance from healthcare personnel. Additional triage should be completed to distinguish the level of acuity and severity of complications each patient is experiencing. In all exposures, post-exposure surveillance can be helpful to monitor for delayed complications (Suchard, 2019). In the event of exposure to a nuclear or radiological event that involves radioactive iodine, potassium iodine may be administered prophylactically to reduce the risk of developing thyroid cancer. When biological weapons are utilized, vaccination, antibiotics, or other antidotes may be utilized dependent upon the culprit of the exposure (International Committee of the Red Cross, 2014). With vesicants, the primary method of decontamination is to utilize water or dilute hypochlorite solution in addition to supportive care such as hydration, burns care, and analgesia to manage skin lesions (Romano and Newmark, 2022). Unfortunately, once a victim develops symptoms, it is unlikely that decontamination will benefit the patient, however, it can minimize spread to others (Suchard, 2019). Similar decontamination strategies apply to those who are exposed to nerve agents. Solutions that release chlorine (e.g., household bleach) and alkaline solutions inactivate nerve agents. As with the vesicants, a dilute hypochlorite solution may be utilized, and this may be compounded by using a 1:10 dilution of household bleach in water. Soap and water or water alone are acceptable alternatives, with priority being placed on urgent washing of the exposed areas (Suchard, 2019). In addition to decontamination, antidotes exist for the management of exposure to nerve agents. The muscarinic effects that ensue such as defecation, diaphoresis, urination, miosis, bradycardia, bronchorrhea, emesis, lacrimation, and salivation may be treated using the antimuscarinic agent, atropine. Nicotinic effects such as fasciculations, weakness, flaccid paralysis will not be reversed by atropine, however, may be treated using the oxime agent, pralidoxime (2-PAM). These antidotes are available as a combination product under the commercially available name Duodote™ and in the military known as Antidote Treatment Nerve Agent Autoinjector (ATNAA). To be efficacious, pralidoxime must be administered before irreversible dealkylation, also known as “aging,” of the organic phosphorus compound-cholinesterase complex forms (Suchard, 2019). Once a complex becomes “aged,” it is negatively charged, and oximes are unable to reactive the negatively charged complex (Romano and Newmark, 2022). This process varies based on the agent. Aging half-life of nerve agents include: soman—2 to 6 min, sarin—3 to 5 h, tabun—14 h, and VX—48 h. For an agent such as soman, pralidoxime is unlikely to benefit the victim due to its short aging half-life. Although tabun has a much longer aging half-life, studies have also shown that pralidoxime is ineffective in treating tabun exposures due to a mechanistic issue. If patients experience convulsions benzodiazepines are first line (Suchard, 2019). Treatment of those exposed to blood agents such as cyanide involves decontamination, oxygen supplementation, and antidote administration. There are two options for cyanide antidotes: a twostep regimen consisting of sodium nitrite and sodium thiosulfate or as an alternative, hydroxocobalamin (Romano and Newmark, 2022). Exposure to choking agents are primarily managed through decontamination and supportive care such as providing respiratory support. Incapacitating agents may take days to recover from, however, for products such as fentanyl, naloxone may be utilized to reverse its effects. Victims who have been exposed to irritants (riot control agents) may be treated by water irrigation of the skin and ocular decontamination utilizing anesthetic eye drops and ophthalmic irrigation (Suchard, 2019).

Chemical properties/environmental fate Volatility is inversely related to persistence, thus the lower the volatility, the higher the environmental persistence. Most chemical gases and vapors are denser than air and collect in low lying areas. Nerve agents are mostly clear and colorless. The G agents (tabun (GA), sarin (GB), and soman (GD)) are volatile, evaporate quickly, and are a vapor hazard. Sarin is the most volatile while VX is an oily liquid with low volatility. Vesicants possess low volatility and high environmental persistence. These products tend to have an odor, making them slightly more easily detected compared to nerve agents (Suchard, 2019). Blood agents are gases or liquids close to their boiling points at room temperature. Cyanide is reported to smell like bitter almonds, but only approximately 50% of the population are able to smell it (Romano and Newmark, 2022). Choking agents exist as a true gas, usually possess an odor, and while some present as a color, others are colorless. Riot control agents are solids at normal temperatures, however, they are dispersed as aerosols or liquid sprays (Suchard, 2019). A comparison of chemical agent properties such as onset and potency can be found in Table 2.

Table 2

Comparison of the effects of scheduled chemical warfare agents.

OPCW schedule

1

3-Quinuclidinyl benzilate 3

Code

Tabun ¼ O-ethyl N,N-dimethyl phosphoramidocyanidate

GA

Sarin ¼ O-isopropyl methylphosphonofluoridate Soman ¼ O-pinacolyl methylphosphonofluoridate O-ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate. Sulfur mustards: bis(2-chloroethyl)sulfide 2-Chloroethylchloromethylsulphide Bis(2-chloroethylthio)methane Sesquimustard ¼ 1,2-bis(2-chloroethylthio) ethane 1,3-Bis(2-chloroethylthio)-n-propane 1,4-Bis(2-chloroethylthio)-n-butane 1,5-Bis(2-chloroethylthio)-n-pentane Bis(2-chloroethylthiomethyl)ether O-mustard ¼ bis(2-chloroethylthioethyl)ether Lewisite 1 ¼ 2-chlorovinyldichloroarsine Lewisite 2 ¼ bis(2-chlorovinyl)chloroarsine Lewisite 3 ¼ tris(2-chlorovinyl)arsine Nitrogen mustard 1 ¼ bis(2-chloroethyl) ethylamine Nitrogen mustard 2 ¼ bis(2-chloroethyl) methylamine Nitrogen mustard 3 ¼ tris(2-chloroethyl)amine Saxitoxin

GB GD VX H (HD when distilled)

Primary effect(s)

Paralysis (nerve)

Blister (vesicant)

Onset

Potency Inhalation LCt50 mg min−1 m−3

Dermal LD50 mg kg−1

100–200

14–57

70–100 70 39–70

28 5 0.142

4–24 h

300–1500

20–100

10–20 s

1500

37

Via flechettes, iv ¼ 0.57 mg kg−1 Via flechettes, iv ¼ 5 mg kg−1 Irritant, poorly absorbed

Seconds (fatal 1–10 m after inhaling, 1–2 h after dermal)

HK Q

T L1 L2 L3 HN1

1–12 h

HN2 HN3 TZ

Paralysis (nerve)

Minutes (fatal after r 15 m)

5

Ricin

W

Gut lining (cytotoxic)

3–24 h

40

1,1,3,3,3-Pentafluoro-2-(trifluoromethyl)-1propene BZ

PFIB

Choking (pulmonary)

5 m–4 h

320

0.5–20 h

3800–200,000 (ICt50 ¼ 110–112)

Poorly absorbed

Phosgene ¼ carbonyl dichloride

Mental confusion (incapacitating), but effects are unpredictable CG

Choking (pulmonary)

20 m–24 h

120–3200

Cyanogen chloride Hydrogen cyanide

CK AC

Asphyxiation (metabolic) Asphyxiation (metabolic)

15 s (fatal after 30 s–30 m)

Chloropicrin ¼ trichloronitromethane

PS

Choking (pulmonary)

3–30 s

11,000 1000–5000, (NB. detoxified at 10 mg kg−1 min−1) 2000

Potent irritant, poorly absorbed

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Where: s, seconds; m, minutes; and h, hours. NB. VG Amiton ¼ O,O-diethyl S-[2-(diethylamino)ethyl] phosphorothiolate) has similar properties to VX although with one-tenth the potency and hence is classified as schedule 2. From Burr S.A. (2014) Chemical warfare delivery systems. In: Wexler P. (ed.) Encyclopedia of Toxicology, 3rd edn., vol. 1, pp. 817–821. Cambridge, MA: Academic Press.

Potent irritant, poorly absorbed 100

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Name

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Conclusion Warfare agents are available in a variety of forms and may be disseminated through various mechanisms. They may be categorized as biological, nuclear, radioactive, or chemical. Chemical agents are further classified as vesicants, nerve agents, blood agents, choking agents, incapacitating agents, and irritants/riot control agents. Toxicity may occur via radiation, inhalation, dermal contact, or ingestion. The main factors impacting ability to induce harm include toxicity, latency, persistency, and transmissibility. Volatility of these products plays a key role in environmental persistence, with lower volatility resulting in higher environmental persistence. While some antidotes exist, treatment of victims exposed to these agents prioritizes decontamination and limiting further spread and contamination.

References International Committee of the Red Cross (2014) Chemical, Biological, Radiological and Nuclear Response – Introductory Guidance. Geneva: Switzerland. Available at: https://shop.icrc. org/download/ebook?sku¼4175/002-ebook. Romano JA Jr. and Newmark J (2022) Chemical terrorism. In: Loscalzo J, Fauci A, Kasper D, Hauser S, Longo D, and Jameson J (eds.) Harrison’s Principles of Internal Medicine, 21st ed. New York, NY: McGraw Hill Available at: https://accesspharmacy.mhmedical.com/content.aspx?bookid¼3095§ionid¼265477318. Suchard JR (2019) Chemical weapons. In: Nelson LS, Howland M, Lewin NA, Smith SW, Goldfrank LR, and Hoffman RS (eds.) Goldfrank’s Toxicologic Emergencies, 11th ed. New York, NY: McGraw Hill Available at: https://accesspharmacy.mhmedical.com/content.aspx?bookid¼2569§ionid¼210264855.

Further reading Burr SA (2014) Chemical warfare delivery systems. In: Wexler P (ed.) Encyclopedia of Toxicology. 3rd ed., vol. 1, pp. 817–821. Cambridge, MA: Academic Press. Ganesan K, Raza SK, and Vijayaraghavan R (2010) Chemical warfare agents. Journal of Pharmacy & Bioallied Sciences 2(3): 166–178. Available at https://doi.org/10.4103/09757406.68498.

Relevant websites https://www.cdc.gov/nceh/demil/overview.htm :Centers for Disease Control and Prevention (2013) Overview of U.S. Chemical Weapons Elimination. https://www.cdc.gov/nceh/dls/radiologic_threat_agents.html :Centers for Disease Control and Prevention (2019) Radiologic Threat Agents. https://programs.fas.org/bio/chemweapons/delivery.html :Federation of American Scientists (2013) Chemical Weapons Delivery. https://irp.fas.org/threat/mctl98-2/p2sec04.pdf :Federation of American Scientists (2013) Chemical Weapons Technology. https://nuke.fas.org/guide/usa/cbw/cw.htm :Federation of American Scientists (2018) Weapons of Mass Destruction Around the World.

Chemical warfare delivery systems Steven A Burr, Peninsula Medical School, University of Plymouth, Plymouth, United Kingdom © 2024 Elsevier Inc. All rights reserved. This is an update of S.A. Burr, Chemical Warfare Delivery Systems, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 817–821, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01002-2.

Chemical agent properties and targeted dispersion Manual delivery Cylinders Hand bombs Landmines Projectile delivery Mortars Artillery shells Self-propelled projectiles Air delivery Unitary bomb Cluster bombs Aircraft spray tanks Binary glide bomb Improvised weapons Storage, transport, and safe handling Detection Decommissioning, neutralization, and incineration References Further reading

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Abstract Various devices have been engineered to maximize targeted dispersion. Accurate information to compare volatility, vapor density, color, odor, environmental half-life and persistence can be collated with difficulty but often has unverifiable military provenance. Recent work focuses on safe handling, storage and transport, detection, decommissioning, neutralization and incineration.

Keywords Anthrax; Arsenical vomiting agents; Bio warfare and terrorism; Blister agents/vesicants; Botulinum toxin; Chemical warfare; Chlorine; Cyclosarin; G-series nerve agents; Lewisite; Nerve agents; Nitrogen mustards; Sarin; Soman; Tabun; V-series nerve agents; VX

Key points

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Nations previously developed highly sophisticated methods of delivery to maximize impact. Terrorists seek improvised approaches that present significant hazard to the user and deliver sub-optimal exposures to targets. Known physicochemical characteristics enable specific targeting by using technology to control dispersal, penetration, adhesion, persistence, and detection. Delivery can be by manual contact, projectile, explosive, or an improvised transfer agent. Methods have been developed to ensure safe handling for storage, transport and manipulation prior to delivery. Emphasis is now on detection and decommissioning.

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Chemical agent properties and targeted dispersion The suitability of a chemical agent for delivery as a weapon depends on the effect required. To cause personnel casualties, the agent needs the appropriate volatility and density for adequate dispersal in the target environment (e.g., hydrogen cyanide is lighter than air and quickly lost unless used in an enclosed space). Non-persistent gases such as chlorine are useful for areas that require rapid occupation. Persistent liquids such as VX need to be non-volatile and oily, with a larger droplet size to contaminate surfaces, and thickened (e.g., with an acryloid copolymer) to make them gelatinous and sticky to present a contact hazard. Accurate topographical maps and weather forecasting increase the success of agent laydown on target. Vegetation can absorb agents. Atmospheric pressure, temperature, humidity, and wind can affect vaporization. Wind speed, direction and consistency, and terrain can all divert delivery. Recognition of color, odor, and early onset warning signs and symptoms may enable protective countermeasures to be employed (e.g., the smell of phosgene is not unusual nor unpleasant and rapidly fades, while the onset of adverse effects may be delayed for several hours after exposure, by which time it is too late to avoid or use protection). Explosive detonation of a central ‘burster’ charge can be used to spread a small volume of agent. Explosive delivery methods are noticeable, but exposure is near instantaneous. However, it is difficult to optimize the size of droplets produced explosively. Some agent may be lost due to projection into surfaces and incineration, whereas flammable agents such as VX may ignite and be lost due to flashing. In contrast, non-flammable agents such as BZ may depend on thermal aerosolization. All agents need to be sufficiently stable to deliver predictable concentrations after storage, transportation, and delivery. Corrosive properties complicate the design of delivery systems (e.g., sarin is slightly corrosive to steel, and phosgene is highly corrosive in the presence of moisture). Unstable agents with short shelf-lives may require the addition of stabilizers (e.g., tributylamine to prevent hydrolysis of sarin, or diisopropylcarbodiimide when containing sarin in aluminium). Rapid environmental degradation enables takeover of the affected area (e.g., saxitoxin or ricin) via flechettes, whereas slow degradation requires personal protection or environmental decontamination to overcome exclusion areas (e.g., later, nitrogen mustards were developed for volatility, potency, and longer persistence). Persistence can be affected by many factors such as atmospheric transport, sedimentary binding, and microorganism biodegradation, in addition to reactions with water and photochemically produced hydroxyl radicals (see Table 1; and Marrs et al., 2007).

Manual delivery Manual delivery systems have the most efficient ’chemical agent fill’ to ’delivery system’ weight ratio. However, such systems also pose the greatest risk to friendly personnel due to close proximity during release and were largely replaced by projectile- and air-delivery systems after World War I.

Cylinders Fritz Haber personally directed the first chlorine gas attack in 1915, releasing 180,000 kg from 5730 cylinders, which then successfully drifted over entrenched French and Algerian troops at Ypres. Cylinder release is highly dependent on wind, which can reverse and return gas to the attacker.

Hand bombs The 2.7 kg (British 6 lb) ground bomb had a 2 min delay fuse with an ejection charge to blow out the end of the bomb and release 1.6 kg of mustard gas.

Landmines The 15 kg KhF-1/KhF-2 (Russian) bounding gas mine (345
150 mm) was electronically triggered by remote operator or tripwire to fire the mine vertically out of its container into the air and after a delay of 1.5 s to fragment at a height of 4–8 m and release 4.5 l of agent to cover a 10 m diameter area. The 4.9 kg M1 landmine (204
75 mm) consisted of a fuel can that released 4.5 kg of mustard gas after detonation of an external bursting charge. The 10 kg M23 landmine (330 mm diameter by 127 mm deep) was activated by a pressure-plate triggering a fuse to detonate an internal burster charge that ruptured the mine and heated 4.7 kg of VX for dispersal as an aerosol mist.

Table 1

Comparison of factors affecting agent dispersion, detection, and persistence.

Name

Tabun ¼ O-ethyl N,N-dimethyl phosphoramidocyanidate Sarin ¼ O-isopropyl methylphosphonofluoridate Soman ¼ O-pinacolyl methylphosphonofluoridate O-ethyl S-2-diisopropylaminoethyl methyl phosphonothiolate Sulfur mustards: bis(2-chloroethyl) sulfide 2-Chloroethylchloromethylsulphide Bis(2-chloroethylthio)methane (HK) Sesquimustard ¼ 1,2-bis (2-chloroethylthio) ethane 1,3-Bis(2-chloroethylthio)-n-propane 1,4-Bis(2-chloroethylthio)-n-bulane 1,5-Bis(2-chloroethylthio)-n-pentane Bis(2-chloroethylthiomethyl)ether O-mustard ¼ bis(2-chloroethylthioethyl)ether Lewisite 1 ¼ 2-chlorovinyldichloroarsine Lewisite 2 ¼ bis(2-chlorovinyl)chloroarsine Lewisite 3 ¼ tris(2-chlorovinyl)arsine Nitrogen mustard 1 ¼ bis (2-chloroethyl) ethylamine Nitrogen mustard 2 ¼ bis (2-chloroethyl) methylamine Nitrogen mustard 3 ¼ tris(2-chloroethyl)amine Saxitoxin

Code

Boiling point/state (at 20  C)

Vapor density (relative to air)

Color

GA

240/Liquid

5.6

Pale amber

GB GD VX

147/Liquid 167/Liquid 298/Liquid

4.86 6.3 9.2

None Amber

H (HD when distilled)

215–7/Oily liquid

5.4

Pale yellow

190/Oily liquid

7.1

194/Liquid

7.1

Water soluble solid (melts at 236) Water soluble solid (melts at 230) 7/Gas

NA

Odor/threshold (mg m–3)

Environmental T1/2 at 20  C & pH 7 photolysis/hydrolysis

Persistence At 4–16  C

At 21–32 C

None (mild fruity almond if impure)

8 h/8.5 h

1–2 d

2–4 d

None-mild fruity/1.5 Camphor/3.3 None/3.9

9.6 h/75 h 8 h/82 h 2.5 h/57 h

0.5–24 h 1–2 d 10–30 d

24–36 h 2–4 d 30–90 d

Garlic/0.015 – desensitizes within 3–8 m

50 h/4–16 m

1–2 d

2–4 d

Mild geramiums/0.14

1.3 m/1.2 m

18–36 h

2–3 d

Dark-pale yellow

Fishy-soapy/0.6

24–36 h/9 d

1–3 d

2–6 d

White

NA

9–28 d in water

Aerosol decays 17% m–1, 12 w for particulates 7–14 d

HK Q

T L1 L2 L3 HN1 HN2 HN3 TZ W

1,1,3,3,3-Pentafluoro-2-(trifluoromethyl)-1propene 3-Quinuclidinyl benzilate

PFIB

Phosgene ¼ carbonyl dichloride Cyanogen chloride

BZ

6.9

Note

None (metallic taste)

14 h on wet metal, >14 d on dry concrete 5.7 d/rapid

Highly stable in the absence of water 10–20 d 30–60 d

White

NA

3–4 w in moist air

3.4 1.98

Pale yellow Note

Hay/1.5 – desensitizes rapidly Acrid/2–2.5

30 m 15–30 m

Hydrogen cyanide

AC

25.6/Liquid-gas

0.94

Almond/0.9

Chloropicrin ¼ trichloronitromethane

PS

112/Oily liquid

5.7

Very pale bluewhite Very pale yellow

44 y/0.026 s Not susceptible/5.25 h to AC unless alkaline 14–22 w/slow

Flypaper/7.96

3–18 w/11 y

1 d–10 w

60 m 30–60 m

771

NA

CG CK

Water soluble solid (melts at 164) 8.2/Gas 13/Gas

Where: m ¼ minutes, h ¼ hours, d ¼ days, w ¼ weeks and y ¼ years. Source: Encyclopedia of Toxicology (2014), Volume 1 https://doi.org/10.1016/B978-0-12-386454-3.01002-2, 817.

12–19 d

Chemical warfare delivery systems

Ricin

772

Chemical warfare delivery systems

Projectile delivery Mortars The 190 mm Livens Projector combined the gas volume of a cylinder with the range of artillery, firing a 14 kg, 550 mm long cylinder (Livens drum) up to 1.5 km. It was inaccurate but simple and inexpensive, enabling use en masse (the use of 1000s delivering saturation coverage of high gas concentrations) in an electrically triggered simultaneous barrage of projectiles that burst on impact to disperse the agent (most commonly phosgene or chloropicrin were employed by the British between 1916 and 1918). The 107 mm diameter (11.3 kg) 60 mm M2 mortar (range 4 km) was capable of delivering a M2A1 2.7 kg distilled mustard, 60% mustard to 40% o-mustard mix (for higher volatility), phosgene, or white phosphorous ammunition round.

Artillery shells The 155 mm (44 kg, 68 cm long) shell (range 14–30 km) had several variants, adapted to deliver various chemicals: M104 for 5.3 kg of mustard or distilled mustard, M110 for 7 kg of mustard, distilled mustard, or white phosphorus, and M121A1 for 2.9 kg of sarin or 2.7 kg of VX. The 105 mm (17.6 kg, 53 cm long) M60 shell (range 11–17 km) contained 1.4 kg of distilled mustard, and was adapted as the (14.5 kg, 50 cm long) M360 containing 725 g of sarin. The 203 mm (90 kg, 89 cm long) M426 shell (range 17–34 km) contained 7.2 kg of sarin or 6.4 kg of VX. Shells usually comprise a hollow one-piece steel shell that is press-fitted with a burster casing. Detonation depends on the fuse type selected. Fuse functioning detonates an explosive charge, which ruptures the projectile and heats and disperses the agent as an aerosol. The 155 mm (60 cm long) M687 shell (range 14–30 km) contained GB2: two canisters separated by a rupture disk (breached by acceleration when fired, with the spinning of the shell causing mixing, and sarin being produced in flight), one canister contained methylphosphonyl difluoride (DF in M20 canister) and the other contained isopropyl alcohol and isopropyl amine (OPA in M21 canister). Shells are typically more robust and safer to handle, store, and transport than other chemical munitions. Conventional artillery equipment can be used without specialist adaptation and can maintain a sustained rate of fire. Range and trajectory can be used to calculate the fuse time delay needed to cause detonation at the desired altitude.

Self-propelled projectiles The 115 mm (25 kg, 2 m long) M55 rocket contained 4.8 kg of sarin or 4.5 kg of VX, delivered up to a range of 6.5 km, M55 rockets could be fired individually or via an M91 trailer mounted multiple-rocket launcher, simultaneously firing 45 M55s (arranged 5
9). M55s were dangerous in long-term storage as the degradation of the propellant stabilizer could lead to auto-ignition and degraded sarin corroded the casing, causing leaks. The tactical ballistic missiles (0.88
11.35 m) R-300 Elbrus/R-17 Elbrus (employed by the former USSR during the Cold War) and Hwasong-5 (a copy, reverse-engineered by Korea) were assigned by NATO as SS-1c Scud-B, with SS-1d Scud-C and SS-1e Scud-D variants having improved guidance control mechanisms. Following a 1 h launch sequence these ‘Scuds’ could deliver 555 kg VX warheads up to 500 km range. Missiles deliver the equivalent to an artillery barrage in a single strike and without the need for closer positioning and repeated firing of multiple artillery units. The reduced warning minimizes the likelihood of a target implementing effective protective countermeasures.

Air delivery Unitary bomb A fuse detonates an internal burster charge, which ruptures the bomb and heats the agent for dispersal as an aerosol mist. The M47 required anticorrosion linings to carry chemical agents and was developed by the United States as the M47A1 for white phosphorus or M47A2 for 161 kg of mustard. The 329 kg MC-1 (41
127 cm) was a dumb bomb developed to deliver 100 kg of sarin, whereas the 200 kg Mark-94 (28
152 cm) could deliver 49 kg of sarin. The 238 kg Mark-116 Weteye (36
216 cm) was developed to deliver 157 kg of liquid sarin and was originally intended as a guided bomb.

Cluster bombs The 453 kg M34 (36
218 cm) contained 76 (arranged 4
19) 4.5 kg cylindrical 92 mm M125 E54R6 bomblets each containing 1.2 kg of sarin, delivering a total payload of 91 kg. Three hundred and fifty-six spherical 115 mm M134 E130R1 bomblets were developed for use in the 2640 kg (8.3 m long) MGR-1 M190 Honest John rocket, to deliver a total payload of 210 kg sarin up to 32 km. Later, 52 spherical M139 E130R2 bomblets were developed for use in the (4.4 m long) MGR-3 M206 Little John rocket, to deliver a total payload of 31 kg sarin up to 24 km. Finally, 330 M139s could be carried by the 4590 kg M212 MGM-29 Sergeant rocket, to deliver a total payload of 195 kg sarin up to 140 km. The M139 bomblet contained 590 g of sarin in two compartments around a central burster charge, with a 22 degree glide angle; en masse the bomblets could saturate a 1 km diameter area. The 340 kg M43 cluster bomb (41
228 cm) contained 57 (3 banks of 19) 72 mm diameter 4.5 kg M138 bomblets each containing 180 g of BZ mixed with an equal quantity of incendiary agent (as pyromix), which was ignited by a fuse following impact, to thermally

Chemical warfare delivery systems

773

aerosolize 10.3 kg of BZ, covering an area of 0.1–0.9 ha. The 79 kg M44 general cluster bomb (38
152 cm) contained 3 M16 generators, each of which in turn contained 42 M6 canisters (arranged 3
14), and a total of 126 M6s each containing 142 g of BZ (for a total M44 payload of 17.8 kg BZ). Such devices were developed for use against intelligence targets or when friendly and enemy forces co-occupied areas (e.g. hostage rescue situations). However, BZ has a very slow onset and is visible as a white cloud, enabling avoidance measures and consequently decreasing the effectiveness of the agent.

Aircraft spray tanks Spraying overcomes the drawbacks of detonation. However, spraying is affected by speed and direction of both wind and aircraft, with delivery usually needing to occur within the boundary layer (i.e. below an altitude of 300 ft) to ensure the target is reached. Knowledge of fluid dynamics and aerodynamics is needed to facilitate production of optimal droplet size and dispersal as a mist. Polymeric thickening may be required so that the liquid forms large enough droplets so as to not evaporate before it reaches the ground. The 878 kg (4.7 m long) TMU-28 was developed to deliver 615 kg of VX.

Binary glide bomb The contents of two compartments would mix after bomb release to produce an agent that is then sprayed during descent. Such devices were technically challenging due to pressure build-up and variable delivery associated with changing altitude. The 270 kg (2.28 m long) VX2 BLU-80 Bigeye was developed to combine NE (elemental sulfur) with QL (isopropyl aminoethylmethyl phosphonite) during flight and deliver 82 kg of VX.

Improvised weapons Any nation with an active military development program has the capability to convert conventional munitions to carry chemical agents. Terrorist groups with sufficient financial backing who are also able to persuade both a chemist to synthesize a suitable agent and an engineer to manufacture a delivery device could also produce a sophisticated chemical weapon. However, the simplest device could just be a sealed container (e.g., plastic bag) that is opened (e.g., punctured by a sharp object) via a timer-triggered mechanism on location. Such a weapon is likely to be manually placed in a densely populated area or to disrupt an essential service. Alternative methods of delivery include aerial release (e.g. from radio-controlled drones, balloons, or private light/microlight aircraft), or a shuttle vehicle that transfers painted or powdered agent by contact (e.g., contamination of currency, post, public transport, food or water sources). Unconventional chemical agents could include misappropriated pharmaceuticals (e.g., psychiatric or anesthetic drugs), industrial waste (e.g., carcinogenic or endocrine disrupting chemicals), or biologically produced toxins (e.g. botulinum).

Storage, transport, and safe handling Ton containers were used for the bulk storage and transport of chemical agents. Ton containers were made of steel (2.1 m long, 725 kg when empty) and had fittings to permit the closed-system transfer of chemical agents. Delivery systems under development can be tested using chemical agent simulants (e.g., methylacetoacetate or di(2-ethylhexyl)phthalate replacing sarin). Binary systems facilitate easier handling and transport by having two safer agents that when combined produce the final agent, given the designation ‘-2’ (e.g., GB2 for binary sarin). The Chemical Agent Transfer System (CHATS) is a protective, airtight glove box for manually draining agent from a ton storage container by attaching a pump followed by triple rinsing the container interior using spray nozzles. When dry, the container interior could be checked using a borescope before removal from the CHATS for reuse or disposal.

Detection Active release or leakage can be detected by use of remote air sampling, optical detection (e.g., using a Raman spectrometer), and identification of biomarkers (e.g., degradation or metabolic sequelae). Passive evaluation of indeterminate munitions can be achieved by portable digital radiography and computed tomography, using X-rays to scan a suspected chemical munition placed vertically on a rotating platform. The resulting digital image can be reviewed to determine whether the contents are a liquid chemical agent. In addition, portable isotopic neutron spectroscopy enables the identification of chemical agents within closed munitions by using gamma rays. Detection of characteristic gamma-ray peaks reveals the presence and concentration of specific chemical agents (Kim et al., 2011).

Decommissioning, neutralization, and incineration Tens of thousands of tons of chemical munitions were disposed of by dumping at sea (from 1946 to 1970), until chemical neutralization and incineration took over. Single Chemical Agent Identification Set (CAIS) Access and Neutralization System (SCANS) is a hand-held 3.8 l container designed to access and treat CAIS items containing up to 100 g of mustard or lewisite.

774

Chemical warfare delivery systems

The Explosive Destruction System (EDS) uses cutting charges to explosively access chemical munitions, eliminating their explosive capacity before the chemical agent is neutralized. A sealed steel vessel contains the blast, vapor, and fragments. The EDS is transportable for onsite treatment of chemical munitions. Larger scale destruction of chemical munitions by incineration requires three furnaces and pollution abatement. The Liquid Incinerator Chamber destroys liquid chemical agent and contaminated liquids after their removal from munitions or following decontamination processes. The liquid is pumped from a tank and sprayed into the incinerator through injection nozzles, for burning in a primary chamber at 2700 degrees F followed by a secondary chamber at 2000 F. The Deactivation Furnace System is a rotary kiln for incineration of chopped rockets, propellants, explosives, and residual chemical agents. The Metal Parts Furnace decontaminates the metal from emptied munitions and other containers at 1500 F (with a 2000 F afterburner) before recycling or disposal. The Pollution Abatement System is a two tower wet scrubber system to cool gases and remove pollutants from gases following incineration. First, the quench tower uses water to lower gas temperatures from 2000 to 150 F. The scrubber tower then uses sodium hydroxide to neutralize acids. Two Venturi filters remove large particles (before and after the scrubber tower), then a mist eliminator removes fine particles, and finally a carbon filter removes any trace amounts of metal and organic material (Kim et al., 2011).

See also: Arsenical vomiting agents; Blister agents; BZ (3-quinuclidinyl benzilate) a psychotomimetic agent; Chemical warfare; Cyclosarin (GF); G-series nerve agents; Lewisite—A toxic warfare agent; Nerve agents; Nitrogen mustards; Sarin; Soman; Tabun; V-series nerve agents other than VX; VX

References Kim K, Tsay OG, Atwood DA, and Churchill DG (2011) Destruction and detection of chemical warfare agents. Chem. Rev. 111(9): 5345–5403. Marrs TC, Maynard RL, and Sidell FR (2007) Chemical Warfare Agents: Toxicology and Treatment. Chichester, UK: John Wiley & Sons, Ltd.

Further reading Bigley AN and Raushel FM (2019) The evolution of phosphotriesterase for decontamination and detoxification of organophosphorus chemical warfare agents. Chemico-Biological Interactions 308: 80–88. Capoun T and Krykorkova J (2021) Comparison of selected methods for individual decontamination of chemical warfare agents: Recent development. Current Advances in Chemistry and Biochemistry 5: 87–105. Crane CC and Crane CC (2002) “No practical capabilities”: American biological and chemical warfare programs during the Korean war. Perspectives in Biology and Medicine 45(2): 241–249. Coleman K (2005) A History of Chemical Warfare. New York: Palgrave Macmillan. Crowley M, Dando M, and Shang L (eds.) (2018) Preventing Chemical Weapons: Arms Control and Disarmament as the Sciences Converge. Royal Society of Chemistry. Dong J, Lv H, Sun X, Wang Y, Ni Y, Zou B, et al. (2018) A versatile self-detoxifying material based on immobilized polyoxoniobate for decontamination of chemical warfare agent simulants. Chemistry–A European Journal 24(72): 19208–19215. Ebrahim AM, Plonka AM, Tian Y, Senanayake SD, Gordon WO, Balboa A, et al. (2019) Multimodal characterization of materials and decontamination processes for chemical Warfare protection. ACS Applied Materials & Interfaces 12(13): 14721–14738. Eubanks LM, Dickerson TJ, and Janda KD (2007) Technological advancements for the detection of and protection against biological and chemical warfare agents. Chemical Society Reviews 36(3): 458–470. Franca TC, Kitagawa DA, Cavalcante SFDA, da Silva JA, Nepovimova E, and Kuca K (2019) Novichoks: The dangerous fourth generation of chemical weapons. International Journal of Molecular Sciences 20(5): 1222. Grissom TG, Plonka AM, Sharp CH, Ebrahim AM, Tian Y, Collins-Wildman DL, et al. (2020) Metal–organic framework-and polyoxometalate-based sorbents for the uptake and destruction of chemical warfare agents. ACS Applied Materials & Interfaces 12(13): 14641–14661. Guyana H and Tanzania Y (2021) Chemical Weapons: Destruction and Conversion. vol. 12, 193. Harvey A (2019) Detection and Identification of Chemical Warfare Agents and Explosives in Complex Matrices. (Doctoral dissertation, University of York. Hou Y, An H, Zhang Y, Hu T, Yang W, and Chang S (2018) Rapid destruction of two types of chemical warfare agent simulants by hybrid polyoxomolybdates modified by carboxylic acid ligands. ACS Catalysis 8(7): 6062–6069. Johnson NH, Larsen JC, and Meek EC (2020) Historical perspective of chemical warfare agents. In: Handbook of Toxicology of Chemical Warfare Agents, pp. 17–26. Academic Press. Kangas MJ, Ernest A, Lukowicz R, Mora AV, Quossi A, Perez M, et al. (2018) The identification of seven chemical warfare mimics using a colorimetric array. Sensors 18(12): 4291. Kloske M and Witkiewicz Z (2019) Novichoks: The A group of organophosphorus chemical warfare agents. Chemosphere 221: 672–682. Li Y, Chen C, Meshot ER, Buchsbaum SF, Herbert M, Zhu R, et al. (2020) Autonomously responsive membranes for chemical warfare protection. Advanced Functional Materials 30(25): 2000258. Moran CM (2021) The future of chemical warfare: How urbanization and proliferation of delivery mechanisms create the need for in-situ Defence. In: Proliferation of Weapons-and Dual-Use Technologies, pp. 93–109. Cham: Springer. Nawała J, Józ´wik P, and Popiel S (2019) Thermal and catalytic methods used for destruction of chemical warfare agents. International Journal of Environmental Science and Technology 16(7): 3899–3912. Norrrahim MNF, Ahmad Shah NA, Jamal SH, Yunus W, Wan MZ, Ernest VFKV, and Kasim NAM (2021) Nanocellulose-based filters as novel barrier systems for chemical warfare agents. Solid State Phenomena. vol. 317, pp. 180–186. Trans Tech Publications Ltd. Salem H, Ternay AL Jr., and Smart JK (2019) Brief history and use of chemical warfare agents in warfare and terrorism. In: Chemical Warfare Agents, pp. 3–15. CRC Press. Škoro N, Puac N, Živkovic S, Krstic-Miloševic D, Cvelbar U, Malovic G, and Petrovic ZL (2018) Destruction of chemical warfare surrogates using a portable atmospheric pressure plasma jet. The European Physical Journal D 72(1): 1–8. Timperley CM, Forman JE, Abdollahi M, Al-Amri AS, Alonso IP, Baulig A, et al. (2018) Advice on chemical weapons sample stability and storage provided by the Scientific Advisory Board of the Organisation for the Prohibition of Chemical Weapons to increase investigative capabilities worldwide. Talanta 188: 808–832.

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Weetman C, Notman S, and Arnold PL (2018) Destruction of chemical warfare agent simulants by air and moisture stable metal NHC complexes. Dalton Transactions 47(8): 2568–2574. Zhao S, Xi H, Zuo Y, Han S, Zhu Y, Li Z, et al. (2019) Rapid activation of basic hydrogen peroxide by borate and efficient destruction of toxic industrial chemicals (TICs) and chemical warfare agents (CWAs). Journal of Hazardous Materials 367: 91–98. Zhou C, Zhang S, Pan H, Yang G, Wang L, Tao CA, and Li H (2021) Synthesis of macroscopic monolithic metal–organic gels for ultra-fast destruction of chemical warfare agents. RSC Advances 11(36): 22125–22130.

Relevant websites http://www.fas.org/nuke/guide/usa/cbw/cw.htm :Federation of American Scientists. http://www.fas.org/programs/bio/chemweapons/index.html :Federation of American Scientists. http://toxnet.nlm.nih.gov :TOXNET, Toxicology Data Network, National Library of Medicine, Search for Chemical Weapon. http://www.cma.army.mil :US Army Chemical Materials Agency, Search for Fact File.

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Chemicals alternatives assessments Margaret H Whittaker, ToxServices LLC, Washington, DC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of MH Whittaker, Chemicals alternatives assessments, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 782–786, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01239-2.

Introduction CAA serves a critical role that goes beyond regulation The role of CAA in avoiding regrettable substitution and promoting sustainable product design Evolution of CAA frameworks Published CAA reviews CAA case studies and online resources OECD SAAT SUBSPORTplus Interstate Chemicals Clearinghouse (IC2) Swedish Center for Chemical Substitution Professional societies, associations, and organizations supporting CAA Association for the Advancement of Alternatives Assessment (A4) Society of Toxicology’s Sustainable Chemicals through Contemporary Toxicology Specialty Section (SCCT) Conclusion References Further reading

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Abstract Chemical Alternatives Assessment (CAA) is a process for identifying and comparing potential chemical and non-chemical alternatives to replace chemicals or technologies of concern on the basis of hazard, performance, and economic viability. Almost two dozen CAA frameworks have been published to date since The University of Massachusetts’ Lowell Center for Sustainable Production first issued their Alternatives Assessment framework in 2006. The practice of CAA continues to expand around the world due to its incorporation in public and private sector initiatives intended to promote toxics reduction and circular product design. In a CAA, alternatives are assessed to ensure that they are safer, perform well for specific applications, are cost effective, and ideally, have no adverse social, environmental, and/or economic impacts associated with their use.

Keywords Alternatives assessment; Chemical hazard assessment; Chemicals alternatives assessment; Green chemistry; Regrettable substitution; Sustainability

Glossary Chemical Alternatives Assessment Process for identifying and comparing potential chemical and non-chemical alternatives to replace chemicals or technologies of concern on the basis of hazard, performance, and economic viability. Chemical Hazards Assessment Classifies hazards for a broad range of human, environmental, and physical hazard endpoints using frameworks that allow for comparison between chemical alternatives. Green Chemistry Design of new chemistries and chemical products that reduce or eliminate the use or generation of hazardous substances.

Key points

• • •

CAA is a distinct decision-making framework from life cycle analysis and risk assessment. CAA targets chemical and product combinations and informs actionable decisions during product design (and reformulation) phases. CAA is intended to avoid regrettable substitution, which may occur when a chemical of concern is restricted or banned, resulting in its replacement with a different chemical that has similar, or even dissimilar but undesirable, hazard properties.

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.01026-5

777

778

Chemicals alternatives assessments

Abbreviations CAA CHA GHS LCA

Chemicals Alternatives Assessment Chemicals Hazard Assessment Globally Harmonized System of Classification and Labeling of Chemicals Life cycle assessment

Introduction Chemical Alternatives Assessment (CAA) is a process for identifying and comparing potential chemical and non-chemical alternatives to replace chemicals or technologies of concern on the basis of hazard, performance, and economic viability. The first CAAs were published in the late 1990s in the United States as frameworks upon which to avoid chemical hazards and promote safer alternatives. Prior to the inception of CAA, life cycle assessment (LCA) and risk assessment were the two primary tools used to select safer alternatives. Ideally, a CAA provides a science-based solution that identifies and characterizes chemical hazards, promotes the selection of less hazardous chemical ingredients, and avoids unintended consequences of switching to a poorly characterized and more hazardous chemical substitute or technology (also known as regrettable substitutions). CAAs are used to evaluate and manage hazards and subsequent health risks through the informed choice of safer chemicals and/or technologies. Governments and international organizations around the world increasingly recognize the ability of CAA to advance safer chemicals and materials. For example, the European Commission’s Chemicals Strategy for Sustainability includes a safe and sustainable-by-design platform that specifies the use of CAA as a means to avoid use of chemicals that may be harmful to human health or the environment (European Commission (EC), 2000). When practiced well, CAAs empower companies to avoid chemicals of concern, drive innovation, and ultimately, benefit the health of humans and the environment (European Chemicals Agency (ECHA), 2023a). As discussed in this chapter, professional societies such as the Association for the Advancement of Alternatives Assessment (A4) dedicated to CAA and informed substitution have been established to build expertise and the capacity among alternatives assessors around the world, and there is an abundance of online tools, case studies, and training to support CAA.

CAA serves a critical role that goes beyond regulation Laws are in force around the world that mandate the registration of chemicals with government bodies, such as the United States Toxic Substances Control Act (TSCA) of 1976 (USC, 1976), amended by the Frank R. Lautenberg Chemical Safety for the 21st Century Act (United States Code (U.S.C.), 2016), and the European Union’s (EU’s) Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) Regulation (European Union (EU), 2023). However, such regulations rarely result in the restriction of large numbers of chemicals of concern (ChemSec, 2023a, 2023b). As an example, since the inception of the EU’s REACH Regulation in 2006, more than 22,000 substances imported or manufactured at more than 1000 metric tons per year in the EU have been registered with the European Chemicals Agency (European Chemicals Agency (ECHA), 2023b); however, fewer than 100 of these registered substances have been restricted under Annex XVII of the REACH Regulation (European Union (EU), 2023). Examples of substances restricted in the EU include the hazardous solvents dimethyl fumarate (CAS#624-49-7) and 1-methyl-2-pyrrolidone (CAS#872-50-4) (European Union (EU), 2023). CAA fills a critical need to support informed substitution and safer chemical selection, ideally by avoiding use of a regrettable chemical or technological substitution.

The role of CAA in avoiding regrettable substitution and promoting sustainable product design Over the past quarter century, professionals such as toxicologists, chemists, and circular design engineers have utilized CAA frameworks during the design or redesign of chemicals, materials, and products. A CAA is intended to avoid regrettable substitution, which may occur when a chemical of concern is restricted or banned, resulting in its replacement with a different chemical that has similar, or even dissimilar but undesirable, hazard properties. Examples of regrettable substitutions are shown in Table 1, and include flame retardants, solvents, and plastics and coatings ingredients (Tickner et al., 2022; Heine and Whittaker, 2021). Hazards of regrettable substitutes may harm human health, the climate, and/or organisms in the environment. Besides serving to avoid regrettable substitutions, CAAs work well to advance sustainable design principles. It is estimated that more than 80% of a product’s environmental impact is determined at the design stage (Ellen MacArthur Foundation, 2023), underscoring the important role of CAA in advancing safer and sustainable materials and products. Unlike green chemistry, which focuses on the design of new chemistries and chemical products that reduce or eliminate the use or generation of hazardous substances (Tickner et al., 2020), a CAA evaluates alternatives to ensure that assessed alternatives are not only less hazardous, but work for specific applications, are cost effective and commercially available, and are not associated with adverse social,

Chemicals alternatives assessments Table 1

779

Examples of regrettable substitutions.

Ingredient Category

Chemical of Concern (Primary hazards)

Regrettable Substitute (Primary hazards)

Flame retardants

Polybrominated diphenyl ethers (PBDEs), including DecaBDE (Neurotoxicity, reproductive toxicity, and/or carcinogenicity). Trichloroethylene (Carcinogenicity) Chlorofluorocarbon (CFCs) such as R-12 (Dichlorodifluoromethane) (Ozone depletion, Greenhouse gas) Bisphenol A (BPA) (Endocrine disruption)

Tris(2,3-dibromopropyl)phosphate (Carcinogenicity, aquatic toxicity) Bromopropane (Carcinogenicity, neurotoxicity) Hydrofluorocarbons (HFCs) such as HFC-23 (aka Fluoroform) (Greenhouse gas) Bisphenol S (BPS) (Endocrine activity)

Solvents Commercial Refrigerants Plastics and Coatings Ingredients

Alternatives Assessment Outcomes

Existing solutions responding to regulatory or market drivers Criteria for defining safer

Green Chemistry Principles

Development of a safer chemical, process, or technology, to replace a chemical of concern

Discovery driven innovation Criteria for design

Fig. 1 The Nexus Between Alternatives Assessment and Green Chemistry (Tickner et al., 2020). Modified from Tickner J, Jacobs M and Whittaker MH (2022) Chapter 14: Addressing the limits of risk assessment by focusing on safer alternatives risk assessment for environmental health. In: Robson MG, Toscano WA, Meng Q, Kaden DA (eds.), Risk Assessment for Environmental Health. 2nd edn., pp. 279–316, Boca Raton: CRC Press.

environmental, or economic impacts during any life cycle stage. This comparison in methodology and the inter-relationship of CAA and Green Chemistry Principles is illustrated in Fig. 1. The book Making Better Environmental Decisions is often credited as being one of the first publications extolling the virtues of CAA over other decision-making frameworks such as risk assessment (O’Brien, 2000). If asked, many CAA practitioners are able to quote the following sentence from O’Brien’s book: “One of the most essential, and powerful steps to change is understanding that there are alternatives (O’Brien, 2000).”

Evolution of CAA frameworks More than 20 CAA frameworks have been published to date, and share traits such as problem scoping, hazard assessment, exposure characterization, technical feasibility, economic feasibility, decision analysis, and increasingly, life cycle analysis (Jacobs et al., 2016; Tickner et al., 2019; Fantke et al., 2020; Whittaker and Heine, 2023). Unfortunately, none of the CAA frameworks are automated, and available CAA frameworks do require that the person performing the CAA be skilled in toxicology, chemistry, ecotoxicology, and environmental science, with a solid understanding of life cycle concepts. The earliest published CAA framework is generally identified as The University of Massachusetts Lowell Center for Sustainable Production (2006). This was followed in 2010 by the U.S. EPA’s Design for the Environment (DfE) Alternatives Assessment Partnerships and the associated DfE Program AA Criteria for Hazard Evaluation (United States Environmental Protection Agency (U.S. EPA), 2010, United States Environmental Protection Agency (U.S. EPA), 2011, Lavoie et al., 2010), followed by CAA guidance from the U.S. National Research Council (NRC) and the Interstate Chemicals Clearinghouse (National Research Council (NRC), 2014, Interstate Chemicals Clearinghouse (IC2), 2014, Interstate Chemicals Clearinghouse (IC2), 2017). More recently, the OECD issued CAA guidance focusing more on single chemical substitutions and not functional substitution involving technology or product changes (Organisation for Economic Co-operation and Development (OECD), 2021). CAA frameworks share many attributes by placing initial emphasis on the assessment of hazards, with LCA and risk-related metrics such as exposure assessment, occurring later in the process. Core elements of a CAA comprise chemical hazard assessment (CHA), life cycle thinking, exposure assessment, technical/functional assessment, economic assessment, and social impact assessment. An example of a CAA framework is shown in Fig. 2, which illustrates the NRC’s CAA framework (National Research Council (NRC), 2014). The NRC CAA framework defines key elements of a CAA as part of a 13-step workflow used to guide decision making.

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1.Identify Chemical of Concern

13. Research / De novo Design

2a. Scoping and 2b. Problem Formulation

3. Identify Potential Alternatives No Alternatives Innovation Required

4. Determine if Alternatives are Available

Alternatives available

5. Assess Physicochemical Properties

6.1 Assess Human Health Hazards

6.3 Conduct Comparative Exposure Assessment

6.2 Assess Ecotoxicity (Chemical Hazards)

7. Integration of Information to Identify Safer Alternatives

Alternatives not safer

Alternatives safer 8. Life Cycle Thinking

9.1 Additional Life Cycle Assessment including. for example, Evaluation of Broader Environmental Impacts (e.g. energy, resources) and Social Impacts.

9.2 Performance Assessment

10. Identify Acceptable Alternatives

9.3 Economic Assessment

Alternatives have unacceptable trade-offs

Acceptable alternatives 11. Compare Alternatives

12. Implement Alternative(s)

Indicates optional steps

Fig. 2 The NRC Framework to Guide Selection of Chemical Alternative (National Research Council (NRC), 2014). From National Research Council (NRC) (2014) A Framework to Guide Selection of Chemical Alternatives. Washington, DC: The National Academies Press.

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The NRC Framework recommends where assessment steps should be completed sequentially, in parallel, or iteratively. The NRC framework was intended to provide flexibility to facilitate use for a diversity of applications.

Published CAA reviews Jacobs et al. (2016) reviewed 20 published CAA frameworks, while publications from the Organization for Economic Cooperation and Development (OECD) and the United States National Research Council (NRC) review 8 and 10 CAA frameworks, respectively (Organisation for Economic Co-operation and Development (OECD), 2013, National Research Council (NRC), 2014). More recently, the Organisation for Economic Co-operation and Development (OECD) (2023a) published a report that discusses government initiatives (both regulatory and non-regulatory), third-party certifications (including Cradle to Cradle Certified® and U.S. EPA Safer Choice), and industry approaches (including Estée Lauder’s Green Score methodology) used around the world to support alternatives assessments and substitution. According to the results of an OECD questionnaire sent out to country members of the OECD Working Party on Risk Management, voluntary approaches to support substitution continue to outweigh regulatory approaches, and the OECD reported that only 12 of the 38 approaches detailed in questionnaire responses were of a regulatory nature (Organisation for Economic Co-operation and Development (OECD), 2023a). In another recent publication, the Organisation for Economic Co-operation and Development (OECD) (2023c) summarizes 33 different approaches developed and implemented by third-party organizations across OECD member countries to support the substitution of chemicals of concern, and categorizes these approaches into six different categories:

• • • • • •

Tools, methods, and other technical resources Technical assistance and training Ecolabels Advocacy and awareness-raising Education and professional associations, and Retailer strategies

Both Organisation for Economic Co-operation and Development (OECD) (2023b, 2023c) reports are treasure-trove for both nascent and experienced alternatives assessors who want to stay on top of informed substitution issues.

CAA case studies and online resources One of the primary means to learn how to undertake a CAA as a decision-making framework is to review published case studies that utilize CAA principles to make informed substitution decisions, as well as utilize on-line training and educational resources intended to educate users in each step of a CAA. Most of the case studies and resources are publicly accessible, with examples of a number of these resources provided below.

OECD SAAT An online index of published CAA frameworks described earlier in this chapter can be found within the OECD’s on-line Substitution and Alternatives Assessment Toolbox (SAAT). As of date of publication of this chapter, the OECD SAAT provides a brief overview and links to 24 different CAA frameworks (Organisation for Economic Co-operation and Development (OECD), 2023b). The OECD SAAT also links to tools, data sources, and case studies that support the practice of CAA in both regulatory and non-regulatory contexts. Examples of CAA case studies that are accessible through the OECD SAAT include the more than dozen case studies, including two widely cited CAAs:

• •

The University of Massachusetts Lowell Toxics Use Reduction Institute (2006) o This set of CAAs assessed the availability of technically and economically feasible safer alternatives for five hazardous chemicals (i.e., lead, formaldehyde, perchloroethylene, hexavalent chromium, and di(2-ethylhexyl)phthalate). U.S. EPA Design for the Environment’s (DfE)‘s Alternatives Assessment Project on Alternatives to Decabromodiphenyl ether (decaBDE) (United States Environmental Protection Agency (U.S. EPA), 2014). o This project assessed the toxicity and environmental fate of flame retardant chemicals that are potential alternatives to decaBDE when used in materials and products where decaBDE was used as a flame retardant.

SUBSPORTplus The German Federal Institute for Occupational Safety and Health maintains the SUBSPORTplus website. SUBSPORTplus identifies chemicals of concern as well as safer chemicals, and also provides updated news regarding recently completed CAA initiatives and publications (with a particular focus on Europe). SUBSPORTplus maintains a database of case studies that has grown to more than 450 case studies exampling different applications of informed substitution (Substitution Support Portal (SUBSPORTplus), 2023). All SUBSPORTplus case stories are available in English, with approximately 90 cases translated into German, and all case studies can be searched by product sector, function, or process.

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Interstate Chemicals Clearinghouse (IC2) The Interstate Chemicals Clearinghouse (IC2), an association of state, local, and tribal governments in the United States, maintains an alternatives assessment library that examples about two dozen CAAs that have been performed in the United States (Interstate Chemicals Clearinghouse (IC2), 2023).

Swedish Center for Chemical Substitution The Swedish Center for Chemical Substitution maintains a dynamic website designed to support businesses (particularly small and medium sized enterprises) and the public (Swedish Center for Chemical Substitution, 2023a) in the substitution of hazardous chemicals. Resources include updated news pertaining to CAA, online training videos, links to restricted chemical lists such as the International Chemical Secretariat’s Chemsec SIN List comprising more than 1500 chemicals of concern (ChemSec, 2023a, 2023b), and case studies from the commercial sector (e.g., Ikea, Peak Performance) documenting success phasing out chemicals of concern such as fluorocarbons. In January 2023, the Swedish Center for Chemical Substitution (2023b) published a compilation of more than 50 websites of positive lists that identify safer alternatives to hazardous chemicals, materials, or products, such as the TCO Accepted Substances List (TCO, 2023), the United States Environmental Protection Agency’s Safer Chemicals Ingredients List (United States Environmental Protection Agency, 2023), and the ChemFORWARD Registry (ChemFORWARD, 2023).

Professional societies, associations, and organizations supporting CAA The execution of a CAA requires expertise in multiple disciplines. Participation in professional societies and associations is critical to hone skills needed to undertake robust CAAs. A number of the leading societies, associations, and organizations supporting CAA are presented in this section.

Association for the Advancement of Alternatives Assessment (A4)

The Association for the Advancement of Alternatives Assessment (A4) is a professional association formed in 2018, and is designed to foster a community of practice to accomplish the following goals related to CAA (Association for the Advancement of Alternatives Assessment (A4), 2023): 1. Accelerate the pace of CAA methods development 2. Create robust and consistent CAA approaches and tools; and 3. Promote high standards of quality and expand CAA and informed substitution practice. The vision of the A4 is that every function performed by a chemical, material, process, or product is achieved with safe and sustainable solutions (Association for the Advancement of Alternatives Assessment (A4), 2023). Membership is available to all and does not require a recommendation from an existing A4 member. A4 members vote for officers, serve on committees, organize, participate, and/or attend quarterly webinars, and participate in CAA-related symposia that take place every other year.

Society of Toxicology’s Sustainable Chemicals through Contemporary Toxicology Specialty Section (SCCT)

The Society of Toxicology’s Sustainable Chemicals through Contemporary Toxicology Specialty Section (SOT-SCCT) is one of SOT’s newest specialty sections. The Sustainable Chemicals through Contemporary Toxicology Specialty Section (also known as SCCT) was formed in 2020 by SOT members who were interested in supporting scientific activities designed to reduce the likelihood of regrettable substitution (phasing out one chemical of concern for one equally or more hazardous) throughout the supply chain through interdisciplinary partnerships. The SOT SCCT public website posts a public resources list to tools, courses, webinars, podcasts, and associations related to informed substitution and safer chemicals selection (Society of Toxicology Sustainable Chemicals Through Contemporary Toxicology Specialty Section (SOT-SCCT), 2023a). SCCT membership is available to SOT members, and individuals wanting to join the SOT SCCT are encouraged to contact current SCCT officers through the SOT contact email identified on the SOT SCCT website (Society of Toxicology Sustainable Chemicals Through Contemporary Toxicology Specialty Section (SOT-SCCT), 2023b) for assistance obtaining letters of recommendation to join SOT and the SOT SCCT Specialty Section.

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Conclusion CAAs are in use around the world to assist in the selection of safer chemicals, materials, or technologies at the design phase, as well as empower substitution decisions to phase out chemicals of concern throughout the supply chain. Since its creation in the early 2000s in the United States, CAA frameworks have undergone refinement to incorporate not only hazard considerations, but now include performance, cost, and societal tradeoffs, and are in use around the world to benefit the health of humans, our climate, and the entire ecosystem around the world.

References Association for the Advancement of Alternatives Assessment (A4) (2023) About A4. Available: https://saferalternatives.org/about/about-a4 ChemFORWARD (2023) ChemFORWARD Registry. Available: https://www.chemforward.org/ ChemSec (2023a) The Investor’s Guide to Hazardous Chemicals. Available: https://chemsec.org/app/uploads/2023/01/The-Investors-Guide-to-Hazardous-Chemicals.pdf ChemSec (2023b) The SIN List. Available: https://sinlist.chemsec.org/ Ellen MacArthur Foundation (2023) What is the Circular Economy. Available: https://www.ellenmacarthurfoundation.org/circular-economy/what-is-the-circular-economy European Chemicals Agency (ECHA) (2023a) Substances of Concern: Why and How to Substitute. Available: https://echa.europa.eu/documents/10162/24152346/why_and_how_ to_substitute_en.pdf/93e9c055-483c-743a-52cb-1d1201478bc1 European Chemicals Agency (ECHA) (2023b) REACH Registration Statistics. Available: https://echa.europa.eu/documents/10162/2741157/registration_statistics_en.pdf/58c2d7bd2173-4cb9-eb3b-a6bc14a6754b?t¼1649160655122 European Commission (EC) (2000) Chemicals Strategy for Sustainability Towards a Toxic-Free Environment. Brussels, 14.10.2020 COM(2020) 667 final, 2020. Available: https://ec. europa.eu/environment/pdf/chemicals/2020/10/Strategy.pdf European Union (EU) (2023) Substances Restricted Under Reach. Available: https://echa.europa.eu/en/substances-restricted-under-reach Fantke P, Huang L, Overcash M, Griffing E, and Jolliet O (2020) Life cycle based alternatives assessment (LCAA) for chemical substitution. Green Chemistry 22: 6008–6024. Available: https://pubs.rsc.org/en/content/articlepdf/2020/gc/d0gc01544j. Heine L and Whittaker MH (2021) Determining the true value of a sustainable chemical toxicology. In: How to Commercialize Chemical Technologies for a Sustainable Future, pp. 31–54. Chichester, UK: John Wiley & Sons, Ltd. ch. 3. Interstate Chemicals Clearinghouse (IC2) (2014) IC2 Alternatives Assessment Guide. Version 1 January 2014. Available: http://theic2.org/article/download-pdf/file_name/IC2_AA_ Guide_Version_1.0.pdf Interstate Chemicals Clearinghouse (IC2) (2017) IC2 Alternatives Assessment Guide. Version 1.1. January 2017. Available: http://theic2.org/article/download-pdf/file_name/IC2_AA_ Guide_Version_1.1.pdf Interstate Chemicals Clearinghouse (IC2) (2023) Alternatives Assessment Library. Available: http://theic2.org/aa_library#gsc.tab¼0 Jacobs MM, Malloy TF, Tickner JA, and Edwards S (2016) Alternatives assessment frameworks: Research needs for the informed substitution of hazardous chemicals. Environmental Health Perspectives 124(3): 265–280. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786344/pdf/ehp.1409581.pdf. Lavoie ET, Heine LG, Holder H, Rossi MS, Lee RE II, Connor EA, Vrabel MA, DiFiore DM, and Davies CL (2010) Chemical alternatives assessment: Enabling substitution to safer chemicals. Environmental Science & Technology 44(24): 9244–9249. https://doi.org/10.1021/es1015789. National Research Council (NRC) (2014) A Framework to Guide Selection of Chemical Alternatives. Washington, DC: The National Academies Press. https://doi.org/10.17226/18872. O’Brien M (2000) Making better environmental decisions. In: An Alternative to Risk Assessment, p. 283. Cambridge, MA: MIT Press. Organisation for Economic Co-operation and Development (OECD) (2013) Current Landscape of Alternatives Assessment Practice: A Meta-Review. Series on Risk Management No. 26. ENV/JM/MONO(2013)24. Available: https://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote¼ENV/JM/MONO%282013%2924&docLanguage¼En Organisation for Economic Co-operation and Development (OECD) (2021) Guidance on Key Considerations for the Identification and Selection of Safer Chemical Alternative, OECD Series on Risk Management, No. 60, Environment, Health and Safety, Environment Directorate. Available: https://www.oecd.org/chemicalsafety/risk-management/guidance-onkey-considerations-for-the-identification-and-selection-of-safer-chemical-alternatives.pdf Organisation for Economic Co-operation and Development (OECD) (2023a) Cross Country Analysis: Approaches to Support Alternatives Assessment and Substitution of Chemicals of Concern, In: Series of Risk Management, 2nd edn. Available: https://www.oecd.org/chemicalsafety/risk-management/cross-country-analysis-approaches-alternativesassessment-and-substitution-second-edition.pdf. Organisation for Economic Co-operation and Development (OECD) (2023b) OECD Substitution and Alternatives Toolbox (SAAT). Available: https://www.oecd.org/chemicalsafety/riskmanagement/substitution-of-hazardous-chemicals/ Organisation for Economic Co-operation and Development (OECD) (2023c) Lessons Learned From Third-Party Approaches That Support Substitution of Chemicals of Concern, OECD Series on Risk Management, No. 78, Environment, Health and Safety, Environment Directorate. https://www.oecd.org/chemicalsafety/risk-management/lessons-learned-fromthird-party-approaches-support-substitution-of-chemicals-of-concern.pdf Society of Toxicology Sustainable Chemicals Through Contemporary Toxicology Specialty Section (SOT-SCCT) (2023a) SCCT Resources List. Available: https://www.toxicology.org/ groups/ss/SCCT/docs/Resources-SCCT.pdf Society of Toxicology Sustainable Chemicals Through Contemporary Toxicology Specialty Section (SOT-SCCT) (2023b) SOT SCCT Executive Officers. Available: https://www.toxicology. org/groups/ss/SCCT/officers.asp Substitution Support Portal (SUBSPORTplus) (2023) SUBSPORT. Available: https://www.subsportplus.eu/subsportplus/DE/Home/Home_node.html Swedish Center for Chemical Substitution (2023a) Swedish Centre for Chemical Substitution website. Available: https://www.ri.se/en/centre-chemical-substitution Swedish Center for Chemical Substitution (2023b) Positive List Compilation. January 2023. Available: https://www.ri.se/en/centre-chemical-substitution/tools-and-databases/positive-lists TCO (2023) TCO Accepted Substances List (ASL). Available: https://tcocertified.com/industry/accepted-substance-list/ The University of Massachusetts Lowell Center for Sustainable Production (2006) Alternatives Assessment Framework. Version 1.0. July 2006. M Rossi, J Tickner, K Geiser (eds.), Lowell Center for Sustainable Production. Available: http://www.chemicalspolicy.org/downloads/FinalAltsAssess06.pdf The University of Massachusetts Lowell Toxics Use Reduction Institute. (2006) Five Chemicals Alternatives Assessment Study. Available: https://www.turi.org/TURI_Publications/TURI_ Guides_to_Safer_Chemicals/Five_Chemicals_Alternatives_Assessment_Study._2006 Tickner J, Jacobs M, Malloy T, Buck T, Stone A, Blake A, and Edwards S (2019) Advancing alternatives assessment for safer chemical substitution: A research and practice agenda. Integrated Environmental Assessment and Management 15(6): 855–866. https://doi.org/10.1002/ieam.4094. Tickner JS, Simon RS, Jacobs M, Pollard LD, and van Bergen SK (2020) The nexus between alternatives assessment and green chemistry: Supporting the development and adoption of safer chemicals. Green Chemistry Letters and Reviews 14(1): 21–42. https://doi.org/10.1080/17518253.2020.1856427. Tickner J, Jacobs M, and Whittaker MH (2022) Addressing the limits of risk assessment by focusing on safer alternatives risk assessment for environmental health. In: Robson MG, Toscano WA, Meng Q, and Kaden DA (eds.) Risk Assessment for Environmental Health, 2nd edn. pp. 279–316. Boca Raton: CRC Press. ch. 14. United States Code (U.S.C.) (1976) Toxic Substances Control Act. 15 U.S.C. }2601. Available: https://www.govinfo.gov/content/pkg/COMPS-895/pdf/COMPS-895.pdf

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United States Code (U.S.C.) (2016) Frank R. Lautenberg Chemical Safety for the 21st Century Act. Available: https://www.congress.gov/114/plaws/publ182/PLAW-114publ182.pdf United States Environmental Protection Agency (2023) Safer Chemicals Ingredients List. Available: https://www.epa.gov/saferchoice/safer-ingredients United States Environmental Protection Agency (U.S. EPA) (2010) DfE Alternatives Assessment Criteria for Hazard Evaluation, version 1. Office of Pollution Prevention & Toxics. United States Environmental Protection Agency (U.S. EPA) (2011) DfE Alternatives Assessment Criteria for Hazard Evaluation. Version 2. Office of Pollution Prevention & Toxics. Available: https://www.epa.gov/sites/production/files/2014-01/documents/aa_criteria_v2.pdf. United States Environmental Protection Agency (U.S. EPA) (2014) DfE Alternatives Assessment Project on Alternatives to Decabromodiphenyl ether (decaBDE). Available: https://www. epa.gov/sites/default/files/2014-05/documents/decabde_final.pdf Whittaker MH and Heine LH (2023) Chemicals alternatives assessment. In: Paustenbach D, Farland B, and Klaunig J (eds.) Patty’s Toxicology, 7th edn. Hoboken, NJ: Wiley Publishing. in press.

Further reading European Commission (EC) (2006) Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006 concerning the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC, 2006. Available: https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri¼CELEX:32006R1907 O’Brien M (2000) Making better environmental decisions. In: An Alternative to Risk Assessment, p. 283. Cambridge, MA: MIT Press. University of Massachusetts, Lowell Center for Sustainable Production (2006) Alternatives Assessment Framework of the Lowell Center for Sustainable Production. Version 1. July, 2006. Available: https://www.uml.edu/docs/alternatives%20assessment%20framework_tcm18-229886.pdf Whittaker MH and Heine LH (2023) Chemicals alternatives assessment. In: Paustenbach D, Farland B, and Klaunig J (eds.) Patty’s Toxicology, 7th edn. Hoboken, NJ: Wiley Publishing. in press.

Relevant websites https://saferalternatives.org/about/about-a4 :Association for the Advancement of Alternatives Assessment (A4). https://www.subsportplus.eu/subsportplus/DE/Home/Home_node.html :Substitution Support Portal (SUBSPORTplus). https://www.ri.se/en/centre-chemical-substitution :Swedish Center for Chemical Substitution. https://www.oecd.org/chemicalsafety/risk-management/substitution-of-hazardous-chemicals/ :Organisation for Economic Co-operation and Development (OECD).

Chemicals in consumer products L Molander, Stockholm University, Stockholm, Sweden © 2014 Elsevier Inc. All rights reserved. This is a reproduction of L. Molander, Chemicals in Consumer Products, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 801–804, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00452-8.

Chemicals in Consumer Products – A Global Concern Production of Chemicals and Consumer Products is Increasing The Life Cycle Perspective Association with Adverse Outcomes Risk Reduction Strategies Regulatory Instruments Voluntary Approaches Urgent Problems and Challenges Further reading

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Abstract Health and environmental risks associated with emissions of hazardous chemicals from consumer products such as clothes, toys, and electronics have become widely acknowledged internationally during the last decade. This article provides an introduction to the key concerns, an overview of regulatory and voluntary strategies used for managing these risks, and finally some emerging issues with regard to chemicals in consumer products are identified.

Keywords Consumer products; Environment; Hazardous chemicals; Health; Information; Regulatory toxicology; Risk assessment; Risk management; Substances of very high concern; Substitution

Chemicals in Consumer Products – A Global Concern Production of Chemicals and Consumer Products is Increasing During the second half of the twentieth century, the global chemical production increased from around 7 million tons to over 400 million tons per year, and it is expected to continue to grow. In 2001, the Organisation for Economic Co-operation and Development (OECD) calculated that global chemical production would show an 85% increase between 1995 and 2020. It is estimated that over 100 000 chemical substances are commercially available on the global market. Chemical production has to a large extent shifted from the OECD countries to low-income countries and economies in transitions, mainly to Brazil, Russia, India, and China, over the last decade. One important explanation for the increasing production of chemicals is the rapidly increasing production of consumer products, such as textiles, toys, and electrical and electronic equipment. The international trade with products has tripled since the 1970s. Chemicals are present in products for different reasons. They can, for example, be used as constituents for the manufacturing of materials, such as plastics, or added to the material for it to achieve certain functions or properties. Examples of such chemicals are perfluorinated compounds (PFCs), which are water and grease repelling, and phthalates, which are used as plasticizers. Other applications include the treatment of products with biocides and finishing with paints and lacquers. Traces of chemical substances used in the manufacturing process may unintentionally remain in the finished product where they no longer serve any purpose. The increasing production of consumer products is closely related to our lifestyles. As our way of living and consuming has changed much during the last 50 years, chemical exposure has also changed. There has been a shift from exposure to a limited number of substances, mainly in the occupational setting, to exposure to numerous chemicals at the same time, where indoor environments and food have become important sources.

The Life Cycle Perspective Chemicals can be released from consumer products during all steps of the life cycle – manufacturing, use, waste handling and disposal, and recycling – thereby posing a potential risk to human health and the environment. During the use phase, chemicals can be released from products through leakage of additive substances, washing and wearing, or via the formation of small particles. Humans and nontarget organisms in the environment may subsequently be exposed via several different routes. Humans can be orally exposed via food and drink, for example, to chemicals that have migrated from food contact Encyclopedia of Toxicology 4th Edition

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materials. Chemicals that come into contact with the skin may result in dermal absorption. Human exposure also occurs via inhalation of particles in air and dust. Organic chemicals, such as brominated flame retardants (BFRs) which can be released from electronic devices and textile products, have been found to accumulate in indoor dust. Children who spend much time close to the floor are especially exposed to chemicals in dust. Even if the chemical emission from a single type of product may be insignificant, the total emission of one chemical from several sources or the combined emissions of several chemicals may be important sources to human exposure and environmental pollution. Emissions from consumer products incorporated or treated with hazardous chemicals may result in long-term exposure to humans and the environment. However, knowledge about the mechanisms involved in the diffuse emissions of substances from products and consequent exposures of humans and the ambient environment is currently insufficient. The fast turnover of consumer products leads to increased resource consumption, generates massive amounts of wastes, and prompts the need for safe and efficient recycling. Hazardous substances in waste may be released and pose risks via incineration or landfills as well as in the recycling process. Risks to human health and the environment are difficult to assess due to the lack of information about the presence of hazardous substances in consumer products. The information that is available is often not disseminated from producers and importers to the waste stage. Thus, hazardous chemicals may be reintroduced to the market via reused and recycled products and materials. Manufacturing of many materials and consumer products that are sold and consumed on the EU or US markets takes place in countries having less restrictive and comprehensive chemical rules for protecting human health and the environment. The manufacturing of consumer products may therefore result in high occupational exposures and environmental releases of dangerous chemicals. Another stage of the life cycle chain closely associated with health and environmental problems in low-income countries is the waste stage. A recognized problem is that waste electrical and electronic equipment is being exported to countries where there are few risk management measures in place for minimizing negative impacts of its chemical content on human health or the environment.

Association with Adverse Outcomes Humans of all ages, including children, unborn babies, and other sensitive subpopulations, are continuously exposed to multiple chemicals at the same time, many of which are commonly used in consumer products. Biomonitoring studies of human chemical exposure have found that numerous chemicals representing different chemical classes are present in the human body at various levels. These include chemicals commonly incorporated in, and known to be released from, consumer products, such as BFRs, PFCs, bisphenol A, and phthalates as well as banned but still widespread persistent environmental contaminants, for example, polychlorinated biphenyls. Analyses conducted on blood samples from three generations in 13 EU member states showed, for example, that BFRs are detected in higher levels and more frequently in the blood of younger generations than in older generations. Research on male reproductive health conducted in Denmark and Finland has indicated a relationship between levels of a group of BFRs, polybrominated diphenyl ethers, in mothers’ breast milk and cryptorchidism in their sons, that is, the testes have not descended in the scrotum by the time of birth. Significant adverse effects of a number of chemicals used in consumer products are reported from both in vitro and in vivo toxicity and ecotoxicity experimental studies, but many relationships remain to be supported by epidemiological studies. However, it is very difficult to link health impacts to exposure to a specific substance, or a mixture of substances, in epidemiological studies due to statistical constraints and the many confounding factors. As the assessment of health and environmental impacts of chemicals involves many complex parameters and uncertainties, assessments are sometimes subject to both scientific and policy debates. Current such topics include the reliability and relevance of so-called low-dose effects (different definitions of low-dose exist, but often it refers to doses below the no observed adverse effect level or to doses in the range of typical human or environmental exposures) of endocrine-disrupting chemicals (EDCs) reported in experimental studies for human health risk assessment, mixture toxicity and risks of nanomaterials.

Risk Reduction Strategies Both regulatory and voluntary strategies are used for managing health and environmental risks posed by hazardous chemicals emitted from consumer products. These may include bans or restrictions on certain substances, mixtures, or uses, requirements such as to disseminate information, and economic incentives for promoting substitution of hazardous chemicals to safer alternatives.

Regulatory Instruments While some chemical sectors are relatively well regulated, regulations of industrial chemicals, and in particular the use of chemicals in consumer products, have been criticized for not being protective enough with regard to human health and the environment. In the last decade, the EU chemicals legislation has essentially been completely renewed. When the industrial chemicals legislation REACH (Registration, Evaluation, Authorisation, and restriction of CHemicals) went into force on 1 June 2007, it replaced about 40 pieces of chemicals legislation. Important reasons behind the development of REACH were that data on chemical properties should be required for all industrial chemicals, despite the date of their entry to the market, as well as shifting the responsibility for assessing the safety of the chemicals from authorities to the chemical producers and importers.

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The trade with consumer products is global and thus chemicals control within the EU affects and is affected by international conditions. The US national chemicals legislation, the Toxic Substances Control Act (TSCA), is also currently under debate concerning its renewal. The Safe Chemicals Act of 2011 is being proposed as a law for addressing problems identified with TSCA. It aims, for example, to improve the safety of chemicals used in consumer products and to increase public access to information on chemical safety. Increasingly more of the production of consumer products is located in areas with fast economic growth, such as parts of Asia and Latin America, where chemicals control is less restrictive than in the EU. China is an important producer and supplier of consumer products to the northwestern part of the world. As a response to the regulatory gap created by the introduction of REACH, Chinese chemicals regulations were revised in 2010 and 2011 in order to improve human health and environmental protection and to overcome trade barriers. In the case of electrical and electronic equipment, the EU directive on Restriction of the use of certain Hazardous Substances (RoHS) has led to the implementation of similar legislations in other parts of the world. The EU RoHS Directive has thereby contributed to a more protective standard on hazardous substances in electronics on a global level. A central problem in chemicals control is that data on toxicological and ecotoxicological properties are lacking or are too insufficiently required for many chemicals for enabling a robust health or environmental risk assessment. In the EU, what data are required by REACH to be submitted to the European Chemicals Agency (ECHA) is volume dependent; the higher the production volume of the substance, the more information about the substance is required. For chemical substances produced or imported in less than 1 ton per year no data are required, and for substances in the tonnage band between 1 and 10 tons the data requirements are very limited. This has the implication that a great number of chemicals cannot be adequately risk assessed or classified according to the hazard criteria as set out in the European regulation on classification, labeling, and packaging of substances and mixtures (CLP). The CLP hazard classifications are central in EU chemicals policy as they are often used as a basis for priority of substances for restrictions and requirements. Another problematic issue for risk assessment and risk management is that information about the chemical content of consumer products is rarely available to regulators, professional buyers, or consumers. The assessment of health and environmental risks associated with the use of chemicals in consumer products is thus often hampered by the lack of important information. The candidate list under REACH is the basis for the authorization process and a tool for increasing and disseminating information on the presence of substances of very high concern (SVHCs) in products, particularly in the supply chain, but also to consumers upon request. Since many supply chains are global, the information requirements connected to the SVHCs will have impacts also outside the EU. As the candidate list is regularly updated, it has been identified to promote chemical companies to work proactively with substitution and to find out the chemical content of their products. However, considering the large number of chemicals in commercial use, the authorization and information requirements currently apply only to a very small share of the chemicals on the EU market; approximately 0.1% has been identified as an SVHC. Decisions on what risk management measures to take, for example, what substances to select for authorization, is not solely based on health or environmental risk assessment conclusions, but also takes into account political, social, economic, and technological implications, including the availability of feasible and less toxic substitute substances.

Voluntary Approaches To accelerate the work towards achieving a safe and sustainable use of chemicals in consumer products, voluntary approaches can be used to complement regulatory restrictions and bans. Such approaches include, for example, different kinds of information efforts. Common voluntary information instruments are targeted information campaigns or positive labeling. These may increase the receivers’ knowledge and perception of hazards and risks, and potentially lead to changes in attitudes and behavior. Consumers who are provided with information about content of hazardous chemicals in products in a user-friendly format may thus change their consumption patterns. Increased knowledge will enable consumers and purchasers, for example, in procurement, to make more informed choices, take precautionary actions, and ask for alternatives. This will put pressure on producers and suppliers of consumer products and may ultimately lead to the phase-out of chemicals with unwanted properties. Along with the increasingly global trade of consumer products, the need for international cooperation increases with regard to the management of associated chemicals risks. The United Nation’s Strategic Approach to International Chemicals Management (SAICM) is an international policy framework, which has identified chemicals in products as an ‘emerging policy issue.’ The overall aim of SAICM is to achieve the goal agreed upon in Johannesburg in 2002 at the World Summit on Sustainable Development that by 2020 chemicals should be “used and produced in ways that lead to minimization of significant adverse effects of human health and the environment.” An important step toward this goal is that all actors, including consumers, have increased access to information on chemicals in products throughout the products’ entire life cycle, including the waste stage. Economic instruments have proven to sometimes constitute effective incentives for reaching environmental goals. The use of economic incentives such as taxes and fees has, however, been practiced only to a limited extent for minimizing the use of hazardous or untested chemicals. Internationally, existing chemicals control through economic incentives mainly concerns waste, packaging, and single substances. The use of economic instruments for managing risks associated with the use of chemicals in different consumer products is also possible, although it is more complicated than for single substances. The challenge is due to the limited knowledge of which products contain hazardous chemicals and in what concentrations.

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Urgent Problems and Challenges Emerging issues with regard to chemicals in consumer products include the need to increase the requirements on the generation of effect and exposure data, information on the chemical content of products, and information dissemination in the supply chain. This will facilitate substitution of hazardous chemicals to safer alternatives and enable supply chain actors and other stakeholders to improve the management of risks. Furthermore, the life cycle perspective in chemicals control needs to be extended. To obtain improved resource efficiency and a sustainable development, it is necessary to minimize the input of hazardous chemicals into products. The majority of the commercially available chemicals are not restricted for use in consumer products, including substances that are considered especially hazardous, such as carcinogens, mutagens, and reprotoxicants; EDCs; persistent, bioaccumulative, and toxic substances; and strongly sensitizing chemicals. Lack of protective legislations can cause problems in all life cycle stages of a product. It has therefore been urged that hazardous chemicals should be avoided already at the stage of production. The EU environmental policy states that environmental damage should be rectified at source and that preventive and precautionary actions should be taken. Translated into the context of chemicals in consumer products, those principles could arguably hold that the input of hazardous chemicals into products should be avoided or minimized in order to prevent problems from arising at the end of pipe, such as difficulties in achieving environmental and health goals.

See also: Bisphenol A; Candidate list of substances of very high concern (SVHC), reach; Chemical safety assessment and reporting tool (Chesar), REACH; Electronic and packaging waste; Environmental risk assessment, cosmetic and consumer products; European Classification and Labeling (C&L) Inventory; Green chemistry; Hazardous waste; Import/export of hazardous chemicals; Mixture, toxicology, and risk assessment; PBT (persistent, bioaccumulative, and toxic) chemicals; Phthalates; Polybrominated diphenyl Ethers; REACH; Risk management; Strategic Approach to International Chemicals Management (SAICM); Surfactants, perfluorinated (PFAS, PFOS, PFOA); Toxic Substances Control Act (TSCA) US; Toy safety and hazards; Uncertainty analysis.

Further reading Articles Beronius A, Rudén C, Håkansson H, and Hanberg A (2010) Risk to all or none? A comparative analysis of controversies in the health risk assessment of bisphenol A. Reprod. Toxicol. 29: 132–146. Hansson SO, Molander L, and Rudén C (2011) The substitution principle. Regul. Toxicol. Pharmacol. 59: 454–460. Molander L and Rudén C (2012) Narrow-and-sharp or broad-and-blunt – regulations of hazardous chemicals in consumer products in the European Union. Regul. Toxicol. Pharmacol. 62: 523–531. Molander L, Breitholtz M, Andersson PL, Rybacka A, and Rudén C (2012) Are chemicals in articles an obstacle for reaching environmental goals? – Missing links in EU chemical management. Sci. Total Environ. 435–436: 280–289. Ruden C and Hansson SO (2010) Registration, Evaluation, and Authorization of Chemicals (REACH) is but a first step – how far will it take us? Six further steps to improve the European chemicals legislation. Environ. Health Perspect. 118(1): 6–10. Tsydenova O and Bengtsson M (2011) Chemical hazards associated with treatment of waste electrical and electronic equipment. Waste Manag. 31: 45–58. Reports Centers for Disease Control and Prevention (CDC) (2009) Fourth National Report on Human Exposure to Environmental Chemicals. Available at:http://www.cdc.gov/exposurereport accessed 17.06.2013. Massey RI, Hutchins JG, Becker M, and Tickner J (2008) Toxic Substances in Articles: The Need for Information. The Nordic Council of Ministers: Copenhagen. TemaNord596. WHO/UNEP (2013) The State-of-the-Science of Endocrine Disrupting Chemicals – 2012. In: Bergman Å, Heindel JJ, Jobling S, Kidd KA, and Zoeller RT (eds.) . ISBN: 978 92 4 150503 1.

Relevant Websites http://www.saicm.org :Strategic Approach to International Chemicals Management (SAICM), 2012. Introducing SAICM (accessed 17.06.2013). http://echa.europa.eu/web/guest/regulations/reach :European Chemicals Agency (ECHA), 2013. REACH (accessed 17.06.2013).

Chemicals of Environmental Concern OI Kalantzi and AA Kanelli, University of the Aegean, Mytilene, Greece © 2024 Elsevier Inc. All rights reserved. This is an update of M.M. Schultz, Chemicals of Environmental Concern, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 805–809, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01004-6.

Introduction Mineral and energy exploration Fossil fuel combustion Agriculture and forestry Industrial production Consumerism Consumerism and chemicals of emerging environmental concern Conclusion References Further reading

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Abstract Chemicals of environmental concern or environmental pollutants are any physical, chemical, biological, or radiological substance or matter that has an adverse effect on air, water, soil, or living organisms. Environmental pollutants may enter the environment through a variety of sources, including from mining and energy exploration, fossil fuel combustion, agriculture and forestry, industrial production, and consumerism. Conventional or historical pollutants discussed include organochlorine pesticides, metals, polyaromatic hydrocarbons, polychlorinated biphenyls, dioxins and polybrominated biphenyl ethers. Chemicals of emerging environmental concern include pharmaceuticals and personal care products, perfluorinated chemicals, and phthalates. Some chemicals of environmental concern may also be classified as endocrine-disrupting chemicals.

Keywords Atrazine; Bisphenol-A (BPA); Chlorofluorocarbons (CFCs); Dichlorodiphenyltrichloroethane (DDT); Dioxins; Endocrine; Disrupting chemicals; Greenhouse gases; Organochlorine insecticides; Perfluorinated chemicals (PFCs); Pharmaceuticals and personal care products (PPCPs); Phthalates; Polychlorinated biphenyls (PCBs); Polycyclic aromatic hydrocarbons (PAHs); Volatile organic compounds (VOCs)

Introduction Chemicals of environmental concern are any physical, chemical, biological, or radiological substance or matter that has an adverse effect on air, water, soil, or living organisms. A reasonable definition of a pollutant is a substance present in greater than normal concentration as a result of human activity and having a detrimental effect on its environment or on something of value in that environment. Contaminants, which are not classified as pollutants unless they have some detrimental effect, cause deviations from the normal composition of an environment. Pollutants can enter through direct dumping, piped outflow, and channeled waste streams as localized point sources, or as diffuse nonpoint sources they can enter rivers, lakes, streams, and groundwater through runoff and soil percolation. Nonpoint sources are considered to be major contributors to air, water, and soil pollution that include runoff from paved streets and parking lots, agricultural lots, soil erosion from logging, and atmospheric deposition of acidic or toxic air pollutants. The source is particularly important, because it is the logical place to eliminate pollution. Common sources of contaminants to the environment include mining and mineral processing, fossil fuel combustion, agricultural and forestry, industrial production, and consumerism (Table 1). After a pollutant is released from a source, it may act on a receptor. The receptor is anything, both biotic and abiotic, that is affected by the pollutant. Humans whose eyes water from atmospheric oxidants are receptors. Juvenile trouts that die after exposure to pesticides in water are also receptors. Eventually, if the pollutant is long lived, it may be deposited in a long-term sink such as aquatic sediments and soils.

Mineral and energy exploration The largest quantitative source of contamination derives from mining and energy extraction. Mining and mineral processing use a variety of chemicals for extraction, ore processing, water treatment, and many other supporting activities such as overburden removal. Mining and energy extraction generate large volumes of waste and have the potential to cause a number of environmental

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Chemicals of Environmental Concern Common sources of contaminants to the environment.

Sources

Contaminants

Mining and mineral processing Fossil fuel combustion Agriculture and forestry Industrial production

Heavy metals, chemicals via cyanide and acids, hydrocarbon products resulting from spills and coal mining, and metallic salts

Consumerism

Sulfur dioxide, carbon dioxide, nitric oxide, ozone, acids, polycyclic aromatic hydrocarbons, and volatile organic compounds Pesticides, nitrates, phosphates, greenhouse gases, and mineral salts Numerous synthetic organic and inorganic compounds, organochlorines, dioxins, heavy metals, hydrocarbons, chlorinated phenols, sulfates, sulfides, surfactants, solvents, acids, bases, salts, pharmaceuticals, plastics, resins, explosives, and natural organics Residential and commercial chemicals, pesticides, fertilizers, hydrocarbons, solvents, surfactants, paints, sealants, pharmaceuticals and personal care products, volatile organic compounds, resins, plastics, metals, salts, acids, and bases

problems if improperly managed. Water and soil degradation can result from salinization, acidification, and chemical contamination. Streams and rivers can also experience severe siltation. Coarse tailings and rock blasting produce large amounts of dust and mobilize heavy metal contaminants, such as lead, copper, aluminum, and zinc, which can leach into the surface and subsurface waters. Cyanide and mercury are used to extract gold from soil and pulverized rock. Mineral processing generates a great deal of particulate matter released from bauxite and coal processing. Acid leaching into soil, groundwater, and riparian environments from mine wastes is common. Acid drainage from mine tailings, ore, and waste dumps contains sulfur and sulfides such as iron sulfide, which can be converted to acids through bacterial oxidation in the presence of moisture and oxygen. Acid mine water drainage also leads to selenium accumulation into surface water and soil-plant system. Selenium is considered as an important nutrient; however excessive concentration poses threats to both the environment and human health. Extreme selenium levels in plants interfere with chlorophyl combination and nitrate accumulation and change the structure and function of proteins. Major health problems include respiratory, gastrointestinal, cardiovascular difficulties, hypertension, mental problems, damages in hair, nails and teeth (Etteieb et al., 2020; Lemly, 2002; Gebreeyessus and Zewge, 2019). Metal contaminants may become mobilized under acidic conditions to cause potential health and environmental problems resulting from leaching into soil, water, and sediment. Acid mine drainage and slag leachate can contain high concentrations of heavy metals and acids. Sulfuric acid can be formed via oxidation of sulfides. As a great deal of attention is paid to the containment and remediation of acid mine drainage, the neutralization of acid pH usually results in the precipitation of many contaminants, usually as metallic salts. These salts would then become soluble and may enter surface and groundwater. Oil spills and coal mining command considerable attention from the media because they are often large scale and visually very dramatic. Nothing seems worse than a mass of toxic crude oil and tarry hydrocarbons smeared over the natural habitats of some foreshore or the sight of strip mining operations. As a result, there is a massive public response and a frenzy of activity by agencies, community groups, and politicians. For many spills, however, the ecological issues are different from those being touted in public discussion. There is, in fact, plenty of evidence that chronic, low-level contamination of habitats by complex exogenous agents may be more compromising in terms of environmental outcomes. In addition, coupled with the destruction, deterioration, and fragmentation of natural habitats, there exists considerably greater threats to long-term sustainability of coastal biodiversity. Attempts to disperse oil spills with surfactants may be potentially hazardous. Hydraulic fracturing, or more commonly known as fracking, has been used commercially since the 1940s to extract petroleum and natural gas from fractures in the Earth’s rock layer that result from the injection of pressurized fracturing fluids. Proponents of hydraulic fracturing argue that this extraction process allows access to fossil fuels that are otherwise inaccessible. However, hydraulic fracturing has raised environmental and regulatory concerns. These concerns have included groundwater contamination and risks to air quality resulting from methane leaks originating from wells. Other toxic hydrocarbons, including benzene, xylene, and naphthalene, have also been detected in air and water near hydraulic fracturing sites. Additional environmental concerns with hydraulic fracturing include water consumption (especially in arid regions); migration of gases and hydraulic fracturing chemicals to the surface and subsequent contamination of surface waters and soil; waste disposal of flowback, which may include brines, heavy metals, radionuclides, and organic chemicals; and the health effects due to exposure of all these chemicals.

Fossil fuel combustion Humanity’s major sources of energy are derived from fossil fuels, principally oil, gas, coal, and wood. The major combustion by-products of fossil fuel burning include sulfur dioxide (SO2), carbon dioxide (CO2), and nitrogen oxides (NOx), and partially oxidized hydrocarbons. The process of burning fossil fuels in thermal power plants, factories, homes, and motor vehicles emits enormous amounts of the aforementioned pollutants. The most important environmental concerns resulting from fossil fuel use are climate change, acid rain, surface ozone, and particulate- and aerosol-bound toxins. Over 99% of climate scientists agree on human-caused contemporary climate change. A component of the climate warming observed since the 1880s is likely attributed to increases in the concentration of ‘greenhouse gases’ such as the fossil fuel combustion product CO2 in the atmosphere. Another side effect of fossil fuel burning is acid rain. In the process of burning organic fuels, some gases, in particular SO2 and NO2, combine with atmospheric water vapor to form sulfuric and nitric acids. Acidified rainwater can attain pH values below 3. Acid rain can cause damage to plant life, in some cases seriously affecting the growth of forests and lakes due to acid-stimulated metal leaching from soils and rock.

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Besides gaseous fossil fuel emissions that contribute to global climate change and acid rain, emissions of particulate matter from incomplete burning also contribute to poor air quality, acidification of ecosystems, degradation of biodiversity and depletion of nutrients in soil. Especially PM2.5 are linked to pulmonary and cardiovascular diseases, type II diabetes, dementia and loss of cognitive function. Coal burning and diesel engines are a major source of particulate organic matter. Additionally, fuel combustion and evaporative emissions from motor vehicles are also major sources of anthropogenic volatile organic compounds (VOCs). Motor vehicles account for a considerable fraction of the total emissions of nitrogen oxides, particulate hydrocarbons, and VOCs in developed countries. Of particular concern is the production of polycyclic aromatic hydrocarbons (PAHs) resulting from incomplete combustion of fossil fuels. These compounds, especially diesel soot emissions, contain some of the most potent mutagens and carcinogens known to humankind such as benzo(a)pyrene. Compared with solid fossil fuels, natural gas and oil are less polluting. Natural gas is the least polluting fossil fuel. The main environmental problems resulting from the production and transportation of primary energy are related to mining of solid fuels (mainly coal) and oil transportation. Coal mining operations produce large amounts of slag wastes and result in acid water drainage. The continuous acid discharges from mines seriously affect aquatic ecosystems, since acid waters containing heavy concentrations of dissolved heavy metals will support only limited water flora, and will not sustain fish and many invertebrates. The major impacts from oil are associated with accidental spillages during transportation both at sea and on land. The resultant damage to coastal areas and marine life can be dramatic in the short term and may also have long-term consequences. Solid wastes and ash disposal (spoil tips) from coal mines lead to the contamination of water percolating through slag heaps that cause groundwater and soil pollution. The combustion of liquefied petroleum gas causes the problems of liquid residual disposal.

Agriculture and forestry The global concentration of greenhouse gases has increased measurably over the past 250 years, partly due to land use activities such as agriculture and forestry. According to the fifth assessment report of the Intergovernmental Panel on Climate Change, carbon dioxide, methane, and nitrous oxide emissions have increased by  40, 150, and 20%, respectively, since 1750. Agriculture and forestry practices have contributed to trends in emissions of these greenhouse gases through fuel consumption, land use conversions, cultivation and fertilization of soil, production of ruminant livestock, and management of livestock manure. Additionally, the irrigation of formerly arid lands leaches minerals from soils at accelerated rates resulting in toxic concentrations of agricultural pollutants, which include nutrients (nitrogen and phosphorus), pesticides, pathogens, selenium, and salts. While farmers do not intend for these materials to move from the field or enterprise, they often do, carried by rainfall, snowmelt, or irrigation water. A wide variety of pesticides are applied to agricultural crops to control insect pests (insecticides), weeds (herbicides), fungi (fungicides), and rodents (rodenticide). Pesticide residues reaching surface water systems may harm freshwater and marine organisms, damaging recreational and commercial fisheries. Pesticides in drinking water supplies may also pose risks to human health. Long-lived pesticides such as dichlorodiphenyltrichloroethane (DDT), aldrin, dieldrin, and mercuric and arsenic compounds still persist in the environment. Shorter lived pesticides such as chlorpyrifos, methyl parathion, 2,4-D herbicides, and numerous new compounds are a global concern. Atrazine, a commonly used, relatively short-lived herbicide (half-life in soil ranges from 13 to 261 days), is at the center of a regulatory battle in the United States. It has been used in the United States for over 50 years and is primarily applied to corn and sorghum fields to increase their crop yields. Atrazine is banned in the European Union due to persistent groundwater contamination and because research has suggested that atrazine is an endocrine-disrupting chemical that induces the production of aromatase, an enzyme that converts androgens (male sex hormones) to estrogens (female sex hormones). Atrazine exposure has also been linked to birth defects, low birth weights, cancer, and fertility problems. Residues of artificial growth hormones, antibacterial compounds (ACs) and antibacterial resistant genes (ARGs) are released into the environment through veterinary practices, the use of other antibiotics during agricultural activities and manufacturing processes, thus contaminating soil, groundwater, surface water and sediment. Regarding ACs and ARGs, these are acknowledged as a serious threat to human health and are associated with the global antibacterial resistance problem. Agriculture is also seen as a potential significant source of nano contaminants in the future, as the nanopesticide and nanomedicine markets are further developed (Snow et al., 2020; Manyi-Loh et al., 2018).

Industrial production The global expansion of industrial and consumer-oriented societies is linked to large-scale industrial production and consumerism that use a vast array of numerous chemical compounds. The listings of such chemicals are too vast to present here but some examples are discussed here. Environmental contaminants in nature typically involve complex mixtures, partitioning factors, chemical transformations, and abiotic and biotic interactions. The biological and environmental effects are complex and may be additive, synergistic, and even antagonistic in nature. Pulp and paper mill sludge is a complex and changeable mixture of dozens or even hundreds of compounds. Some are well known, like natural wood extractives, organochlorines, organosulfides, and dioxins. Priority pollutants and chemicals of concern that must be analyzed in pulp mill residues include heavy metals, chlorinated hydrocarbons, chlorobenzenes, PAHs, chlorinated phenols, chlorinated catechols, chlorinated guaiacols, phthalates, resin acids, alkylphenols and alkylphenol ethoxylates, and plant sterols. In 1775, PAHs were the first group of compounds known to cause cancer in humans. Nowadays, many of these compounds are well-known carcinogens in humans and animals. PAHs are produced in the environment as the result of heating organic matter to high temperatures like tobacco smoke, soot, coal tar, creosote production, wood burning, smoked foods, roasted coffee, charbroiled

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Chemicals of Environmental Concern

meat, and fossil fuel combustion exhaust. However, the major environmental source comes from asphalt, tar, used motor oil, diesel exhaust, and coal burning. Dioxins, a by-product of herbicide and pulp and paper production, are highly toxic members of a class of organochlorine chemicals including polychlorinated dibenzo-p-dioxins (PCDDs), polydibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), polybrominated dibenzo-p-dioxins (PBDDs), polybrominated dibenzofurans (PBDFs), and polychlorinated pesticides. Dioxins and its related compounds are cytotoxic and genotoxic, and have hormonal effects that may disrupt the endocrine system and cellular signaling pathways in wildlife and humans. Dioxins have both estrogenic and antiestrogenic effects, depending on the organ or tissue affected. Exposure to relatively low levels of these chemicals has had catastrophic effects on populations of Beluga whales, alligators, turtles, mink, otters, bald eagles, osprey, cormorants, terns, herring gulls, migratory birds, chickens, lake trout, chinook and coho salmon, etc. throughout the United States and Canada. PCDFs are formed as inadvertent by-products in the production and use of PCBs, formerly used as an insulator in electrical transformers and, in combination with PCDDs, in the production of chlorophenols and have been detected as contaminants in these products. PCDFs and PCDDs also may be produced in thermal processes such as incineration and metal processing and in the bleaching of paper pulp with free chlorine. PCDFs are also found in residual waste from the production of vinyl chloride and the chloralkali process for chlorine production. The relative amounts of PCDF and PCDD congeners produced depend on the production or incineration process and vary widely. Like PCDDs, PCDFs are ubiquitous in soil, sediments, and air. Excluding occupational or accidental exposures, most background human exposure to PCDFs occurs as a result of eating meat, milk, eggs, fish, and related products, as PCDFs are persistent in the environment and accumulate in animal fat. High exposures have occurred in relation to incidents in Japan (yusho) and Taiwan (yucheng) involving contamination of rice oil and in accidents involving electrical equipment containing PCBs. Occupational exposures also may occur in metal production and recycling, and in the production and use of chlorophenols and PCBs. Chemical wood preservatives account for the single largest pesticide use in the United States and one of the greatest pesticide threats to public health and the environment. Wood preservatives protect wood products from fungus and insect decay. The three principal wood preservatives include chromated copper arsenate (CCA), pentachlorophenol (penta), and creosote. The US Environmental Protection Agency (EPA) has classified many chemicals and even certain heavy metal contaminants as known or probable carcinogens, teratogens, cellular toxins, endocrine disrupters, and reproductive toxins. The arsenic in CCA, certain PAHs, and dioxins are known human carcinogens and are linked to disorders and birth defects.

Consumerism Everything we put down the drain or flush down the drain ends up in our watersheds via wastewater treatment, which can affect the health of terrestrial and aquatic wildlife, plants, the atmosphere, and the water quality in our area. Large amounts of pharmaceuticals and personal care products (PPCPs) are released into the environmental every day through wastewater treatment by way of domestic waste from human excretion, direct disposal of unused or expired drugs in toilets, or rinsing of personal care products down the drain. Additional sources of consumer environmental contaminants include cleaning agents, surfactants, pesticides, fertilizers, lawn and garden treatments, paints, and sealants. It is imperative that those seeking a healthy lifestyle and reduction in pollutant exposure choose with care the products they use to clean and maintain their homes, yards, and pets. When one purchases a hazardous product for the home, it creates a market for these toxic chemicals. Once we use the hazardous substance, the vapors released or the water contaminated pose a risk to ecosystems and human populations alike. Early examples of consumer-based environmental contaminants include the invention of chlorofluorocarbons (CFCs) in the late 1920s and early 1930s. CFCs were developed in response to the need for safer alternatives to the sulfur dioxide and ammonia refrigerants used at the time. Chlorofluorocarbons were chosen for their safety and for their advantageous chemical properties. These compounds are low in toxicity, nonflammable, noncorrosive, and nonreactive with other chemical species, and have desirable thermal conductivity and boiling point characteristics. These features led to increased demand as more applications arose for CFC use. However, CFCs are human-made substances that release chlorine atoms which destroy ozone in the atmosphere, specifically in the stratosphere, which causes an increase in UV radiation at the ground level. The amount of CFCs produced (and therefore likely released into the atmosphere) steadily increased over several decades until an international agreement called the Montreal Protocol was signed in September 1987. In this agreement, the world’s nations agreed to phase out CFC production. As a result, most stratospheric CFC concentrations have leveled off or are decreasing. To date, the Montreal Protocol is considered the most successful international environmental agreement. In 1962, Rachel Carson’s novel, Silent Spring, implicated pesticides such as DDT for the decline of some wildlife populations. Continued research in the late 1960s and 1970s discovered elevated use of manufactured organic chemicals in industrial and domestic applications. Chemicals of concern included PCBs, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), and related dioxin and furan congeners, PAHs, and various organic solvents. Regulatory efforts by the EPA and international efforts (e.g., Stockholm Convention of Persistent Organic Pollutants) resulted in a decreased production of these xenobiotics. During the previous decade attention was shifted to another source of consumer environmental contaminants: single used plastics (SUPs). The latter refer to plastic products intended to be used once or for a small period of time before being thrown away. They have drastic impacts on both the environment and human health. Due to their high resilience disposable plastics decompose slowly, thereby persisting in ecosystems for hundreds of years and eventually becoming microplastics. Moreover, plastic pollution imposes a direct threat to wildlife; animals often consume microplastics or are entangled to them resulting in their injury or even death. SUPs contain chemicals additives enhancing their durability such as PAEs, dimethyl phthalate (DMP), diethyl phthalate

Chemicals of Environmental Concern

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(DEP), benzyl butyl phthalate (BBP), diethylhexyl phthalate (DEHP), antioxidants, heat stabilizers and n-hexane which can be transferred to the packaged product and eventually the human body. Other routes of human exposure to microplastics are food consumption and inhalation (Li and Suh, 2019; Chen et al., 2021).

Consumerism and chemicals of emerging environmental concern An active area of environmental research is in the area of ‘emerging contaminants.’ What is an emerging contaminant? Emerging contaminants are not only chemicals that have recently been released into the environment, but are broadly defined “as any synthetic or naturally occurring chemical or any microorganism that is not commonly monitored in the environment but has the potential to enter the environment and cause known or suspected adverse ecological and (or) human health effects.” The majority of emerging contaminants differ from ‘conventional’ or ‘historical’ environmental chemicals of concern (e.g., pesticides, metals, PAHs, PCBs, dioxins) because many emerging contaminants are present in consumer products and are routinely used in households. Emerging contaminants encompass a wide variety of chemicals, including many pharmaceuticals and PPCPs. Examples of personal care products are fragrances, antimicrobial agents, sunscreens, and fluorescent whitening agents. As already mentioned, a primary route into the environment for PPCPs is via wastewater treatment, because many of these chemicals are flushed or rinsed down the drain and are not removed by conventional wastewater treatment. Since many of these PPCPs are biologically active, they may pose a risk to ecosystems and human populations alike. Since wastewater effluent often discharges to drinking water sources, there is the potential for drinking water to contain PPCPs. Recent studies provide evidence that PPCPs may contaminate drinking water, although it is currently unclear if the concentrations are significant enough to pose any risk to human health. Many chemicals of emerging environmental concern are also potential endocrine-disrupting chemicals. An endocrine-disrupting chemical can be a natural or synthetic chemical that interferes with the hormonal system. Many of the previously discussed chemicals of concern are also classified as endocrine-disrupting chemicals, including DDT, PCBs, dioxins, and pesticides (e.g., dieldrin, atrazine, methoxychlor). Developing organisms are particularly vulnerable to the effects of the endocrine-disrupting chemicals. Exposure early in life (often during a critical or sensitive developmental period) to an endocrine-disrupting chemical can induce permanent, nonreversible effects. These effects often do present themselves until adolescence or adulthood as in the (unfortunate) case of diethylstilbestrol, more commonly known as DES. DES, a synthetic nonsteroidal estrogen, was prescribed to women during the 1940s–1970s to prevent miscarriage. Although there have been few documented adverse effects in the mothers who took DES during their pregnancies, many of their daughters and sons who were exposed to DES in utero developed problems associated with their reproductive organs. Many daughters are sterile and a small percentage have developed a rare form of vaginal or cervical cancer. The prenatally exposed sons have an increased incidence of abnormalities in their sexual organs, decreased sperm counts, and a higher incidence of testicular cancer. Suspected endocrine-disrupting chemicals are found in a wide variety of products, including insecticides, herbicides, fumigants, and fungicides that are used in agriculture as well as in the home. Other endocrine disruptors are found in industrial and consumer chemicals such as detergents, resins, plasticizers, organometals, halogenated aromatic hydrocarbons, monomers in many plastics, and PPCPs. Exposure to these chemicals occurs through direct contact in the workplace or at home, or through ingestion of contaminated water, food, or air. Substances that are considered both chemicals of emerging environmental concern and endocrine disruptors that are of regulatory concern to the EPA include polybrominated diphenyl ethers (PBDEs), long-chain perfluorinated chemicals (PFCs), short-chained chlorinated paraffins (SCCPs), phthalates, bisphenol-A (BPA), and triclosan. PBDEs are flame retardants that are used extensively in consumer products ranging from textiles to electronics. PFCs have widespread applications as liquid repellants for paper, packaging, textile, leather, and carpets, as well as in industrial solvents, additives, coatings, and fire-fighting foams. Among these contaminants perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have received the most attention as they are extremely stable to the environment and biota, and are highly related to health problems such as cancer, reproductive effects and thyroid disruption (Zeng et al., 2019). SCCPs, phthalates, and bisphenol-A are all used as plasticizers to increase the flexibility of the plastic material and are present in countless consumer products, including many children’s toys. Triclosan is an antimicrobial additive in many personal care products that not only include antibacterial soaps, toothpaste, and mouthwash, but increasing use in household items including garden hoses, toys, cutting boards, and furniture. Other pollutants of environmental concern which have remained unidentified due to the limitations of analytical techniques refer to nanomaterials (NMs) or nanoparticles (NPs) and artificial sweeteners. NMs are highly used in the fields of engineering, chemistry, agriculture, electronics and medicines and cosmetic products such as shampoos, toothpastes, detergents, clothing and sunscreens. On the other hand, artificial sweeteners such as sucralose, acesulfame, neotame, cyclamate, saccharine, aspartame and neohesperidine may be safe for human consumption but have confirmed adverse effects on the physiology and locomotion of population species (e.g., crustaceans) (Noguera-Oviedo and Aga, 2016; Richardson and Kimura, 2017; Ramírez-Malule et al., 2020).

Conclusion The 20th century has brought with it tremendous gains in science and technology as well as gains in the quality of human life and longevity. However, these gains have been accompanied by certain hazards, many associated with the 100,000 chemicals that are now commonly in use. As stated earlier, environmental contaminants are materials that can pollute our surroundings and adversely

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impact living organisms. Often these pollutants are chemical compounds produced by human endeavors, although environmental contamination can also come from nonhuman sources such as naturally occurring metals, animal waste, oil seeps, and algal blooms. Environmental contaminants may pollute soil, surface water, or aquatic sediments. Many compounds also leach through soils into groundwater, potentially impacting drinking water supplies. Numerous pollutants are discharged directly into the atmosphere by human industry, where winds may transport them to Earth’s most remote corners. It is important, however, to note that industry is not the sole source of contaminants; individuals also contribute to this problem through the use of household pesticides and fertilizers, improper disposal of hazardous materials (e.g., used motor oil, paints, cleaning products), and by using many consumer products. Consequently, sites with one predominant contaminant are a rarity; complex mixtures and subsequent exposures define the real world.

See also: Atrazine; Bisphenol A; Carbon dioxide; DDT (dichlorodiphenyltrichloroethane); Estrogens IV: Estrogen-like pharmaceuticals; Perfluorooctanoic acid; Persistent organic pollutants; Pesticides and its toxicity; Pharmaceuticals effects in the environment; Pollution Prevention Act, United States; Pollution, indoor air; Pollution, soil; Pollution, water; Polybrominated diphenyl Ethers; Polychlorinated biphenyls (PCBs); Polycyclic aromatic hydrocarbons (PAHs); TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin); Toxic Substances Control Act (TSCA) US; Volatile organic compounds

References Chen Y, Awasthi AK, Wei F, Tan Q, and Li J (2021) Single-use plastics: Production, usage, disposal, and adverse impacts. Science of the Total Environment 52: 141772. https://doi. org/10.1016/j.scitotenv.2020.141772. Etteieb S, Magdouli S, Zolfaghari M, and Brar SK (2020) Monitoring and analysis of selenium as an emerging contaminant in mining industry: A critical review. Science of the Total Environment 698: 134339. https://doi.org/10.1016/j.scitotenv.2019.134339. Gebreeyessus GD and Zewge F (2019) A review on environmental selenium issues. SN Applied Sciences 1(1): 1–19. https://doi.org/10.1007/s42452-018-0032-9. Lemly AD (2002) Selenium pollution around the world. In: Selenium Assessment in Aquatic Ecosystems. Springer Series on Environmental Management. New York, NY: Springerhttps://doi.org/10.1007/978-1-4613-0073-1_1. Li D and Suh S (2019) Health risks of chemicals in consumer products: A review. Environment International 123: 580–587. Manyi-Loh C, Mamphweli S, Meyer E, and Okoh A (2018) Antibiotic use in agriculture and its consequential resistance in environmental sources: Potential public health implications. Molecules 23(4). https://doi.org/10.3390/molecules23040795. Noguera-Oviedo K and Aga DS (2016) Lessons learned from more than two decades of research on emerging contaminants in the environment. Journal of Hazardous Materials 316: 242–251. https://doi.org/10.1016/j.jhazmat.2016.04.058. Richardson SD and Kimura SY (2017) Emerging environmental contaminants: Challenges facing our next generation and potential engineering solutions. Environmental Technology and Innovation 8: 40–56. https://doi.org/10.1016/j.eti.2017.04.002. Snow DD, Cassada DA, Biswas S, et al. (2020) Detection, occurrence, and fate of emerging contaminants in agricultural environments. Water Environment Research 92(10): 1741–1750. https://doi.org/10.1002/wer.1429. Zeng Z, Song B, Xiao R, et al. (2019) Assessing the human health risks of perfluorooctane sulfonate by in vivo and in vitro studies. Environment International 26: 598–610. https://doi. org/10.1016/j.envint.2019.03.002.

Further reading Daughton CG and Ternes TA (1999) Pharmaceuticals and personal care products in the environment: Agents of subtle change? Environmental Health Perspectives 107: 907–938. Grossman E (2009) Chasing Molecules. Washington: Island Press. Hites RA (2011) Dioxins: An overview and history. Environmental Science & Technology 45: 16–20. Katsikantami I, Sifakis S, Tzatzarakis MN, Vakonaki E, Kalantzi OI, Tsatsakis AM, and Rizos AK (2016) A global assessment of phthalates burden and related links to health effects. Environment International 97: 212–236. https://doi.org/10.1016/j.envint.2016.09.013. Kolpin DW, Furlong ET, Meyer MT, et al. (2002) Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. Streams, 1999–2000: A national reconnaissance. Environmental Science & Technology 36: 1202–1211. Ramírez-Malule H, Quiñones-Murillo DH, and Manotas-Duque D (2020) Emerging contaminants as global environmental hazards. A bibliometric analysis. Emerging Contaminants 6: 179–193. https://doi.org/10.1016/j.emcon.2020.05.001. Richardson SD and Ternes TA (2011) Water analysis: Emerging contaminants and current issues. Analytical Chemistry 83: 4614–4648. Sagasta JM, Zadeh SM, and Turral H (eds.) (2018) More People, More Food, Worse Water? A Global Review of Water Pollution from Agriculture. More People, More Food, Worse Water?. Rome: A Global Review of Water Pollution From Agriculture. Snyder SA, Westerhoff P, Yoon Y, and Sedlak DL (2003) Pharmaceuticals, personal care products, and endocrine disruptors in water: Implications for the water industry. Environmental Engineering Science 20: 449–469. Steingraber S (2010) Living Downstream: An Ecologist’s Personal Investigation of Cancer and the Environment, 2nd edn. Philadelphia: Da Capo Press.

Relevant websites https://dtsc.ca.gov/environmental-chemistry-lab/chemicals-of-emerging-concern/ :California Department of Toxic Substances Control: Chemicals of Emerging Concern. https://www.norman-network.net/ :Network of reference laboratories, research centers and related organizations for monitoring of emerging environmental substances.

Chernobyl RK Chesser and BE Rodgers, Texas Tech University, Lubbock, TX, USA © 2014 Elsevier Inc. All rights reserved. This is a reproduction of R.K. Chesser, B.E. Rodgers, Chernobyl, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 822–829, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00080-4.

Background Social, Health, and Environmental Impacts: Dose Estimates Environmental and Genetic Impacts: Empirical Evidence Lessons from the Chernobyl Accident Further Reading

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Abstract The accident at the Chernobyl nuclear power plant is considered to be the worst nuclear accident in history. At 1.24 a.m. on 26 April 1986, a large explosion and fire occurred at reactor four of the Chernobyl nuclear power plant, releasing massive amounts of nuclear fuel into the surrounding environment. Radiation releases from the accident continued for about 10 days, and the fallout spread from Chernobyl to the surrounding territories and across the globe. Following the Chernobyl accident, about 350 000 residents of Ukraine, Belarus, and Russia were evacuated and resettled in other areas of the Former Soviet Union. Sixty-four human deaths have been attributed to acute radiation sickness resulting from the accident, and estimates of ultimate excess cancer deaths are estimated to be 4000–9335, predominantly in workers who assisted with the cleanup of the Chernobyl power plant. The heaviest amount of radiation was released into the first two plumes that killed about 400 ha of pine forest and likely eliminated resident wildlife populations in a 2
10 km area west of the reactor. Only about 1% of the radiation originally released still remains due to the rapid decay of most isotopes. Although there are reports of some genetic damage in organisms living in the contaminated habitats, there is no evidence of mutations that have been transferred to progeny. Evaluations in the aftermath of the Chernobyl accident led to numerous refinements to reactor operations and designs as well as methods to protect human populations.

Keywords EcotoxicologyFukushima Daiichi (Japan); Ionizing radiation; Radiation toxicology; Radioactive iodine; Radiocesium; Three Mile Island

Background Chernobyl’s nuclear reactor number four was a graphite-moderated, 3140-megawatt (MW) thermal (1000-MW electric) power facility located 180 km north-north west of Kiev, Ukraine. The reactor was commissioned for operation by the Soviet Union in December 1983. The reactor had 1659 fuel rods containing 172 550 kg of uranium enriched to 2.1% 235U. A significant power surge occurred during an experiment conducted in April 1986, which resulted in a loss of reactor control and inadequate flow of coolant water. Generation of hydrogen gas caused a series of explosions at 1.24 a.m. on 26 April 1986 that breached containment and spewed radioactive particles into the surrounding environment (Figure 1). The meltdown, explosion, and 10-day graphite fire at the Chernobyl nuclear power plant reactor four was the worst nuclear disaster in history. The initial hydrogen explosion destroyed the reactor containment vessel and ejected over 6000 kg of nuclear fuel particles and graphite into the environment along a narrow, westerly path of intense radiation. Fuel elements remaining in the core were resuspended into the atmosphere on the second day by drops of sand from multiple helicopter sorties. After the first day, the heat buildup in the exposed reactor core was sufficient to propel aerosols and oxidized volatile elements such as 134,137Cesium, 131Iodine, and 135Xenon high into the stratosphere. These volatile nuclides were dispersed over a very broad geographic area, primarily affecting Ukraine, Belarus, Russia, and Scandinavia (Figure 2). The total radioactivity released by the accident was 3.7–5.5
1018 Becquerels (Bq), or 100–150 million Curies (Ci). Thirty-one deaths were officially attributed to the explosion and radiation exposure to the personnel working in or near the facility. In 2008, the United Nations’ Scientific Council on Effects of Atomic Radiation (UNSCEAR) updated the mortality estimates to a total of 64 deaths of persons diagnosed with acute radiation syndrome from Ukraine and Belarus. The impact of the Chernobyl accident was exacerbated by the lack of a secondary containment structure in the RBMK-1000 reactor facility. The hydrogen explosion dislodged the 5000 metric ton biological shield and exposed the nuclear fuel to the environment. Fuel rods nearing the end of their fuel cycle, such as at Chernobyl, have accumulated over 100 different radionuclides as a product of 3 years of fission and neutron activation. Expulsion of nuclear fuel into the environment at Chernobyl was the main reason that this accident contributed significantly greater number of radioactive nuclides and higher radiation levels than at Fukushima, Japan, in 2011. At Fukushima, the nuclear fuel was damaged by partial meltdown, but primary and secondary

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Figure 1 Aerial photograph of Chernobyl reactor four on 3 May 1986.

containment structures prevented dispersion by explosive fragmentation. Venting of hydrogen gas at Fukushima did create explosions that damaged the superstructure of the facilities, but prevented rupture of the containment safety measures. Escaping gas and coolant water nevertheless contained considerable quantities of radioactive cesium, iodine, and xenon that had aerosolized from the damaged fuel. Although estimates of total releases are technically difficult, it appears that the total radiation release at Fukushima was 100–150 times less than that at Chernobyl. The accident at Japan’s Fukushima Daiichi facility is estimated to have released about 40
1015 Bq contributed principally by the volatile elements (cesium, iodine, and xenon). The release of radiocesium at Fukushima, however, was more comparable, reaching 20–40% of that released at Chernobyl. Design inadequacies associated with the RBMK-type reactors led to the closure of the Chernobyl nuclear power plant in 2000. The aftermath of the Chernobyl accident helped shape safety measures in nuclear power plants and advance our understanding of the consequences of exposure to radiation.

Social, Health, and Environmental Impacts: Dose Estimates Evacuation of the 45 000 citizens of the city of Pripyat, Ukraine, located only 2.9 km from Chernobyl’s reactor four, commenced at 2.30 p.m. on 27 April, using buses sequestered from surrounding cities (Figures 3 and 4). Prior to that time, plumes of radiation had already been deposited and heavy contamination enveloped the city. Although citizens of Pripyat accumulated an average external radiation dose of only 0.0115 Sieverts (Sv) over their 37 h of exposure, their doses would have been much greater had the initial plume moved directly through the city. All 90 000 inhabitants within a 30-km radius around the reactor (exclusion zone) were evacuated from 3 to 5 May. After 1986, about 220 000 additional persons were relocated, and relocation continued until 1992. In all, over 350 000 persons were resettled. It was estimated that the later (non-Pripyat) exclusion zone evacuees received average and maximum external doses of 0.00182 and 0.383 Sv, respectively, before they left the exclusion zone. Efforts were made to limit doses to Chernobyl liquidators, those who assisted with the cleanup of the Chernobyl power plant, to a total of 0.25 Sv. However, many were exposed to much higher doses as a consequence of their increased proximity to the reactor as well as possible ingestion or inhalation of contamination. Radiation doses to firemen and reactor personnel exposed shortly after the explosion reached as

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portions of their normal life cycles. Documenting the doses received at Chernobyl is an important step in performing empirical studies for observing and noting the responses to exposure to the radiation (Figure 7). There have been no documented instances of radiation-induced genetic variations that have transcended generations in Chernobyl wildlife. While damage to genetic material (DNA strand breakage, micronuclei, chromosomal aberrations) has been shown to be significantly elevated in some species in radioactive environments, the impacts are ephemeral and apparently do not persist in affected lineages. Efforts to document adaptation of individuals to the radioactive environment have also shown no significant results; however, some anecdotal conclusions to the contrary have been reported. The vast literature on wildlife exposed to radiation at Chernobyl contains conclusions ranging from significant individual and population impacts to no measurable effects. Conclusions drawn from the results of field studies should be considered carefully. Readers should consider whether the authors documented absorbed radiation dose and how the dose was calculated and confirmed. Experimental design is extremely important in such studies and although controls may be difficult, researchers should address the confounding factors of naturally occurring geographic and temporal variation in their reporting. There is little doubt that radiation doses immediately following the accident induced measurable genetic damage in exposed wildlife. However, it appears that the biological impact on wildlife species near Chernobyl has been subtle and difficult to document at either the individual or the population level. Although present-day radiation doses experienced by wildlife species in limited areas near Chernobyl are unprecedented, they are nevertheless well below those documented to cause substantial genetic damage and are not sufficient to limit population growth. Lifetime doses in all but a very narrow tract of the Western Trace are below 0.1 Sv and are thereby classified as low-dose radiation. In fact, population recovery has likely been hastened by the evacuation of 135 000 people from the exclusion zone. Ecological recovery of habitats previously used as pasturelands and orchards has led to a rebound of many species to levels higher than before the accident. The Chernobyl exclusion zone is now being used in conservation efforts to recover the endangered Prezwalski’s horse and European bison. Populations of these species have been released into the zone and are free from interference from excessive human activity.

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Figure 7 Graphics showing the radiation dose rates (in 2005) in the Western Plume (A and B) and in the Northern Plume (C and D). From Chesser, R.K., Rodgers, B.E., 2008. Near-field particle dynamics and empirical fallout patterns in Chernobyl’s Western and Northern Plumes. Atmospheric Environment 42: 5124–5139.

Lessons from the Chernobyl Accident In the aftermath of the Chernobyl accident there have been many valuable refinements to the design and operation of nuclear reactors. Refinements of operating power, the number and design of control elements, inclusion of automated emergency procedures, and enhanced training of operators have improved the safety of nuclear reactors. The RBMK-type reactor, although still in operation in some areas of the Russian Federation and Eastern Europe, is considered to be unstable due to its inability to withstand loss of cooling water and general lack of sufficient containment structures. It was clear after Chernobyl that there was insufficient planning for a nuclear emergency involving substantial releases of radiation that could affect human populations. The 45 000 citizens of the city of Pripyat, Ukraine, were spared massive doses of radiation only by the fortuitous directions of the wind during the 37 h prior to evacuation. Because radioactive particles and aerosols of volatile elements may flow for very long distances from the source, it is imperative that communications of releases be accurately and immediately transmitted across political borders to permit protective actions to be put into place. Prophylaxes, such as potassium iodide and cesium chloride tablets, should be immediately available to potentially affected communities. The studies at Chernobyl show that human and environmental health is affected predominantly during the first few days/weeks after a major radiation release by a reactor breach. Rapid decay of short-lived radionuclides, while producing large radiation doses over a short time period, will decay rapidly enough to sufficiently reduce exposure risks in the long term. At Chernobyl, even the most contaminated habitats began to recover most of their ecological diversity within a few years after the accident.

See also: Aerosols; Apoptosis; Carcinogenesis; Cesium; Chromosome aberrations; Ecotoxicology; Emergency response and preparedness; Genetic toxicology; Hormesis; Iodine; LD50/LC50 (lethal dosage 50/lethal concentration 50); Plutonium; Radiation Toxicology, Ionizing and Nonionizing; Risk Characterization; Strontium; Three Mile Island; Uranium.

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Further Reading Cardis E (2003) Reconstruction of doses for Chernobyl liquidators. OECD Papers 3(1) OECD, 2003. Chesser RK, Rodgers BE, Wickliffe JK, et al. (2001) Accumulation of 137cesium and 90strontium from abiotic and biotic sources in rodents at Chornobyl. Ukraine. Environ. Toxicol. Chem. 20: 1927–1935. Chesser RK, Sugg DW, DeWoody AJ, et al. (2000) Concentrations and dose rate estimates of 134,137cesium and 90strontium in small mammals at Chornobyl. Ukraine. Environ. Toxicol. Chem. 19: 305–312. Chesser RK and Baker RJ (2006) Growing up with Chernobyl. Am. Sci. 94: 542–549. Chesser RK and Rodgers BE (2008) Near-field particle dynamics and empirical fallout patterns in Chernobyl’s western and northern plumes. Atmos. Environ. 42: 5124–5139. Chumak VV, Likhtarjov IA, and Riepin VS (1996) Irradiation doses of evacuated population. In: Baryakhtar VG (ed.) Chornobyl Catastrophe. 1997 National Academy of Sciences of Ukraine, pp. 422–424. Kiev, Ukraine: Kyiv Editorial House. Howell EK, Gaschak SP, Griffith KDW, and Rodgers BE (2011) The effects of environmental low-dose irradiation on tolerance to chemotherapeutic agents. Environ. Toxicol. Chem. 30(3): 640–649. Imanaka T and Koide H (2000) Assessment of external dose to inhabitants evacuated from the 30-km zone soon after the Chernobyl accident. Radiatsionnaya biologiya. Radioecologiya 20(5). Kashparov VA, Lundin SM, Zvarych SI, Yoshchenko VI, Levchuk SE, Khomutinin YV, Maloshtan IM, and Protsak VP (2003) Territory contamination with the radionuclides representing the fuel component of Chernobyl fallout. Sci. Total Environ. 317(1–3): 105–119. Likhtarev IA, Chumack VV, and Repin VS (1994) Retrospective reconstruction of individual and collective external gamma doses of population evacuated after the Chernobyl accident. Health Phys. 66: 643–652. Rodgers BE and Holmes KM (2008) Radio-adaptive response to environmental exposures at Chernobyl. Dose Response 6(2): 209–221. Rodgers BE, Baker RJ, et al. (2000) Frequencies of micronuclei in bank voles from zones of high radiation at Chornobyl. Ukraine. Environ. Toxicol. Chem. 19: 1644–1649. Saenko V, Ivanov V, Tsyb A, Bogdanova T, Tronko M, Demidchik Y, and Yamashita S (2011) The Chernobyl accident and its consequences. Clin. Oncol. 23(4): 234–243. Talerko N (2005) Reconstruction of (131) I radioactive contamination in Ukraine caused by the Chernobyl accident using atmospheric transport modelling. J. Environ. Radioact. 84(3): 343–362. UNDP and UNICEF, 2002. The Human Consequences of the Chernobyl Nuclear Accident. UNSCEAR 2008 Report. Volume II. Effects of Ionizing Radiation. United Nations, Vienna, Austria. Wickliffe JK, Bickham AM, Rodgers BE, et al. (2003) Exposure to chronic, low-dose rate gamma-radiation at Chernobyl does not induce point mutations in big blue mice. Environ. Mol. Mutagen. 42: 11–18.

Relevant Websites http://lowdose.energy.gov :US Dept. of Energy: Low Dose Radiation Research Program. http://www.bellona.org/articles/articles_2011/norwegian_estimates :Bellona: New Norwegian report says Fukushima radiation releases twice initial estimates. http://www.iaea.org/Publications/Booklets/Chernobyl/chernobyl.pdf :International Atomic Energy Agency. http://www.unscear.org/unscear/en/publications/2008_2.html :United Nations Scientific Committee on the Effects of Atomic Radiation. http://www.un.org/ha/chernobyl/docs/report.pdf :United Nations. http://www.who.int/ionizing_radiation/chernobyl/en/ :World Health Organization.

Children’s Environmental Health Kristie Trousdale, Children’s Environmental Health Network, Washington, DC, United States © 2024 Elsevier Inc. All rights reserved. This is an update of P.J.E. Quintana, Children’s Environmental Health, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 830–832, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00801-0.

Introduction Children’s risk of exposures to toxicants Inhalation Ingestion Dermal absorption Children’s environments Prenatal Homes Child care K-12 schools Workplaces Children’s vulnerability to harm from environmental exposures Environmental hazards of significant relevance to children’s health Lead Pesticides Air pollution Endocrine-disrupting chemicals Federal efforts to protect children Institutionalization of children’s environmental health Children’s environmental health data Children’s environmental health research and translation Protecting children from environmental hazards Conclusion References Further reading

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Abstract Children are uniquely vulnerable to environmental health hazards. They experience increased exposures to certain toxicants due to their size and behaviors such as oral exploration. Children’s increased respiration rate, ingestion intake, and activity levels can also increase exposures, and their physiological immaturity may result in higher absorbed doses. Young children lack refined awareness of environmental hazards and the agency to mitigate risk. When exposed during critical windows of development, children may suffer irreversible harm. Toxicants of concern include lead, pesticides, air pollutants, and endocrine-disrupting chemicals. Social determinants of health and climate change contribute to and compound environmental risks to children.

Keywords Air pollution; Child care; Children; Climate change; Endocrine-disrupting chemicals; Environmental justice; Health disparities; Housing; Infants; Lead; Ozone; Particulate matter; Pesticides; Risk assessment; Schools

Key points

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Children have increased exposures to certain toxicants due to physical, physiological, and behavioral characteristics and circumstances unique to their lifestages. Children are at increased risk of harm from exposures to toxicants because their bodies and organ systems are still developing. Major toxicants of concern include lead, pesticides, air pollution, and endocrine-disrupting chemicals. Protecting children’s environmental health requires a systems approach and a paradigm shift that centers the precautionary principle and environmental justice.

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Introduction Children are uniquely susceptible to environmental toxicants. Their distinctive physical, cognitive, physiological, and behavioral traits can increase exposure and absorption risks (Landrigan and Etzel, 2014; Agency for Toxic Substances and Disease Registry (ATSDR), 2022; U.S. Environmental Protection Agency (EPA), 2022a, 2022b, 2022c, 2022d, 2022e, 2022f, 2022g, 2022h, 2022i, 2022j, 2022k, 2022l). Circumstances based on their different lifestages—including the environments where they spend significant amounts of time—result in different patterns of exposure than experienced by adults. In addition, children are at greater risk of harm from toxic exposures because, from conception through adolescence, their bodies are growing and developing, sometimes at rapid rates. The disruption of critical windows of development of organ systems, including the nervous, immune, respiratory, digestive, and reproductive systems, can lead to severe and life-long adverse outcomes (Agency for Toxic Substances and Disease Registry (ATSDR), 2013; Agency for Toxic Substances and Disease Registry (ATSDR), 2020b). Children also have many years of life ahead of them to accumulate a higher body burden of toxicants from chronic exposures. This places them at greater risk of developing diseases with long latency periods such as cancers or Parkinson’s Disease (Agency for Toxic Substances and Disease Registry (ATSDR), 2013). This article explores these issues, highlights some of the most concerning toxicants to children’s health and development, and provides information on efforts spanning research, policy, and practice that have been working to address these issues for improved child health outcomes.

Children’s risk of exposures to toxicants Children can experience greater exposures to toxicants because their bodies’ defenses may be under-developed at the time of exposure. For instance, in developing fetuses and newborns the immature blood-brain barrier may not adequately protect the child’s brain from toxicants circulating in the blood. Their ability to metabolize, deactivate, and excrete certain toxicants may not be as efficient or rapid as that of an adult, and their immature immune systems may not react as quickly to neutralize or remove a foreign harmful substance in the body (Etzel and Balk, 2019). The youngest children lack a refined awareness about environmental hazards and about strategies to protect themselves from these hazards. For example, they lack the power and agency to mandate the adoption of safer household pest control strategies or practices to reduce a parent’s toxicant take-home exposure from the workplace. These and other characteristics, behaviors, and dependence-related circumstances influence children’s patterns and routes (inhalation, ingestion, and dermal and eye absorption) of exposure.

Inhalation Relative to body mass, a child breathes more and thus, is exposed to, a greater dose of air pollutants than an adult. Newborns and infants have immature lungs, weaker respiratory muscles, smaller airways, and a higher basal oxygen demand. During the first year of life, they have a median respiratory rate of 44 respirations per minute. This rate continues to drop throughout childhood, reaching a rate of 12–20 respirations per minute by adulthood (Chourpiliadis and Bhardwaj, 2022). Infants and young children also have lower breathing zones than do adults due to their shorter stature and propensity to crawl, sit, and play on the ground. Some toxicants that are heavier than air, such as mercury vapor, accumulate at higher levels closer to the ground, and others, such as motor vehicle exhaust, are emitted within a young child’s breathing zone. Resuspension of dust near the ground can also be a source of inhalation exposure to contaminant particles. Behaviorally, school-aged children are more likely to spend time outdoors in active play and sports, increasing their respiratory rate and their risk of ambient air pollution inhalation. Research from the University of Southern California found a significant association between playing multiple sports in areas with high ozone levels and the likelihood of developing childhood asthma (McConnell et al., 2002). In addition, the university’s Children’s Health Study found that improved air quality over time-especially lower levels of nitrogen dioxide and particulate matter-was associated with children’s improved lung growth and functioning (Gauderman et al., 2015).

Ingestion Children are growing, and their higher metabolic needs per unit of body weight means that pound for pound, children eat and drink more than adults, which increases their exposure to food and water contaminants. Infants and young children also crawl, roll, sit, and play on the ground and exhibit hand-to-mouth behaviors typical of oral exploration, which can increase their ingestion of toxicants found in soil and that accumulate in dust and settle into carpets and on flooring, such as lead and pesticides. Children also absorb some ingested toxicants differently than adults, which can increase their relative dose. For example, children’s growing bodies have higher calcium needs, which results in increased intestinal absorption of the mineral. Because lead can mimic calcium in the body, it can be absorbed in its place in the gastrointestinal tract. Given similar levels of lead ingestion in a child and an adult, the former will absorb more of the lead. Adults absorb approximately 3–10% of ingested inorganic lead, whereas children absorb approximately 40–50%, after a meal (Agency for Toxic Substances and Disease Registry (ATSDR), 2020a, Agency for

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Toxic Substances and Disease Registry (ATSDR), 2020b). Children who are malnourished, and whose diet is especially lacking in iron and calcium, have increased absorption of ingested lead. Young children with very limited diets also have an elevated exposure risk to some contaminants. For example, infants fed primarily rice cereal will ingest proportionately higher levels of arsenic (a contaminant readily taken up by rice) than will an adult with a more diverse diet. In addition, breast milk and infant formula are potential sources of toxicant exposures unique to newborns and infants. In 2008, a scandal involving intentional melamine contamination of infant formula in China resulted in infant nephrological harm and even death (Wen et al., 2016). In February 2022, the largest manufacturer of infant formula in the U.S. closed one of its facilities, and product recalls were issued, due to bacterial contamination, which resulted in 4 infant illnesses and 2 deaths (Centers for Disease Control and Prevention (CDC), 2022a, 2022b, 2022c, 2022d, 2022e). The water used to mix and prepare formula introduces another potential route of contamination, especially for children in families who are reliant on drinking water from private and unregulated wells or whose municipally-supplied drinking water has elevated levels of biological or chemical contaminants. Maternal exposures can influence that of their children postnatally. Lipid-soluble pollutants can pass from the mother to the nursing newborn or infant through breast milk. For most newborns and infants, the proven benefits of breastmilk outweigh any possible harm from this exposure, but select circumstances may warrant more guidance (CDC). Assessment of contaminants in breast milk can be used as a population-level indicator of exposure, but additional research is needed to better characterize infants’ exposures from breast milk and the associated health risks (Bernasconi et al., 2022; Lehmann et al., 2018).

Dermal absorption Dermal contact with certain substances can result in skin irritation or even damage, and some toxic compounds can cross the skin and be absorbed into the bloodstream and transported to other areas of the body. Newborns and infants have greater skin surface area to body mass ratios compared with adults; their skin is also thinner, with relatively high permeability. Thus, a newborn will absorb a higher dose of toxicant from dermal contact per kilogram of body weight than an adult will (Etzel and Balk, 2019). Children’s dermal exposures result from crawling and playing on contaminated surfaces, contact with baby and children’s products, and topical application of personal care products, such as diapering lotions, shampoos, sunscreen, and other common products. Children exhibit considerable hand-to-face behaviors, including eye rubbing, which can result in absorption of contaminants through the eyes as well.

Children’s environments Prenatal Many toxicants can pass from a mother to her fetus through the placenta, including lipid-soluble compounds such as organophosphate pesticides and elements such as lead and mercury. Researchers at the University of San Francisco analyzed matched maternal and cord blood samples and identified 109 chemicals in pregnant women, 55 of which had never been previously reported in people, and 42 of which were of unknown sources and uses. The chemicals of known origin and uses included pesticides, flame retardants, polyfluoroalkyl substances (PFAS), and other chemicals used in common consumer products such as food packaging, cosmetics, and furnishings. Many of these chemicals were detected in both the mothers and the newborns, indicating placental transfer (Wang et al., 2021). The most sensitive organogenesis occurs during fetal development and the perinatal period, as is described more fully in the following section, “Children’s vulnerability to harm from environmental exposures”. There is also increasing interest among researchers in both maternal and paternal preconception exposures, heritable epigenetic effects, and their potential impacts to the health of future offspring (Montjean et al., 2022).

Homes Approximately 45 million metropolitan homes in the U.S. have one or more health and safety hazards, such as water leaks, problems with heating, peeling paint, and signs of rodents (National Center for Healthy Housing (NCHH), 2020). This problem disproportionately affects people and families of color. Black Americans are nearly twice as likely to live in unsafe or unhealthy homes compared with the general population (U.S. Census Bureau, 2019). Newborns and infants spend most of their time in the home, and prolonged periods within a relatively narrow range of living space, such as in a crib, due to limited mobility. If a newborn’s nursery has dampness and mold, poor air filtration and ventilation, or other hazards, that newborn is likely exposed to the hazard for most of the day, every day, during a vulnerable period of development. Infants also spend a great deal of time on the floor as they gradually acquire mobility, putting them into significant contact with any hazardous chemicals in the carpeting or flooring materials or with toxicants present in settled dust such as lead-contaminated dust. The National Center for Healthy Housing, in partnership with the American Public Health Association, developed the Healthy Housing Standard, a resource that provides minimum performance standards in housing and building codes for safe and healthy homes. The health-based standards can be adapted for use in local ordinances, regulations, and other housing-related policies (National Center for Healthy Housing (NCHH) and American Public Health Association (APHA), 2014). In addition to poorer housing quality, families of color are more likely than white families to live in closer proximity to environmental hazards such as polluting industrial facilities and hazardous waste sites due to segregation borne of racist housing and land use policies.

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Child care Millions of children under age 6 attend home-based and center-based child care in the U.S., many of whom are in attendance 40+ hours a week (Agency for Toxic Substances and Disease Registry (ATSDR), 2017). Common environmental hazards in and around center-based child care facilities include pesticide residues, lead paint, dust, and soil hazards, and volatile organic compounds (Viet et al., 2013; Quirós-Alcalá et al., 2016), and programs serving children of color are more likely to be located in closer proximity to polluting industrial facilities, hazardous waste sites, or high-traffic roadways. Child care licensing is primarily handled at the state level, and environmental health considerations beyond basic smoking restrictions, lead-based paint hazard reduction, and proper storage of toxic chemicals are not explicitly or thoroughly addressed in most states’ licensing regulations (Environmental Law Institute (ELI), 2015). The American Academy of Pediatrics, the American Public Health Association, and the National Resource Center for Health and Safety in Child Care and Early Education published a special collection of environmental health best practices for child care (American Academy of Pediatrics (AAP), American Public Health Association (APHA), National Resource Center for Health and Safety in Child Care and Early Education (NRC), 2019). Improper siting of a child care facility can expose the children in the program to legacy contaminants or current pollution in or adjacent to the structure. In 2006 a child care center in Gloucester County, New Jersey closed down upon discovery of very high levels of mercury vapor in the facility. The center was located in a former mercury thermometer manufacturing plant that had never been decontaminated, and a third of the children tested had abnormally elevated mercury levels in their bodies (New Jersey Department of Health (NJ DOH), 2017). The Agency for Toxic Substances and Disease Registry (ATSDR) established the Choose Safe Places for Early Care and Education program in 2017 to provide resources and support to states to assist with the development of safe siting action plans. In 2020, ATSDR released the Disaster Recovery Supplement to provide guidance on reducing environmental hazards to children in child care programs post-disaster.

K-12 schools Children are legally mandated to attend schools in the U.S. and spend approximately 15,600 h at school over the course of their elementary and secondary education (Eitland et al., 2017). Many school buildings throughout the country are old and in disrepair, especially those in rural or under-resourced areas and in communities of color (American Society of Civil Engineers (ASCE), 2021). Approximately 41% of U.S. schools need new or updated heating, ventilation, and air conditioning (HVAC) systems. The importance of these systems in maintaining safe and healthy indoor air quality has been underscored by the highly virulent airborne transmission of COVID-19. In addition, the ailing infrastructure of many schools present unabated legacy hazards, including lead, asbestos, and polychlorinated biphenyls (PCBs). Many schools are located in close proximity to Superfund sites, major roadways, industrial facilities, and agricultural fields where industrial pesticides are applied. These education settings are also of relevance to pre-conception and prenatal exposures, as the majority of teachers, and school and child care staff, are women-many of whom are of reproductive age. Moreover, school facilities serve as community hubs. They are often used as shelters or gathering places during crises, as well as for additional activities, including Early Head Start and Head Start programs, before-and-after-school child care programs, county sports programs, club meetings, and other activities outside of school hours. EPA’s Healthy School Environments provides assessment tools, training webinars, guidelines and best practices, checklists, a model program, and more resources to help schools establish and enhance healthy environments (EPA).

Workplaces Approximately 35% of U.S. children aged 16–19 are part of the workforce (Hamilton Project and Brookings Institution, 2019). Adolescent jobs are varied; common opportunities include work in retail and food establishments, lifeguarding, and self-employed landscaping. Outside of physical hazards, such as extreme heat or other weather concerns, injury, and noise, chemical hazards in these occupational settings can include pesticides, solvents, cleaning and disinfecting products, and phthalates. Of particular concern for the adolescent worker are exposures to carcinogens, teratogens, and endocrine-disrupting chemicals. Teens have more years of life ahead of them within which to develop environmental diseases, such as cancers, after long latency periods. Acute exposures to reproductive toxicants can have adverse effects on adolescents who are pregnant, and chronic exposures can accumulate over time to impact their reproductive health and future offspring. As many as 500,000 children work in agriculture in the U.S., where the minimum age requirement to work is 12 years (Association of Farmworker Opportunity Programs (AFOP), 2007). For farms employing under 7 people per year, children younger than 12 years of age can conduct farm work, and studies have found children as young as 7 years at work in agriculture (Human Rights Watch, 2014). Many of these children are from marginalized backgrounds. They are more likely to be children of color, migrants, living in poverty, linguistically isolated, and undocumented. They can face exposure to pesticides directly from their own work, from para-occupational exposures, from pesticide drift and volatilization, from food and drinking water, and from residential pesticide use. Behavioral differences factor into children’s occupational risks as well. Due to the still- developing brain and reasoning abilities of youth, older children and adolescents are more likely to take risks and less likely than adults to adhere to occupational safety measures, such as wearing personal protective equipment (Salazar et al., 2004; Wickman et al., 2008).

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Children’s vulnerability to harm from environmental exposures Greater harm can befall a child exposed to the same level of toxicant exposure as an adult because the child’s body and organ systems are still developing. There are especially critical windows of development during the prenatal and perinatal periods, which if disrupted, could lead to life-long adverse health or developmental outcomes. Two organ system examples are provided below. Prenatal neurotoxicant exposures during weeks 3 through 8 of gestation can result in major morphological abnormalities of the central nervous system, such as neural tube defects. When exposed during weeks 20–36, the child is more likely to develop minor congenital defects or functional deficits, such as learning disabilities (U.S. Environmental Protection Agency (EPA), 2022h). While these earliest windows of development are critical, it is important to note that biological and functional brain development continues throughout childhood and into early adulthood. Children’s developing respiratory systems are also sensitive to toxicant exposure. Significant lung development begins in the womb, and the full maturation process continues into adolescence. Children are born with fewer alveoli or air sacs in their lungs, compared with adults. The air sacs not only proliferate throughout early childhood, but they, along with the lungs and airways, continue to grow into the teen years, allowing for increased lung volume and capacity (American Academy of Pediatrics Council on Environmental Health, 2004). Furthermore, as previously mentioned, children have many years of life ahead of them to accumulate greater body burdens of toxicants, which presents increased risk for diseases. They also have a higher likelihood of developing diseases resulting from early exposures that have long induction and latency periods.

Environmental hazards of significant relevance to children’s health Lead Lead is a naturally occurring metallic element used in a wide variety of applications and products. Health and medical experts have known for over a century about lead’s neurotoxic effects, and there is no level of exposure considered safe. Even at very low levels it can impair a child’s cognitive development, with lasting effects, including reduced IQ and problems with attention and behavior. Chronic exposure contributes to kidney and heart disease in adulthood. Pregnant individuals exposed to very high levels of lead have greater risk of miscarriage, stillbirth, preterm birth, and low birth weight. At very high levels, lead can cause seizures, coma, and death. Children are exposed through ingesting lead in dust, soil, and drinking water. The primary source of exposure is lead-based paint hazards, especially in homes built before 1978 (the year lead-based paint was banned for residential purposes). The Centers for Disease Control and Prevention (CDC) estimate that 2.6 million homes with young children in the U.S. have lead-based paint hazards. Another important source of exposure is lead in drinking water, which can leach from drinking water service lines and indoor plumbing fixtures made from lead. The risk from lead in drinking water is of highest concern for prenatal and infant exposure. Infants who are fed powdered formula mixed with tap water can receive 40–60% of their lead exposure from drinking water, compared with approximately 20% for a person on a more diverse diet (EPA). In 2014 it came to light that the municipal drinking water in Flint, Michigan had levels of lead that exceeded U.S. Environmental Protection Agency (EPA) standards, and higher body burdens of lead in the city’s children. Flint’s water distribution system included thousands of aging lead service lines into residents’ homes. The city had switched to a water source with highly corrosive water and failed to take the necessary steps to prevent lead from leaching from the pipes into the water (Kennedy, 2016). The resulting crisis in Flint illuminated the fact that up to 10 million homes in communities across the country receive drinking water via lead service lines, and many communities struggle with elevated lead levels. The federal Infrastructure Investment and Jobs Act of 2021 directed an unprecedented $15 billion to states for lead service line replacement—one component of the Biden Administration’s Lead Pipe and Paint Action Plan (The White House, 2021a; The White House, 2021b). Additional sources of lead exposure include industrial air emissions, aviation gas, and lead in baby food and juices, some imported ceramic cookware and pottery, personal care products, inexpensive metal jewelry, and old or antique toys. In 2021 CDC lowered the blood lead reference value (BLRV) from 5 mg/dL to 3.5 mg/dL (Centers for Disease Control and Prevention (CDC), 2022b). This BLRV is based on the 97.5th percentile of blood lead levels (BLLs) in children ages 1–5 years, meaning that 2.5% of these children have BLLs at or above 3.5 mg/dL. The value is intended for surveillance and to target public health interventions, not to imply BLLs below 3.5 mg/dL are safe. The CDC estimates that 500,000 children ages 1 to 5 in the U.S. have elevated levels of blood lead. However, many children are not tested, and some states do not regularly report testing results to the CDC. One study estimated that the number of impacted children is closer to 1.2 million.

Pesticides Pesticides encompass a wide range of compounds designed to kill, repel, or impede unwanted “pests”; they include insecticides, herbicides, fungicides, rodenticides, and antimicrobials, and they are ubiquitous in indoor and outdoor environments where children spend time. Children are exposed to pesticides via multiple sources, including residues on food, in drinking water, dust, soil, household surfaces, spray drift, older wooden structures treated with wood preservatives, pet flea treatments, insect repellents,

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and head lice treatments. They are exposed through inhalation, ingestion, and dermal routes, and they may not be able to metabolize and excrete pesticides as effectively or quickly as adults. Research has demonstrated that newborns and young children have lower paraoxonase (PON1) activity than adults (Huen et al., 2009). PON1 is the enzyme that detoxifies organophosphate pesticides. Over 31,000 calls were made to U.S. Poison Control Centers about pesticide exposure concerns for children 5 years of age and under in 2020 (Gummin et al., 2021). Acute exposures to pesticides can cause severe illness and even death in young children. Children’s chronic exposure, including prenatal exposure, to pesticides are also associated with adverse health outcomes. During the prenatal period, both residential proximity to agricultural pesticide applications and parental indoor insecticide use have been linked with increased risk for childhood leukemia (Chen et al., 2015; Park et al., 2020), and organophosphate and organochlorine pesticides are associated with neurodevelopmental and behavioral problems (Bennett et al., 2016; Eskenazi, 2008). Despite most organochlorines being banned and no longer used in the U.S., the compounds persist in the environment, and children can be exposed through food, dust, and soil. Additional impacts linked to pesticide exposure include: adverse birth outcomes such as low birth weight, preterm birth, and birth defects; and risk for developing asthma, and exacerbation of existing asthma (American Academy of Pediatrics Council on Environmental Health, 2012). Under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), manufacturers must register pesticides not considered “minimum risk” pesticides with the EPA. The Agency includes instructions on storage, use, and disposal in registered products’ labeling; use deviating from the directions is illegal. However, misuse of pesticides is a common occurrence, and incidents can occur even when following labeling instructions. The Food Quality Protection Act (FQPA) required that the EPA default to an added uncertainty factor—a 10-fold margin of safety—when setting values of acceptable pesticide levels in food residues, to account for the unique vulnerabilities of infants and children. However, even this is not always protective enough nor applied in practice (Naidenko, 2020). In 2021 the EPA issued a final rule to ban the use of chlorpyrifos, a neurotoxic organophosphate pesticide from use on food crops, after many years of review, pressure from health advocates, and after several states initiated their own bans (U.S. Environmental Protection Agency (EPA), 2022a; U.S. Environmental Protection Agency (EPA), 2022f). A number of other organophosphate pesticides remain in use, and the agency is currently reviewing their safety.

Air pollution Outdoor air pollution includes six common criteria pollutants associated with adverse health outcomes—ground-level ozone, particle pollution (i.e., particulate matter), nitrogen oxides, sulfur oxides, carbon monoxide, and lead—and 188 hazardous air pollutants (HAPs) including benzene, mercury, and dioxin. These pollutants are associated with preterm birth, adverse respiratory and neurodevelopmental impacts, and increased risk of cancer and reproductive health problems (EPA). Both stationary sources, such as coal-fired power plants, waste incinerators, dry cleaners, and smelters, and mobile sources, such as vehicles, contribute to outdoor air pollution. Since EPA began regulating ambient air pollution under the Clean Air Act of 1970, air quality in the U.S. has significantly improved overall. However, approximately 31 million American children live in counties where ozone and/or particle pollution levels exceed national standards (American Lung Association (ALA), 2022). Families of color and those with lower incomes disproportionately live in communities with higher levels of air pollutants (Jbaily et al., 2022). Outdoor air infiltrates indoors, where the pollutants it introduces can accumulate at high levels along with existing indoor air contaminants. In fact, indoor air quality can be 2–5 times more polluted than outdoor air (U.S. Environmental Protection Agency (EPA), 1987). This is of particular relevance because children in the U.S. spend an estimated 80–90% of their time inside (Etzel and Balk, 2019). Indoor air contaminants include environmental tobacco smoke, carbon monoxide, and radon. In addition, gas stoves and wood stoves can produce unhealthy levels of particulate matter, nitrogen dioxide, and formaldehyde in homes, and water incursion problems and dampness lead to increased mold and airborne mold spores. Other sources of indoor air pollution include use of pesticide sprays and foggers, household cleaners and aerosols, and common building materials and furnishings—such as flooring materials or upholstery that off-gas volatile organic compounds. These contaminants can build up to hazardous levels, especially in homes and buildings with poor ventilation and filtration systems and practices.

Endocrine-disrupting chemicals There are over 86,000 industrial chemicals registered for use in the U.S. marketplace (EPA). Many are synthetic and used in common consumer products such as furnishings, cookware, clothing, personal care products, toys, and baby products. An estimated 9.5 trillion pounds of over 40,000 of these chemicals are produced each year (Koman et al., 2019). An analysis of 19 countries and regions indicates that over 350,000 chemicals and chemical mixtures are registered for production and use globally (Wang et al., 2020). Over the life cycle of manufacturing, use, and disposal, these compounds are widely dispersed into the environment, where many persist for decades, and some are ubiquitous in the bodies of most humans. Several classes of these chemicals, particularly the persistent organic pollutants, are endocrine disruptors. These include bisphenol A (BPA), brominated and chlorinated flame retardants, fluorinated organic compounds such as per- and polyfluoroalkyl substances (PFAS), organochlorine pesticides, and PCBs. Exposure to endocrine-disrupting chemicals (EDCs) are associated with a number of health and developmental endpoints, including low birth weight and preterm birth, male reproductive health abnormalities, precocious puberty, adverse neurodevelopmental outcomes, diabetes, and cancers (Meeker, 2012).

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EPA has developed several guidance documents specific to assessing risks to children from environmental hazards, including Guidance on Selecting Age Groups for Monitoring and Assessing Childhood Exposures to Environmental Contaminants (U.S. Environmental Protection Agency EPA, 2005), A Framework for Assessing Health Risk of Environmental Exposures to Children (U.S. Environmental Protection Agency EPA, 2006), and Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens U.S. Environmental Protection Agency (EPA), 2005. They are meant to account for children’s unique susceptibility at various life stages. For example, EPA recommends the use of age dependent adjustment factors in cancer risk assessments of carcinogens acting through a mutagenic mode of action (U.S. Environmental Protection Agency (EPA), 2005). A factor of 10 is used for newborns and infants less than 2 years old, a factor of 3 is used for children aged 2 to less than 16, and no factor is used for people who are age 16 or older. EPA’s guidance has no legally binding effect on the agency or any other regulatory entity. In addition, the agency’s guidance and current risk assessment methods are outdated and do not adequately account for variability and uncertainty. The Program on Reproductive Health and the Environment at the University of California at San Francisco coordinated a team of scientific experts who published a 5-article series with recommendations for updating chemical risk assessments to improve decision-making and protect public health (Maffini et al., 2023; Nielsen et al., 2023; Vandenberg et al., 2023; Varshavsky et al., 2023; Woodruff et al., 2023). The 1976 Toxic Substances Control Act (TSCA) is the legal mandate to EPA to regulate the production, importation, use, and disposal of chemical substances other than pesticides, food, drugs, cosmetics, and some others (U.S. Environmental Protection Agency (EPA), 2022i). It has proven ineffective; since the law was enacted, a very small percentage of chemicals in the marketplace have been tested for safety to children’s health, and only a handful of those have been partially regulated (Koman et al., 2019). The 2016 Frank R. Lautenberg Chemical Safety for the 21st Century Act was the first significant update to TSCA (U.S. Environmental Protection Agency (EPA), 2022j). The legislation strives to close some critical loopholes in TSCA and explicitly requires the consideration and protection of pregnant individuals and children when implementing chemical safety policy. While it does not require the use of a 10-fold uncertainty factor similar to the FQPA to protect infants and children, EPA was given increased authority to require the testing of new chemicals prior to their introduction to the marketplace and to ban or limit the use of chemicals that pose risks to human health (EPA). However, based on the testing schedule adopted by the agency, it will take decades or even centuries for EPA to review and possibly regulate the thousands of chemicals that are currently in use in addition to newly introduced chemicals. States lead the way in enacting more protective chemical safety policy, and state-level actions have in numerous cases, had national impacts. As of July 2022, over 280 laws that ban or restrict certain toxicants have been adopted in 38 states (Safer States, n.d.). One noteworthy example is the Safer Products for Washington Act of 2019, which restricted 4 chemical classes in 10 product categories, including certain uses of bisphenols, PFAS, phthalates, and other EDCs. Washington state regulators banned entire chemical classes in order to prevent instances of regrettable substitution, whereby manufacturers replace a hazardous chemical with one from the same class that is subsequently found to be equally or more hazardous. Some states have passed legislation specific to children’s health concerns, such as New York’s 2019 law regulating chemicals in children’s products and Vermont’s 2019 law strengthening and expanding its existing chemical disclosure program for children’s products. Each year, California publishes an updated list of toxic chemicals regulated under the state’s 1986 law, Proposition 65; as of February 2022, over 1000 chemicals are on the list (California Office of Environmental Health Hazard Assessment (CA OEHHA), 2022). The law strives to protect residents from these chemicals in drinking water and consumer products through prohibited discharge and required warnings, respectively.

Federal efforts to protect children Institutionalization of children’s environmental health President Clinton issued Executive Order (EO) 13,045: Protection of Children from Environmental Health Risks and Safety Risks in 1997. One of the EO requirements was the creation of the President’s Task Force on Environmental Health Risks and Safety Risks to Children (PTF), an inter-agency body charged with coordinating the federal government’s efforts to protect children’s environmental health. Also in 1997, the EPA established its Office of Children’s Health Protection (OCHP), tasked with ensuring that children’s unique vulnerabilities and needs are considered throughout the agency’s work. The Director of OCHP and a senior staff member of the U.S. Department of Health and Human Services co-chair the Steering Committee of the PTF. The EPA reaffirmed its commitment to protecting children’s health in a 2021 release of the agency’s updated policy specific to children. In addition, EPA identified children’s health as one of 4 cross-agency strategies to guide the implementation of its FY 2022–2026 strategic plan.

Children’s environmental health data EPA’s America’s Children and the Environment (ACE) report is a collection of national children’s environmental health indicators (environmental, biomonitoring, and health outcomes) first published in 2000. The second and third editions were published in 2003 and 2013, and a selection of the indicators were updated and released in a special report in 2019. The ACE report can be used to identify trends in children’s environmental health and track the effectiveness of EPA’s policies and decisions. State and local data informs more targeted research and policies. The Centers for Disease Control and Prevention (CDC) launched the online information and visualization platform—the Environmental Public Health Tracking Network—in 2009. It is a

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network of federal, state, and local tracking and surveillance systems. Included are environmental and climate hazards, population biomonitoring data, community design elements, population characteristics—including age, income and race/ethnicity—and health outcomes such as asthma, autism, and cancer. The information has been data linked, standardized, and presented in maps, charts, and tables to better study relationships between the various indicators. Currently 25 states and 1 city are funded by CDC to establish and maintain a tracking program as part of this network. Tracking and surveillance serve to identify areas warranting closer investigation, generate scientific hypotheses, and inform interventions and policy at state and local levels. Some environmental justice screening and visualization tools include children as a vulnerable group. EPA developed a nationally consistent environmental justice screening and mapping tool (EJSCREEN) to improve equitable public health protection and released it to the public in 2015 for stakeholder use. Users can develop reports or maps for a geographic area of 12 environmental justice indexes (combined environmental and demographic indicators) or layer individual indicators, including an indicator for the percentage of children under age 5, on side-by-side maps to identify areas for further investigation or potential prioritization of efforts. In August of 2022, CDC/ATSDR released the Environmental Justice Index, a place-based tool that ranks all U.S. census tracts on 36 environmental burden, social vulnerability, and health vulnerability indicators and assigns each community a single score. It provides decision-makers with information on communities’ cumulative environmental health burden using a health equity lens. One of the social vulnerability indicators included in the tool is the percent of population age 17 or younger.

Children’s environmental health research and translation For over 20 years the EPA and the National Institute of Environmental Health Sciences (NIEHS) partnered to advance research on children’s environmental health via joint funding a network of extramural research centers throughout the U.S. —the NIEHS/EPA Children’s Environmental Health and Disease Prevention Research Centers. EPA discontinued funding the centers in 2019, ending the program, but the contributions of these centers to scientific knowledge about children’s environmental health was significant, and produced insights to inform improved detection, treatment, and prevention of deleterious health outcomes including asthma, cancer, and adverse neurodevelopmental and birth outcomes. NIEHS canceled the ambitious but unwieldy National Children’s Study (NCS) in 2014, but remains committed to children’s environmental health research. The agency has made over 250,000 data and samples collected as part of the NCS initial studies available to researchers as part of its archive. In 2016 it launched a 7-year program called Environmental Influences on Child Health Outcomes which leverages existing longitudinal birth cohorts to collect and standardize environmental, exposure, and health outcome data for over 50,000 children of diverse backgrounds. It also supports intervention studies in under-resourced and rural areas. The birth cohort protocols and harmonized data, in addition to study findings, will enhance continued research. Increased federal investments can address remaining research gaps, such as investigation of cumulative exposures including chemical and non-chemical stressors, elucidation of mechanistic pathways, and the many environmental exposures and health outcome relationships still unexplored or in need of continued study. However, there is also a great deal of knowledge ready to move into practice, policy, and programs to protect children. In 2022 NIEHS launched its Collaborative Centers in Children’s Environmental Health Research and Translation (CEHRT), a network of 6 institutional centers and a coordinating center. One of the CEHRT Network’s primary goals is to translate children’s environmental health research findings into actionable steps such as tools and interventions for relevant stakeholders, especially in the communities most impacted by disproportionate exposures to pollutants. An example of a federal approach to move research into translation is the 1998 creation of the Pediatric Environmental Health Specialty Units (PEHSUs), a national network of experts in the prevention, diagnosis, management, and treatment of children’s environmental health issues funded by EPA and ATSDR. PEHSU experts provide education, training, and outreach to pediatricians, health profession students, and community members about children’s environmental health and ways to protect children from toxicants.

Protecting children from environmental hazards To best safeguard the health and well-being of children and that of future generations, a paradigm shift is needed, in which policymakers and regulators adopt a precautionary approach—focusing on hazard rather than risk reduction. Children’s environments, lifestyles, and exposures are complex and changing rapidly over time. They are exposed to mixtures of environmental agents—including chemical, biological, and physical, and they present with individual susceptibility factors from genetic makeup, nutritional status, presence of biological infection, psychosocial stressors, limited resources, and other considerations. In addition, climate change impacts (e.g., extreme heat events and droughts, increased severity of storms and flooding, increased wildfire activity) magnify environmental hazards through increased air pollution and contaminated ground and surface waters (Helldén et al., 2021). For example, wildfire activity is associated with greater PM2.5 exposure and increased pediatric asthma hospitalizations (Henry et al., 2021), and wildfires are increasing in frequency, duration, size, and severity (Westerling, 2016). Thawing permafrost, disaster-breached industrial plants, and hazardous waste sites release chemical toxicants, which can be transported to other areas via flooding, and higher temperatures may increase the volatilization of persistent organic pollutants (Balbus et al., 2013). Research and current projections underscore the intergenerational inequities of climate change, including

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estimates that children born in 2020 will experience up to 7 times the number of extreme climate change-related events as those born in 1960 (Thiery et al., 2021). These impacts exacerbate environmental injustices and health disparities for children of color and children living in poverty. Meaningful progress in protecting children from climate change impacts and environmental hazards necessitates addressing social determinants of health, including racism and poverty. In 2021 the Biden Administration established the Justice40 initiative, a whole-of-government effort directing 40% of total benefits from federal investments in climate, clean energy, and environmental cleanup to disadvantaged communities. Hundreds of federal programs under multiple agencies are covered under this initiative. EPA’s FY 2022–2026 strategic plan includes a goal centered on advancing environmental justice and civil rights. The agency also developed the E.O. 13,985 Equity Action Plan that identified priority actions needed to achieve the objectives of that goal.

Conclusion Children, especially children of color and children in under resourced communities, are uniquely susceptible to environmental health hazards. They may experience heightened and distinctive exposures to toxicants, and their developing bodies are more likely to suffer lasting harm from these exposures. Climate change impacts increase environmental health hazards and leave the most disadvantaged children even more vulnerable to disease and disability. Enhanced and expanded pediatric tracking and surveillance, robust coordinated research, and cumulative risk assessment frameworks that integrate multiple data streams, climate drivers, and nonchemical factors are needed to inform the development, implementation, and evaluation of policy, regulatory or public health actions to address children’s environmentally-mediated diseases. Where protective policies and regulations are lacking, equitable and accessible community and stakeholder education on actionable steps to reduce children’s exposures to toxicants is critical. Cross-agency and cross-sector coordination and collaboration, including innovative public-private partnerships, are needed at all levels of government, along with the funding and capacity necessary for sustainability. Many working to improve children’s environmental health are taking a systems approach towards transformative change, and addressing the root causes of racism and poverty.

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(2018) Environmental dhemicals in breast milk and formula: Exposure and risk assessment implications. Environmental Health Perspectives 126: 096001. https:// doi.org/10.1289/EHP1953. Maffini MV, Rayasam SDG, Axelrad DA, et al. (2023) Advancing the science on chemical classes. Environmental Health 21(Suppl. 1): 120. https://doi.org/10.1186/s12940-02200919-y. McConnell R, et al. (2002) Asthma in exercising children exposed to ozone: A cohort study. Lancet 359(9304): 386–391. https://doi.org/10.1016/S0140-6736(02)07597-9. Meeker JD (2012) Exposure to environmental endocrine disruptors and child development. Archives of Pediatrics & Adolescent Medicine 166(6): E1–E7. https://doi.org/10.1001/ archpediatrics.2012.241. Montjean D, et al. (2022) Impact of endocrine disruptors upon non-genetic inheritence. International Journal of Molecular Sciences 23(3350). https://doi.org/10.3390/ijms23063350. Naidenko OV (2020) Application of the Food Quality Protection Act children’s health safety factor in the U.S. EPA pesticide risk assessments. Environmental Health 19(16). https://doi. org/10.1186/s12940-020-0571-6. National Center for Healthy Housing (NCHH) (2020) State of Healthy Housing. Available at: https://nchh.org/tools-and-data/data/state-of-healthy-housing/ (Accessed 12 June, 2022). National Center for Healthy Housing (NCHH) and American Public Health Association (APHA) (2014) National healthy housing standard. Available at: https://nchh.org/resource-library/ national-healthy-housing-standard.pdf (Accessed 28 March, 2022). New Jersey Department of Health (NJ DOH) (2017) Kiddie Kollege Mercury Investigation. Available at: https://www.nj.gov/health/ceohs/environmental-occupational/hazardous-wastesites/gloucester/kiddiekollege.shtml (Accessed 12 June, 2022). Nielsen GH, Heiger-Bernays WJ, Levy JI, et al. (2023) Application of probabilistic methods to address variability and uncertainty in estimating risks for non-cancer health effects. Environmental Health 21(Suppl. 1): 129. https://doi.org/10.1186/s12940-022-00918-z. Park A, et al. (2020) Prenatal pesticide exposure and childhood leukemia—A California statewide case-control study. International Journal of Hygiene and Environmental Health 226: 113486. https://doi.org/10.1016/j.ijheh.2020.113486. Quirós-Alcalá L, et al. (2016) Volatile organic compounds and particulate matter in child care facilities in the District of Columbia: Results from a pilot study. Environmental Research 146: 116–124. https://doi.org/10.1016/j.envres.2015.12.005. Safer States (n.d.) States are leading the way to safer chemicals. Available at: https://www.saferstates.org/ (Accessed 12 July, 2022). Salazar MK, et al. (2004) Hispanic adolescent farmworkers’ perceptions associated with pesticide exposure. Western Journal of Nursing Research 26(2): 146–166. The White House (2021a) Justice40: A Whole-of-Government Initiative. Environmental Justice. Available at: https://www.whitehouse.gov/environmentaljustice/justice40/ (Accessed 31 July, 2022). The White House (2021b) Fact Sheet: The Biden-Harris Lead Pipe and Paint Action Plan. Available at: https://www.whitehouse.gov/briefing-room/statements-releases/2021/12/16/ fact-sheet-the-biden-harris-lead-pipe-and-paint-action-plan/ (Accessed 6 July, 2022). Thiery W, et al. (2021) Intergenerational inequities in exposure to climate extremes. Science 374(6564): 158–160. https://doi.org/10.1126/science.abi7339. U.S. Census Bureau (2019) American Housing Survey. Available at: https://www.census.gov/programs-surveys/ahs/data.html (Accessed 12 June, 2022). U.S. Environmental Protection Agency (EPA) (1987) The Total Exposure Assessment Methodology (TEAM) Study: Summary and Analysis. EPA/600/6-87/002a Washington, DC: EPA. U.S. Environmental Protection Agency (EPA) (2005) Supplemental Guidance for Assessing Susceptibility from Early-life Exposure to Carcinogens. EPA 630-R-03-003F. Available at: https://www.epa.gov/sites/production/files/2013-09/documents/childrens_supplement_final.pdf (Accessed 16 January, 2023). U.S. Environmental Protection Agency (EPA) (2022a) About the TSCA Chemical Substance Inventory. TSCA Chemical Substance Inventory. Available at: https://www.epa.gov/tscainventory/about-tsca-chemical-substance-inventory (Accessed 12 July, 2022). U.S. Environmental Protection Agency (EPA) (2022b) America’s Children and the Environment. Available at: https://www.epa.gov/americaschildrenenvironment (Accessed 12 July, 2022). U.S. Environmental Protection Agency (EPA) (2022c) Basic Information about Lead in Drinking Water. Ground Water and Drinking Water. Available at: https://www.epa.gov/groundwater-and-drinking-water/basic-information-about-lead-drinking-water (Accessed 16 January, 2023). U.S. Environmental Protection Agency (EPA) (2022d) EJScreen: Environmental Justice Screening and Mapping Tool. Available at: https://www.epa.gov/ejscreen (Accessed 12 July, 2022). U.S. Environmental Protection Agency (EPA) (2022e) Environments and Contaminants—Hazardous Air Pollutants. America’s Children and the Environment (ACE). Available at: https:// www.epa.gov/americaschildrenenvironment/environments-and-contaminants-hazardous-air-pollutants (Accessed 16 January, 2023).

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U.S. Environmental Protection Agency (EPA) (2022f ) Frequent Questions about the Chlorpyrifos 2021 Final Rule. Available at: https://www.epa.gov/ingredients-used-pesticideproducts/frequent-questions-about-chlorpyrifos-2021-final-rule (Accessed 16 January, 2023). U.S. Environmental Protection Agency (EPA) (2022g) Healthy School Environments. Available at: https://www.epa.gov/schools (Accessed 16 January, 2023). U.S. Environmental Protection Agency (EPA) (2022h) Protecting Children’s Environmental Health. Available at: https://www.epa.gov/children/children-are-not-little-adults (Accessed 16 January, 2023). U.S. Environmental Protection Agency (EPA) (2022i) Summary of the Toxic Substances Control Act. Laws and Regulations. Available at: https://www.epa.gov/laws-regulations/ summary-toxic-substances-control-act (Accessed 16 January, 2023). U.S. Environmental Protection Agency (EPA) (2022j) The Frank R. Lautenberg Chemical Safety for the 21st Century Act. Assessing and Managing Chemicals under TSCA. Available at: https://www.epa.gov/assessing-and-managing-chemicals-under-tsca/frank-r-lautenberg-chemical-safety-21st-century-act (Accessed 12 July, 2022). U.S. Environmental Protection Agency (EPA) (2022k) EPA Takes Next Step to Keep Chlorpyrifos Out of Food, Protecting Farmworkers and Children’s Health. Available at: https://www. epa.gov/newsreleases/epa-takes-next-step-keep-chlorpyrifos-out-food-protecting-farmworkers-and-childrens (Accessed 16 January, 2023). U.S. Environmental Protection Agency (EPA) (2022l) FY22-26 EPA Strategic Plan. Available at: https://www.epa.gov/system/files/documents/2022-03/fy-2022-2026-epa-strategicplan.pdf (Accessed 1 June, 2022). U.S. Environmental Protection Agency (EPA) (2005) Guidance on Selecting Age Groups for Monitoring and Assessing Childhood Exposures to Environmental Contaminants. EPA 630P-03-003F. https://www.epa.gov/sites/default/files/2013-09/documents/agegroups.pdf. Accessed 12 February 2023. U.S. Environmental Protection Agency (EPA) (2006) A Framework for Assessing Health Risk of Environmental Exposures to Children. https://ordspub.epa.gov/ords/eims/eimscomm. getfile?p_download_id¼459047. Accessed 12 February 2023. Vandenberg LN, Rayasam SDG, Axelrad DA, et al. (2023) Addressing systemic problems with exposure assessments to protect the public’s health. Environmental Health 21(Suppl. 1): 121. https://doi.org/10.1186/s12940-022-00917-0. Varshavsky JR, Rayasam SDG, Sass JB, et al. (2023) Current practice and recommendations for advancing how human variability and susceptibility are considered in chemical risk assessment. Environmental Health 21(Suppl. 1): 133. https://doi.org/10.1186/s12940-022-00940-1. Viet S, et al. (2013) Lead, allergen, and pesticide levels in licensed child care centers in the United States. Journal of Environmental Health 76(5): 8–14. Wang Z, et al. (2020) Toward a global understanding of chemical pollution: A first comprehensive analysis of national and regional chemical inventories. Environmental Science & Technology 54(5): 2575–2584. https://doi.org/10.1021/acs.est.9b06379. Wang A, et al. (2021) Suspect screening, prioritization, and confirmation of environmental chemicals in maternal-newborn pairs from San Francisco. Environmental Science & Technology 55(8): 5037–5049. https://doi.org/10.1021/acs.est.0c05984. Wen JG, et al. (2016) Melamine-contaminated milk formula and its impact on children. Asia Pacific Journal of Clinical Nutrition 25(4): 697–705. https://doi.org/10.6133/ apjcn.072016.01. PMID: 27702712. Westerling AL (2016) Increasing western US forest wildfire activity: Sensitivity to changes in the timing of spring. Philosophical Transactions of the Royal Society B 371: 20150178. https://doi.org/10.1098/rstb.2015.0178. Wickman ME, Anderson NLR, and Smith Greenberg C (2008) The adolescent perception of invincibility and its influence on teen acceptance of health promotion strategies. Journal of Pediatric Nursing 23(6): 460–468. Woodruff TJ, Rayasam SDG, Axelrad DA, et al. (2023) A science-based agenda for health-protective chemical assessments and decisions: Overview and consensus statement. Environmental Health 21(Suppl. 1): 132. https://doi.org/10.1186/s12940-022-00930-3.

Further reading National Association of State Boards of Education (NASBE) (2021) How states are handling lead in school drinking water. Available at: https://www.nasbe.org/how-states-are-handlinglead-in-school-drinking-water/ (Accessed 12 June, 2022). Poison Control (2020) Poison Statistics: National Data 2020. Available at: https://www.poison.org/poison-statistics-national (Accessed 12 July, 2022). Salas RN, Jacobs W, and Perera F (2019) The case of Juliana v. U.S. —Children and the health burdens of climate change. New England Journal of Medicine 380: 2085–2087. Zhang Y, Bi P, and Hiller JE (2007) Climate change and disability-adjusted life years. Journal of Environmental Health 70(3): 32–36.

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Chloral hydrate Emily Kershner and Michelle Troendle, Department of Emergency Medicine, Division of Medical Toxicology, Virginia Commonwealth University Health System, Richmond, VA, United States © 2024 Elsevier Inc. All rights reserved. This is an update of M. Troendle, B.K. Wills, Chloral Hydrate, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 833–834, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00708.

Chemical profile Background Uses Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Acute and short-term toxicity Animal Human Chronic toxicity Human Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Exposure of standards and guideline References Further reading

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Abstract This article reviews chloral hydrate. It is used as a sedative hypnotic and a drug of abuse. Its effects cause sedation and respiratory depression, which are more profound with concomitant alcohol use. It is metabolized in the liver to trichloroethanol, an active metabolite with a half-life of 4–12 h. Trichlorethanol has similar effects to halogenated hydrocarbons and can cause myocardial sensitization after chloral hydrate overdose. Overdose is typically managed by supportive care, including intubation for airway protection.

Keywords Chloral hydrate; Chloralex; CNS depression; Hydrated chloral; Knockout drops; Mickey Finn; Noctec; Sedative hypnotic; Trichloroacetic acid; Trichloroethanol; Urochloralic acid

Chemical profile

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Name: Chloral Hydrate. Chemical Abstracts Service Registry Number: 302-17-0. Synonyms: Mickey Finn, Knockout drops, Noctec, Hydrated chloral, Chloralex. Molecular Formula: CCl3CH(OH)2

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Chemical Structure:

Background Chloral hydrate was first synthesized in 1832 by Justus von Liebig and was the first synthetic central nervous system (CNS) depressant. It was used to treat delirium tremens, insomnia, and anxiety, although it is considered an unapproved drug by the United States Food and Drug Administration. Initially considered to be a safer alternative to opium, it was noted to produce rapid unconsciousness when combined with ethanol. Physical dependence can occur with chronic use.

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Uses Chloral hydrate is used as a sedative hypnotic, more commonly in pediatrics for procedural sedations. With the advent of newer sedative hypnotics, its use has significantly decreased. Italy and France banned its medical use in 2006. It is also a drug of abuse, particularly in combination with ethanol to produce an amnestic effect in an individual who ingests it unknowingly.

Environmental fate and behavior Chloral hydrate has been detected at 5 mg L−1 in the United States drinking water supply. Although chloral hydrate does not exist naturally, it can be produced as a by-product of chlorination of water at water treatment facilities, specifically in exposed water with high amounts of humic and fulvic substances.

Exposure and exposure monitoring Most exposures are through ingestion. It is available as tablets, capsules, oral solution, and rectal suppositories. Toxicity may occur in patients as a result of unknowingly ingesting chloral hydrate or through iatrogenic dosing errors during pediatric procedural sedations (Nordt et al., 2014). Cases are often with a co-ingestion of ethanol and/or other sedatives.

Toxicokinetics Chloral hydrate is rapidly and well absorbed in the gastrointestinal (GI) tract. It is lipid soluble with an onset of action of approximately 30 min and half-life of only a few minutes. Chloral hydrate has a volume of distribution of 0.6–0.75 L kg−1 and is rapidly metabolized by hepatic alcohol dehydrogenase to trichloroethanol. This active metabolite is responsible for the hypnotic effects and has a plasma half-life of 4–12 h. Trichloroethanol includes three elimination pathways. It can be conjugated with glucuronic acid to urochloralic acid, which is then renally excreted. Trichloroethanol can also be oxidized by aldehyde dehydrogenase to trichloroacetic acid, an inactive metabolite. Less than 10% of trichloroethanol is excreted unchanged by the kidneys.

Acute and short-term toxicity Animal Chloral hydrate is used in veterinary medicine as a sedative hypnotic, and toxic effects are similar in humans and animals.

Human Chloral hydrate is irritating to the GI tract, resulting in nausea, vomiting, and hemorrhagic gastritis. While GI irritation is typically mild, there are rare reports of gastric and intestinal necrosis, that have led to perforation and stricture formation (Lin and May, 2006). The major cause of death is believed to be secondary to cardiac dysrhythmias. Chloral hydrate and its metabolites can decrease myocardial contractility, shorten the refractory period, and increase sensitivity to catecholamines. Serious dysrhythmias can include ventricular fibrillation, ventricular tachycardia, and torsades de pointes. Respiratory failure can develop as a result of therapeutic use. As expected, patients can develop significant CNS depressions. Interestingly, case reports of seizure activity and a paradoxical central stimulant reaction has been reported.

Chronic toxicity Human Chronic use may result in renal damage, skin eruptions, and gastritis. Physical dependence can also occur and withdrawal symptoms can be severe, including seizures and delirium.

Reproductive toxicity Animal studies have shown an adverse effects on the fetus, but there is not enough research on humans to determine risk.

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Genotoxicity Chloral hydrate has been shown to cause aneuploidy and stop mitosis.

Carcinogenicity Chloral hydrate has not been identified as a carcinogen. Studies in rats given high doses during the lifetime did not produce carcinogenic concerns. However, chloral hydrate is structurally similar to known carcinogens. A 2006 human study demonstrated an association with short-term chloral hydrate prescriptions and the development of prostate cancer (Haselkorn et al., 2006). However, there was not enough evidence to support a causal relationship.

Clinical management Activated charcoal can be used if the patient has a normal level of consciousness and no signs of esophageal irritation. There is no specific antidote and supportive care is the mainstay of treatment. While flumazenil administration is generally not recommended due to the risk of precipitating seizures in chronic users, rare cases of successful reversal of CNS depression in acute toxicity have been reported (Donovan and Fisher, 1989). Intubation should be performed if the patient is obtunded with loss of protective airway reflexes. If there are any signs concerning for possible esophageal injury, endoscopy should be performed. Few hospital-based laboratories have the ability to rapidly detect chloral hydrate or its metabolites. Hypotension should be managed with intravenous crystalloids and vasopressors. Dysrhythmias may be refractory to standard treatments. There have been reported cases of patients in refractory ventricular fibrillation who have been successfully treated with b-blockers (like propranolol or esmolol) (Shakeer et al., 2019). There is no role for forced diuresis. Hemodialysis and hemoperfusion could theoretically be useful in severe cases, although they have been rarely employed.

Exposure of standards and guideline The World Health Organization recommends concentrations limited to 10 mg L−1. The US Environmental Protection Agency limits drinking water concentrations to 60 mg L−1.

References Donovan KL and Fisher DJ (1989) Reversal of chloral hydrate overdose with flumazenil. BMJ 298(6682): 1253. Haselkorn T, Whittemore AS, Udaltsova N, and Friedman GD (2006) Short-term chloral hydrate administration and cancer in humans. Drug Safety 29(1): 67–77. Lin YC and May JY (2006) Severe esophageal burn following chloral hydrate overdose in an infant. Journal of the Formosan Medical Association 105(3): 235–237. Nordt SP, Rangan C, Hardmaslani M, Clark RF, Wendler C, and Valente M (2014) Pediatric chloral hydrate poisonings and death following outpatient procedural sedation. Journal of Medical Toxicology 10(2): 219–222. Shakeer SK, Kalapati B, Al Abri SA, and Al Busaidi M (2019) Chloral hydrate overdose survived after cardiac arrest with excellent response to intravenous b-blocker. Oman Medical Journal 34(3): 244–248.

Further reading Han P, Song H, Yang P, Xie H, and Kang YJ (2011) Cardiac arrhythmias induced by chloral hydrate in rhesus monkeys. Cardiovascular Toxicology 11(2): 128–133. Pershad J, Palmisano P, and Nichols M (1999) Chloral hydrate: the good and the bad. Pediatric Emergency Care 6: 432–435. Salmon AG, Kizer KW, Zeise L, Jackson RJ, and Smith MT (1995) Potential carcinogenicity of chloral hydrate–A review. Journal of Toxicology. Clinical Toxicology 33(2): 115–121. Review. Sing K, Erickson T, Amitai Y, and Hryhorczuk D (1996) Chloral hydrate toxicity from oral and intravenous administration. Journal of Toxicology. Clinical Toxicology 1: 101–106.

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Chlorambucil Maria Chiara Astuto and Catalina Manieu, European Food Safety Authority, Parma, Italy © 2024 Published by Elsevier Inc. This is an update of N. Gupta, J.R. Salvatore, Chlorambucil, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 835–836, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00478-4.

Chemical profile Background Uses Exposure Pharmacokinetics Mechanism of toxicity Acute and short-term toxicity Chronic toxicity Immunotoxicity Reproductive and developmental toxicity Carcinogenicity and genotoxicity Interactions Pharmacogenomics Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines References Further reading

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Abstract Chlorambucil is nitrogen mustard derivative with a broad spectrum of antitumor activity used to treat chronic lymphocytic anemia, Hodgkin's and non-Hodgkin's lymphomas, and other conditions. The mechanism of action is linked to its behavior as alkylating agent, and it is considered carcinogenic via genotoxic mechanism. In short term, consistently reported findings are vomiting, ataxia, seizures, coma, and pancytopenia, whereas one of the most common long-term effects is severe bone marrow suppression. Reproductive effects reported seem to be related to its mutagenic and teratogenic activity. Scarce information is available on ecotoxicology, however, it appears highly unstable in water.

Keywords Alkylating agent; Antineoplastic; Chemotherapy; Chronic lymphocytic anemia; Nitrogen mustard

Chemical profile Chlorambucil is a butanoic acid substituted at position 4 by a 4-[bis(2-chloroethyl)amino]phenyl group. It is an organochlorine compound, an aromatic amine, a tertiary amino compound, and a monocarboxylic acid (ChEBI database, n.d., https://www.ebi.ac. uk/chebi).

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Synonyms: 4-{4-[Bis(2-chloroethyl)amino]phenyl}butanoic acid, Chlorambucyl, Chloraminophen, Chlorbutin, Ambochlorin, Leukeran CAS Number: 305-03-3 Molecular Formula: C14H19Cl2NO2

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Chemical Structure:

Background Chlorambucil is a nitrogen mustard that acts as alkylating agent. Originally produced as potential chemical warfare agents during World War I and II, nitrogen mustard compounds, including Chlorambucil, were soon marketed as pharmaceuticals (CDC website, n.d., www.cdc.gov). Chlorambucil is currently used as antineoplastic drug to treat lymphomas and a type of chronic leukemia (Amjad and Kasi, 2020). Chlorambucil is a harmful if swallowed incidentally; can cause skin and eye irritation and may cause respiratory irritation; it is classified as carcinogen, and might be toxic for the reproduction, according to the UN Global Harmonized System (GHS) for classification and labeling of hazardous substances.

Uses Chlorambucil is a pharmaceutical agent originally approved by the Food and Drug Administration (FDA) in 1957 due to its alkylating/antineoplastic activity for the treatment of chronic lymphocytic leukemia (CLL), Hodgkin’s and non-Hodgkin’s lymphomas, and mycosis fungoides (Cramer et al., 2016; Rai et al., 2000). Chlorambucil has also been used to treat other conditions such as polycythemia vera, autoimmune uveitis, rheumatoid arthritis, ovarian carcinoma, Waldenstrom’s macroglobulinemia and for autoimmune and inflammatory conditions, such as nephrotic syndrome (FDA Orange Book, n.d., www.fda.gov; Lepretre et al., 2015; Takimoto and Calvo, 2008; Vidal et al., 2016).

Exposure Chlorambucil is administered orally in a dose of 2–10 mg per day for the average adult patient. For those adults with severe renal failure, a dose reduction by 25% with creatinine clearance (CrCL) from 10 to 50 mL min−1, and by 50% if CrCL is less than 10 mL min−1 is recommended. Maintenance doses for Chlorambucil may be as low as 0.03 mg kg−1 per day but not to exceed 0.1 mg kg−1 per day. Pulse dosing is sometimes done up to 0.4 mg kg−1. (FDA Orange Book, n.d., www.fda.gov). Possible human exposure includes inhalation, incidental ingestion, and dermal contact. Chlorambucil has been classified as a skin sensitizer, and the use of splash goggles, lab coat, dust respirator and gloves as Personal Protective Equipment (PPE) is recommended while manipulating (Aydogdu et al., 1997; Steinritz et al., 2019). No information on environmental levels of exposure is available, however, due to its extensive metabolism in the treated patients, environmental emissions are considered of low concern.

Pharmacokinetics Chlorambucil is rapidly and completely absorbed in the gastrointestinal tract, however, concomitant food intake with Chlorambucil, delays its absorption and reduces its plasma levels (Adair and McElnay, 1986; Silvennoinen et al., 2000). It is extensively metabolized in the liver to phenylacetic acid mustard, and its metabolites are extensively bound to plasma and tissue proteins. After 24 h of a single dose intake, about 15–60% of Chlorambucil appears in the urine. However, less than 1% is intact drug and overall has low urinary excretion (Adair et al., 1986; Mitoma et al., 1977; Singh and Malhotra, 2004; Yasuda et al., 2021).

Mechanism of toxicity As alkylating agent and aromatic nitrogen mustard derivative, Chlorambucil induces modifications by alkylation and cross-linking of DNA, eventually inhibiting RNA transcription and proteins synthesis (Amjad and Kasi, 2020). Further, Chlorambucil impairs angiogenesis, by inhibiting the early vascular endothelial cell migration, as well as wound healing process (Steinritz et al., 2015).

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Acute and short-term toxicity In the short-term, Chlorambucil causes several side effects, such as nausea, vomiting, diarrhea, ataxia, seizures, coma, pancytopenia and hypersensitivity reactions. Chlorambucil neurotoxicity might be revealed by seizures occurrence, myoclonus, muscular twitching, tremors and agitation (Salloum et al., 1997). Anemia, leukopenia, neutropenia, and myelosuppression might indicate hematologic toxicity. Other potential adverse effects are: dermatologic toxicity such as skin sensitization, gastrointestinal discomfort, respiratory effects as acute interstitial pneumonitis, renal and hepatotoxicity, and immunologic effects (Aydogdu et al., 1997; Giles et al., 1990; Shafqat and Olszewski, 2014; Steinritz et al., 2014).

Chronic toxicity One of the most common side effects of Chlorambucil is the severe bone marrow suppression, which is reversible upon drug withdrawal. Long-term use of Chlorambucil has been associated to the development of secondary cancers, such as acute myelocytic leukemia (Haznedaro
glu et al., 1997; Kauppi et al., 1996; Palmer and Denman, 1984).

Immunotoxicity Immune hypersensitivity reaction and drug fever have been shown (Steinritz et al., 2019).

Reproductive and developmental toxicity Black-boxed warning for human fertility effects related to the mutagenic and teratogenic action of this alkylating agent (Mirkes and Greenaway, 1982). Reproductive effects including infertility, sexual dysfunction, and disorder of menstruation have been reported. It has been reported malformations such as renal agenesis upon exposure to Chlorambucil during the first trimester (Steege and Caldwell, 1980). Fertility is affected in both females and males; however, gender differences have been identified, where the overall mutagenesis rate for female mature gametes is one order of magnitude below male mature gametes. Moreover, the type of mutations in the DNA as well as the rate of occurrence between female and male gametes is gender-dependent (Blumenfeld, 2007; Russell et al., 1996). It is suggested that because of the detrimental effect in the DNA, Chlorambucil can produce long-term azoospermia (Meistrich, 2009), however, a case report suggests that spermatogenesis might return, years after the end of the treatment (Marmor et al., 1992).

Carcinogenicity and genotoxicity Chlorambucil has been reevaluated by International Agency for Research on Cancer (IARC) in 2012 (IARC, 2012). It is considered as a direct-acting alkylating agent that is carcinogenic via a genotoxic mechanism. According to IARC, there is sufficient evidence in humans and experimental animals regarding the carcinogenicity of Chlorambucil, and therefore it is concluded that Chlorambucil is carcinogenic to humans (Group 1) (IARC, 2012). This has been reported by several case reports where patients with Chronic Lymphocytic Leukemia or with autoimmune diseases treated with Chlorambucil, in the long-term, have developed Acute Myeloid Leukemia (Haznedaro
glu et al., 1997; Kauppi et al., 1996; Lerner, 1978; Palmer and Denman, 1984; Ramadan et al., 2012).

Interactions Evidence shows that pharmaceuticals like cyclophosphamide, busulfan and melphalan, may affect the transport of DNA-alkylating agents, such as Chlorambucil, to lymphoma and leukemia cell lines, possibly affecting their final bioavailability. The interaction seems explained by a common drug transport-mediated mechanism (Valdez et al., 2017). Chlorambucil also showed synergistic interactions with olaparib, a drug for the treatment of BRCA-mutated advanced ovarian cancer (Evers et al., 2010). Furthermore, a nano-enabled preparation with paclitaxel, a chemotherapeutic agent to treat breast cancer, revealed a synergistic inhibition of Lung Adenocarcinoma cell-line growth in vitro (Fan and Li, 2021). Likewise, a conjugate of floxuridine and Chlorambucil exhibits a high synergistic effect in the inhibition of ovarian cancer cells growth (Huang et al., 2020).

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Pharmacogenomics Pharmacogenomics approaches were used to investigate a pharmaceutical strategy to be used for counteracting Chlorambucilresistant ovarian carcinoma cells. Microarray gene expressions followed by gene network analysis showed that ARHGEF6 signaling may have a role in induction of apoptosis in ovarian carcinoma cells (Maiti, 2010).

Clinical management There is no specific antidote for Chlorambucil, and it is not dialyzable. For oral/parenteral exposure, induced emesis with ipecac is not recommended due to chance of seizures. However, activated charcoal or gastric lavage can be performed. Seizures toxicity may be managed with intravenous benzodiazepine. Pancytopenia should be monitored for up to 3 weeks, and fluid/electrolyte replacement in persistent vomiting. (FDA Orange Book, n.d., www.fda.gov; EMA Article 57 database, n.d. www.ema.europa.eu).

Environmental fate and behavior Chlorambucil is an off-white to pale slight odorous powder, insoluble in water. It is slightly dispersible in diethyl ether and acetone. It has a melting point of 69
C, boiling point of 424
C, and 5.75 pKa. The partition coefficient is 4.07 and has a molecular weight of 304.22 g mol−1 (EPA Comptox, n.d. https://comptox.epa.gov). A study shows that Chlorambucil rapidly degraded in water losing a hydroxyl group and forming a bioactive transformation product (Gómez-Canela et al., 2015).

Ecotoxicology Scarce information is available on ecotoxicology. Evidence suggests that Chlorambucil might enhance multixenobiotic resistance (MXR) mechanism transporter activity in Daphnia Magna (Campos et al., 2014). However, Chlorambucil appears highly unstable in water and the transformation product, generated by degradation, decreases its concentration within 24 h, suggesting exponential loss of toxic activity toward Daphnia Magna survival (Gómez-Canela et al., 2015).

Exposure standards and guidelines FDA and EMA regulate Chlorambucil prescription drug labeling, together with other specific requirements. The reader is referred to the ‘Exposure’ section for more information on this aspect (‘FDA Orange Book,’ www.fda.gov; ‘EMA Article 57 database,’ www.ema. europa.eu). Pubchem: https://pubchem.ncbi.nlm.nih.gov/compound/2708 EPA Comptox: https://comptox.epa.gov/dashboard/dsstoxdb/results?search¼DTXSID7020263

References Adair CG and McElnay JC (1986) Studies on the mechanism of gastrointestinal absorption of melphalan and chlorambucil. Cancer Chemotherapy and Pharmacology 17(1): 95–98. Adair CG, Bridges JM, and Desai ZR (1986) Can food affect the bioavailability of chlorambucil in patients with haematological malignancies? Cancer Chemotherapy and Pharmacology 17(1): 99–102. Amjad MT and Kasi A (2020) ‘Cancer chemotherapy’. [Update 2020 Nov 24]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing. 33232037. Available from: https:// www.ncbi.nlm.nih.gov/books/NBK564367/. Aydogdu I, Ozcan C, Harputluoglu M, Karincaoglu Y, Turhan O, and Ozcanu A (1997) Severe adverse skin reaction to chlorambucil in a patient with chronic lymphocytic leukemia. Anti-Cancer Drugs 8(5): 468–469. Blumenfeld Z (2007) Gender difference: fertility preservation in young women but not in men exposed to gonadotoxic chemotherapy. Minerva Endocrinologica 32(1): 23–34. Campos B, Altenburger R, Gomez C, Lacorte S, Pina B, Barata C, and Luckenbach T (2014) First evidence for toxic defense based on the multixenobiotic resistance (MXR) mechanism in Daphnia magna. Aquatic Toxicology 148: 139–151. https://doi.org/10.1016/j.aquatox.2014.01.001. CDC website, www.cdc.gov. Accessed on July 2021. ChEBI database. https://www.ebi.ac.uk/chebi. Accessed on July 2021. Cramer P, Hallek M, and Eichhorst B (2016) State-of-the-art treatment and novel agents in chronic lymphocytic leukemia. Oncology Research and Treatment 39(1–2): 25–32. EMA Article 57 database. www.ema.europa.eu. Accessed on July 2021. EPA Comptox’. https://comptox.epa.gov. Accessed on July 2021. Evers B, Schut E, van der Burg E, Braumuller TM, Egan DA, Holstege H, Edser P, Adams DJ, Wade-Martins R, Bouwman P, and Jonkers J (2010) A high-throughput pharmaceutical screen identifies compounds with specific toxicity against BRCA2-deficient tumors. Clinical Cancer Research 16(1): 99–108. Fan ML and Li JP (2021) A novel combinational nanodrug delivery system induces synergistic inhibition of lung adenocarcinoma cells in vitro. Letters in Drug Design & Discovery 18(1): 104–110. FDA Orange Book, https://www.fda.gov/. Accessed on July 2021. Giles FJ, Smith MP, and Goldstone AH (1990) Chlorambucil lung toxicity. Acta Haematologica 83(3): 156–158.

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Gómez-Canela C, Campos B, Barata C, and Lacorte S (2015) Degradation and toxicity of mitoxantrone and chlorambucil in water. International journal of Environmental Science and Technology 12: 633–640. Haznedaro
glu _lC, Savas¸ MC, Benekli M, Sayinalp NM, Özcebe OI, and Dündar S (1997) Acute lymphoblastic leukemia occurring in a case of chronic lymphocytic leukemia. Hematology 2(1): 87–89. Huang P, Wang G, Wang Z, Zhang C, Wang F, Cui X, Guo S, Huang W, Zhang R, and Yan D (2020) Floxuridine-chlorambucil conjugate nanodrugs for ovarian cancer combination chemotherapy. Colloids and Surfaces. B, Biointerfaces 194: 111164. IARC (2012) Monographs on the evaluation of carcinogenic risks to humans. Pharmaceuticals, Chlorambucil, vol. 100A. https://publications.iarc.fr/Advanced-Search?q¼chlorambucil. Kauppi MJ, Savolainen HA, Anttila VJ, and Isomäki HA (1996) Increased risk of leukaemia in patients with juvenile chronic arthritis treated with chlorambucil. Acta Paediatrica 85(2): 248–250. Lepretre S, Dartigeas C, Feugier P, Marty M, and Salles G (2015) Systematic review of the recent evidence for the efficacy and safety of chlorambucil in the treatment of B-cell malignancies. Leukemia & Lymphoma 57(4): 852–865. Lerner HJ (1978) Acute myelogenous leukemia in patients receiving chlorambucil as long-term adjuvant chemotherapy for stage II breast cancer. Cancer Treatment Reports 62(8): 1135–1138. Maiti AK (2010) Gene network analysis of oxidative stress-mediated drug sensitivity in resistant ovarian carcinoma cells. The Pharmacogenomics Journal 10(2): 94–104. Marmor D, Grob-Menendez F, Duyck F, and Delafontaine D (1992) Very late return of spermatogenesis after chlorambucil therapy: case reports. Fertility and Sterility 58(4): 845–846. Meistrich ML (2009) Male gonadal toxicity. Pediatric Blood & Cancer 53(2): 261–266. Mirkes PE and Greenaway JC (1982) Teratogenicity of chlorambucil in rat embryos in vitro. Teratology 26(2): 135–143. Mitoma C, Onodera T, Takegoshi T, and Thomas DW (1977) Metabolic disposition of chlorambucil in rats. Xenobiotica 7(4): 205–220. Palmer RG and Denman AM (1984) Malignancies induced by chlorambucil. Cancer Treatment Reviews 11(2): 121–129. Rai KR, Peterson BL, Appelbaum FR, Kolitz J, Elias L, Shepherd L, Hines J, Threatte GA, Larson RA, Cheson BD, and Schiffer CA (2000) Fludarabine compared with chlorambucil as primary therapy for chronic lymphocytic leukemia. The New England Journal of Medicine 343(24): 1750–1757. Ramadan SM, Fouad TM, Summa V, Hasan S, and Lo-Coco F (2012) Acute myeloid leukemia developing in patients with autoimmune diseases. Haematologica 97(6): 805–817. Russell LB, Hunsicker PR, and Shelby MD (1996) Chlorambucil and bleomycin induce mutations in the specific-locus test in female mice. Mutation Research 358(1): 25–35. Salloum E, Khan KK, and Cooper DL (1997) Chlorambucil-induced seizures. Cancer 79(5): 1009–1013. Shafqat H and Olszewski AJ (2014) Chlorambucil-induced acute interstitial pneumonitis. Case Reports in Hematology 2014: 575417. Silvennoinen R, Malminiemi K, Malminiemi O, Seppälä E, and Vilpo J (2000) Pharmacokinetics of chlorambucil in patients with chronic lymphocytic leukaemia: comparison of different days, cycles and doses. Pharmacology & Toxicology 87(5): 223–228. Singh BN and Malhotra BK (2004) Effects of food on the clinical pharmacokinetics of anticancer agents: underlying mechanisms and implications for oral chemotherapy. Clinical Pharmacokinetics 43(15): 1127–1156. Steege JF and Caldwell DS (1980) Renal agenesis after first trimester exposure to chlorambucil. Southern Medical Journal 73(10): 1414–1415. Steinritz D, Schmidt A, Simons T, Ibrahim M, Morguet C, Balszuweit F, Thiermann H, Kehe K, Bloch W, and Bölck B (2014) Chlorambucil (nitrogen mustard) induced impairment of early vascular endothelial cell migration - effects of a-linolenic acid and N-acetylcysteine. Chemico-Biological Interactions 219: 143–150. Steinritz D, Schmidt A, Balszuweit F, Thiermann H, Ibrahim M, Bölck B, and Bloch W (2015) Assessment of endothelial cell migration after exposure to toxic chemicals. Journal of Visualized Experiments (101): e52768. Steinritz D, Lang S, Popp T, Siegert M, Rothmiller S, Kranawetvogl A, Schmidt A, John H, Gudermann T, Thiermann H, and Kehe K (2019) Skin sensitizing effects of sulfur mustard and other alkylating agents in accordance to OECD guidelines. Toxicology Letters 314: 172–180. Takimoto CH and Calvo E (2008) Principles of oncologic pharmacotherapy. In: Pazdur R, Wagman LD, Camphausen KA, and Hoskins WJ (eds.) Cancer Management: A Multidisciplinary Approach, 11th ed. Valdez BC, Hassan M, and Andersson BS (2017) Development of an assay for cellular efflux of pharmaceutically active agents and its relevance to understanding drug interactions. Experimental Hematology 52: 65–71. Vidal L, Gurion R, Ram R, Raanani P, Bairey O, Robak T, Gafter-Gvili A, and Shpilberg O (2016) Chlorambucil for the treatment of patients with chronic lymphocytic leukemia (CLL)—A systematic review and meta-analysis of randomized trials. Leukemia & Lymphoma 57(9): 2047–2057. Yasuda H, Yasuda M, and Komatsu N (2021) Chemotherapy for non-Hodgkin lymphoma in the hemodialysis patient: A comprehensive review. Cancer Science 112(7): 2607–2624.

Further reading LiverTox: Clinical and research information on drug-induced liver injury. Chlorambucil. Bethesda (MD): National Institute of Diabetes and Digestive and Kidney Diseases [Updated 2017 Oct 5]. Available from: https://www.ncbi.nlm.nih.gov/books/NBK548207/.

Relevant websites https://www.accessdata.fda.gov/drugsatfda_docs/label/2011/010669s032lbl.pdf :US FDA: Prescribing information for LeukeranW (chlorambucil) tablets. https://ntp.niehs.nih.gov/ntp/roc/content/profiles/chlorambucil.pdf :US National Institutes of Health: Report on Carcinogens—Chlorambucil.

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Chloramphenicol Besen Sanga and Madan K Kharel, School of Pharmacy and Health Professions, University of Maryland Eastern Shore, Princess Anne, MD, United States © 2024 Elsevier Inc. All rights reserved. This is an update of M. Abdollahi, S. Mostafalou, Chloramphenicol, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 837–840, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00709-0.

Chemical profile Background (significance/history) Uses Mechanism of action Environmental fate and behavior Routes and pathways relevant physicochemical properties Partition behavior in water, sediment, and soil Environmental persistency (degradation/speciation) Long-range transport Bioaccumulation and biomagnifications Exposure and exposure monitoring Environmental release and exposure Human exposure Toxicokinetics Toxicity Mechanism of toxicity Acute and short-term toxicity (to include irritation and corrosivity) Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive toxicity Neurotoxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Other hazards Exposure standards and guidelines Bacterial chloramphenicol resistance References

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Abstract Chloramphenicol is a broad-spectrum antimicrobial agent. It acts by inhibiting protein synthesis. Its high lipid solubility results in rapid absorption, a large volume of distribution, and accelerated penetration into almost all tissues. Aplastic anemia and bone marrow suppression are chloramphenicol’s most serious adverse effects in humans. Circulatory system collapse, also known as “gray baby syndrome” in newborn infants, is associated with high serum levels of chloramphenicol. Chloramphenicol is also associated with the development of optic neuritis, scotoma with failing vision, and cleft lip. Chloramphenicol is classified as FDA pregnancy category C and requires caution if used during gestation and lactation.

Keywords Antibacterial; Antibiotic; Antimicrobial; Aplastic anemia; Bacteriostatic; Bone marrow suppression; Gray baby syndrome; Resistance; Streptomyces venezuelae

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Name: Chloramphenicol Chemical Abstracts Service Registry Number: CAS 56-75-7

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Chloramphenicol

Mixture Name: Chloromyxin; Elase-Chloromycetin; Ophthocort Synonyms: Amphicol, Amseclor, Aquamycetin, Biocetin, Biophenicol, Chlomycol, Chloramex, Chloramficin, Chloramsaar, Chlorocaps, Chlorocid, Chlorocol, Chloromax, Chloromycetin, Chloronitrin, Chloramex, Chloroptic, Chlornitromycin, Cloramfenicol, Cloramficin, Cloramicol, Cloromisan, Cylphenicol, Detreomycin, Enteromycetin, Farmicetina, Fenicol, Globenicol, Ismicetina, Kemicetine, Klorocid S, Leukomyan, Leukomycin, Levomicetina, Micloretin, Novomycetin, Ophthochlor, Paraxin, Quemicetina, Romphenil, Septicol, Sintomicetina, Tevcocin, Tifomycine, Unimycetin Chemical Class: Antibiotic with both bactericidal and bacteriostatic properties Molecular Formula: C11H12Cl2N2O5 Chemical Structure:

Background (significance/history) Chloramphenicol was first isolated from cultures of the soil bacterium Streptomyces venezuelae in 1947 but is now produced synthetically. Due to its broad-spectrum coverage against Gram-positive and Gram-negative bacteria, the antibiotic gained popularity shortly after its discoveries. The FDA approved using chloramphenicol in 1950 to treat serious infections for which less potentially dangerous drugs are ineffective or contraindicated. It became one of the top-selling antibiotics in the United States in the 1950s. The drug produced several severe adverse effects causing a limited use of Chloramphenicol in recent years.

Uses Chloramphenicol is active against gram-positive and gram-negative bacteria and anaerobic microorganisms. It was initially introduced as a treatment for typhoid, but now it is rarely used for this purpose because of the prevalence of multiple drug-resistant Salmonella typhi. Its ability to penetrate the blood-brain barrier has made this antibiotic an effective choice for treating brain abscesses caused by staphylococci and mixed or unknown microorganisms (Wiest et al., 2012). Chloramphenicol is also used to treat meningitis caused by Haemophilus influenzae, Streptococcus pneumonia and Neisseria meningitidis when other safer antibiotics are not appropriate. Chloramphenicol is also effective against Vancomycin-Resistant Enterococci. The antibiotic is also used in Veterinary to treat pulmonary, urinary tract infections caused by a variety of organisms such as Staphylococcus aureus, Streptococcus pyogenes and Brucella bronchiseptica, Escherichia coli, Proteus vulgaris, and Corynebacterium renale.

Mechanism of action Chloramphenicol readily diffuses through the bacterial cell wall and binds with the 50 S ribosomal subunit. The binding of the drug at the ribosome blocks peptidyl transferase activity. This causes a halt in the transfer of amino acids to the growing peptide chain- a crucial event of the translation process-thereby inhibiting protein synthesis.

Environmental fate and behavior Routes and pathways relevant physicochemical properties Chloramphenicol exists in white to grayish-white or yellowish-white fine crystalline powder or fine crystals, needles, or elongated plates form. It has a bitter taste. Chloramphenicol has a solubility of 25,000 mg L−1 (25 mg mL−1) in water at 25  C, and it is very soluble in methanol, ethanol, butanol, ethyl acetate, acetone, and chloroform. Waste streams of drug industries producing chloramphenicol can be the source of its release to the environment. Estimated vapor pressure of 1.7  10–12 mmHg at 25  C indicates that chloramphenicol will exist solely in the particulate phase. Chloramphenicol is stable for several years at room temperature. The antibiotic is relatively stable at high pH. However, exposure of this molecule to a pH over 10 can result in degradation.

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Partition behavior in water, sediment, and soil In the aquatic system, chloramphenicol is not expected to adsorb to suspended solids and sediments given by the Koc (Soil Organic Carbon–Water Partitioning Coefficient) value of 99.

Environmental persistency (degradation/speciation) Chloramphenicol solutions are susceptible to direct photolysis by sunlight or high temperatures and decompose to form hydrochloric and dichloric acid. Hydrolysis of chloramphenicol is not anticipated under environmental conditions because it lacks a functional group to hydrolyze. Chloramphenicol has been reported to degrade 86.2% with a biodegradation rate of 3.3 mg COD per gram per hour using adapted activated sludge as the inoculums (NCBI, 2022). Bacteria can degrade the antibiotic to produce a variety of degradation products (Zhang et al., 2020).

Long-range transport The Koc value of 99 suggests that chloramphenicol has high mobility in the soil in terrestrial systems.

Bioaccumulation and biomagnifications An estimated bioconcentration factor 200 mg L−1, and in cats is 100 mgm−3. No reliable dermal or eye irritation studies are available (ATSDR, 2018). The targets of chlordane toxicity after short-term inhalation and oral exposures are the liver, nervous and immune systems, and the developing offspring. Clinical signs of neurotoxicity included, salivation, abnormal respiratory movements, hyperexcitability, convulsions, and paralysis (ATSDR, 2018).

Human Deaths from accidental dermal exposure or intentional ingestion of chlordane are reported. Some of the oral poisoning cases have been from mislabeled or unlabeled pesticide bottles (Dadey and Kammer, 1953). The estimated acute oral lethal dose is 25–50 mg kg−1 (ATSDR, 2018). Initial signs involved confusion and convulsions.

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Non-lethal acute signs of poisoning are headaches, tremors, seizures, numbness, nausea, fatigue, gastrointestinal symptoms, skin, and eye irritation and unconsciousness. The exposure level or duration is generally unknown. Incoordination, excitability, hyporeflexia, and convulsions occur at about 0.15 mg kg−1 (USEPA, 1997).

Chronic toxicity Animal

Mortality occurred in rats after 50–163 days at 6–32 mg kg−1 day−1 and in mice within 42 days to 18 months at 4–21 mg kg−1 day−1. Rapid weight loss and convulsions were reported in rats prior to death (ATSDR, 2018). Liver is the most common non-lethal target in 28 days to 2 years oral and inhalation studies in rats and mice. Increased liver weights, changes in serum liver enzyme activities and triglycerides, hypertrophy, necrosis, fat in the hepatocytes and fatty degeneration occurred at the LOELs of 0.06–8 mg kg−1 day−1 and 1–28 mg m−3. Similar effects were reported for oxychlordane and heptachlor epoxide. The LOELs for other tissue toxicity included 6 mg kg−1 day−1 for decreased thymus weight of rats, 16–50 mg kg−1 day−1 for reduced body weight in rats and 1.7–50 mg kg−1 day−1 for neurological disturbance (e.g., hyperexcitability, hypersensitivity to touch, tremors, seizures, and convulsions) in rats and mice. Liver tumors occurred in mice at 1.2–8.3 mg kg−1 day−1 (ATSDR, 2018; USEPA, 1997).

Human Adverse health outcomes are described in humans who had increased blood and fat levels of oxychlordane, heptachlor and transnonachlor. Neurological effects in apartment occupants exposed for up to 7 years to airborne chlordane at about 0.0005 mg m−3 included slowing of reaction time, balance dysfunction, reduction in cognitive function and deficit of immediate and delayed recall (USEPA, 1997). Exposure to oxychlordane is associated with an increased waist circumference and an increased prevalence of diabetes, possibly through adipocyte dysfunction (Mendes et al., 2021).

Immunotoxicity Animal Oral studies in mice showed altered immune system from chlordane exposure during gestation days (GDs) 1–18 and throughout lactation period. The LOEL for suppression of cell-mediated immunity (depressed delayed-type hypersensitivity reaction), granulocyte-macrophage and spleen-forming stem cells in the bone marrow was 4–8 mg kg−1 day−1 (Barnett et al., 1990b). Reduced thymus weight and increased leukocyte counts occurred in rats exposed to 1–28 mg m−3 chlordane in air for 28–90 days. Altered levels of serum immunoglobulin M, G1, and G2 were reported in rats received 25 mg kg−1 day−1 chlordane orally for 28 days. In the same study, the component nonachlor at 2.5–25 mg kg−1 day−1 decreased the ability to combat bacterial infections (Tryphonas et al., 2003). In vitro, chlordane impaired the lymphocyte functions in monkeys (Chuang et al., 1992).

Human Chlordane (oxychlordane and cis- and trans-nonachlor) in umbilical cord blood from 300 newborns were associated with lower levels of the pro-inflammatory cytokine IL-1b (Neta et al., 2011). Studies using trans-nonachlor and chlordane in fat biopsies as markers of chlordane exposure at home or at work revealed an association with immunological disregulation (distribution of lymphocytes and decreased response to foreign antigens). The exposure was 3 days to 15 months and the immunological test was followed for 4 months to 10 years, indicating lasting impact on immune functions (McConnachie and Zahalsky, 1992; ATSDR, 2018).

Reproductive and developmental toxicity Animal Reduced size of seminiferous tubules, degeneration of spermatogenic epithelium and increased androgen receptor sites in ventral prostate were reported in mice treated orally with 100–300 mg kg−1 day−1 and in rats treated with 20 mg kg−1 day−1 for 30 days (ATSDR, 2018). Decreased pup survival (up to 55%) was observed when pregnant rats received 21–28 mg kg−1 day−1 chlordane orally on GD 6–19 and pregnant mice at 8 mg kg−1 day−1 throughout gestation (GD 1–18). Plasma corticosterone in the surviving offspring was drastically elevated at the 0.16 mg kg−1 day−1, possibly due to diminished liver metabolic activity (ATSDR, 2018). Mice fed chlordane during late gestation (GD 12–19) exhibited normal pregnancy, offspring and lactation, however, pups at 38 days of age showed depressed acquisition of avoidance response, increased seizure threshold and exploratory activity at the LOEL of 1 mg kg−1 day−1 (ATSDR, 2018).

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Human A study with 163 men recruited through fertility clinics showed positive association between serum oxychlordane levels and decreased sperm motility (odds ratio ¼ 1.98,95% CI; 1.07–3.69), suggesting an increased risk of subfertility (20 years in soil. The degradation rate under field conditions is 4–28% per year. In river water under sunlight, 85% of chlordane persisted to the end of the 8-week experiment. Abiotic degradation (hydrolysis, oxidation, dechlorination or direct photolysis) is also not an important environmental fate process. The estimated Log Koc of 3.5–4.6 predicts strong soil adsorption and resistant to leaching to groundwater (ATSDR, 2018).

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Chlordane in soil and sediment can be taken up by organisms and plants. Due to its stability, it can travel long distances and contaminate area remote from its use. The measured Log Kow of 5.54 indicates extensive bioaccumulation in food chains (ATSDR, 2018).

Ecotoxicology Chlordane is highly toxic to aquatic life (IPCS, 1984). The 96-h LC50 for fresh water invertebrates and fish ranged widely. The mean values from all data for each of the following species are: 0.058 mg L−1 in Daphnia magna, 0.0063 mg L−1 in freshwater shrimp, 0.037 mg L−1 in fathead minnow, 0.003 mg L−1 in carp, 0.025 mg L−1 for rainbow trout, and 0.056 mg L−1 in salmon, 0.059 mg L−1 in bluegill, and 0.19 mg L−1 in guppy. The LC50 for saltwater species also ranged widely; 0.0004 mg L−1 in pink shrimp, 0.006 mg L−1 in easter oyster, 0.012 mg L−1 in striped bass, 0.018 mg L−1 in sheepshead minnow, and 0.12 mg L−1 in threespine stickleback. Chronic toxicity data are sparse. Sheepshead minnow did not survive after 10 days at 7.1 mg L−1. The LOEC from life-cycle test is 0.32 mg L−1 for brook trout, 1.22 mg L−1 for blue gill, and 1.7 mg L−1 for midges. The 8-day dietary LD50 of chlordane is 331 mg kg−1 in bobwhite quail, 430 mg kg−1 in pheasant, and 858 mg kg−1 in mallard ducks. Chlordane is highly toxic to bees and earthworms (IPCS, 1984).

Exposure standards and guidelines

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American Conference of Governmental Industrial Hygienists (ACGIH) Threshold Limit Value (TLV): 0.5 mg m−3 (30 ppb) TWA; [skin]; A3—Confirmed animal carcinogen with unknown relevance to humans (ACGIH, 2018). National Institute for Occupational Safety and Health (NIOSH) Recommended Exposure Level (REL): 0.5 mg m−3 (30 ppb) (up to 10-h workday during a 40-h workweek TWA), Skin; A potential occupational carcinogen (NIOSH, 2003). National Institute for Occupational Safety and Health (NIOSH) Immediately Dangerous To Life or Health Concentration (IDLH): 100 mg m−3 (6 ppm); a potential occupational carcinogen (NIOSH, 2003). Occupational Safety and Health Administration (OSHA) Permissible Exposure Limit (PEL): 0.5 mg m−3 (30 ppb) (8-h Time weighted average, TWA); Skin—potential for dermal absorption (29 CFR 1910.1000, 2004). United States Environmental Protection Agency (US EPA) Drinking water Maximum Contaminant Level (MCL): 0.002 mg L−1 (20 ppb) (USEPA, 2018). World Health Organization (WHO) Acceptable Daily Intake (ADI): 0.5 mg kg−1 (WHO, 2004). United States Environmental Protection Agency (US EPA)—Reference Dose (chronic oral) 0.0005 mg kg−1 day−1; Reference Concentration (chronic) 0.0007 mg m−3; Oral slope factor 0.35 per mg kg−1 day1; Inhalation Unit Risk 0.0001 per mg m−3 day−1 (USEPA, 1997). Office of Environmental Health Hazard Assessment (OEHHA) Proposition 65 Listing (Cancer)—No Significant Risk Levels (NSRL) 0.5 mg day−1 (OEHHA, 1988).

Acknowledgment Authors would like to thank Dr. Shelley DuTeaux for her helpful discussions and review of this paper.

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Environment International 84: 154–160. DPR (2021) Pesticide Illness Surveillance Program. California Pesticide Illness Query (CalPIQ). Sacramento, CA: Department of Pesticide Regulation. Duteaux S (2020) RE: Personal Communication with Green Leaf Lab S0J0021. Certificate Analysis for Back Country Organics (COnfidential; Dunsmuir, CA). Type to DUTEAUX, S. November 17, 2020. EFSA (2007) Chlordane as undesirable substance in animal feed, scientific panel on contaminants in the food chain. The EFSA Journal 582: 1–53. FDA (2020) In: Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration (ed.) Guidance for Industry: Action Levels for Poisonous or Deleterious SUbstances in Human Food and Animal Feed. U.S. Food and Drug Administration. IARC (1991) IARC Monographs on the Evaluation of Carcinogenic Risk to Human. Volume 53 Occupational Exposures in Insecticide Application, and Some Pesticides. Chapter Chlordane and Heptachlor. 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Toxicological Sciences 153: 409. Kania-Korwel I, Hornbuckle KC, Peck A, Ludewig G, Robertson LW, Sulkowski WW, Espandiari P, Gairola CG, and Lehmler HJ (2005) Congener-specific tissue distribution of aroclor 1254 and a highly chlorinated environmental PCB mixture in rats. Environmental Science & Technology 39: 3513–3520. Lackovic M and Sejud J (2002) In: Louisiana Department of Health And Hospitals, Office of Public Health (ed.) Summary of Health-Related Pesticide Incidents Reported in Louisiana From October 1995 Through September 2000. Baton Rouge, LA: Louisiana Department of Health And Hospitals, Office of Public Health. Lucier G, Mcdaniel O, Williams C, and Klein R (1972) Effects of chlordane and methylmercury on metabolism of carbaryl and carbofuran in rats. Pesticide Biochemistry and Physiology 2: 244–255. Mcconnachie PR and Zahalsky AC (1992) Immune alterations in humans exposed to the termiticide technical chlordane. Archives of Environmental Health 47: 295–301. Mendes V, Ribeiro C, Delgado I, Peleteiro B, Aggerbeck M, Distel E, Annesi-Maesano I, Sarigiannis D, and Ramos E (2021) The association between environmental exposures to chlordanes, adiposity and diabetes-related features: A systematic review and meta-analysis. Scientific Reports 11: 14546. Mills PK and Yang R (2005) Breast cancer risk in Hispanic agricultural workers in California. International Journal of Occupational and Environmental Health 11: 123–131. Neta G, Goldman LR, Barr D, Apelberg BJ, Witter FR, and Halden RU (2011) Fetal exposure to chlordane and permethrin mixtures in relation to inflammatory cytokines and birth outcomes. Environmental Science & Technology 45: 1680–1687. NIOSH (2003) Chlordane (CAS RN: 57-74-9). In: NIOSH Pocket Guide to Chemical Hazards & Other Databases CD-ROM. Department of Health & Human Services, Centers for Disease Prevention & Control. National Institute for Occupational Safety & Health. NIOSH (2007) NIOSH Pocket Guide to Chemical Hazards. 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In: Aldrin, Dieldrin, Endrin, Chlordane, Heptachlor, Hexachlorobenzene, Mirex, Toxaphene, Polychlorinated Biphenyls, Dioxins and Furans. International Programme on Chemical Safety (IPCS). Rought SE, Yau PM, Chuang LF, Doi RH, and Chuang RY (1999) Effect of the chlorinated hydrocarbons heptachlor, chlordane, and toxaphene on retinoblastoma tumor suppressor in human lymphocytes. Toxicology Letters 104: 127–135. Shigenaka G (1990) Chlordane in the marine environment of the United States: Review and results from the National Status and trend program. In: NOAA Technical Memorandum NOS OMA 55. Seattle, Washington: National Oceanic and Atmospheric Administration. Tekpli X, Rivedal E, Gorria M, Landvik NE, Rissel M, Dimanche-Boitrel MT, Baffet G, Holme JA, and Lagadic-Gossmann D (2010) The B[a]P-increased intercellular communication via translocation of connexin-43 into gap junctions reduces apoptosis. Toxicology and Applied Pharmacology 242: 231–240. Tryphonas H, Bondy G, Hodgen M, Coady L, Parenteau M, Armstrong C, Hayward S, and Liston V (2003) Effects of cis-nonachlor, trans-nonachlor and chlordane on the immune system of Sprague-Dawley rats following a 28-day oral (gavage) treatment. Food and Chemical Toxicology 41: 107–118. UNEP (2001) Stockholm Convention Secretariat. http://chm.pops.int/. Accessed September 2021. U.S. Environmental Protection Agency (2021) Comptox chemicals dashboard. In: Chlordane. U.S. Environmental Protection Agency. (accessed August 10, 2021). USEPA (1997) Toxicological review of chlordane (Technical). In: Support of Summary Information on the Integrated Rink Information System (IRIS). Washington DC: U.S Environmental Protection Agency. USEPA (2018) Drinking Water Standards and Health Advisories Tables, 2018 edn. Washington, DC: Office of Water, U.S. Environmental Protection Agency. WHO (2004) Chlordane in Drinking Water: Background Document for Development of WHO Guidelines for Drinking-water Quality. Yang C and Chen S (1999) Two organochlorine pesticides, toxaphene and chlordane, are antagonists for estrogen-related receptor alpha-1 orphan receptor. Cancer Research 59: 4519–4524.

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Relevant websites https://pubchem.ncbi.nlm.nih.gov :Pubchem https://comptox.epa.gov/dashboard :ToxCast and EDSP21 dashboard http://npic.orst.edu :National Pesticide Information Center http://www.epa.gov :United States Environmental Protection Agency http://www.osha.gov :United States Occupational Safety and Health Administration

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Chlordecone Nilank Shaha, Hayeon Chungb, and Kaylin Huitsingb, aDepartment of Medical Physiology and Pharmacology, Touro College of Osteopathic Medicine, Middletown, NY, United States; bTouro College of Osteopathic Medicine, Middletown, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S. Biswas, B. Ghosh, Chlordecone, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 846–848, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00275-X.

Chemical profile Properties Exposure limit Uses Background Exposure routes and pathways Toxicokinetics Mechanism of toxicity Acute exposure toxicity Animal Chronic exposure toxicity (animal/human) Animal Human Clinical management Ecotoxicology Summary References Further readings

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Abstract Chlordecone (Kepone), an insecticide and fungicide, which was first introduced commercially in 1958. However, in 1975 due to its toxic effects on workers from industrial exposure, it was banned in the United States. Tremor and hyper-excitability are the principal features of chlordecone toxicity in humans, which occurs due to the alteration in neurotransmitter activity in dopaminergic, serotonergic and a-noradrenergic systems. Incidence of carcinogenicity in chlordecone-exposed humans has been extensively studied and some findings showed connections between chlordecone and prostate cancer while others found no correlations. Chlordecone passes through the GI tract initially and concentrates mainly in the liver when ingested. Main route of elimination is through via fecal excretion. Chlordecone does not get degraded in the environment, however, slow degradation of chlordecone by microorganisms has been observed.

Keywords Alcohol; Cancer; Chlordecone; Exposure; Hydrate; Insecticide; Intoxication; Occupational; Toxicity; Tremor; Workers

Key points

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Although manufacturing of chlordecone was banned decades ago, it still persists in the environment, soil and ecosystem in particular. It bioaccumulates in aquatic organisms (such as fish) and consumable animal stocks. EPA classifies chlordecone as a probable human carcinogen and multiple studies on chlordecone and prostate cancer have shown that there is a risk. However, recent research publications have shown that no significant correlation exist between prostate cancer and chlordecone exposure. Studies show no correlation and reported results are conflicting.

Encyclopedia of Toxicology 4th Edition

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Chemical profile

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Name: Chlordecone Chemical Abstracts Service Registry Number: CAS 143-50-0 Synonyms: 1,3,4-Methano-2H-cyclobuta-(cd)pentalen-2-one; 1,1a,3,3a,4,5,5,5a,5b,6-Decachloroctahydro; Kepone; GC1189; Ciba8514; ENT16,391; NCI-C00191; Decachloroketone; Decachlorotetracyclodecanone; Decachlorotetrahydro4,7-methanoindeneone Chemical/Pharmaceutical/Other Class: Polycyclic chlorinated hydrocarbons Chemical formula: C10Cl10O Chemical Structure:

Properties Chlordecone is a non-volatile, tan to white, crystalline, odorless solid. It has low solubility in water, slight solubility in benzene and light petroleum; and soluble in acetone, ketones, alcohols, and acetic acid.

Exposure limit National Institute for Occupational Safety and Health (NIOSH) Recommended exposure limit (REL) ¼ 0.001 mg/m3 listed as potential occupational carcinogen

Uses Chlordecone had wide application as organochlorine pesticide and fungicide. Specific applications include control of the rust mites in non-fruit-bearing citrus trees, wireworms in tobacco fields, glass mole cricket, slugs, nails and fire ants.

Background Chlordecone (aka kepone), was first produced in the United States in the early 1950s and introduced commercially in 1958 as an insecticide. A large quantity of this compound (approx. 3.6 million pounds) was manufactured in the US between 1951 and 1975. Production of chlordecone stopped in 1975 after the incidence of severe toxicity was reported in industrial workers only chlordecone manufacturing plants. Estimated average annual production before the shutdown of kepone was 882,000 pounds (NCBI, 2023). While production of chlordecone ceased in 1975, Mirex, another insecticide, had continued production through 1977 which produces chlordecone upon degradation. During 1974–1975, the Life Science Products associated with Allied Chemical Corporation, who was the sole producer of kepone in the USA experienced kepone-related intoxication over half of their 133 employees. Illegally discharged kepone in the nearby James river by the factory, also resulted in extensive contamination in the water and marine life throughout the tide-water region in Virginia. The product plant was officially shut down in 1975. Although the ban of chlordecone was implemented within the United States by 1975, use of this insecticide continued throughout the West Indies from 1973 to 1993 in banana fields (Multigner et al., 2016). Typical symptoms of chlordecone intoxication include nervousness, headache and tremor. Most of these effects were described in this review (Biswas and Ghosh, 2014).

Exposure routes and pathways Ingestion is the most common route of exposure. However, inhalational, and dermal routes are also common. Lactating women excrete substantial amounts of chlordecone through breast milk (Barlow and Sullivan, 1982). Chlordecone is very well absorbed following oral exposure, which initially passes through the GI tract, then gets highly concentrated in the liver.

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Toxicokinetics Chlordecone absorption in humans via ingestion, inhalation and dermal contacts has been investigated by the measurement of chlordecone concentration in blood, subcutaneous (s.c) fat and other body fluids and tissues. The half-life of chlordecone in blood and fat tissue is 165 days and 125 days, respectively (NCBI, 2023). Chlordecone is readily absorbed from the GI tract and establishes equilibrium of distribution among most tissues within 24–48 h. In one experiment, male Sprague-Dawley rats received a single dose of 40 mg/Kg [14C]-labeled chlordecone orally in corn oil solution resulting in 10% of the total radioactivity excreted in the first day indicating approximately 90% of the dose was absorbed in the body. A complete biodistribution study done in 32 workers exposed to chlordecone for a period of 3–16 months indicated that high concentrations of chlordecone were present in blood, liver and subcutaneous fat tissues whereas modest amount of chlordecone was found in muscle, gallbladder, bile and stool. In addition, trace amounts were detected in aqueous body fluids such as urine, saliva, gastric juice, cerebrospinal fluids, and blood. Chlordecone showed strong affinity for fat tissues. The high lipophilicity of chlordecone results in a fat to blood ratio of 7:1. In addition, it has been reported that 75% of chlordecone in blood binds with albumin and high-density lipoproteins, decreasing the free toxic concentrations. Main deposition organ for chlordecone is the liver, although it can be found in the brain, kidney and fat (NCBI, 2023). Preferential uptake and slow elimination from the liver was observed in laboratory animals. A study looking at the distribution of chlordecone in rats after a single oral dose of 40 mg/Kg [14C]-labeled chlordecone in corn oil showed initial distribution of high-level radioactivity in adrenal gland followed by liver, lung and the fat tissues. However, 72 h later, the highest concentration of chlordecone was found in the liver throughout the 182-day study period. Liver was found to have the lowest elimination rate compared to other tissues. The preferential binding of chlordecone to albumin and HDL was demonstrated in human, rat, and pig plasma. The study explained preferential retention of chlordecone by the liver is due to its binding to plasma proteins and lipoproteins (Soine et al., 1982). Metabolites of chlordecone include glucuronide conjugates and chlordecone alcohol (Guzelian, 1982; Emond and Multigner, 2022). Metabolism of chlordecone-to-chlordecone hydrate and then to a more stable metabolite, chlordecone alcohol are initiated by the hydration of ketone group by aldo-keto reductase enzyme. Chlordecone is taken up by the liver where it gets reduced to chlordecone alcohol and conjugated with glucuronic acid to form glucuronide conjugate. 72% of unconjugated form of chlordecone, 9% of conjugated form, and 19% of stable polar metabolites resistant to beta-glucuronidase were measured in the bile of occupationally exposed workers (ATSDR, 1995). Activity of reductase enzyme was detected in liver cytosol of humans, rabbits and gerbils, but was absent in laboratory animals including rats, mice, hamsters and Guinea pigs. A study with gerbil showed an existence of species-specific reductase (catalyzing dehydrogenation of chlordecone alcohol to chlordecone) because gerbils exclusively eliminated chlordecone alcohol in the stool and more than twice of chlordecone alcohol was excreted in bile compared to chlordecone and glucuronide conjugate. The ratio of chlordecone-to-chlordecone alcohol in gerbil stool was 10 times higher than the ratio in human stool (Houston et al., 1981). There was a 38% increase in chlordecone reductase activity in gerbils, pre-treated with a single oral dose of chlordecone. Induction of chlordecone reductase activity in pigs was suggested when an increase in the ratio of chlordecone alcohol to chlordecone was observed in the gallbladder bile. Chlordecone induces cytochrome P450 mixed function oxidase enzyme systems in rats. In humans, the hydrate and alcohol-form of chlordecone undergoes glucuronide conjugation by a-D-glucuronic acid. Metabolites of chlordecone do not impart significant toxicity. It is not subjected to metabolism in laboratory animals. Chlordecone is excreted in bile in humans as a reduced metabolite (chlordecone alcohol) and in the form of a glucuronide conjugate (Multigner et al., 2016). Fecal excretion is the main route of elimination, while minimal amounts are eliminated through the kidney. It was shown that by 84 days, 65% of the dose is excreted through feces whereas only 1.6% through urine.

Mechanism of toxicity Cardinal features of chlordecone intoxication in humans are tremor, which occurs due to the alteration in neurotransmitter activity in dopaminergic, serotonergic, and a-noradrenergic systems. At the cellular levels, changes in ATPase activity and calcium homeostasis in the nervous system were observed. Calcium is a vital ion within the central nervous system. It plays a major role in neurotransmitter synthesis, neurotransmitter release via neuronal synapses, and neuronal excitability (Gareri et al., 1995). Calcium uptake in animals exposed to chlordecone decreased following a single oral dose of 40 mg/Kg. Moreover, there was a decreased total protein-bound, myelin, and synaptosomal calcium level following eight consecutive daily oral doses of 25 mg/kg-day in 4- to 6-week-old male ICR mice (Toxicological Review of Chlordecone (Kepone), 2009). In vitro study results suggested alteration of calcium regulation as the main reason for neurological disorder. Chlordecone-induced inhibition of brain mitochondrial and synaptosomal membrane-bound Na+, K+, -ATPase and oligomycin-sensitive Mg2+-ATPase activity. This may result in blocked cellular uptake and storage of neurotransmitters such as catecholamines and g-aminobutyric acid leading to neurotoxicity. Significant decrease in the level of dopamine in the whole brain and striatum was seen in animals exhibiting tremor. Chlordecone-induced hepatic biliary dysfunction could be due to the inhibition of Mg2+-ATPase resulting in decreased hepatic mitochondrial energy production. Chlordecone, at high doses, induces hepatic microsomal drug metabolizing systems. It decreases the tolerance of carbon tetrachloride and a 67-fold increase in the toxicity of a non-lethal dose of carbon tetrachloride in laboratory rats was observed. The hepatotoxicity could be due to decreased energy owing to the disrupted intracellular calcium homeostasis.

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Chlordecone was also found to translocate estrogen receptors from the cytosolic to the nuclear fraction in both isolated rat uteri and ovariectomized immature rats, which showed a decrease in the uterine levels of cAMPs. It shows relatively higher affinity for recombinant human estrogen and progesterone receptors including influencing the cell signaling mechanism in the human body (Ray et al., 2007). Moreover, a study suggested histone methylation alteration with exposure to chlordecone. It showed that exposure to chlordecone during the crucial developmental window (embryonic days E6.5-E15.5) induces an increase in prostatic intraepithelial neoplasia phenotype (PIN) in both F1 (exposed progeny) and F3 (non-exposed progeny) generation as well as decreased histone occupancy of H3K4me3 histone in the promoter of some genes from the sperm of F1 and F3 mice (Legoff et al., 2021).

Acute exposure toxicity Animal Tremor and hyper-excitability are the main neurological symptoms observed in chlordecone treated mice. Animals can also experience loss of body weight owing to decreased water and food consumption. Reversal of the condition in surviving animals is observed following withdrawal of chlordecone administration. Mortality is observed in animals treated with chlordecone with the rate of mortality being dose dependent. The oral LD50 values for chlordecone is 71, 126, 250 and 480 for rabbits, rats, dogs and chickens respectively. Dermally administered chlordecone demonstrates an LD50 value of 434 mg/Kg.

Chronic exposure toxicity (animal/human) Animal Chronic exposure of chlordecone in the diet for 80 weeks at the dose of 0, 15, 30, or 60 ppm for male rats and 0, 30 and 60 ppm for female rats demonstrated clinical signs such as rough hairs, dermatitis and anemia. These results were low to moderate during the first year, but gradually increased during the second year of study. Hepatocellular carcinomas which demonstrated well-differentiation without vascular invasion and metastasis occurred in high dose dependent rats. Liver toxicity was observed in rats, quail, mice and dogs chronically treated with chlordecone. In vitro studies also found similar results (Mehendale and Ray, 1990). Incidence of tumors in other organs, endocrine and reproductive systems were observed. However, incidence rates for all tumor types were not statistically increased as compared to control and no dose-response trend was observed. Growth retardation of the fetus was observed in chlordecone fed pregnant animals given 2 mg/Kg-day, 40 ppm, 75 ppm, and 300 ppm in rats, mice, laying hen, and quail respectively.

Human Health effects of occupational workers (133 men) who were working only in chlordecone manufacturing plants have been extensively studied. It has been found that out of 133 men, 76 experienced neurological symptoms such as nervousness, headache, tremors collectively called, “Kepone Shake Syndrome”, which persisted for 9–10 months after cessation of exposure. Individuals exposed to large doses experienced additional symptoms such as skin rashes, hepatomegaly, joint pain, and oligospermia. Sperm count and motility had returned to normal 5–7 years following cessation of exposure along with treatment of cholestyramine. Hepatomegaly was noticed in 20 out of 32 workers with high blood level (>0.6 mg/mL) of chlordecone. However, no evidence of significant liver toxicity such as liver neoplasia, fibrosis, cholestasis, hepatocellular necrosis was observed. Among factory workers experiencing neurological symptoms such as nervousness, tremors, and ataxia, blood concentrations of chlordecone were 0.009 and 11.8 ppm for subjective and objective reports. In workers without neurological symptoms, blood concentrations ranged between 0.003 and 4.1 ppm. Blood concentrations of 0.005 to 0.0325 ppm were reported in surrounding communities’ residents experiencing neurological symptoms from local contamination of soil and drinking water (Cannon et al., 1978). Currently, there are no reported deaths due to acute or long term chlordecone exposure. However, there have been conflicting reports suggesting a link between cancer and exposure to chlordecone. A study investigating 623 men with prostate cancer and 671 controls in Guadeloupe found that an increase in plasma chlordecone concentration or cumulative exposure index was associated with risk of prostate cancer (Multigner et al., 2010). Organochlorine (OCP), which has been found to be associated with CYP17A1, also increases the risk of non-Hodgkin’s lymphoma (NHL) by hormonal modulation (Dhalla, 2014). Moreover, elevated risk of stomach cancer was shown in several studies involving agricultural women workers with long term chlordecone exposure compared to the general population (Luce et al., 2020). Nonetheless, a meta-analysis research showed no evidence of an association between specific organochlorine pesticides and increased incidence of prostate cancer (Lewis-Mikhael et al., 2016). To further investigate, a study measured organochlorine pesticides (including chlordecone) content in two different ethno-geographical origins: Mainland France or French West Indies. This study measured the OCPs concentrations in periprostatic adipose tissue (PPAT), which reflects cumulative exposure and prostate cancer aggressiveness (Multigner et al., 2016). They found that Chlordecone was only found in the African-Caribbean population and most OCP concentrations were positively correlated with age, and some with BMI. They concluded that there was no significant association between OCPs content and risk of prostate

Chlordecone Table 1

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Outlines the symptomatology of Chlordecone toxicity in humans.

Organ system

Toxic effect

Neurological

Irritability Poor Short-Term Memory Gait Ataxia Slurred Speech Tremors Weakness Oligospermia Hepatomegaly

Musculoskeletal Reproductive Gastrointestinal

disease after adjusting for age, BMI, and polyunsaturated fatty acid composition of PPAT (Antignac et al., 2023). Prenatal exposure to chlordecone and development of obesity in children was studied in Guadalupe. The study measured 575 children’ adiposity indicators at age seven and chlordecone concentrations in cord blood at birth and in the children’s blood at 7 years of age. It showed that prenatal chlordecone exposure tended to be associated with increased adiposity at 7 years of age, particularly in boys, but the data was not statistically significant to link the findings together (Costet et al., 2022). Although direct carcinogenesis has not been a feature seen with chlordecone exposure in humans, it should be further investigated (Table 1).

Clinical management Due to the enterohepatic circulation of chlordecone and naturally low secretion rates, the first step for toxicity treatment would be removing the individual from the source. In case of ingestion, emesis could be induced followed by administration of activated charcoal and cathartics. Oil-based cathartics are avoided. To prevent the re-absorption of chlordecone into the GI tract, cholestyramine treatment can be used for chlordecone intoxication. Cholestyramine is an anion-exchange resin that binds to chlordecone resulting in a non-absorbable complex. This non absorbable complex increases the fecal excretion thus preventing its reabsorption in the GI tract. Cholestyramine treatment given to the industrial workers exposed to chlordecone was found to reduce the average t1/2 of chlordecone in the blood from 165 to 80 days and from 125 days to 64 days in fat tissue. Clinical manifestations subsided as the chlordecone was cleared in the body. More recent studies show carbonaceous materials as an efficient decrease of bioavailability of chlordecone in the soil (El Wanny et al., 2022; Comte, 2022). A significant decrease in relative bioaccessibility and CD concentrations in liver were observed when 5 carbonaceous materials (ORBO, DARCO, Coco CO2, Oak P1.5, Sargasso biochar) were tested and used to amend Guadeloupe nitisol soil at 2% (Feidt et al., 2021).

Ecotoxicology Estimated half-life of chlordecone in soils is between 1 and 2 years, whereas in air, it is up to 50 years (UNEP, 2003). Chlordecone does not get degraded in the environment and does not undergo hydrolysis or photolysis. However, microorganisms can degrade chlordecone slowly. In a study observing the effects of chlordecone in sandy loam soil compared to silty loam soil, it was discovered that gram negative bacteria (b- and ɣ- Proteobacteria, Planctomycetes, and Bacteroidetes) was significantly modified in sandy loam. Chlordecone induced changes in microbial community taxonomic composition and function of the soil suggested microbial toxicity of chlordecone (Merlin et al., 2016). Chlordecone gets absorbed into soil and sticks to suspended particulate matters in water. In the French West Indies, animals may be exposed to chlordecone from the contaminated soil (Deyris, 2023). Chlordecone ingestion study involving Caribbean region pigs manipulated chlordecone levels in the soil and calculated how much chlordecone the pigs ingested by looking at their perirenal adipose tissue and fecal samples. Different breeds of pigs ingested about half a kg of dry soil per animal per day. Simulated results showed that when CD soil is 0.1 mg kg−1, CD concentrations in perirenal adipose tissue exceeded the maximum residue limit set at 0.02 mg CD per kg of fat regardless of breed and soil ingestion. Drinking water can also expose animals to pollutants such as Chlordecone (Collas et al., 2023). Chlordecone also significantly accumulates in fish and other aquatic organisms and consumption of contaminated fish and seafood is the main route of chlordecone exposure in the general population. During a study examining micronucleus frequency in fish exposed to chlordecone, the level of genotoxicity to cichlid fish erythrocytes was evaluated. This examination evaluated the clastogenic and eugenic agents that have the potential to induce abnormal chromosomal damage. It was determined that chlordecone exposure at sublethal levels significantly increased the frequency of micronuclei formation in erythrocytes. The level of damage correlated with the level of exposure. The significant presence of micronuclei formation due to chlordecone exposure raises the concern of toxic symptoms present in organisms consuming large quantities of chlordecone contaminated aquatic animals in environments where there is heavy chlordecone pollution (Asifa et al., 2019).

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Summary Chlordecone is a toxic pesticide which was banned in the US in 1975 for its detrimental effects on humans. However, chlordecone after-effects are still well studied and seen in Guadalupe and the French West Indies area where they used chlordecone until 1993. The intoxication mechanism, adverse effects, and degradation has been well studied in both animals and humans, however, the carcinogenic effects of chlordecone needs to be further investigated to conclude its effect on certain types of cancers (Deloumeaux and Bhakkan-Mambir, 2022).

See also: Occupational toxicology; Neurotoxicity; Organochlorine insecticides

References Antignac JP, Figiel S, Pinault M, Blanchet P, Bruyère F, Mathieu R, et al. (2023) Persistent organochlorine pesticides in periprostatic adipose tissue from men with prostate cancer: Ethno-geographic variations, association with disease aggressiveness. Environmental Research 216: 114809. Asifa KP, Vidya PV, and Chitra KC (2019) Effect of chlordecone on the reproductive potential of the cichlid fish, pseudetroplus maculatus (Bloch, 1795). Journal of Aquatic Research and Marine Sciences 2(2): 163–177. https://doi.org/10.29199/2637-9309/arms-202033. ATSDR, U (1995) Toxicological profile for MIREX and chlordecone. US Department of Health and Human Services. August 1995. Barlow SM and Sullivan FM (1982) Chlordecone. Reproductive Hazards of Industrial Chemicals. Biswas S and Ghosh B (2014) Chlordecone. Encyclopedia of Toxicology, 3rd edn, 2014: 846–848. Cannon SB, Veazey JM Jr., Jackson RS, Burse VW, Hayes C, Straub WE, et al. (1978) Epidemic Kepone poisoning in chemical workers. American Journal of Epidemiology 107(6): 529–537. Chemicals, UNEP (2003) Regionally based assessment of persistent toxic substances: Global report 2003. Collas C, Gourdine JL, Beramice D, Badot PM, Feidt C, and Jurjanz S (2023) Soil ingestion, a key determinant of exposure to environmental contaminants. The case study of chlordecone exposure in free-range pigs in the French West Indies. Environmental Pollution 316: 120486. Comte I, et al. (2022) Long-term pollution by chlordecone of tropical volcanic soils in the French West Indies: New insights and improvement of previous predictions. Environmental Pollution (Barking, Essex: 1987) 303. https://doi.org/10.1016/j.envpol.2022.119091. 119091. Costet N, Lafontaine A, Rouget F, et al. (2022) Prenatal and childhood exposure to chlordecone and adiposity of seven-year-old children in the Timoun mother-child cohort study in Guadeloupe (French West Indies). Environmental Health : A Global Access Science Source 21(1): 42. https://doi.org/10.1186/s12940-022-00850-2. Deloumeaux J, Bhakkan-Mambir B, Desroziers, et al. (2022) Urological cancers in French overseas territories: A population-based cancer registry pooled analysis in Martinique, Guadeloupe and French Guiana (2007–2014). Journal of Epidemiology and Global Health 12(3): 232–238. Deyris PA, et al. (2023) Efficient removal of persistent and emerging organic pollutants by biosorption using abundant biomass wastes. Chemosphere 313: 137307. https://doi.org/ 10.1016/j.chemosphere.2022.137307. Dhalla A (2014) Interactions between Cytochrome P450 Genetic Polymorphisms and Plasma Organochlorines in Non-Hodgkin Lymphoma. Doctoral dissertation University of British Columbia. El Wanny N, Le Roux Y, Fournier A, Baroudi M, Woignier T, Feidt C, and Delannoy M (2022) Organochlorine POPs sequestration strategy by carbonaceous amendments of contaminated soils: Toward a better understanding of the transfer reduction to laying hens. Journal of Hazardous Materials 434: 128871. Emond C and Multigner L (2022) Chlordecone: development of a physiologically based pharmacokinetic tool to support human health risks assessments. Archives of Toxicology 96(4): 1009–1019. Feidt C, El Wanny N, Ranguin R, Gaspard S, Baroudi M, Yacou C, et al. (2021) In vitro and in vivo assessment of a CLD sequestration strategy in Nitisol using contrasted carbonaceous materials. Environmental Geochemistry and Health 1–10. Gareri P, Mattace R, Nava F, and De Sarro G (1995) Role of calcium in brain aging. General Pharmacology 26(8): 1651–1657. https://doi.org/10.1016/0306-3623(95)00043-7. PMID: 8745152. Guzelian PS (1982) Comparative toxicology of chlordecone (kepone) in humans and experimental animals. Annual Review of Pharmacology and Toxicology 22: 89–113. Houston TE, Mutter LC, Blanke RV, and Guzelian PS (1981) Chlordecone alcohol formation in the Mongolian gerbil (Meriones unguiculatus): A model for human metabolism of chlordecone (kepone). Toxicological Sciences 1(3): 293–298. Legoff L, D’Cruz SC, Lebosq M, Gely-Pernot A, Bouchekhchoukha K, Monfort C, et al. (2021) Developmental exposure to chlordecone induces transgenerational effects in somatic prostate tissue which are associated with epigenetic histone trimethylation changes. Environment International 152: 106472. Lewis-Mikhael AM, Bueno-Cavanillas A, Guiron TO, Olmedo-Requena R, Delgado-Rodríguez M, and Jiménez-Moleón JJ (2016) Occupational exposure to pesticides and prostate cancer: a systematic review and meta-analysis. Occupational and Environmental Medicine 73(2): 134–144. Luce D, Dugas J, Vaidie A, Michineau L, El-Yamani M, and Multigner L (2020) A cohort study of banana plantation workers in the French West Indies: first mortality analysis (2000–2015). Environmental Science and Pollution Research 27(33): 41014–41022. Mehendale HM and Ray SD (1990) Inhibition of cell division in hepatoma cell cultures by chlordecone and carbon tetrachloride combination. Toxicology In Vitro 4(3): 179–183. https:// pubmed.ncbi.nlm.nih.gov/20837413/. Merlin C, Devers M, Béguet J, et al. (2016) Evaluation of the ecotoxicological impact of the organochlorine chlordecone on soil microbial community structure, abundance, and function. Environmental Science and Pollution Research 23: 4185–4198. https://doi.org/10.1007/s11356-015-4758-2. Multigner L, Ndong JR, Giusti A, and et. Al. (2010) Chlordecone exposure and risk of prostate cancer. Journal of Clinical Oncology 28(21): 3457–3462. Multigner L, Kadhel P, Rouget F, Blanchet P, and Cordier S (2016) Chlordecone exposure and adverse effects in French West Indies populations. Environmental Science and Pollution Research International 23(1): 3–8. https://doi.org/10.1007/s11356-015-4621-5.

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National Center for Biotechnology Information (2023) PubChem Compound Summary for CID 299, Chlordecone. Retrieved February 10, 2023 from https://pubchem.ncbi.nlm.nih.gov/ compound/Chlordecone. Ray S, Xu F, Li P, Sanchez NS, Wang H, and Das SK (2007) Increased level of cellular Bip critically determines estrogenic potency for a xenoestrogen Kepone in the mouse uterus. Endocrinology 148(10): 4774–4785. Soine PJ, Blanke RV, Guzelian PS, and Schwartz CC (1982) Preferential binding of chlordecone to the protein and high density lipoprotein fractions of plasma from humans and other species. Journal of Toxicology and Environmental Health, Part A Current Issues 9(1): 107–118. Toxicological Review of Chlordecone (Kepone) (2009) US. Environmental Protection Agency. Washington, DC. EPA/635/R-07/004F. http://www.epa.gov/iris/toxreviews/1017tr.pdf.

Further readings Dromard CR, et al. (2022) Temporal variations in the level of chlordecone in seawater and marine organisms in Martinique Island (Lesser Antilles). Environmental Science and Pollution Research International 29(54): 81546–81556. https://doi.org/10.1007/s11356-022-21528-9. Fiedler H, Li X, and Zhang J (2023) Persistent organic pollutants in human milk from primiparae - correlations, global, regional, and national time-trends. Chemosphere 313: 137484. https://doi.org/10.1016/j.chemosphere.2022.137484.

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Chlordimeform Lucio G Costa, Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA, United States © 2024 Elsevier Inc. All rights reserved. This is an update of L.G. Costa, Chlordimeform, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 849-850, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00112-3.

Chemical profile Introduction Uses Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Animal Human Chronic toxicity Animals Humans Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Exposure standards and guidelines Summary and conclusion References

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Abstract Chlordimeform is a formamidine insecticide/acaricide, once used extensively on fruits and cotton. Its insecticidal action is due to activation of octopamine receptors, and its toxicity in mammals is mediated by activation of alpha2-adrenergic receptors. Chlordimeform is well absorbed and rapidly metabolized to compounds of higher toxicity. Demethylated chlordimeform has a higher acute toxicity and higher affinity for alpha2-adrenoceptors, while 4-chloro-o-toluidine has carcinogenic properties. Chlordimeform is a probable human carcinogen and has been withdrawn from the market since the early 1990s.

Keywords 4-Chloro-o-toluidine; Acaricide; Alpha2-adrenergic receptors; Bladder cancer; Carcinogen; Chlordimeform; Formamidine; Hypotension; Insecticide

Chemical profile

• • • • •

Chemical Abstract Service Registry Numbers: CAS 6164-98-3 (base); CAS 19750–95-9 (hydrochloride salt) Synonyms: Chlorphenamidine; ENT 27335 (base); ENT 27567 (salt); Chlorphenamide; Galecron™ Chemical Formula: C10H13ClN2 Chemical Name: N0 (4-chloro-2-methylphenyl)-N,N-dimethylformamidine Chemical structure: CH3 Cl

N

CH

N CH3

CH3

Introduction Chlordimeform is a member of the formamidine family of insecticides/acaricides, now represented by Amitraz [N0 -2,4-(dimethylphenyl)-N-N ((2,4-dimethylphenyl) imino) methyl-N-methanimidamide], as chordimeform was withdrawn from the market in Encyclopedia of Toxicology 4th Edition

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1992 due to potential carcinogenicity. Formamidines are a unique class of insecticides as they target the adrenergic nervous system. In insects they activate the octopamine receptors, while in mammals a primary target is represented by the alpha2-adrenergic receptors (Costa, 2020).

Uses Chlordimeform was used as a broad spectrum acaricide/insecticide. It is particularly effective against mites and ticks, and some Lepidoptera insects. It was extensively used in agriculture, particularly in fruits such as apples, cherries, and strawberries, as well as in rice and cotton. It was also used in veterinary medicine as an acaricide. In 1976 its use was suspended because of suspected carcinogenicity, but it was re-introduced in most countries in 1978 for insect control in cotton. In the late 1980s-early 1990s, production and use ceased worldwide because of carcinogenicity of one of its major metabolites.

Environmental fate and behavior Chlordimeform has a relatively high volatility, and low water solubility, though the salt is highly water soluble. Chlordimeform undergoes rapid microbial degradation in soil and is rapidly degraded in plants. There is no evidence of bioaccumulation of chlordimeform in the food chain.

Exposure and exposure monitoring Exposure to chlordimeform occurred in occupational settings (manufacture, formulation, use) and was primarily due to dermal exposure with minor contribution from inhalation. Some exposure may have occurred in the general population upon aerial spraying in cotton fields. Exposure also occurred because of residues present in raw and processed foods. Cases of accidental or intentional oral exposure to chlordimeform have also been reported, particularly in China.

Toxicokinetics Chlordimeform is well absorbed from all routes of exposure. The base is better absorbed dermally than the hydrochloride salt. Chlordimeform undergoes significant metabolism and is rapidly excreted, primarily through the urine. Chlordimeform’s metabolism plays a most relevant role in its toxicity, as the two demethylated metabolites demethylchlordimeform and didemethylchlordimeform have higher acute toxicity than the parent compound. Two other metabolites of chlordimeform, 4-chloro-o-toluidine and N-formyl-4-chloro-o-toluidine, are believed to be responsible for the carcinogenic effects of chlordimeform (Popp et al., 1992). Neither chlordimeform nor any of the metabolites accumulate in tissues. However, despite its discontinued use since 1993, low levels of chlordimeform have been found in human adipose tissue in residents of Southeast China as late as 2008.

Mechanism of toxicity Earlier studies indicated that a primary mechanism of chlordimeform’s toxicity was inhibition of monoamine oxidase; however, subsequent findings suggested that this biochemical effect does not play a significant role in its acute toxicity. Other reported effects of chlordimeform include inhibition of oxidative phosphorylation and inhibition of calcium channels. The chemical structure of chlordimeform is similar to the structure of norepinephrine and of other sympathetic amines. In insects, chlordimeform exerts its toxicity by activating octopamine receptors. The equivalents of the latter in mammals are the alpha2-adrenergic receptors. Chlordimeform acts as an agonist at alpha2-adrenoceptors, and its demethylated metabolite, which is more acutely toxic, is also 400-fold more potent toward alpha2-adrenoceptors (Costa et al., 1988). Signs and symptoms of chlordimeform’s acute exposure can be explained by activation of alpha2-adrenergic receptors (Costa, 2020).

Acute and short-term toxicity Animal Chlordimeform has a moderate acute toxicity in rodents (oral LD50 ¼ 200–300 mg/kg b.w.). Main signs upon acute exposure are hypothension, hypothermia, hyperglycemia, and bradycardia. Anemia, and kidney and liver damage have also been reported in short-term animal studies.

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Human Effects of chlordimeform in humans include severe hypotension, cardiac toxicity, nausea and vomiting; central nervous system depression, and blurred vision. In some cases, impairments of kidney and liver functions have also been reported.

Chronic toxicity Animals The main adverse effect of chlordimeform is carcinogenicity.

Humans Cases of haematuria were reported in workers in a chlordimeform packaging plant. Various studies in individuals exposed to chlordimeform indicate an increased risk of bladder cancer, primarily when exposure to 4-chloro-o-toluidine also occurred (Popp et al., 1992). Chlordimeform is classified by IARC and by EPA as a probable human carcinogen (Group 2A and B2, respectively), based on the findings in mice and on limited information in humans.

Immunotoxicity There is no evidence that chlordimeform induces significant immunotoxic effects.

Reproductive toxicity No significant treatment-related effects were found in multigenerational reproductive studies in rodents. Chlordimeform does not appear to be a teratogen.

Genotoxicity Chlordimeform is negative in most in vitro tests for mutagenicity and genotoxicity in both bacterial and mammalian systems. In contrast, 4-chloro-o-toluidine tested positive in various in vitro tests for DNA damage and cell transformation.

Carcinogenicity The main adverse effect of chlordimeform upon chronic exposure is carcinogenicity, and two metabolites, 4-chloro-o-toluidine and N-formyl-4-chloro-o-toluidine, appear to be responsible for this effect. 4-Chloro-o-touidine in particular, is a more potent carcinogen than chlordimeform, and is believed to be responsible for the tumors observed in rodents and in humans. The exact mechanism of carcinogenicity by 4-chloro-o-toluidine is unclear but may involve genotoxic mechanisms (IPCS, 1998). In mice, a dose-dependent incidence of hemangioendotheliomas, particularly in liver, kidney, and spleen, have been found in two studies. Hemangiomas and hemangiosarcomas were also found in mice upon chronic dietary exposure to the chlordimeform metabolite 4-chloro-o-toluidine. No treatment related increase in tumor incidence was reported in rats.

Clinical management Treatment for chlordimeform intoxication is supportive and symptomatic. Adequate ventilation should be supported. Diazepam may be used for seizures, and atropine for bradycardia. Hypotension may respond to the use of an inotrope. Gastric lavage may be considered shortly after ingestion, and oral activated charcoal would be appropriate later. Antagonists of alpha2-adrenergic receptors, such as yohimbine, have been shown to reverse acute toxicity in animals, but their efficacy has not been assessed in humans.

Ecotoxicology Limited studies indicate that chlordimeform has moderate to low toxicity toward fish and avian species.

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Exposure standards and guidelines The last available temporary Acceptable Daily Intake (ADI) for chlordimeform was 0.0001 mg/kg/day, established in 1978. The temporary ADI was withdrawn in 1987.

Summary and conclusion Chlordimeform was a formamidine insecticide/acaricide whose insecticidal action is due to activation of octopamine receptors, while its toxicity in mammals is mediated by activation of alpha2-adrenergic receptors. Chlordimeform is well absorbed and rapidly metabolized to compounds of higher toxicity. Demethylated chlordimeform has a higher acute toxicity and higher affinity for alpha2-adrenoceptors, while 4-chloro-o-toluidine has carcinogenic properties. Chlordimeform is a probable human carcinogen and has been withdrawn from the market since the early 1990s.

References Costa LG (2020) Neurotoxicity of amitraz, a formamidine pesticide. In: Aschner M and Costa LG (eds.) Neurotoxicity of Pesticides. Advances in Neurotoxicology. vol. 4, pp. 255–276. San Diego: Academic Press. Costa LG, Olibet G, and Murphy SD (1988) Alpha2-adrenoceptors as a target for formamidine pesticides: In vitro and in vivo studies in mice. Toxicology and Applied Pharmacology 93: 319–328. IPCS (International Programme on Chemical Safety) (1998) Chlordimeform (Environmental Health Criteria). vol. 199, Geneva: World Health Organization159. Popp W, Schmieding W, Speck M, Vahrenholz C, and Norpoth K (1992) Incidence of bladder cancer in a cohort of workers exposed to 4-chloro-o-toluidine while synthesizing chlordimeform. British Journal of Industrial Medicine 49: 529–531.

Chlorfenvinphos Mitra Geier and Svetlana E Koshlukova, Department of Pesticide Regulation, California Environmental Protection Agency, Sacramento, CA, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S.E. Koshlukova, N.R. Reed, Chlorfenvinphos, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 851–854, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01110-6.

Chemical profile Background Regulatory history Illness reports SENSOR illness report data Spanish poison control center database California Department of Pesticide Regulation Pesticide Illness Surveillance Program Uses/occurrence Exposure and exposure monitoring Toxicokinetics (ADME) Mechanism of toxicity New approach methodologies Toxicity forecaster (ToxCast) Endocrine disruption screening program for the 21st century (EDSP21) Physiologically based pharmacokinetic-pharmacodynamic (PBPK-PD) modeling Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive and developmental toxicity Animal Human Developmental neurotoxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Toxicogenomics Clinical management Environmental fate and behavior Ecotoxicology Exposure standards and guidelines Acknowledgment References Further reading

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Abstract Chlorfenvinphos (CAS 470-90-6) is a chlorinated organophosphorus ester used as an insecticide and acaricide. Its primary mode of action is inhibition of the enzyme acetylcholinesterase in insect and mammalian nervous systems leading to cholinergic hyperstimulation. It is a direct-acting cholinesterase inhibitor and does not require metabolic activation to yield anticholinesterase activity. Chlorfenvinphos represents the oldest generation of OPs that exhibit marked mammalian toxicity. In mammals, chlorfenvinphos also causes reproductive and developmental toxicity, and alters the immune response. Chlorfenvinphos is highly toxic to fish, birds, bees and aquatic invertebrates. Based on its physiochemical properties, chlorfenvinphos is expected to leach into groundwater, but not bioaccumulate. Occupational exposure may occur through inhalation or skin contact with treated animals, and the general public may be exposed through groundwater leaching or contact with contaminated soils. Concerns regarding high acute toxicity and developmental and reproductive effects led to the cancelation of all its uses in the United States, Canada, European Union, Cambodia, India, and Lao PDR, and restriction to veterinary uses in Australia. The World Health Organization has classified chlorfenvinphos as “extremely hazardous”.

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Chlorfenvinphos Keywords 2-[Chloro-1-(2,4-dichlorophenyl)ethenyl] diethyl phosphate; Acetylcholinesterase; Atropine; Carcinogen; Chlorfenvinphos; Cholinergic effects; Developmental toxicity; Immunotoxicity; Neurotoxicity; Organophosphate; Organophosphorus; Oxidative dealkylation; Oxime; Pesticide

Key points

• • • • • •

Chlorfenvinphos is a chlorinated organophosphorus ester. It primarily causes toxicity through inhibition of the enzyme acetylcholinesterase in the nervous system. Chlorfenvinphos causes neurotoxicity, and reproductive and developmental toxicity in mammals. Chlorfenvinphos is highly toxic to fish, birds, aquatic invertebrates and honey bees. It is classified by the World Health Organization as “extremely hazardous”. Concerns regarding human and ecological risks have resulted in complete and partial bans around the world. However, chlorfenvinphos is still used in many countries on crops and as a veterinary medicine for combating ectoparasites.

Abbreviations ChE CYP JMPR KOC KOW LC50 LD50 LOEC LOEL MRL NOEL OP ppm RBC USEPA

Cholinesterase Cytochrome P450 Enzyme Joint FAO/WHO Meeting on Pesticide Residues Soil Organic Carbon-Water Partition Coefficient Octanol-Water Partition Coefficient Median Lethal Concentration Median Lethal Dose Lowest Observed Effect Concentration Lowest Observed Effect Level Minimal Risk Level No Observed Effect Level Organophosphorus Parts Per Million Red Blood Cell United States Environmental Protection Agency

Chemical profile

• • • • • • • •

Name: [(EZ)-2-chloro-1-(2,4-dichlorophenyl)ethenyl] diethyl phosphate Synonyms: Chlorfenvinphos, Chlorfenvinfos, Chlofenvinfos, Chlorphenvinfos Chlorphenvinphos, Chlorofenvinphos, 2Chloro-1-(2,4-dichlorophenyl)vinyl diethyl phosphate, Chlorfenvinphos solution, Clofenvinfos (INN), Dermaton (TN), Supona (TN), AC1NTBS1, AC1Q3MCM, 2-Chloro-1-(2,4-dichlorophenyl)ethenyl diethyl phosphate CAS Number: CAS 63-25-2 Chemical class: Organophosphate; Insecticide, Acaricide Molecular formula: C12H14Cl3O4P Molecular weight: 359.57 g mole−1 Density: 1.53 g cm−3 at 25
C Vapor pressure: 0.000008 mmHg at 25
C

Chlorfenvinphos

•

Chemical Structure:

• • • • • •

Boiling point: 120
C Melting point: −23 to −19
C Flash point: No data. Conversion Factor (ppm to mg m−3): 1 ppm ¼ 14.7 mg m−3 at 25
C Appearance: Colorless to amber liquid Odor: Mild odor

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Background Chlorfenvinphos is a chlorinated organophosphorus ester manufactured as an insecticide and acaricide. Like other organophosphorus insecticides (OPs), its most prominent toxicity is inhibition of the enzyme acetylcholinesterase (AChE) in insects and mammals. Chlorfenvinphos represents the oldest generation of OPs that exhibit marked mammalian toxicity. It is a direct-acting cholinesterase inhibitor and does not require metabolic activation to yield anticholinesterase activity (ATSDR, 1997; Hutson et al., 1967). First sold in 1963, chlorfenvinphos has been used extensively to control insect pests on domestic animals, and in households and animal buildings (NRA, 2000). Formulations include emulsifiable concentrates, topical aerosol sprays, wound dressings, and dip and jetting liquids (NRA, 2000). It continues to be used in several countries as an important veterinary medicine for ectoparasites in livestock (APVMA, 2021; NRA, 2000).

Regulatory history Originally manufactured by Shell International Chemical Company Ltd., Ciba AG and Allied Chemical Corporation in 1963, chlorfenvinphos was extensively used to control insect pests on domestic animals, in households, and animal buildings (ATSDR, 1997; NRA, 2000). Concerns regarding high acute toxicity and developmental and reproductive effects led to the cancelation of all its uses in the U.S. in 1991, cancelation of all uses in Canada in 1995, and classification of chlorfenvinphos as extremely hazardous by the World Health Organization (WHO) in 1992 (ATSDR, 1997; Koshlukova, 2021; NRA, 2000). Chlorfenvinphos was banned in the European Union (EU) in 2006, and has been banned from use in Cambodia, India, and Lao PDR (European Commission, 2002; Food and Agriculture Organization of the United Nations, 2015; NRA, 2000). In 2000, the Australian Pesticides and Veterinary Medicines Authority (APVMA) recommended canceling agricultural uses of chlorfenvinphos and restricting its veterinary uses (NRA, 2000). As of 2021, chlorfenvinphos remains in use in Australia as an ectoparasite and fly strike control agent in cattle and sheep. The Agency for Toxic Substances and Disease Registry (ATSDR) reported in 1997 that chlorfenvinphos was detected in the soil, surface and groundwater samples in at least one of the 1428 National Priorities List (NPL) sites (the most hazardous waste sites) in the U.S. No information is available on current detections or the number of sites evaluated for chlorfenvinphos since 1997 (ATSDR, 1997). It is regulated under “The Emergency Planning and Community Right-to-Know Act of 1986” (EPCRA) which requires manufacturers or users to report annual release to any environmental media (ATSDR, 1997; Code of Federal Regulations, 2020). Chlorfenvinphos is also one of the 38 high priority hazardous chemicals and chemical warfare agents for which the United States Environmental Protection Agency (US EPA) is developing advisory levels for exposure in case of large scale disasters or environmental contamination (US EPA, 2011).

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Illness reports Data on illnesses or injuries associated with exposure to chlorfenvinphos were identified from three sources.

SENSOR illness report data In the United States, the Sentinel Event Notification System for Occupational Risk Pesticides (SENSOR-Pesticides) program, administered by the National Institute for Occupational Safety and Health (NIOSH), maintains a database of acute pesticide illness reports in participating states. Thirteen states participate in the program’s data collection: California, Florida, Illinois, Iowa, Louisiana, Michigan, Nebraska, New Mexico, New York, North Carolina Oregon, Texas, and Washington. Between 1998 and 2011, two cases of acute chlorfenvinphos-related illness were reported in Florida:

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In 2006, a 64-year-old was reported to have inhaled a mixture containing chlorfenvinphos and hypochlorous acid, and exhibited symptoms including throat irritation and burning lungs. In 2007, a 65-year-old was reported to have ingested chlorfenvinphos and exhibited vomiting and diarrhea.

Spanish poison control center database Over 200 records of non-occupational poisonings are available during 1995–2010 from the Spanish Poison Control Center Database. The majority were accidental oral exposure from chlorfenvinphos veterinary products (93.4% chlorfenvinphos) (Martínez and Ballesteros, 2012). No information was available on the symptoms or health effects associated with the exposure.

California Department of Pesticide Regulation Pesticide Illness Surveillance Program DPR’s Pesticide Illness Surveillance Program (PISP) maintains a database of pesticide-related illnesses and injuries reported in California. Case reports are received from physicians and Workers’ Compensation records. There was one report linked to chlorfenvinphos between 1992 and 2017 (the most recent available data) (DPR, 2021). The health effects attributed to exposure to chlorfenvinphos alone was related as definite for the case. Exposure occurred from ingestion. Clinical signs of chlorfenvinphos exposure included irritated throat, nausea, vomiting, weakness, shortness of breath, coughing, and diaphoresis.

Uses/occurrence Chlorfenvinphos has been used extensively in veterinary products (dip, dust and collars) for flea and tick control on pets and domestic animals, and in dairy barns, milk rooms, poultry houses and other animal buildings (APVMA, 2021). Agriculturally, it was used on potatoes, rice and maize, and for control of soil insects and nematodes (APVMA, 2021).

Exposure and exposure monitoring Insecticidal use of chlorfenvinphos can result in human exposure. Dermal and inhalation pathways are likely to dominate workers’ exposures from handling treated cattle, sheep and wool wax, or re-entering treated fields (Ames et al., 1989). Exposure of the general public may occur from ingestion of imported foods and lanolin-containing pharmaceutical products (NRA, 2000). In countries like the U.S. where chlorfenvinphos is not in use, exposures may continue from run off and leaching from hazardous waste disposal sites (ATSDR, 1997). Occupational exposure remains possible for workers at contaminated waste sites or in legal production. Exposure is also likely for members of the general public living near hazardous waste sites, where chlorfenvinphos has been detected in surface and groundwater and soil samples (ATSDR, 1997). At a given exposure concentration, children generally have higher body burden due to their higher intake (inhalation volume, amount of food intake) or contact on a per body weight basis.

Toxicokinetics (ADME) The estimated oral absorption of chlorfenvinphos is 86–94% in rats, dogs and humans (Hutson et al., 1967). In rats, peak blood level of chlorfenvinphos and its metabolites occurs within 1 h of dosing (Ikeda et al., 1992). Dermal and inhalation absorption of chlorfenvinphos in humans is evident in the detection of the parent compound and metabolites in various tissues and organs, and by the inhibition of AChE activities (NRA, 2000). In rats, dogs, and humans, chlorfenvinphos is extensively metabolized through oxidative dealkylation by the liver cytochrome P450 enzymes (CYP) and produces 2-chloro-1-(2,4-dichlorophenyl) vinylethylhydrogen phosphate, 2,4-dichloromandelic acid and acetaldehyde possibly through epoxide intermediates (Hutson et al., 1967; Ikeda et al., 1991). In humans, chlorfenvinphos and its metabolites are found in serum, cervical mucus, follicular and sperm fluids, and milk (Wagner et al., 1990). It also binds to plasma proteins, e.g., albumin (Tarhoni et al., 2008). Chlorfenvinphos is detected in omental fat of sheep 3 days after applications

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in a dip or spray race (Robinson et al., 1966). Chlorfenvinphos metabolism in liver slices is more rapid in dogs, rabbits and mice than in rats (Donninger et al., 1972). Urine is the main route of elimination; 86–89% of a single oral dose is excreted in urine over 4 days in rat and dogs, and 94% is excreted within 24 h in humans (Hutson et al., 1967; Wouters and Van Hoeven-Arentzen, 1994). About 2–16% is excreted in the feces (Hutson et al., 1967). An elimination half-life (t1/2) of 24 h is reported in rabbits (Wouters and Van Hoeven-Arentzen, 1994). Chlorfenvinphos was detected in cow milk (Wouters and Van Hoeven-Arentzen, 1994). The major metabolites in excreta are 2-chloro-1-(2,4-dichlorophenyl) vinylethylhydrogen phosphate, 1-(2,4-dichlorophenyl) ethanol, 1-(2,4-dichlorophenyl) ethanediol, 2,4-dichloromandelic acid, 2,4-dichlorobenzoyl glycine and glucuronide conjugates (Hutson et al., 1967).

Mechanism of toxicity Chlorfenvinphos causes toxicity primarily by binding and inhibiting the serine hydrolase AChE. In the nervous system, AChE hydrolyzes the neurotransmitter ACh thereby terminating its synaptic action. AChE inhibition increases the availability of acetylcholine at the neural synapse, leading to cholinergic overstimulation, autonomic and neuromuscular disfunction, and at higher levels, resulting in coma and death. Chlorfenvinphos’ major metabolites do not inhibit ChE (Hutson et al., 1967). Chlorfenvinphos also inhibits butyrylcholinesterase, which may function as a molecular scavenger for anticholinesterase compounds in the blood or substitute for AChE where it is low (Roszczenko et al., 2013). Acute exposure to chlorfenvinphos and the resulting ChE inhibition may result in long term hyperactivity of the cholinergic system. This can lead to persistent alterations of the brain cholinergic-dopamine balance and a prolonged reduced sensitivity to psychostimulants such as amphetamine (Gralewicz et al., 2011; Gralewicz et al., 2000; Gralewicz et al., 2010; Gralewicz et al., 2002; Lutz et al., 2006). Other non-ChE targets of chlorfenvinphos include hepatic CYPs, lipid metabolism, oxidative stress, aromatic amino acid transferases, and cytotoxicity (Ikeda et al., 1991; Lukaszewicz-Hussain, 2008; Roszczenko et al., 2013). Chlorfenvinphos may also act via central noradrenergic mechanisms to induce hypotension by accelerating the noradrenaline turnover in the brain (ATSDR, 1997; Puzay nska, 1984). An adverse outcome pathway (AOP) was developed for AChE inhibitors (OPs and carbamates, see Fig. 1) (US EPA, 2016a). The initiating event for both classes of pesticides is inhibition of AChE, which leads to accumulation of acetylcholine and ultimately to neurotoxicity (US EPA, 2016a).

New approach methodologies New Approach Methodologies (NAMs) utilizing in vitro and alternative methods to replace conventional mammalian testing are increasingly explored to inform chemical hazard in risk assessment. This is an important methodological development toward reducing the total number of animals used in toxicity testing. A large number of studies have been performed in primary and immortal cell lines and automated screening technologies with chlorfenvinphos. Various applications of these studies with chlorfenvinphos are described below.

Toxicity forecaster (ToxCast) Chlorfenvinphos was included in the ToxCast program by the US EPA, which leverages high-throughput screening technologies to produce a large database of in vitro toxicity data and generate predictive models for thousands of chemicals in the environment (US EPA, 2016b). The results from ToxCast assays are used to prioritize chemicals for subsequent animal toxicity testing, to inform on chemical hazards, and to develop further technologies for high-throughput screening and predictive toxicity modeling. Chlorfenvinphos was active in 141 of 630 tested assays (US EPA, 2021), but many active assays were either flagged for poor quality or contained AC50 values higher than 10 mM, values generally considered false positives (Judson et al., 2016). The 14 active assays that met quality criteria included CYP2C19, estrogen receptor, glucocorticoid receptor, progesterone receptor, vitamin D receptor, thromboxane A2, and cholesterol transit (TSPO). COMPARA modeling predicted chlorfenvinphos acts as an androgen antagonist.

Neuronal tissue dose

AChE inhibition

ACh accumulation

Neurotoxicity

Fig. 1 Adverse outcome pathway for acetylcholinesterase (AChE) inhibition by organophosphates like chlorfenvinphos.

Clinical signs and/or death

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Endocrine disruption screening program for the 21st century (EDSP21) Part of the ToxCast assay suite, EDSP assays are specific for interactions at the thyroid, androgen and estrogen receptors and for steroidogenesis processes. Chlorfenvinphos was active in 5/13 assays for estrogen (3/5 assays had precautionary flags or had borderline activity), 4/9 assays for androgen (2/4 assays had precautionary flags or had borderline activity), 2/8 assays for thyroid (1/2 assays had precautionary flags or had borderline activity), and 7/26 assays for steroidogenesis (3/7 assays had precautionary flags or had borderline activity) (accessed 23 August 2021).

Physiologically based pharmacokinetic-pharmacodynamic (PBPK-PD) modeling A physiologically based pharmacokinetic analysis was performed using data from Fischer 344 rats to predict changes in chlorfenvinphos body burden after pre-treatment followed by a second exposure to chlorfenvinphos (Ikeda et al., 1992). Oral pre-treatment of rats with 15 mg kg−1 chlorfenvinphos reduced plasma concentrations for oral exposure with 30 mg kg−1 chlorfenvinphos by 4- to 10-fold over 6 h compared to naïve controls, but did not have an effect on plasma concentrations for 5 mg kg−1 intravenous secondary exposure. Conversely, oral pretreatment with 15 mg kg−1 chlorfenvinphos significantly reduced liver concentrations for 5 mg kg−1 intravenous secondary exposures. Oral secondary exposures exhibited transiently decreased liver concentrations compared to controls until 4 h post treatment. The US EPA Computational Toxicology (CompTox) Dashboard used in vitro to in vivo extrapolation (IVIVE) modeling to predict steady-state human plasma concentrations (0.68 mg L−1) and pharmacokinetic t1/2 (1.99 h).

Acute and short-term toxicity Animal There are clear differences in the acute oral LD50 values of chlorfenvinphos across species. These are 9.7–39 mg kg−1 for rats, 117 mg kg−1 for mice, 125–500 mg kg−1 for guinea pigs, 300 mg kg−1 for rabbits, and >5000 mg kg−1 for dogs (Ambrose et al., 1970; ATSDR, 1997; Ikeda et al., 1992; NRA, 2000; Puzay nska, 1984; Takahashi et al., 1991; Wouters and Van Hoeven-Arentzen, 1994). The species sensitivity may reflect the rate of metabolic detoxification of chlorfenvinphos to 2-chloro-1-(2,4-dichlorophenyl) vinylethylhydrogen phosphate, which was lower in rats than dogs in vivo and lower in rats than mouse, rabbit, and dog in vitro (Donninger et al., 1972; Hutson et al., 1967). Rats were shown to be protected from chlorfenvinphos acute toxicity by CYP inducers (e.g., dieldrin), possibly through enhanced detoxification (ATSDR, 1997). In rats, the dermal LD50 is 30 mg kg−1 day−1 and the inhalation LC50 is 0.133 mg L−1 (NRA, 2000). Chlorfenvinphos is not irritating to the eye and is a weak skin sensitizer (NRA, 2000). The main target of chlorfenvinphos toxicity after short-term oral exposure is the nervous system. Muscarinic and nicotinic cholinergic syndromes include hypersalivation, respiratory distress, miosis, muscular twitches, tremors, ataxia, diarrhea and vomiting (Ikeda et al., 1992; Takahashi et al., 1991). Other non-lethal effects are metabolic and liver enzyme changes, elevation of plasma corticosteroids, hypotension, alteration in noradrenaline levels and aromatic amino-transferase activity in the brain and sleep disturbance (ATSDR, 1997; Gralewicz et al., 2011; Ikeda et al., 1992; Lukaszewicz-Hussain, 2008; Puzay nska, 1984; Takahashi et al., 1991). Chlorfenvinphos has not been tested for delayed neuropathy in hens.

Human Human deaths have occurred from intentional ingestion of chlorfenvinphos. Pulmonary congestion and edema were reported in one case at about 830 mg kg−1 (Martínez and Ballesteros, 2012). Postmortem, chlorfenvinphos was detected in the stomach, liver, blood and urine. Unconsciousness, absence of tendon reflexes, severely inhibited blood and red blood cell (RBC) ChE, respiratory failure and hypersecretion occurred after a single oral dose of about 360 mg kg−1 (ATSDR, 1997). In clinical studies with adults, the lowest observed effect level (LOEL) for plasma and RBC ChE inhibition is 1 mg kg−1 orally and 5 mg kg−1 dermally (NRA, 2000). The most common clinical signs of poisoning are those associated with AChE inhibition: lacrimation, salivation, tremors, nausea, miosis and muscle incoordination. For additional details on acute exposures, see the “Clinical management” section below.

Chronic toxicity Animal Reproductive 2- and 3-generation studies in rats (see section “Reproductive and developmental toxicity”) showed decreased pup survival at 10 mg kg−1 day−1 (Ambrose et al., 1970). Non-lethal LOELs reported in 30 days to 2 years oral studies with chlorfenvinphos included:

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0.8 mg kg−1 for gastrointestinal effects in rats, 1.5–7 mg kg−1 day−1 for increased liver weight and decreased thymus and kidney weight in rats and mice

Chlorfenvinphos

• • •

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93 mg kg−1 day−1 for hypertrophy and hyperplasia of adrenal cortex in mice 1.9–10 mg kg−1 day−1 for neuromuscular effects and decreases in body weight, bodyweight gains and food consumption in rats 0.5–0.7 mg kg−1 day−1 for 17–48% plasma, RBC and brain ChE inhibition in rats and dogs after 1 week- 2 years of oral exposures (Ambrose et al., 1970; ATSDR, 1997; Kowalczyk-Bronisz et al., 1992; NRA, 2000)

Human Adverse health outcomes are described in humans occupationally exposed to chlorfenvinphos for up to 15 years. Workers involved in production of chlorfenvinphos showed impaired immune system, respiratory muscle, olfactory, and liver function, inhibition of plasma ChE, and changes in electromyographic voltage measured in the ulnar nerve region (Konieczny et al., 1999; Kossmann et al., 1997; Kossmann and Konieczny, 2001; Liska-Markiel et al., 1990). Most of these studies involved exposures to a combination of pesticides and cannot be ascribed to chlorfenvinphos alone.

Immunotoxicity Chlorfenvinphos administered to mice, rats and rabbits for 84–90 days at 1.5–10 mg kg−1 day−1 produced various immunological/ lymphoreticular changes (Abadin et al., 2007; ATSDR, 1997). The LOEL for reduction of thymus weight, involution of the thymus, and stimulation of spleen colonies was 1.5 mg kg−1 day−1 in mice (Kowalczyk-Bronisz et al., 1992). This is the basis for ATSDR to establish the Minimal Risk Level (MRL) for intermediate-duration oral exposure to chlorfenvinphos, in addition to setting the acute and chronic MRLs based on ChE inhibition (Abadin et al., 2007; ATSDR, 1997). Decreased numbers of hemolysin-producing cells and E-rosette-forming cells, increased interleukin-1 activity, and delayed hypersensitivity reaction occurred at higher doses (Abadin et al., 2007). In rats and rabbits, reduced spleen weight, spleen cytomorphology changes, elevated serum hemagglutinin and hemolysin activity, and increased number of nucleated lymphoid cells producing hemolytic antibody to sheep erythrocytes occurred at 3–10 mg kg−1 day−1 (ATSDR, 1997).

Reproductive and developmental toxicity Animal The available data suggest that chlorfenvinphos affects reproduction and pre- and post-natal development. In prenatal developmental studies, pregnant rats received chlorfenvinphos up to 3 mg kg−1 day−1 orally on GD 6–15 and pregnant rabbits received up to 100 mg kg−1 day−1 on GD 6–18 (Ambrose et al., 1970; NRA, 2000). In rabbits, there was significant inhibition of plasma and erythrocyte ChE and an increased incidence of hydrocephalus at the LOEL of 25 mg kg−1 day−1 (NRA, 2000). Chlorfenvinphos increased the incidence of open eyes and edema in hamster dams and decreased the weight and length of hamster fetuses at 50 mg kg−1 day−1 (Dzierzawski and Minta, 1979). In 2- and 3-generation studies, rats fed 0.05–30 mg kg−1 day−1 chlorfenvinphos mated normally and exhibited normal pregnancy. However, pup viability and lactation index were severely impacted at 5–10 mg kg−1 day−1 (Ambrose et al., 1970). Marked decreases in fertility (50–84%) and birth index, reduction in maternal and pup body weight gain, and inhibition of plasma and brain ChE had the lowest LOEL of 0.5 mg kg−1 day−1 (Ambrose et al., 1970).

Human No developmental toxicity data on chlorfenvinphos are available in humans.

Developmental neurotoxicity No developmental neurotoxicity data on chlorfenvinphos are available in laboratory animals or in humans.

Genotoxicity Chlorfenvinphos shows mostly negative results in genotoxicity tests. It was negative for mutation in Escherichia coli (WP2 hcr), Bacillus subtilis (H17 Rec+ and M45 Rec-), and in multiple studies in multiple strains of Salmonella typhimurium (ATSDR, 1997). The only positive report in S. typhimurium was in the TA100 strain, with decreased potency in the presence of metabolic activation (ATSDR, 1997). A pesticide mixture containing 0.3% chlorfenvinphos tested negative in a male rat bone marrow micronucleus assay from oral exposures and for chromosomal aberration in human lymphocytes in vitro (Dolara et al., 1993).

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Carcinogenicity No carcinogenicity data on chlorfenvinphos are available in laboratory animals or in humans.

Organ toxicity Chlorfenvinphos affects the nervous system, respiratory system, cardiovascular system, immune system, reproductive system, skin, blood, and liver (ATSDR, 1997).

Interactions Concomitant exposure to other OPs and carbamates with similar mechanism of action may result in cumulative toxicity. Children are more sensitive to acute chlorfenvinphos toxicity due to their lower CYP expression. People with low plasma ChE are also more sensitive because plasma ChE acts to reduce the availability of chlorfenvinphos to neuromuscular tissue (US EPA, 2002). In animals, chlorfenvinphos toxicity is decreased by CYP inducers and is increased by agents that inhibit liver metabolic enzymes (ethylisocyanide and halogenated alkanes and alkenes) (ATSDR, 1997).

Toxicogenomics Curated chlorfenvinphos data were retrieved from the Comparative Toxicogenomics Database (Davis et al., 2021) (accessed 24 August 2021). The most commonly identified interaction with chlorfenvinphos was with AChE (12 interactions). Additionally identified gene interactions included genes involved in xenobiotic response (ɣ-glutamyltransferase 1 (GGT1), 2 interactions; CYP3A4 and NR1I2, 1 interaction each). Chlorfenvinphos also interacted with Est-6, a gene involved in pheromone response in Drosophila.

Clinical management As with many OP poisonings, mild to moderate oral exposure to chlorfenvinphos can result in bradycardia, salivation, lacrimation, diaphoresis, vomiting, diarrhea, urination, and miosis. Nicotinic effects of tachycardia, hypertension, mydriasis, and muscle cramps may also be present. Ingestion of large quantities of chlorfenvinphos can result in muscle fasciculations, weakness, respiratory failure, giddiness, agitation, confusion, and delirium. Hypotension, ventricular dysrhythmias, metabolic acidosis, pancreatitis, and hyperglycemia can also develop. Coma and seizures may result in the most severe cases. Importantly, delayed effects (up to 4 days post-exposure) may develop, including respiratory paralysis as well as motor paralysis of neck flexor muscles and the proximal limbs. This may be followed by the development of delayed peripheral neuropathy. Patients may exhibit the inability to lift the neck, slowed eye movements, facial weakness, difficulty swallowing, and generalize limb weakness. Following acute inhalation exposure, chlorfenvinphos vapors may cause mucous membrane and upper airway irritation, bronchospasm and dyspnea. Acute respiratory insufficiency is the main cause of death in acute inhalation poisonings. Long-term sequelae for either route may include subtle neuropsychological deficits. Management of moderate to severe toxicity includes airway management and simple decontamination (i.e., removal of contaminated clothes, wash skin with soap and water). Administration of atropine is useful for muscarinic effects and pralidoxime is effective for nicotinic manifestations. Seizures and agitation respond well to benzodiazepines (WEBWISER, 2021).

Environmental fate and behavior Chlorfenvinphos is soluble in organic solvents (e.g., ethanol, acetone) and has moderate solubility in water (145 mg L−1 solubility at 23
C). The calculated Henry’s law constant of 0.00000002 atm m3 mole−1 indicates that surface water volatilization is unlikely an important fate process. The estimated t1/2 for reacting with photochemically generated hydroxyl radicals in the air is 7–92 h. Photolysis of chlorfenvinphos produces dechlorinated products or isomerization of the Z- to the E-isomer(NRA, 2000). Chlorfenvinphos undergoes abiotic hydrolysis, photodegradation, and biotic degradation in soil and water (ATSDR, 1997). Depending on the soil type and climate, its soil persistence varies from 14 days to over 210 days (NRA, 2000). In river water, t1/2 ranges from 13 to 51 days (Medina et al., 1999). Chlorfenvinphos hydrolysis increases with alkalinity and persistence reaches years under acidic environments (ATSDR, 1997). The main degradates are 2,4-dichloro-1-(1 hydroxyethyl)benzene, 2,4-dichlorochloromethyl ketone, 2,4-dichlorobenzoic acid and 2-hydroxy-4-chlorobenzoic acid (Beynon and Wright, 1967). The estimated Log KOC of 2.45 indicates moderate soil adsorption and the potential for groundwater leaching.

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Chlorfenvinphos in soil and sediment can be taken up by organisms and plants. Based on the measured Log KOW of 3.81–4.22, chlorfenvinphos is not expected to bioaccumulate in food chains. Australian studies reported a t1/2of 57 days in greasy wool of merino sheep with over 29% of the applied pesticide recovered in the grease to be processed into lanolin products (NRA, 2000).

Ecotoxicology Chlorfenvinphos is highly toxic to freshwater Daphnia, with reported acute LC50 at 250 mg/kg/day by gavage for 13 weeks showed thymic necrosis and lymphoid or myeloid depletion of bone marrow, spleen, or thymus. While histopathologic evidence suggests that chlorobenzene is immunotoxic (CDC, 2020).

Reproductive toxicity There is limited data on the reproductive toxicity of Chlorobenzene. In a two-generation study of rats exposed intermittently to chlorobenzene vapor indicated increased occurrences of degenerative testicular changes in males of both generations at 450 ppm. (6/30 versus 1/30 among controls; P ¼ 0.051) (Nair et al., 1987).

Endocrine toxicity Endocrine disruptive behavior of chlorobenzene has been extensively reported (Satoh et al., 2008; Kabir et al., 2015; Witczak et al., 2021; Qi et al., 2022).

Genotoxicity The metabolites of chlorobenzene, 3,4- and 2,3-epoxides of chlorobenzene, are epoxides that are able to covalently bind to DNA, RNA and proteins. Genotoxicity of chlorobenzene has been evaluated in many in vivo studies and a few in vitro assays. In vitro studies demonstrate negative results in the majority of the studies on gene mutation, chromosomal aberration, DNA damage and UDS and in vivo SCE experiments. However, in vivo results indicate that chlorobenzene may induce genotoxic effects. From overall evaluation of these results, chlorobenzene is considered not to be genotoxic (Faisal et al., 2006; CDC, 2020).

Toxicogenomics Meaningful extrapolation of data from animals to humans has been difficult due to a large number of variables between humans and experimental animals. Humans show higher levels of soluble metabolites and although the formation of covalently bound products is lower compared to rodents. Interestingly, a 10-fold difference in the rate of metabolism of chlorobenzene in different human livers indicates possible differences in drug metabolizing enzyme phenotypes. There are also significant species and sex differences in the metabolism of chlorobenzene with markedly higher rates of oxidation in male mice than in male rats and female mice (ATSDR, n.d.; Nedelcheva et al., 1998).

Clinical management Treatment is symptomatic and supportive. For ocular contact, the eyes should be irrigated immediately with abundant running water. If the material contacts the skin, the affected areas should be washed with soap and water promptly. If inhalation exposure occurs, the exposed person should be moved to fresh air immediately and should be provided with respiratory support (oxygen or artificial respiration) if necessary. Bronchospasm can be treated with inhaled b2-agonist and oral or parenteral corticosteroids (Pravasi, 2014).

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If the material has been ingested, vomiting should not be induced. For ingestion, gastric lavage (followed by saline catharsis) should be performed or activated charcoal should be administered. The trachea should be protected from aspiration. Renal and hepatic function should be monitored and supported if necessary. Patients should be interviewed for signs and symptoms of neurotoxicity (Nagyeri et al., 2012).

Ecotoxicology In acute toxicity of chlorobenzene to algae, a 96-h EC50 (growth inhibition) for freshwater alga was 12.5 mg/L. The acute toxicity of chlorobenzene to invertebrates is reported in freshwater and seawater crustaceans. A 48-h EC50 (immobilization) for the freshwater water flea was 0.59 mg/L (USEPA, 2000). The acute toxicity of chlorobenzene to fish is reported in rainbow trout, bluegill and fathead minnow, and the 96-h LC50 values were 4.7 mg/L for the rainbow trout, 7.4 mg/L for the bluegill and 7.7 mg/L for the fathead minnow. The long-term toxicity to fish in the early life stage has been reported in rainbow trout, goldfish and largemouth bass, and the reliable lowest LC50 was the 7.5-day LC50 of 0.05 mg/L for 4-day posthatch of the largemouth (USEPA, 2000).

Other hazards Chlorobenzene is highly flammable, and the vapors are heavier than air. They will spread along the ground and collect in low or confined areas. The lower flammable limit is 1.8%, the upper flammable limit is 9.6%, and the flash point is 851F (29.21C closed cup). The explosive limit value ranges from 7.1% to 1.3% at 150  C (Pubchem, 2022a, 2022b). Combustion of chlorobenzene can form phosgene and hydrogen chloride gases. Chlorobenzene reacts with strong oxidizing materials, powdered sodium, and phosphorus trichloride and sodium (Pubchem, 2022a, 2022b).

Exposure standards and guidelines Based on the results of a chronic toxicity study in rats, the US EPA classified chlorobenzene as group ‘D’ (not classifiable as to carcinogenicity in humans). In addition, the American Conference of Governmental Industrial Hygienists classified chlorobenzene as ‘A3’ (confirmed animal carcinogen with unknown relevance to humans) (USEPA, 2000). Summary of exposure criteria for chlorobenzene:

Agency

Criteria

Averaging Time

ACGIH NIOSH OSHA

TLV—TWA, 10 ppm IDLH, 1000 ppm PEL (TWA), 75 ppm (350 mg/m3)

8 h/40 h week NA 8 h/40 h week

Conclusion Chlorobenzene (CB) is considered an ingredient of OCPs (organochlorine pesticides) is readily absorbed from the respiratory and GI tracts. Primary exposure routes are via inhalation and orally. Upon exposure, chlorobenzene is widely distributed in the blood, but may accumulate to some extent in adipose tissue due to its lipophilic nature. Exposure to high doses of CB can cause multiorgan toxicity, primarily the liver. Most chlorobenzene is metabolized via a chlorobenzene 3,4-epoxide pathway to ultimate form glucuronide or sulfate conjugates which are excreted via the kidneys. Urinary excretion of chlorobenzene metabolites is the major route of excretion. Several publications have emerged in recent years regarding its influence on the endocrine system, which is a concern.

See also: Benzene; Bromobenzene; Dichlorobenzene; Hexachlorobenzene

References ATSDR (n.d.) https://www.atsdr.cdc.gov/ToxProfiles/tp131-c3.pdf CDC (2020) https://www.atsdr.cdc.gov/ToxProfiles/tp131.pdf

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Chemical Risk Information Platform (CHRIP) (2014) Biodegradation and Bioconcentration. Tokyo, Japan: National Institute of Technology and Evaluation. Available from http://www. safe.nite.go.jp/english/db.html/. Committee on Acute Exposure Guideline Levels, Committee on Toxicology, Board on Environmental Studies and Toxicology, Division on Earth and Life Studies, and National Research Council (2012) Acute Exposure Guideline Levels for Selected Airborne Chemicals. vol. 12. Washington, DC: National Academies Press (US). 3, Chlorobenzene: Acute Exposure Guideline Levels. Available from: https://www.ncbi.nlm.nih.gov/books/NBK201466/. Faisal SM, et al. (2006) Micronuclei induction and chromosomal aberrations in Rattus norvegicus by chloroacetic acid and chlorobenzene. Ecotoxicology and Environmental Safety 65(2): 159–164. Hu H, et al. (2015) Determination of benzene series compounds and chlorobenzenes in water sample by static headspace gas chromatography with flame ionization detection. Journal of Separation Science 38(11): 1916–1923. https://pubmed.ncbi.nlm.nih.gov/25802171/. Kabir ER, et al. (2015) A review on endocrine disruptors and their possible impacts on human health. Environmental Toxicology and Pharmacology 40(1): 241–258. https://pubmed. ncbi.nlm.nih.gov/26164742/. Lehmann I, Röder-Stolinski C, Nieber K, and Fischäder G (2008) In vitro models for the assessment of inflammatory and immuno-modulatory effects of the volatile organic compound chlorobenzene. Experimental and Toxicologic Pathology 60(2–3): 185–193. Nagyeri G, Valkusz Z, Radacs M, et al. (2012) Behavioral and endocrine effects of chronic exposure to low doses of chlorobenzenes in Wistar rats. Neurotoxicology and Teratology 34(1): 9–19. Nair RS, Barter JA, Schroeder RE, Knezevich A, and Stack CR (1987) Twogeneration reproduction study with monochlorobenzene vapor in rats. Fundamental and Applied Toxicology 9(4): 678–686. Nedelcheva V, Gut I, Soucek P, et al. (1998) Cytochrome P450 catalyzed oxidation of monochlorobenzene, 1,2- and 1,4-dichlorobenzene in rat, mouse, and human liver microsomes. Chemico-Biological Interactions 115(1): 53–70. https://doi.org/10.1016/s0009-2797(98)00058–1. Pravasi S (2014) Chlorobenzene. In: Encyclopedia of Toxicology, 3rd edn, pp. 870–876. Elsevier. Pubchem (2022a) https://pubchem.ncbi.nlm.nih.gov/compound/Chlorobenzene#section¼Consumption-Patterns Pubchem (2022b) https://pubchem.ncbi.nlm.nih.gov/compound/Chlorobenzene#section¼Non-Human-Toxicity-Excerpts Qi SH, et al. (2022) Effects of organochlorine pesticide residues in maternal body on infants. Frontiers in Endocrinology 13: 890307. PMC9218079. Röder-Stolinski C, Fischäder G, Oostingh GJ, et al. (2008) Chlorobenzene induces the NF-kappa B and p38 MAP kinase pathways in lung epithelial cells. Inhalation Toxicology 20(9): 813–820. Ryan RP, Terry CE, and Leffingwell SS (1999) Toxicology Desk Reference, 5th edn vol. 1, New York: Taylor & Francis. 313–316. Satoh K, Nonaka R, Ohyama K, et al. (2008) Endocrine disruptive effects of chemicals eluted from nitrile-butadiene rubber gloves using reporter gene assay systems. Biological & Pharmaceutical Bulletin 31(3): 375–379. https://pubmed.ncbi.nlm.nih.gov/18310895/. USEPA (2000) https://www.epa.gov/sites/default/files/2016-09/documents/chlorobenzene.pdf Witczak A, et al. (2021) Endocrine-disrupting organochlorine pesticides in human breast milk: Changes during lactation. Nutrients 13(1): 229. https://pubmed.ncbi.nlm.nih.gov/ 33466783/.

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Chlorobenzilate Atoosa Karimi Babaahmadi and Maryam Armandeh, Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of D.M. Janz, Chlorobenzilate, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 874–875, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00113-5.

Chemical profile Introduction Uses Environmental fate and behavior Mechanism of toxicity Exposure and exposure monitoring Toxicokinetics Interactions Acute and short-term toxicity (animal/human) Chronic toxicity (animal/human) Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Other hazards Exposure standards and guidelines References

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Abstract Chlorobenzilate is an organochlorine pesticide currently restricted in the United States and Europe. However, it is infrequently used in other regions of the world. It is almost water-insoluble and is extensively adsorbed to soil particles in the upper layers of soil. This pesticide has low mobility in the soil and only a minor percentage of penetration into groundwater. Although there is no information on whether chlorobenzilate causes cancer in humans, in orally treated rats, chlorobenzilate was found to be carcinogenic, with an increased frequency of liver tumors. This chemical is categorized as Group B2, a probable human carcinogen by the EPA.

Keywords Acaricide; Chlorinated hydrocarbon; Chlorobenzilate; Pesticide

Key points 1. Chlorobenzilate is an organochlorine pesticide that controls mite species. 2. Despite a lack of ecotoxicological information, chlorobenzilate appears to have low acute toxicity to aquatic and terrestrial species. 3. There is insufficient information on the carcinogenicity of chlorobenzilate in humans, although it has been carcinogenic in animals.

Chemical profile

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Name: Chlorobenzilate. Chemical Abstracts Service Registry Number: 510-15-6 (Janz, 2014a). Synonyms: Ethyl-4,4-dichlorobenzilate; Ethyl-4,4-dichlorophenylglycollate; Ethyl 2,2-bis(4-chlorophenyl)-2-hydroxyacetate; Chlorobenzilate.

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Molecular Formula: C16H14Cl2O3 (Janz, 2014a)

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Chemical Structure:

Introduction Chlorobenzilate is a colorless to light yellow solid in its pure state but a brownish liquid in its commercial form (Spencer, 1982; Meister and Sine, 2008; O’Neil, 2001). Chlorobenzilate is an organochlorine pesticide in the same category as dichlorodiphenyltrichloroethane (DDT) (Janz, 2014a). As a pesticide, chlorobenzilate was sprayed on citrus trees and deciduous fruit trees until 1999 (Janz, 2014b). It was initially developed by Ciba-Geigy and introduced in 1952 (International Agency for Research on Cancer, n.d.).

Uses Chlorobenzilate was primarily used as a pesticide against mites species in citrus and deciduous fruit trees (Janz, 2014a). The product has a limited insecticidal effect, killing only mites and ticks (IARC, 1983). Chlorobenzilate was first employed as a DDT synergist https://www.chemicalbook.com/ChemicalProductProperty_EN_CB1431322.htm#:
:text¼Historically%2C%20chlorobenzilate% 20was%20used%20as,than%20citrus%20in%20other%20countries. This pesticide is no longer allowed to be used in the United States and Europe, but it is thought to be applied in other countries on crops other than citrus ().

Environmental fate and behavior Chlorobenzilate is unstable in the soil. After application at 0.5–1.0 ppm, its half-life is 10–35 days, where it undergoes microbial degradation. Chlorobenzilate is practically insoluble in water and is highly adsorbed to soil particles in the upper soil layers. It has little mobility in the soil and penetrates in a small amount into groundwater. It has low water solubility (log Kow ¼ 4.74) and is highly adsorbed to sediments and particulate matter in aqueous media. Due to an estimated Koc of 1500, chlorobenzilate has low soil mobility and is not expected to penetrate groundwater. Neither photolysis nor hydrolysis is capable of decomposing chlorobenzilate (International Agency for Research on Cancer (IARC), 1983). The half-life of chlorobenzilate in loamy and silty clay soils was 10.8–15.1 and 29.5–169.1 days, respectively (The United Nations Environment Programme, n.d.). Water or soil evaporation is not significant due to the fixed estimate of 7.2 atm m3 mol−1. Chlorobenzilate will exist in both vapor and particulate phases upon release into the air. In ambient air, vapor phase chlorobenzilate has a half-life of 3.2 days. Wet or dry deposition is expected to remove chlorobenzilate from the atmosphere in the particulate phase. Bioaccumulation in aquatic organisms is moderate to high due to the biological concentration factor range of 709–224 L/kg in fish (International Agency for Research on Cancer (IARC), 1983).

Mechanism of toxicity Similar to DDT, chlorobenzilate disrupts normal Na+ and K+ flow across axonal membranes in the central nervous system (CNS) and peripheral nervous system. It may also counteract the gamma-aminobenzoic acid inhibitory effect in the CNS, leading to a hyperexcitable condition of neurotransmission (Janz, 2014a).

Exposure and exposure monitoring Workers may be exposed to chlorobenzilate through the skin or inhalation (U.S. Department of Health and Human Services, 1993). Chlorobenzilate may enter the air or water by spray drilling while used on products. Individuals may be exposed by breathing polluted air, drinking contaminated water, or eating contaminated fruits (Janz, 2014a; The United Nations Environment Programme, n.d.). Although these routes of exposure may or may not be present in the United States and Europe due to cessation of use, the current potential use of chlorobenzilate is unknown in other parts of the world (Charistou et al., 2022).

Chlorobenzilate

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Toxicokinetics Chlorobenzilate is easily absorbed from the gastrointestinal tract after oral intake due to its high lipid solubility. After exposure to commercial (oil-based) formulations, dermal absorption also occurs. Following a daily oral dose of 12.8 mg kg−1 for 35 weeks, there was no substantial accumulation of chlorobenzilate in the adipose tissue of male mongrel dogs. When chlorobenzilate was incubated in the presence of rat liver homogenates, the primary metabolites generated were dichlorobenzilic acid, dichlorobenzhydrol, chlorobenzene acid, and dichlorobenzophenone. After oral dosing, dogs and rats showed urinary excretion of these metabolites and considerable excretion of unaltered chlorobenzilate in the feces. Despite its chemical similarity to DDT, chlorobenzilate appears to be eliminated significantly more quickly in mammals after absorption.

Interactions Chlorobenzilate was tested to suppress gap junctional intercellular communication in the Chinese hamster V79 metabolic co-operation assay and the scrape-loading/dye-transfer assay in WB-F344 rat liver epithelial cells due to its high lipid solubility. In nitrosamine-initiated male Sprague-Dawley rats, the pesticide was tested to increase the formation of gammaglutamyltranspeptidase-positive altered hepatic foci and induce cytochrome P450 monooxygenase isoenzymes. In vitro tests revealed that chlorobenzilate is an effective inhibitor of cell-cell communication in both test systems, leading to foci development. In the in vivo investigation, similar results were obtained. Chlorobenzilate was found to stimulate the phenobarbital-inducible cytochrome P450 isoenzyme and produce hepatomegaly. There was no precise association between cytochrome P450b induction/ liver growth and the effect associated with tumor promotion in vivo and in vitro for this pesticide (Flodstorm et al., 1990).

Acute and short-term toxicity (animal/human) Muscle pain, ataxia, mild delirium, and fever have been reported in workers exposed to chlorobenzilate (International Agency for Research on Cancer (IARC), 1983). In rodents, acute exposure to chlorobenzilate causes tears, salivation, diarrhea, and rapid, deep breathing. Intestinal irritation and bleeding in the lungs have also been reported (Janz, 2014a).Oral LD50 of chlorobenzilate in rats, mice, and hamsters is between 700 and 729 mg kg−1, and cutaneous LD50 is more than 10,000 mg kg−1 in mice and rabbits (Smith, 1991).

Chronic toxicity (animal/human) Several symptoms, including anemia, poor appetite, intra-medullary hematopoiesis of the liver and spleen, cardiac changes, and erythroid bone marrow hyperplasia, have been reported in dogs chronically exposed to high levels of chlorobenzilate (U.S. Environmental Protection Agency, 1999; The United Nations Environment Programme, n.d.). Chronic exposure to chlorobenzilate may cause the same symptoms as acute exposure. Disturbed electrical activity in the CNS, conjunctivitis, and dermatitis have been observed following chronic occupational exposure in humans (U.S. National Library of Medicine, 1995). Studies of chronic diet exposure in male and female rats showed a significant reduction in body weight without affecting survival https:// inchem.org/documents/jmpr/jmpmono/v068pr08.htm.

Reproductive toxicity Sperm damage and testicular atrophy have been observed in mice exposed to chlorobenzilate (U.S. Environmental Protection Agency, 1999). No teratogenic effects were observed in the offspring of rats exposed to dietary chlorobenzilate or in rabbits receiving the pesticide by gavage (International Agency for Research on Cancer (IARC), 1983). In a three-generation reproductive study in mice, chlorobenzilate did not affect the number of uterine implants, bed size, and weaning survival (International Agency for Research on Cancer (IARC), 1983).

Genotoxicity Negative results were obtained when examining the mutagenicity of chlorobenzilate by the standard Ames method, using five Salmonella strains in the presence or absence of liver microsomes (Moriya et al., 1983).

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Carcinogenicity No information about the carcinogenic effect of chlorobenzilate is available in humans (U.S. Environmental Protection Agency, n.d.). According to a National Toxicology Program (NTP) study, chlorobenzilate caused liver tumors in orally exposed rats (National Toxicology Program, 1978). Based on insufficient human data and adequate evidence in animals, The Environmental Protection Agency (EPA) has classified chlorobenzilate as a probable human carcinogen in group B2 (U.S. Environmental Protection Agency, 1997).

Clinical management Treatment is limited to symptomatic relief. Ventilation is usually necessary to open the airway. It is important to monitor heart rhythms and treat arrhythmias as needed. In case of eye exposure, the eyes should be rinsed immediately with water or salt water and irrigated during transport. Oral consumption of activated charcoal is recommended after ingestion. After skin contamination, the exposed area should be washed with soap and water (International Agency for Research on Cancer (IARC), 1983).

Ecotoxicology Although there is a lack of ecotoxicological evidence, it appears that chlorobenzilate has a relatively low acute toxicity to aquatic and terrestrial species. In marine invertebrates and fish, acute mortality ranges from 0.55 to 1.0 mg L−1 (48- and 96-h LC50 values). Acute lethality (7- to 8-day LD50 values) in mallard ducks and bobwhite quail after intake of contaminated food ranged from 3375 to 5620 mg kg−1. Bees are practically unaffected by chlorobenzilate. There is no information on chronic toxicity in aquatic or terrestrial vertebrates (Wang et al., 1994).

Other hazards No other hazards have been noted.

Exposure standards and guidelines Chlorobenzilate can be consumed at a daily dose of 0.02 mg kg−1 day−1 (The United Nations Environment Programme, n.d.).

References Charistou A, et al. (2022) Guidance on the assessment of exposure of operators, workers, residents and bystanders in risk assessment of plant protection products. EFSA Journal 20(1). https://doi.org/10.2903/j.efsa.2022.7032. Flodstorm S et al (1990) Carcinigenesis (Eynsham) 11(8): 1413–1418. IARC (1983) Chlorobenzilate (IARC Summary & Evaluation, Volume 30). International Agency for Research on Cancer (n.d.) Chlorobenzilate, International Agency for Research on Cancer. International Agency for Research on Cancer (IARC) (1983) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans: Miscellaneous Pesticides, Vol. 30. Lyon: World Health Organization. Janz DM (2014a) Chlorobenzilate. In: Encyclopedia of Toxicology, pp. 874–875. Elsevier. Janz DM (2014b) Chlorobenzilate. In: Encyclopedia of Toxicology, 3rd edn, vol. 3, pp. 874–875. Elsevier. https://doi.org/10.1016/B978-0-12-386454-3.00113-5. Meister RT and Sine C (2008) Crop Protection Handbook, Vol. 94. Willoughby, OH: Meister Media Worldwide. D 104. Moriya M, Ohta T, Watanabe K, Miyazawa T, Kato K, and Shirasu Y (1983) Further mutagenicity studies on pesticides in bacterial reversion assay systems. Mutation Research/Genetic Toxicology 116(3–4): 185–216. https://doi.org/10.1016/0165-1218(83)90059-9. National Toxicology Program (1978) Bioassay of Chlorobenzilate for Possible Carcinogenicity (CAS No. 510-15-6). TR No.75. Bethesda, MD: U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health. O’Neil MJ (ed.) (2001) The Merck Index—An Encyclopedia of Chemicals, Drugs, and Biologicals, 13th edn, p. 366. Whitehouse Station, NJ: Merck and Co., Inc. Smith AG (1991) Chlorinated hydrocarbon insecticides. In: Hayes WJ Jr and Laws ER Jr (eds.) Handbook of Pesticide Toxicology. New York, NY: Academic Press Inc. 6-3. Spencer EY (1982) Guide to the Chemicals Used in Crop Protection, 7th edn, p. 111. Publication 1093. Research Institute, Agriculture Canada: Ottawa, Canada: Information Canada. The United Nations Environment Programme (n.d.), Operation of the Prior Informed Decision Guidance. The United Nations Environment Programme. U.S. Department of Health and Human Services (1993) Hazardous Substances Data Bank (HSDB). Bethesda, MD: National Toxicology Information Program, National Library of Medicine. U.S. Environmental Protection Agency (1997) Health Effects Assessment Summary Tables. FY 1997 Update. Solid Waste and Emergency Response, Office of Emergency and Remedial Response: Cincinnati, OH. EPA/540/R-97-036. U.S. Environmental Protection Agency (1999) Integrated Risk Information System (IRIS) on Chlorobenzilate. Washington, DC: National Center for Environmental Assessment, Office of Research and Development. U.S. Environmental Protection Agency (n.d.) Health and Environmental Effects Profile for Chlorobenzilate. EPA/600/x-84/210. Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment, Office of Research and Development: Cincinna. U.S. National Library of Medicine (1995) Hazardous Substances DataBank. Bethesda, MD: U.S. National Library of Medicine. 6-18. Wang YS, Chen SW, Yen JH, and Chen YL (1994) Dissipation and movement of acaricide chlorobenzilate in the environment. Ecotoxicology and Environmental Safety 28(2): 193–200. https://doi.org/10.1006/eesa.1994.1045.

Chlorodibenzofurans (CDFs) Amelia B Hizon-Fradejasa, Jeb Reece H Grabatoa, Sofia Angela P Federicoa, and Elmer-Rico E Mojicab, aInstitute of Chemistry, University of the Philippines Los Baños, College, Laguna, Philippines; bDepartment of Chemistry and Physical Sciences, Pace University, New York, NY, United States © 2024 Elsevier Inc. All rights reserved. This is an update of R.D. Kimbrough, Chlorodibenzofurans (CDFs), Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 876–879, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01113-1.

Chemical profile Background Uses/occurrence Exposure and exposure monitoring Toxicokinetics Mechanism of toxicity Human toxicity Animal toxicity Immunotoxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity Organ toxicity Clinical management Environmental fate and behavior Exposure standards and guidelines Further reading

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Abstract Chlorodibenzofurans, or CDFs, are a group of compounds that contain one to eight chlorine atoms connected to the carbon atoms of the parent dibenzofuran. They are mostly produced in small amounts as unwanted derivatives while producing certain chemicals. CDFs can also be released from incinerators and landfill fires along with polychlorinated dibenzopara-dioxins (PCDDs). Similar to PCDDs, CDFs are ubiquitous in soil, sediments, and air. They are persistent in the environment and accumulate in animal tissue lipids. High exposures have occurred in relation to contamination of rice oil incidents in Japan (Yusho) and Taiwan (Yucheng) and in accidents involving electrical equipment containing PCBs.

Keywords Chorodibenzofurans; Polychlorinated dibenzofurans; PCDF occurrence; PCDFs; Toxicity; Yucheng; Yusho

Key points

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Chlorodibenzofurans (CDFs) are a family of chemicals that has one to eight chlorine atoms attach to the carbon atoms of the parent chemical, dibenzofuran. Exposure can occur through oral, inhalation, and dermal contact.

Chemical profile

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Name: Chlorodibenzofurans Synonyms: Chlorinated dibenzofurans (CDFs), polychlorinated dibenzofurans (PCDFs) CAS Number: 136677–10-6 Molecular Formula: C12H8-nCl(n)O; where n can be 1 to 8

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Chlorodibenzofurans (CDFs)

Chemical Structure:

Cl(n) O

Cl(n)

Background CDFs are a class of structurally similar chlorinated hydrocarbons containing two benzene rings fused to a central furan ring. There are 135 different isomers of CDFs most notably known as congeners. These congeners have varying harmful health effects, but CDF congeners containing chlorine atoms at the 2,3,7,8- positions of the dibenzofuran molecule are known to be most harmful.

Uses/occurrence There is no known use for chlorodibenzofurans. However, they may be synthesized in the laboratory for toxicological research purposes. Mostly, they are by-products in the industry that are not deliberately produced. CDFs are by-products of paper and pulp mills bleaching and are also released in incinerators and combustion processes.

Exposure and exposure monitoring CDFs can be released from hazardous waste incineration facilities, fire accidents involving polychlorinated biphenyl (PCB)-filled transformers and capacitors, and high industrial processes like copper smelting and electric arc furnaces in steel mills and other similar practices. It can also come from the combustion of fossil fuels by power plants, home heating, fireplaces, and automobile exhaust. CDFs are present in the environment, mainly in air, soil, and sediment. This chemical is not soluble in water and is more likely to be present in the air as vapors and bind to particles, soil, and sediment. CDFs are known to have large soil adsorption coefficients which means that it has a high tendency to bind to soil. It also has low mobility on soil surfaces causing it to retain and accumulate in the soil. When CDFs are present in water, it is mainly adsorbed to suspended solids and sediments. CDFs are also found in higher concentrations in fishes than in the water or sediment. Humans are exposed to CDFs through inhalation, oral, and dermal exposure. Inhalation exposure is a minor route of exposure for the general population. People can be exposed to CDFs in the air near incinerators or accidental landfill fires. Oral exposure, on the other hand, is a more common route of exposure to CDFs. It is known to contaminate food, such as meat, fish, and dairy products. Lastly, dermal exposure to CDFs is more common in occupational settings. Occupational exposure can be from the production of polychlorinated biphenyls (PCBs), handling and spraying of 2,4,5-T pesticide, metal production and recycling, production of chlorine gas, exposure in bleached pulp mills, production of polyvinyl chloride (PVC), and accidental exposure. CDFs are classified under hazardous wastes constituents which makes sure of its proper treatment and disposal. Furthermore, one can get tested to measure CDF levels in the blood, body fat, and breastmilk. For children and other sensitive human receptors it is best to avoid areas affected by hazardous waste sites to prevent contact with soil that is possibly contaminated with CDFs.

Toxicokinetics CDFs were detected in blood and adipose tissue following inhalation exposures in humans. CDFs are mostly taken through the oral route and are absorbed in the gastrointestinal (GI) tract. Additionally, studies conducted in experimental animals demonstrate that CDFs can be absorbed through the skin. CDFs are lipid soluble and tend to accumulate in tissue lipid. They particularly concentrate on the adipose and liver tissues. It is generally accepted that biotransformation of CDFs occurs primarily in the liver. The major metabolic reactions include hydroxylation with or without dechlorination or migration of substituents from the site of hydroxylation to the adjacent carbon, and oxygen bridge cleavage, followed by glucuronidation. Cytochrome P450 isoenzymes appear to catalyze the metabolic reactions. The elimination of PCDFs, like that of the polychlorinated dibenzo-para-dioxins (PCDD), depends strongly on the position of the chlorine atoms. Those congeners with a 2,3,7,8-chlorine substitution pattern exhibit the slowest elimination rates in all experimental animal species studied. As PCDFs are stored primarily in the liver and adipose tissue, the whole-body half-life of these compounds is governed mainly by the elimination from these two body compartments. Studies conducted in monkeys, mice, and rats indicate that feces are the dominant pathway for excretion of absorbed CDFs. Furthermore, absorbed CDFs, to some extent, can also be eliminated via urine.

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Mechanism of toxicity Several studies suggest that CDFs share the same toxicity mechanism as dioxins since they share similar structures. Most, if not all, of the health effects of CDFs and related compounds are mediated by binding to the Aryl hydrocarbon (Ah) receptor, which regulates the synthesis of a variety of proteins via alterations in gene expression. Oxidative stress is another proposed mechanism for the toxicity of CDFs. Significant increases in the production of superoxide anion and lipid peroxidation were identified in the liver and brain tissues of rats administered with 2,3,4,7,8-pentaCDF. The increases in the biomarkers of oxidative stress were dose-related.

Human toxicity Most of the information on human health effects that pertains to CDFs is from studies of people who ingested contaminated rice oil for up to 10 months during the Yusho (Japan) and Yucheng (Taiwan) poisoning incidents. A mass poisoning, called the Yusho (‘oil disease’) incident, occurred in western Japan in 1968. The disease was caused by ingestion of a specific brand of rice oil that was contaminated not only with PCBs but also with PCDFs and other related substances. In 1979, 11 years after the Japanese Yusho incident, a similar incident occurred in central Taiwan. About 2000 persons were identified as Yucheng patients, primarily from Taichung and Changhwa counties. Oil samples were found to be contaminated with PCBs and PCDFs, like the Yusho oil. Although the health effects cannot be attributed solely to CDFs due to mixed chemical exposure and possible interactions between CDFs, PCBs, and other components of the contaminated rice oils, but there is sufficient evidence that CDFs are the main causal agents. In both groups, the most notable acute effects were dermatological and neurological signs and symptoms of fatigue, headaches and gastrointestinal distress (nausea, vomiting, abdominal pain). Indications of the Yusho and Yucheng incidents include serious health effects such as severe skin lesions (chloracne and hyperpigmentation) and ophthalmological symptoms (like hypersecretion of eyelid glands), increased susceptibility to respiratory infection (e.g., chronic bronchitis), and neurological symptoms and signs (limb numbness, reduced nerve conduction velocities, delayed neurobehavioral development). Less serious effects observed in Yusho and Yucheng patients include mild hematological changes like anemia and clinically insignificant hepatic alterations (e.g., changes in ultrastructure and serum triglycerides). Some of these effects, particularly dermal, ocular, and neurobehavioral manifestations, also occurred in children born of exposed mothers. Babies born to mothers who consumed contaminated oil were characterized at birth by brown pigmentation (‘colacolored babies’) on the skin and the mucous membrane, gingival hyperplasia, very early postnatal eruption of the teeth or natal teeth, calcification of the skull and low birth weight. For additional information resources and updated literature, refer to the further reading section of this chapter.

Animal toxicity The health effects associated with exposure of experimental animals to CDFs are similar across congeners, although there are differences in relative toxicity. At lower doses, the primary targets are the liver, thymus, thyroid hormones, and developing organism following acute- or intermediate-duration exposure. In addition to these targets, chronic exposure also results in damage to the adrenal cortex, kidney, uterus, and gingiva. Chronic oral exposure to 2,3,4,7,8-pentaCDF resulted to cancer to test animals. When Sprague-Dawley rats were given various PCDFs in their diet, body weight loss, thymic atrophy, and depletion of hepatic vitamin A were observed. When the PCDFs were administered as a mixture, it was observed that the individual PCDF toxicity was additive. While oral doses of 30–300 mg/kg bw/day 2,3,7,8-TCDF for 22 days did not induce clinical signs of toxicity in mice, guineapigs given an oral dose of 10 mg/kg or 15 mg/kg 2,3,7,8-TCDF or 2,3,4,7,8-PeCDF died 9–20 days after exposure. The high sensitivity of the guineapig was attributed to the low metabolism rate in this species. Lethality was observed when rhesus monkeys were administered a single dose of 1500 mg/kg 2,3,7,8-TCDF. On the other hand, some monkeys survived when given a dose of 1000 mg/kg (2/4) but developed facial oedema and loss of eyelashes, fingernails and toenails. Mild anemia, relative lymphopenia and marked relative and absolute neutrophilia were discovered when blood analyses were done on the test animals.

Immunotoxicity Clinical observations strongly suggest that the Yusho and Yucheng exposed sub-population groups experienced frequent or more severe skin and respiratory infections and lowered resistance to illness. Various changes in immune status were reported among the Yusho and Yucheng patients, including decreased serum IgA and IgM levels and lymphocytes, diminished phagocyte complement and IgG receptors, and diminished delayed-type skin hypersensitivity response in subpopulations. In a study of the humoral immunosuppressive effect of 1,2,3,4,6,7,8-HpCDF in C57BL/6 mice, the 50% immunosuppressive dose (ID50) was found to be 208 mg/kg, while the ID50 for 1,2,3,4,5,6,7,8-HpCDD was 85 mg/kg. Immunosuppressive effect of PCDFs is less compared to 2,3,7,8-TCDD which has ID50 of 0.74 mg/kg.

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Reproductive and developmental toxicity PCDFs are teratogenic in mice, causing the same spectrum of birth defects and developmental toxicity as 2,3,7,8-TCDD. At doses which are not maternally or fatally toxic, oral administration of PCDFs to the dam caused cleft palate and hydronephrosis.

Genotoxicity There are only limited number of in vitro and in vivo studies that evaluated the genotoxicity of CDFs. OctaCDF and 2,3,7,8-tetraCDF in assays with several strains of Salmonella typhimurium bacteria, were not mutagenic with or without metabolic activation. Similarly, 2,3,7,8-tetraCDF did not induce forward mutations or inter- or intragenic recombinations with the yeast, Saccharomyces cerevisiae, without exogenous metabolic activation.

Carcinogenicity In one study, exposure of rats to 2,3,4,7,8-pentaCDF showed increase incidence of mouth and liver cancer. This is consistent to studies which demonstrated increased risk of cancer for people exposed to elevated levels of CDFs in their diet. The International Agency for Research on Cancer (IARC) established that 2,3,4,7,8-pentaCDF is carcinogenic to humans (Group 1). In contrast, the other CDF congeners are not classifiable as to their carcinogenicity to humans (Group 3).

Organ toxicity Epidemiological and experimental animal studies presented strong indication that the liver is a target of CDF toxicity, showing effects like variations in serum triglycerides and cholesterol levels, increases in liver weight, lipid buildup in the liver, and hepatocellular hypertrophy. On the other hand, the kidney does not appear to be a sensitive target of CDF toxicity following acute or intermediate exposure. Skin effects are the most apparent signs of CDF toxicity in humans and animals. The studies in animals, although limited by number of congeners and species tested, suggest that high doses of 2,3,7,8-tetraCDF and 2,3,4,7,8-pentaCDF cause dermal alterations and that monkeys are more sensitive than rodents. Ocular effects have been observed in humans and monkeys but only when exposed to high doses of CDFs. At lower doses in rodent studies, these effects have not been observed.

Clinical management When CDFs are inhaled, the person affected should be removed from the premises and supplied with oxygen. Skin and eye exposure should be treated by washing the affected part with large amounts of water and soap. Oral ingestion of CDFs is treated through administering charcoal orally.

Environmental fate and behavior In the environment, CDFs are ubiquitous in the soil, sediments and air. CDFs are present in the atmosphere both in the vapor and particulate phase. The lower chlorinated congeners are slightly volatile and degrade in the atmosphere relatively fast, while the higher chlorinated congeners are less volatile but undergo atmospheric degradation gradually and are subject to long-range transport. The transport of atmospheric CDFs to soil and water occurs by dry and wet deposition. Wet deposition happens during precipitation, wherein CDFs are taken from the air by snow or rain. CDFs bind to soil and sediment and are not likely to move into groundwater from soil, since CDFs are lipophilic. CDFs are persistent in the environment and concentrate in animal fat. They accumulate in fish at much higher levels than levels found in the water or sediment. Similar to other highly halogenated substances, higher chlorinated congeners degrade in the environment slowly via microbial means (aerobic biodegradation) and tend to bioconcentrate. CDFs can build up in other animals, birds, and people that are exposed to them in their food.

Exposure standards and guidelines In the US, no specific guidelines or standards have been set for PCDFs by agencies like EPA, OSHA and NIOSH. On the other hand, the regulatory requirements vary widely among the countries of the European Union. In Germany, an occupational technical exposure limit value of 50 pg I-TEQ/m3 in air has been established for PCDDs and PCDFs. With respect to incinerator emissions,

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Germany and the Netherlands have set daily average limit values of 0.1 ng I-TEQ/m3 of exhaust gases for PCDDs/PCDFs from industrial waste incinerators, Sweden 0.1–0.5 ng TEQ/m3, and the United Kingdom 1 ng I-TEQ/m3 with a goal to reduce both PCDD and PCDF emissions from industrial and municipal waste incinerators to 0.1 ng/m. In Japan, a limit of 0.5 ng I-TEQ/m3 2,3,7,8-PCDD/PCDF is recommended for municipal waste incinerators. For dairy products, a maximal tolerable concentration for PCDDs/PCDFs of 17.5 ng I-TEQ/kg fat has been set in the United Kingdom. In Germany, PCDDs/PCDFs must not exceed 5 ng I-TRQ/kg milk fat and, in the Netherlands, they must not exceed. 6 ng I-THQ/kg milk and milk product fat. The Canadian Government has proposed a tolerable daily intake (TDI) value of 10 pg I-TEQ/ kg bw per day for PCDDs and PCDFs.

Further reading Agency for Toxic Substances and Disease Registry (ATSDR) (2022) Toxicological Profile for Chlorodibenzofurans (CDFs) (Draft for Public Comment). Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service. Available at: https://wwwn.cdc.gov/TSP/ToxProfiles/ToxProfiles.aspx?id¼938&tid¼194. Accessed 9 February 2023. Domingo JL (2023) Dioxins and furans in cow milk and dairy products: A review of the scientific literature. International Journal of Dairy Technology. 76(1): 15–27. https://doi.org/ 10.1111/1471-0307.12917. Fernandez-Gonzalez R, Yebra-Pimentel I, Martinez-Carballo E, and Simal-Gandara J (2015) A critical review about human exposure to polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) through foods. Critical Reviews in Food Science and Nutrition 55(11): 1590–1617. González N and Domingo JL (2021) Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in food and human dietary intake: An update of the scientific literature. Food and Chemical Toxicology 157: 112585. Kanan S and Samara F (2018) Dioxins and furans: A review from chemical and environmental perspectives. Trends in Environmental Analytical Chemistry 17: 1–13. Masuda Y (2001) Fate of PCDF/PCB congeners and change of clinical symptoms in patients with Yusho PCB poisoning for 30 years. Chemosphere. 43(4–7): 925–930. National Center for Biotechnology Information (2023) PubChem Compound Summary for CID 568, Dibenzofuran. Retrieved February 9, 2023 from https://pubchem.ncbi.nlm.nih.gov/ compound/Dibenzofuran. National Center for Biotechnology Information (2023) PubChem Compound Summary for CID 32781, 2-Chlorodibenzofuran. Retrieved February 9, 2023 from https://pubchem.ncbi. nlm.nih.gov/compound/2-Chlorodibenzofuran. National Center for Biotechnology Information (2023) PubChem Compound Summary for CID 53154, 4-Chlorodibenzofuran. Retrieved February 9, 2023 from https://pubchem.ncbi. nlm.nih.gov/compound/4-Chlorodibenzofuran. Onozuka D, Nakamura Y, Tsuji G, and Furue M (2021) Cancer-and noncancer-specific cumulative incidence of death after exposure to polychlorinated biphenyls and dioxins: A competing risk analysis among Yusho patients. Environment International 147: 106320. Onozuka D, Nakamura Y, Tsuji G, and Furue M (2020) Mortality in Yusho patients exposed to polychlorinated biphenyls and polychlorinated dibenzofurans: A 50-year retrospective cohort study. Environmental Health 19(1): 1–10. Shen H, Han J, Guan R, Cai D, Zheng Y, Meng Z, Chen Q, Li J, and Wu Y (2022) Use of different endpoints to determine the bioavailability of polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) and polychlorinated biphenyls (PCBs) in Sprague–Dawley rats. Scientific Reports 12(1): 20433. https://doi.org/10.1038/s41598-022-25042-3. Van den Berg M, De Jongh J, Poiger H, and Olson JR (1994) The toxicokinetics and metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and their relevance for toxicity. Critical Reviews in Toxicology 24(1): 1–74. Woolf AD (2022) Japan “Yusho” poisoning, 1968 and Taiwan “Yucheng” poisoning, 1979. In: History of Modern Clinical Toxicology, pp. 121–135. Academic Press.

Relevant websites https://wwwn.cdc.gov/TSP/ToxFAQs/ToxFAQsDetails.aspx?faqid¼937&toxid¼194 :ToxFAQs™ for Chlorodibenzofurans (CDFs) https://www.atsdr.cdc.gov/toxguides/toxguide-32.pdf :ToxGuideTM for Chloro- dibenzofurans (CDFs) – January 2022 https://cameochemicals.noaa.gov/chemical/30022 :Chemical Datasheet – Chlorodibenzofurans https://www.euro.who.int/__data/assets/pdf_file/0017/123065/AQG2ndEd_5_11PCDDPCDF.pdf :Air quality guidelines for Europe. second edition (2000) – Chapter 5.11 Polychlorinated dibenzodioxins and dibenzofurans https://www.ncbi.nlm.nih.gov/books/NBK409967/ :IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Polychlorinated Dibenzo-para-dioxins and Polychlorinated Dibenzofurans. Lyon (FR): International Agency for Research on Cancer; 1997. (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, No. 69.) Polychlorinated Dibenzofurans. http://toolkit.pops.int/Publish/Downloads/UNEP-POPS-TOOLKIT-2012-En.pdf :UNEP (2013) Toolkit for Identification and Quantification of Releases of Dioxins, Furans and Other Unintentional POPs (under Article 5 of the Stockholm Convention), January 2013

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Chloroethane Atoosa Karimi Babaahmadi and Maryam Armandeh, Toxicology and Diseases Group (TDG), Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran, Iran; Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS), Tehran, Iran © 2024 Elsevier Inc. All rights reserved. This is an update of S. Milanez, Chloroethane, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 880–882, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01114-3.

Chemical profile Introduction Use Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Mechanisms of toxicity Interactions Acute and short-term toxicity Animal Human Chronic toxicity Animal Human Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicity Exposure standards and guidelines References Further reading

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Abstract Chloroethane, also known as ethyl chloride, is a versatile organic compound used in gasoline additives, polymers, anesthetics, and as a refrigerant in industry. Exposure to levels of more than 40,000 ppm via inhalation for a few hours causes symptoms of neurotoxicity and pathological alterations in the lungs, heart, liver, kidneys, and spleen, leading to death in some species. Chronic exposure to 15,000 ppm in mice resulted in mild nephrotoxicity. After a few breaths of 20,000–40,000 ppm, in humans, mild eye discomfort, slight stomach pains, nausea, vomiting, and disorientation were noted, while more prolonged exposure caused temporary drowsiness, dizziness, poor coordination, and unconsciousness. Anesthesia doses of chloroethane have induced cardiac depression in some cases due to vagus nerve stimulation, resulting in arrhythmia and death. Abusers who inhaled chloroethane on a regular basis had serious brain damage. Chloroethane is classified as an animal carcinogen with no known human implications.

Keywords Cardiotoxicity; Chloroethane; Ethyl chloride; Nephrotoxicity; Neurotoxicity

Key points 1. 2. 3. 4. 5.

Chloroethane, also known as ethyl chloride, is a chemical compound initially used to produce tetraethyl lead. Chloroethane is an adaptable, organic compound used in diverse applications. Chloroethane presents numerous chemical and physical properties that make it the proper chlorinated solvent. Prolonged contact with chloroethane, can be toxic to both humans and animals in various organs. Chloroethane is not carcinogenic to humans, but its carcinogenic effects have been found in animals.

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Chloroethane

Chemical profile

• • •

Name: Chloroethane. Synonyms: Ethyl chloride; Monochloroethane “https://pubchem.ncbi.nlm.nih.gov/compound/Chloroethane#section¼3DConformer” anodynon, chelen, chlorine, chloroethyl, chloryl, dublofix, hydrochloric ether, kelene, monochloroethane, narcotile (Safe Work Australia, 2019). Molecular Formula: C2H5Cl “https://www.drugbank.ca/drugs/DB13259”

•

Chemical Structure:

Introduction Chloroethane is a colorless gas with a sharp smell. When held in pressurized containers, it is a liquid; nevertheless, it soon evaporates when exposed to air. Chloroethane is easily ignited. It is synthesized using ethylene hydrochloride and as a by-product of polyvinyl chloride production. It is a lipophilic compound with moderate solubility in water but with high solubility in organic solvents “https://wwwn.cdc.gov/TSP/substances/ToxSubstance.aspx?toxid¼161”.

Use Historically, chloroethane was used to make tetraethyl lead (an anti-knock additive to leaded gasoline). Presently, chloroethane is widely used as a blower in foamed plastics. The use of chloroethane in insecticides and as an ethylating agent in the manufacture of dyes and drugs, refrigerants, local anesthetics, paints, chemicals, and pharmaceuticals has also been approved. In medicine, chloroethane is used to reduce pain caused by burns and insect bites, test pulp freshness in dentistry, and as a supplement in treating alopecia areata and creep eruptions as an anti-stimulant to relieve muscle and visceral pain “http://www.npi.gov.au/ resource/chloroethane-ethyl-chloride”.

Environmental fate and behavior Chloroethane is released into the environment during production and consumption as a solvent, refrigerant, anesthetic, and in organic synthesis through various waste streams. The ethyl chloride gas phase is decomposed in the atmosphere by reaction with photochemically produced hydroxyl radicals with a half-life of >23 days. If ethyl chloride is left in the soil, it has very high mobility. When released into water, ethyl chloride is not absorbed into suspended solids and sediments in the water. Since chloroethane is only moderately water-soluble, it can be removed from the air by wet or dry deposition. It is expected to have very high mobility in the soil “http://www.npi.gov.au/resource/chloroethane-ethyl-chloride; https://pubchem.ncbi.nlm.nih.gov/ compound/Chloroethane#section¼Environmental-Fate-Exposure-Summary”. There is a possibility that chloroethane can be released into the environment through its production and usage as a chemical intermediate, by burning plastics and refuse in wastewater treatment facilities, and by leaching from landfills (Anon, 2000).

Exposure and exposure monitoring Chloroethane may be absorbed into the body by inhalation, ingestion, and eye and skin contact. Workers may be exposed to chloroethane through inhalation and dermal contact during its use or productionGeneral public may be exposed to the substance by inhalation, consuming contaminated water, applying consumer products (solvents, paints, etc., and refrigerants), and its use as a topical anesthetic. Even though chemists use gas chromatography to measure ethyl chloride in blood, milk, and urine, no commonly used medical test can determine whether or not the individual has been exposed to ethyl chloride.

Toxicokinetics Chloroethane can easily be absorbed via inhalation. Oral exposure to chloroethane did not lead to its absorption. Based on physical properties, a dermal flux rate of 0.99 mg/cm2/h has been estimated. Once absorbed, chloroethane has more affinity to fat than muscle or liver based on partition coefficients. Studies on the human metabolism of chloroethane have not yet been conducted. Chloroethane’s two most significant metabolism pathways in rats and mice are the production of acetaldehyde by cytochrome P450

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and conversion to S-ethyl-glutathione by glutathione conjugation. Acetaldehyde is rapidly metabolized into acetic acid. Upon further metabolization, glutathione metabolites become S-ethyl-L-cysteine in mice and S-ethyl-N-acetyl-L-cysteine in rats and mice (ATSDR, n.d.).

Mechanisms of toxicity Toxicity mechanism of chloroethane has not been fully elucidated, though it primarily affects the central nervous system (CNS), the heart, liver, and kidneys. Chloroethane is a small lipophilic compound capable of crossing membranes through diffusion. Humans and animals may experience anesthetic effects due to inhaling high concentrations of chloroethane, which interacts with the lipid layer of the cellular membrane or with hydrophobic areas of specific membrane-bound proteins. A reduction in ATP use has been observed in in vitro studies, possibly due to disruption of the lipid structure in the transverse tubule walls when chloroethane is introduced. As a result of inhaling high concentrations of chloroethane, the heart becomes more susceptible to catecholamine effects. Ethyl chloride can cause euphoria, excitement, asphyxia, and hypoxia, resulting in death. There is speculation that chloroethane may cause an atrioventricular block by depressing the atrioventricular nodal conduction (TODD, 1998).

Interactions Chloroethane interacts with Tramadol, Fluoxetine, Haloperidol, Cyclobenzaprine and Ziprasidone. The risk or severity of CNS depression can be increased when Ethyl chloride is combined with Tramadol, Fluoxetine, Haloperidol, and Cyclobenzaprine. Ziprasidone and Ethyl chloride can increase the risk of adverse effects “https://pubchem.ncbi.nlm.nih.gov/compound/ Chloroethane#section¼3D-Conformer”.

Acute and short-term toxicity Animal As a result of vagal nerve stimulation, dogs exposed to anesthetic concentrations of chloroethane had twitching and tremoring muscles and cardiac depression, which caused asystole, ventricular tachycardia, and death (ATSDR, 1998). The liver weight and hepatocellular vacuolation of mice exposed to 5000 ppm continuously for 11 days increased, but other organs showed no significant differences (Landry et al., 1989). The guinea pigs died after exposure of 9 h to 40,000 ppm, with pathological changes in hearts, lungs, kidneys, livers, and spleens and signs of CNS toxicity such as dizziness and ataxia (United States Environmental Protection Agency, 1999).

Human Humans exposed to high levels of ethyl chloride by inhalation experienced temporary drunkenness, dizziness, lack of coordination, and unconsciousness. Its former medical use as an anesthetic during major surgery has caused several accidental deaths (Anon, 2000). Chloroethane vapor irritates the eyes, nose, and throat, and chloroethane liquid irritates the skin and eyes. Subjective intoxication and slowed reaction times were reported after exposure to 13,000 ppm for 12 min. Slight eye irritation, minor abdominal cramps, nausea, vomiting, and dizziness after a few breaths of 20,000–40,000 ppm were reported, but may need 10 min to become intoxicated, confused, and unconscious. Long-term exposure to chloroethane can result in frostbite symptoms due to prolonged skin exposure (ATSDR, 1998).

Chronic toxicity Animal Inhalation exposure to ethyl chloride has been shown to cause effects on the kidneys, lungs, liver, and heart in animals (Anon, 2000). Studies with rats and mice showed that chronic exposure to up to 15,000 ppm chloroethane for 2 years did not cause histopathological changes to the respiratory tract, GI tract, cardiovascular system, or liver. Mice exposed to 15,000 ppm showed mild nephrotoxicity as evidenced by tubular regeneration and minimal glomerulosclerosis in females and slight renal tubular cell nuclei extension in males (ATSDR, n.d.).

Human Some individuals who intentionally inhaled very high concentrations of ethyl chloride for a few months reported neurological symptoms, including sluggish lower limb reflexes, ataxia, tremors, inability to control voluntary movements, slowed reflexes, disorientation, short-term memory loss, seizures, an enlarged liver speech difficulties, hallucinations, and liver effects (U.S. Environmental Protection Agency, 1999).

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Immunotoxicity Evaluation of immunotoxicity for chloroethane has only been performed in studies of its inhaled form. Some inhalation studies have shown negative results for its immunotoxicology. Decreased leukocyte phagocytes in mice and reduced phagocytic activity in humans have been observed in some other studies by examining the neurotoxicity of chloroethane in mice and humans (Haneke, 2002). There have been cases of contact dermatitis after chloroethane has been applied to the skin. Neither continuous inhalation exposure of 4800 ppm for 11 days nor intermittent exposure to up to 15,000 ppm for 2 weeks to 2 years caused adverse effects on the organs or tissues of the immune system of mice. The spleen of guinea pigs exposed to 40,000 ppm chloroethane for 90 min showed anemia or enlargement (Zhang et al., 2009).

Reproductive toxicity Ethyl chloride inhalation exposure has not been associated with human reproductive or developmental effects. There was no evidence of reproductive harm caused by exposure to ethyl chloride. A study on animals showed a decrease in uterine mass. In another study, inhalation exposure to ethyl chloride did not cause evidence of fetal toxicity (increase in unossified skull bones) (Agency for Toxic Substances and Disease Registry (ATSDR), 1998). Animals exposed to chloroethane by inhalation for up to 2 years did not exhibit any pathological changes in their reproductive organs. Mice exposed to chloroethane during gestation days 6–15 did not show any maternal toxicity or adverse effects on reproductive or fetal parameters, except for a small but statistically significant increase in unossified skull bones at the high dose (5000 ppm). Chloroethane anesthesia decreased the motility and muscle tone of the uterus in dogs. The glutathione levels were reduced in several organs in rats and mice exposed to chloroethane, with the uterus showing the most significant reduction. The uterine weight of mice exposed to chloroethane at high concentrations was reduced, and the estrous cycle duration was longer (ATSDR, 1998).

Genotoxicity Inhalation of chloroethane does not appear to result in genotoxic effects in humans. In mice exposed to 25,000 ppm chloroethane for 3 days, the number of micronuclei in bone marrow cells was not increased, or DNA synthesis was not affected. The researchers determined that the dose used in this study had an exposure concentration of approximately 66% of the flammability limit. It was the highest dose that could be administered safely (TODD, 1998). A mutagenic effect at the HPRT locus was observed in Chinese hamster ovary cells from mice exposed to chloroethane but not in hepatocyte primary cultures or micronuclei isolated from mice exposed by inhalation. Two independent experiments caused cytotoxicity in BALB/c-3T3 cells, but only one elicited morphological transformation. TA1535 and TA100 Salmonella typhimurium were mutagenic in the Ames test but not TA98 or TA1537 strains (ICH Harmonised Guideline, 2021).

Carcinogenicity For ethyl chloride, no carcinogenicity data are available. According to The National Toxicology Program (NTP), inhaled chloroethane is carcinogenic to female mice and may also be carcinogenic to rats (Anon, 2000). The number of uterine tumors and hepatocellular tumors significantly increased in female mice, but the male data was lacking due to the low survival rate. Male and female rats were found to have benign and malignant epithelial neoplasms of the skin and three unusual malignant astrocytomas of the brain. Ethyl chloride has not been classified as carcinogenic by The Environmental Protection Agency (EPA) (National Toxicology Program, n.d.).

Clinical management Chloroethane vapors can irritate skin, eyes, and mucous membranes, causing blurred vision, ataxia, CNS depression, headache, dizziness, stupor, vomiting, eczema, allergic contact dermatitis, and abdominal pain cramps, and liver and kidney toxicity. In addition to increasing myocardial sensitivity to catecholamines, chloroethane can also lead to cardiac arrhythmias. People exposed to inhalation should be moved to fresh air and monitored for respiratory effects. It is necessary to remove any contaminated clothes. The afflicted area should be carefully cleaned with soap and water if exposed to the skin. Systemic or topical corticosteroids or antihistamines can treat skin hypersensitivity responses (Pereira et al., 2000). First, remove any contact lenses that are in the victim’s eyes. Immediately flush the victim’s eyes with water or normal saline solution for 20–30 min while calling a hospital or poison control center. Avoid applying ointments, oils, or medications to the victim’s eyes without receiving specific medical instructions “https://pubchem.ncbi.nlm.nih.gov/compound/Chloroethane#section¼3D-Conformer”.

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Ecotoxicity There was limited information available regarding chloroethane’s ecotoxicity. For the algae, Scenedesmus subspicatus and Daphnia Magna, EC50 values of 58 mg/L and 39 mg/L, respectively, were found. The soil bacterium Pseudomonas putida is shown to have an EC10 >140 mg/L (Pereira et al., 2000).

Exposure standards and guidelines Use National Institute for Occupational Safety & Health (NIOSH) and Mine Safety and Health Administration (MSHA) approved supplied-air respirator with a full facepiece, operated in a pressure-demand or other positive pressure mode when exposure may exceed 100 ppm. To provide increased protection, use in conjunction with an auxiliary self-contained breathing apparatus operated in a positive-pressure manner. Exposure to 3800 ppm is immediately hazardous to life and health. To mitigate exposure above 3800 ppm, wear a self-contained breathing apparatus approved by NIOSH/MSHA, with a full facepiece and a pressure-demand or other positive-pressure mode (Anon, 2007). According to ACGIH (2018) and SCOEL (1999), a time-weighted average (TWA) of 100 ppm is recommended based on the NOAEC determined in mice and an uncertainty factor of 10 to account for the short study duration. TWA protects liver, heart, and brain effects and ototoxicity (Safe Work Australia, 2019). Following ACGIH guidelines, the threshold limit value for TWA was set at 100 ppm over 8 h with a skin designation and defined excursion levels “https://www.osha. gov/annotated-pels/table-z-1”. Chloroethane is not assessed for carcinogenicity in the IRIS “https://cfpub.epa.gov/ncea/iris_drafts/ recordisplay.cfm?deid¼12183.”.

References Agency for Toxic Substances and Disease Registry (ATSDR) (1998) Toxicological Profile for Chloroethane (Update). Atlanta, GA: Public Health Service, U.S. Department of Health and Human Services. Anon (2000) Ethyl Chloride (Chloroethane). vol. 1, Environmental Protection Agency, p.3. https://www.epa.gov/sites/default/files/2016-09/documents/ethyl-chloride.pdf. Anon (2007) How to determine if you are being medical testing health hazard information acute Health Effects Cancer Hazard Reproductive Hazard Other Long-Term Effects. ATSDR (1998) Toxicological Profile for Chloroethane. U.S. Department of Health and Human Services Public Health Service Agency for Toxic Substances and Disease Registry. ATSDR (n.d.) The Agency for Toxic Substances and Disease Registry (ATSDR), based in Atlanta, Georgia, is a federal public health agency of the U.S. Department of Health and Human Services. Haneke KE (2002) Toxicological Summary for 4-Phenylcyclohexene. The National Institute of Environmental Health Sciences, pp. 1–53. ICH Harmonised Guideline (2021) Application of the principles of the ICH M7 Guideline to calculation of compound-specific acceptable intakes. Addendum to M7(R2), 6 October 2021, Draft Version, vol. 7, July 2015. Landry TD, et al. (1989) Ethyl chloride: 11-day continuous exposure inhalation toxicity study in B6C3F1 mice. Fundam Appl Toxicol 13(3): 516–522. https://doi.org/10.1016/02720590(89)90288-1. National Toxicology Program (n.d.) Toxicology and Carcinogenesis Studies of Chloroethane (Ethyl Chloride) (CAS No. 75-00-3) in F344/N Rats and B6C3F1 Mice (Inhalation Studies). TR No. 346. U.S. Department of Health and Human Services, Public Health Service. Pereira AMPT, Silva LJG, Laranjeiro CSM, Lino C, Bromatology G and Sciences A (2000) Supporting information for Selected pharmaceuticals in different aquatic compartments: Part II—Toxicity and environmental risk assessment E-mail addresses: Table S1. Ecotoxicological Data on the Selected Pharmaceuticals. Safe Work Australia (2019) Ethyl chloride (75-00-3) Safe Work Australia. U.S. Environmental Protection Agency (1999) Integrated Risk Information System (IRIS) on Ethyl Chloride. National Center for Environmental Assessment, Office of Research and Development: Washington, DC 1999. TODD GD, et al. (1998) Toxicological profile for chloroethane. U.S. Department of Health and Human Services, Public Health Service Agency for Toxic Substances and Disease Registry. United States Environmental Protection Agency (1999) Toxicological Review of Chloroethane (CAS No. 75-00-3) in Support of Summary Information on the Integrated Risk Information System (IRIS). United States Environmental Protection Agency. Zhang EY, Chen AY, and Zhu BT (2009) Mechanism of dinitrochlorobenzene-induced dermatitis in mice: Role of specific antibodies in pathogenesis. PLoS One 4(11): e7703. https:// doi.org/10.1371/journal.pone.0007703.

Further reading RXLIST (n.d.) https://www.rxlist.com/ethyl-chloride-drug.htm#warnings.

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Chlorofluorocarbons WT Tsai, National Pingtung University of Science and Technology, Pingtung, Taiwan © 2024 Elsevier Inc. All rights reserved. This is an update of W.T. Tsai, Chlorofluorocarbons, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 883–884, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.01118-0.

Background Major uses Environmental hazards Exposure routes and pathways Toxicokinetics Acute (short-term) health effects Chronic (long-term) health effects Exposure standards/limits References

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Abstract Chlorofluorocarbons (CFCs) cause significantly stratospheric ozone depletion and global warming relating to the greenhouse effect. CFCs under commercial uses as refrigerants and blowing agents include trichlorofluoromethane (CFC-11), dichlorodifluoromethane (CFC-12), chlorotrifluorocarbon (CFC-13), 1,1,2,2-tetrachloro-1,2-difluoroethane (CFC-112), 2,2-Difluorotetrachloroethane (CFC-112a), 1,1,2-trichloro-1,2,2-trifluoroethane (CFC-113), 1,1,1-trichloro2,2,2-trifluoroethane (CFC-113a), 1,2-dichlorotetrafluoroethane (CFC-114), 1,1-dichlorotetrafluoroethane (CFC-114a), chloropentafluoroethane (CFC-115) and 1,2-dichlorohexafluoro-cyclobutanes (R-316c). Regarding their health effects, CFCs can cause asphyxiation and cardiac sensitization at high concentrations, but their potential for chronic effects is low. Through CFCs have been banned on use due to the issues of ozone layer protection and climate change, the reduction in the environmental and health impacts of these substances should be considered by the occupational exposure limits (OEL) in some occasional cases.

Keywords Aerosol propellant; Blowing agent; Cleaning agent; Exposure risk; Fire extinguisher; Global warming potential; Health effect; Ozone depletion potential; Ozone layer; Refrigerant; Occupational exposure limit

Key points

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Environmental properties of common chlorofluorocarbons (CFCs) are summarized. Health effects of some chlorofluorocarbons (CFCs) are reviewed. Occupational exposure limits (OEL) of some chlorofluorocarbons (CFCs) are surveyed.

Background It is recognized that nontoxic and nonflammable refrigerants (Freon or chlorofluorocarbon (CFC)) were commercialized in the manufacture of chlorofluoro-derivatives of methane and ethane until the 1930s. Since that time, studies on the synthesis of CFCs and their applications have progressed in many directions, such as aerosol, blowing agent for foam manufacture, fire extinguisher, cleaning solvent, and refrigerant. Due to the potential environmental and health effects of ozone depletion and greenhouse effect, the use of Freons has been reduced by international agreements since the end of the 1980s. Under a treaty known as the Montreal Protocol on Substances that Deplete the Ozone Layer, which was first established in 1987, several interim replacements for CFCs were thus developed since the 1990s, i.e., partially or fully fluorinated or partially chlorofluorinated alkanes and olefins, including hydrochlorofluorocarbons (HCFCs), hydrofluorocarbons (HFCs), perfluorocarbons (PFCs), hydrofluoroolefins (HFOs) and hydrochlorofluoroolefins (HCFO) (Sekiya et al., 2006; Rusch, 2018; Sicard and Baker, 2020). Table 1 lists the chemical identities and the information on atmospheric lifetime, ozone depletion potential (ODP), and global warming potential (GWP) of common CFCs (Mackay et al., 2006; United Nations Environment Programme UNEP, 2018; Hodnebrog et al., 2020), including trichlorofluoromethane (CFC-11), dichlorodifluoromethane (CFC-12), chlorotrifluorocarbon (CFC-13), 1,1,2,2-tetrachloro1,2-difluoroethane (CFC-112), 2,2-Difluorotetrachloroethane (CFC-112a), 1,1,2-trichloro-1,2,2-trifluoroethane (CFC-113),

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Chlorofluorocarbons Environmental properties of common chlorofluorocarbons (CFCs).

CFCs (abbrev.)

CAS no.

Molecular formula

Mol. wt. (g/mol)

Lifetime (years)

ODPa

GWPb

Trichlorofluoromethane (CFC-11) Dichlorodifluoromethane (CFC-12) Chlorotrifluorocarbon (CFC-13) 1,1,2,2-tetrachloro-1,2-difluoroethane (CFC-112) 2,2-Difluorotetrachloroethane (CFC-112a) 1,1,2-Trichloro-1,2,2-trifluoroethane (CFC-113) 1,1,1-Trichloro-2,2,2-trifluoroethane (CFC-113a) 1,2-Dichlorotetrafluoroethane (CFC-114) 1,1-Dichlorotetrafluoroethane (CFC-114a) Chloropentafluoroethane (CFC-115) (E )-1,2-Dichlorohexafluoro-cyclobutane (Z )-1,2-Dichlorohexafluoro-cyclobutane

75-69-4 75-71-8 75-72-9 76-12-0 76-11-9 76-13-1 354-58-5 76-14-2 374-07-2 76-15-3 356-18-3 356-18-3

CCl3F CCl2F2 CClF3 CCl4F2 CCl3CClF2 CCl2FCClF2 CCl3CF3 CClF2CClF2 CCl2FCF3 CClF2CF3 (E )-1,2-c-C4F6Cl2 (Z )-1,2-c-C4F6Cl2

137.38 120.91 104.46 203.83 203.83 187.40 187.40 170.93 170.93 154.47 232.94 232.94

52 102 640 63.6 52 93 55 189 105 540 75 114

1.0 0.80 1.0 0.98 0.86 0.81 0.73 0.50 0.72 0.26 0.46 0.54

5160 10,300 13,900 4370 3455 6080 3750 8580 6670 7310 4050 5400

a

Ozone depletion potential (ODP). Global warming potential (GWP) with 100-year time horizon.

b

1,1,1-trichloro-2,2,2-trifluoroethane (CFC-113a), 1,2-dichlorotetrafluoroethane (CFC-114), 1,1-dichlorotetrafluoroethane (CFC-114a), chloropentafluoroethane (CFC-115) and 1,2-dichlorohexafluoro-cyclobutanes (R-316c). It showed that atmospheric lifetimes vary among CFCs. CFC-11 has the shortest lifetime and a lower GWP. These CFCs contain only the elements chlorine, fluorine, and carbon. They are usually colorless gases or liquids that evaporate easily at room temperatures. They are generally unreactive and stable, nontoxic, and nonflammable. It means that the atmosphere is the most likely fate for their accumulations of emissions. CFCs are also a part of the group of chemicals known as the volatile organic compounds. Based on their physicochemical properties, minimizing the impacts of CFCs not only contributes to the ozone layer protection and global warming mitigation, but also takes into account the potential health hazards from occupational and environmental exposures. Factors to reduce waste and minimize the future impact of these substances should be considered.

Major uses The chemical inertness, thermal stability, low toxicity, and non-flammability of these CFCs coupled with their unique physical properties are used in many application fields, including refrigerant for air conditioning, aerosol (propellant), blowing agent for foam manufacture, fire extinguisher, cleaning agent (solvent), dielectric fluid, and ion implantation of semiconductor device. In addition, CFCs (i.e., CFC-11, CFC-12, CFC-113) have been used as environmental tracers in ocean circulation and aquifers investigations. However, it should be noted that the timetable of phase-out of these compounds was regulated by the Montreal protocol (i.e., the production of CFCs ended by 1 January 1996 and their applications banned).

Environmental hazards GWP expresses the relative increase in earthward IR radiation flux due to the emission of organic compounds. Notably, all CFCs have high GWP values relative to the reference compound, carbon dioxide (seen in Table 1). As described above, the most significant environmental hazard associated with CFCs is ozone depletion, which is caused by chlorine molecules in these so-called ozone-depleting substances that migrate to the stratosphere and then react catalytically with ozone, thus destroying it. However, from an air quality perspective, CFCs have been listed as having “negligible photochemical reactivity” and do not contribute to smog formation and ground-level ozone. Accordingly, they are exempt from volatile organic compound regulations according to the US Clean Air Act Amendments of 1990.

Exposure routes and pathways Inhalation (pulmonary route) is the main source of exposure to CFCs. Skin absorption or contact (dermal exposure) and eye contact may also occur. Because they are no longer used as refrigerants and blowing agents, human exposure to CFCs may occur via inhalation from accidental leaks or spills from discarded or waste gas cylinders.

Chlorofluorocarbons

919

Toxicokinetics Humans can be exposed to CFCs via inhalation, ingestion, or dermal contact. Inhalation is exposure pathway is one of the primary route of exposure by which CFCs can be rapidly absorbed into the blood. Due to its rapid absorption properties in blood, CFCs can be found in heart, lungs and muscles. Exhalation is identified as the primary mode of elimination. Based on several experimental studies and human volunteer exposure studies, CFCs can be absorbed across the alveolar membrane, gastro-intestinal tract, or the skin (WHO, 1990). CFCs are characterized by high chemical and thermal stabilities, non-flammability, and low toxicity. Although CFCs are physiologically inert, they can cause cardiac sensitization (i.e., sensitization of the heart to the body’s adrenalin) at high concentrations (e.g., above 10% in air). This can lead to cardiac arrhythmia, resulting in irregular heartbeat and sometimes cardiac arrest (Dekant, 1996).

Acute (short-term) health effects Due to the physiochemical properties of CFCs, there is only a low potential for human toxicity based on the results of mammalian (i.e., rat) tests, and no significant acute health risk is expected. Briefly, the biotransformation of CFCs has been shown to undergo cytochrome P450-catalyzed oxidation or reduction reactions. Acyl halides that are formed are further hydrolyzed to give excretable haloacetic acid (e.g., trifluoroacetic acid) in urine. Although CFCs are physiologically inert, exposure to pressurized CFCs like liquid nitrogen may occur with a refrigerant leak that can cause frostbite to the upper airway if inhaled, possibly resulting in asphyxiation and cardiac sensitization (i.e., sensitization of the heart to the body’s adrenalin) at high concentrations. In occasional cases like the repair of air-conditioning systems, fires of landfill, or thermal destruction of waste CFCs could result in the occupational exposure to decomposition toxic products, including hydrogen fluoride (HF), hydrogen chloride (HCl), carbon monoxide (CO), phosgene (COCl2), and carbonyl fluoride (COF2).

Chronic (long-term) health effects In general, occupational workers exposed to CFCs at the exposure standards/limits (described below) showed no adverse health effects. In experimental animals exposed to CFCs for long-term tests, no significant effects were observed. Regarding the carcinogenic, reproductive, and developmental effects of CFCs, their potential for such chronic effects is extremely low.

Exposure standards/limits American Conference of Governmental Industrial Hygienists (ACGIH) has established threshold limit values (TLV) for some of the CFCs (Table 2). Table 2 also includes information on adverse health effect(s) based on which those TLVs were derived (ACGIH, 2019). OSHA PEL values are also provided for comparison. Table 2

Toxicity data and exposure limits of common chlorofluorocarbons (CFCs).

CFCs

Target organsa

OSHA-PELb (ppm)

ACGIH-TLVc (ppm)

TLV-basis

CFC-11 CFC-12 CFC-112a

Skin, respiratory system, cardiovascular system Cardiovascular system, peripheral nervous system Eyes, skin, respiratory system, central nervous system

1000 1000 500

1000 (Ceiling) 1000 100

CFC-112

Eyes, skin, respiratory system, central nervous system

500

50

CFC-113 CFC-114 CFC-115

Skin, heart, central nervous system, cardiovascular system Respiratory system, cardiovascular system Skin, central nervous system, cardiovascular system

1000 1000 –d

1000 1000 1000

Cardiac sensitization Cardiac sensitization Liver & kidney damage; central nervous system impairment Liver & kidney damage; central nervous system impairment Central nervous system impairment Pulmonary function Cardiac sensitization

a

National Institute for Occupational Safety and Health (NIOSH), NIOSH Pocket Guide to Chemical Hazards. OSHA-PEL: US Occupational Safety and Health Administration permissible exposure limit based on 8-h time-weighted average.

b c

ACGIH-TLV: American Conference of Governmental Industrial Hygienists-Threshold Limit Value based on 8-h time-weighted average.

d

Not available.

920

Chlorofluorocarbons

CFCs have a low potential for toxicity in humans as well as ecological receptors, and are considered not readily biodegradable. TLV basis can provide a reference and health basis for associated symptoms related to exposure. OSHA’s PEL values and ACGIH’s TLVs for most of the CFCs (except for CFC-112,CFC-112a) have been set at 1000 ppm as an 8 h time-weighted average (TWA) based on the health effects such as cardiac sensitization and pulmonary function. However, it should be noted that there are some exceptions as in the cases of CFC-112a and CFC-112. Based on experimental animal studies with repeated exposures at high concentrations, liver and kidney damages were reported. Therefore, TLV-time-weighted average values of 100 ppm and 50 ppm have been set for CFC-112a and CFC-112, respectively.

See also: Aerosols; Global climate change and environmental toxicology: Characterizing interactions between chemicals, species sensitivity, and human behavior; Ozone

References American Conference of Governmental Industrial Hygienists (ACGIH) (2019) Documentation of the Threshold Limit Values and Biological Exposure Indices. Cincinnati, Ohio, USA: ACGIH. Dekant W (1996) Toxicology of chlorofluorocarbon replacements. Environmental Health Perspectives 104(Suppl. 1): 75–83. Hodnebrog Ø, Aamaas B, Fuglestvedt JS, Marston G, Myhre G, Nielsen CJ, Sandstad M, Shine KP, and Wallington TJ (2020) Updated global warming potentials and radiative efficiencies of halocarbons and other weak atmospheric absorbers. Reviews of Geophysics 58: e2019RG000691. https://doi.org/10.1029/2019RG000691. Mackay D, Shiu WY, Ma KC, and Lee SC (2006) Handbook of Physical-chemical Properties and Environmental Fate for Organic Chemicals, 2nd edn. Boca Raton, FL, USA: CRC Press. Rusch GM (2018) The development of environmentally acceptable fluorocarbons. Critical Reviews in Toxicology 48: 615–665. Sekiya A, Yamabe M, Tokuhashi K, Hibino Y, Imasu R, and Okamoto H (2006) Evaluation and selection of CFC alternatives. In: Tressaud A (ed.) Fluorine and the Environment: Atmospheric Chemistry, Emissions, & Lithosphere, pp. 33–87. Amsterdam: Elsevier. Sicard AJ and Baker RT (2020) Fluorocarbon refrigerants and their syntheses: Past to present. Chemical Reviews 120(17): 9164–9303. United Nations Environment Programme (UNEP) (2018) Scientific Assessment of Ozone Depletion: 2018. Geneva: UNEP. WHO (1990) Fully Halogenated Chlorofluorocatbons, Environmental Health Criteria 113. Available at: https://www.inchem.org/documents/ehc/ehc/ehc113.htm#. (Accessed February 12, 2023).

Relevant websites https://www.inchem.org/documents/ehc/ehc/ehc113.htm# :Fully Halogenated Chlorofluorocarbons (Environmental Health Criteria 113). https://pubchem.ncbi.nlm.nih.gov/#query¼Chlorofluorocarbon :PUBCHEM-Chlorofluorocarbon profile. http:// www.cdc.gov/niosh/npg/default.html/ :NIOSH Pocket Guide to Chemical Hazards. https://www.canada.ca/en/environment-climate-change/services/air-pollution/issues/ozone-layer/measures-protect/other-national-initiatives.html :Protecting the ozone layer: other national initiatives-Government of Canada. https://www.epa.gov/ozone-layer-protection/ozone-depleting-substances :United States Environmental Protection Agency-Ozone Depleting Substances and other resources.

Chloroform Eugenio Vilanova, Carmen Estevan, Miguel A Sogorb, and Jorge Estévez, Instituto de Bioingeniería, Unidad de Toxicología y Seguridad Química, Universidad Miguel Hernández de Elche, Elche, Spain © 2024 Elsevier Inc. All rights reserved. This is an update of J. Estévez, E. Vilanova, Chloroform, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 885–890, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00479-6.

Chemical profile Background Uses Environmental fate and behavior Routes and pathways Physical and chemical properties Partition behavior in water, sediment and soil Environmental persistency Bioaccumulation and biomagnification Exposure and exposure monitoring Routes and pathways Human exposure Environmental exposure Occupational exposure Toxicokinetics Mechanism of toxicity Acute and short-term toxicity Chronic toxicity Reproductive toxicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Freshwater/sediment organisms toxicity Marine organisms toxicity Terrestrial organisms toxicity (soil microorganisms, plants, terrestrial invertebrates, terrestrial vertebrates) Other hazards Exposure standards and guidelines References

921 922 922 922 922 923 923 923 923 924 924 924 924 924 924 924 925 925 926 926 926 926 926 926 927 927 928 928 928

Abstract Chloroform is a volatile, heavy, colorless liquid substance. It is used as intermediate in the production of dyes, pesticides, and other substances, as a production and extraction solvent, in the water chlorination and in the pulp and paper bleaching. The tissues that most actively metabolize chloroform (liver, kidney) are also the primary target tissues. There are clear differences between genders, strains, and species in their relative sensitivity to chloroform, and these differences in toxicity correlate with differences in metabolic capacity.

Keywords Chloroform; Cytochrome P-450; Threehalomethanes

Chemical profile

• • •

Name: Chloroform Chemical Abstracts Service Registry Number: 67-66-3 European Inventory of Existing Commercial Chemical Substances Number: 200-663-8

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.01083-6

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922

• • •

Chloroform

Synonyms: Methane trichloride, Trichloromethane, Methenyl trichloride, Methyl trichloride, Trichloroform Molecular Formula: CHCl3 Chemical Structure:

Background Chloroform was discovered in 1831 independently by Liebig J., Soubeiran E., and Guthrie S. Chloroform was used in surgery by Ives E., but Simpson J.Y. first used chloroform in midwifery (1847). The anesthetic properties had been described by Flourens M.J.P. In 1906, Brown used a warmed mixture of nitrous oxide and oxygen, followed by ether and chloroform (Baskerville, 1911; Wawersik, 1997). Very soon sudden deaths were reported and a specific effect on the heart was suspected. In 1911, Levy A.G. proved in experiments with animals that chloroform could cause cardiac fibrillation. Between 1865 and 1920, chloroform was used in 80–95% of all narcosis performed in UK and German-speaking countries. The discovery of hexobarbital in 1932 was the beginning to the gradual decline of chloroform narcosis. The clinical use of chloroform ended in 1976 (Wawersik, 1997). Classification of the chloroform hazard according to the Globally Harmonized System (GHS) is shown in Table 1. It is classified as carcinogen category 2 (suspected of causing cancer) and also for oral acute toxicity, skin irritation, and for long-term effect (ECHA, 2023). Detail toxicological and environmental properties can be consulted in international databases and information in regulatory process (ECHA-ESR, 2007; WHO, 2004; NCBI-PubChem, 2023; EU-ECHA, 2022).

Uses Chloroform is mainly used as a raw material in the production of hydrochlorofluorocarbon-22 (HCFC 22), as a production and extraction solvent especially in the pharmaceutical industry (e.g., in the extraction of penicillin and other antibiotics), and it is also used as a solvent, degreasing agent, or chemical intermediate in industries like adhesives, pesticides, fats, oils, etc. Chloroform is a by-product in the manufacture of vinyl chloride/polyvinyl chloride (VC/PVC, IUPAC name: polychloroethene) products and other chlorinated bulk chemicals. Similarly, chloroform is an important building block for fluorinated polymers and copolymers. Chloroform has been used in the United States as an insecticidal fumigant on stored grains and as mildew fungicide for tobacco seedlings, but these applications are not registered in the European Union (EU). Elsewhere, unintended emissions of chloroform are observed in water chlorination processes or chlorination for paper bleaching.

Environmental fate and behavior Routes and pathways Chloroform in soil or surface water volatilizes readily; at equilibrium, greater than 99% is expected to partition to the atmosphere. Some wet deposition of atmospheric chloroform may occur, but subsequent revolatilization is likely to be extensive. Chloroform is not expected to partition significantly to soils or sediments, because its affinity for organic carbon and lipids is low. Compartmental partitioning has been reported to be 99.1%, 0.9%, 0.01%, and 0.01% in air, water, soil, and sediment, respectively. The preferred target compartment in the environment at equilibrium is the air compartment.

Table 1

Chloroform classification according to the Globally Harmonized System of Classification and Labeling of Chemicals.

Hazard class and category code(s)

Hazard statement code(s) and meaning

Pictogram signal word code(s)

Acute tox. 4 Carc. 2 STOT RE 2 Skin irrit. 2

H302 harmful if swallowed H351 suspected of causing cancer H372 may cause damage to organs through prolonged or repeated exposure H315 causes skin irritation

GHS08 GHS07

Source: Annex VI. Table 3.1 of European Regulation 1272/2008 (ECHA, 2023).

Chloroform Table 2

923

Physical and chemical properties of chloroform.

Property

Value

Molecular weight Relative density Melting point Boiling point Vapor Pressure Relative density Surface tension Partition coefficient octanol water Henry’s law Water solubility Flash point Flammability

119.5 g mol−1 1480 kg m−3 −63.5
C 61.3
C 209 hPa at 20
C 1.48 at 20
C 0.0271 Nm−1 at 20
C Log Kow1.97 constant H ¼ 367 Pa.m3 mol−1 at 25
C 8700 mg L−1 at 23
C none no

Source: European Union Risk Assessment Report of chloroform (ECHA-ESR, 2007). More information in: WHO, 2004; NCBI-PubChem, 2023; EU-ECHA, 2022.

Physical and chemical properties Chloroform is a volatile, heavy, colorless liquid. It is nonflammable and possesses a characteristic sweet odor. The physical and chemical properties of chloroform are summarized in Table 2.

Partition behavior in water, sediment and soil Chloroform is considered as nonbiodegradable in water. Hydrolysis is an unimportant fate process at a neutral pH value and direct photolysis in water is not expected too. In surface water, the main removal process is volatilization with estimated half-lives of 1.5 days and 9–10 days in a river and a lake, respectively. Most studies have indicated little biodegradation up to 25 weeks in aquatic systems under aerobic conditions. The major fate of chloroform at the soil surface is temperature-dependent volatilization due to its volatile nature and low soil adsorption.

Environmental persistency In air, a half-life value of 105 days is estimated. Chloroform emitted to air reacts primarily with photochemically generated hydroxyl radicals in the troposphere. The reaction products include phosgene, dichloromethane, formyl chloride, carbon monoxide, carbon dioxide, and hydrogen chloride. The chemical degradation in sediments and soil is not rapid, except under anaerobic methanogenic conditions. Chloroform biodegradation is observed in anaerobic sediment and a half life in sediment is estimated at 15 days. The major degradation products under anaerobic conditions are carbon dioxide, methane, and hydrogen chloride, with smaller amounts of dichloromethane. A Koc value of 185 L kg−1 has been estimated.

Bioaccumulation and biomagnification The octanol/water partition coefficient and the bioconcentration factor measured for fish (see Table 3) indicate that chloroform is unlikely to bioaccumulate to any significant extent in aquatic biota. Table 3

Results from bioaccumulation experiments in different species of fish studied in Flow through.

Species

Exposure [d]

Water conc. [mg L−1]

Depuration

BCF (bioaccumulation factor)

Cyprinus carpio Cyprinus carpio Oncorhynchus mykiss Lepomis macrochirus Micropterus salmoides Ictalurus punctatusa

42 42 1 1 1 1

1000 100 1000 1000 1000 1000

– – Total depuration within 24 h Total depuration within 24 h Total depuration within 24 h 91% depuration within 26 h

1.4–4.7 4.1–13 3.4–10.4 1.6–2.5 2.1–2.2 3–3.4

a Equilibrium has not been reached. Source: European Union Risk Assessment Report of chloroform (ECHA-ESR, 2007). More information in: WHO, 2004; NCBI-PubChem, 2023; EU-ECHA, 2022.

924

Chloroform

Exposure and exposure monitoring Routes and pathways The environmental exposure of chloroform is based on the expected releases of the substance during the life cycle stages of the production of this substance, the use as an intermediate (HCFC 22 production, dyes, pesticides production, and other), the use as a solvent (chemical and pharmaceutical industry), the unintended formation (by-product during chemical and VC/PVC products manufacturing), water chlorination (drinking water, municipal wastewater, swimming pools, cooling water), the pulp and paper bleaching, atmospheric reaction of high tonnage chlorinated solvents, vehicle emissions, landfills, incineration processes, and natural sources. The major global sources for tropospheric chloroform would be direct emissions from the surface ocean, soils, and fungi, although biological processes are not well defined. Estimated emissions from anthropogenic sources account for 10% of the total emitted and the amounts emitted from fires represented only 0.4%. Chlorination of soil organic matter is one possible source of chloroform.

Human exposure Environmental exposure Based on concentrations determined in Canadian air (national surveys), food in Canada and the United States, and drinking-water, an average intake from food, drinking-water, and air varied from 0.6 to 10 mg kg−1 body weight per day. Upper-bounding estimates were calculated using maximum reported concentrations in water, food, and air and ranged from 40 to 95 mg kg−1 body weight per day. The estimated total daily dose was 54.8 mg kg−1 body weight per day in the EU Risk Assessment Report of chloroform.

Occupational exposure

Mean time-weighted average (TWA) exposures of 13, 2, and 1 mg m−3 for production operators, drummers/bottle fillers, and maintenance/utility personnel at one pesticide plant with levels of 10–1000 mg m−3 in a Polish pharmaceutical plant, an 8-h TWA of 77.4 mg m−3 (range 13–227 mg m−3) in a police forensic laboratory, and (during 1968–72) levels of 34–830 mg m−3 (mean 230 mg m−3, 79 samples) in a film manufacturing plant using a solvent containing 22% chloroform, have been reported (Flanagan and Pounder, 2010).

Toxicokinetics Chloroform is well absorbed, metabolized, and eliminated by mammals after oral, inhalation, or dermal exposure and widely distributed in the entire organism, via blood circulation and preferentially in fatty tissues and in the brain due to its liposolubility. The half-life in humans was 7.9 h following inhalation exposure. An oral-exposure study found most of the chloroform dose being eliminated within 8-h postexposure. Chloroform is mainly metabolized in liver; the major metabolite is carbon dioxide. The oxidative pathway in vivo generates also reactive metabolites including phosgene, whereas the reductive pathway generates the dichloromethylcarbene free radical. Both pathways proceed through a cytochrome P450-dependent enzymatic activation step and their balance depends on species, tissue, dose, and oxygen tension. Phosgene is produced by oxidative dechlorination of chloroform to trichloromethanol, which spontaneously dehydrochlorinates. The chloroform toxicity is due to its metabolites. Transplacental transfer of chloroform has been reported in mice and in the fetal blood in rats and it is expected to appear in human colostrum and is excreted in mature breast milk. Inhalation, dermal, and oral absorptions are considered to be 80%, 10%, and 100%, respectively, in animals and humans.

Mechanism of toxicity Chloroform elicits the same symptoms of toxicity in humans as in laboratory animals. Oral administration to rats and mice resulted in central nervous system depression and nasal lesions in rats and liver damage in both species and in mice by gavage, and in dogs. Rats exposed by inhalation for 4–6 months showed liver damage (necrosis) and increased kidney weight. Cell proliferation was seen in the nasal tissues of rats and mice inhaling chloroform for 13 weeks. Chloroform-induced kidney tumors in male mice exposed by inhalation or by ingestion in a toothpaste vehicle, but not when given in corn oil. Mice and female rats were not susceptible to chloroform-induced kidney cancer. Chronic inhalation caused ossification, necrosis, hyperplasia, and metaplasia in the nasal tissues of rats and mice, but not nasal tumors. Oxidative metabolism of chloroform also produces hydrochloric acid, which may contribute to the toxic effect.

Chloroform Table 4

mice rats

925

Acute toxicity properties of chloroform. Oral LD50 mg kg bw−1

LC50 (6 h inhalation exposure) g m−3

36–1366 450–2000

6.2 9.2

Source: European Union Risk Assessment Report of chloroform (ECHA-ESR, 2007). More information in: WHO, 2004; NCBI-PubChem, 2023; EU-ECHA, 2022.

Acute and short-term toxicity Acute toxicity varies depending upon the strain, sex and vehicle. Table 4 shows LC50 and LD50 values for different species. A systemic and local LOAEL of 1.0 g kg−1 has been reported in rabbits by dermal route based on extensive necrosis of the skin and degenerative changes in the kidney tubules after chloroform exposure under occlusive conditions. An oral NOAEL of 30 mg kg−1 has been reported in rats based on serum enzyme changes indicative of liver damage at highest doses. The mean lethal oral dose for an adult is estimated to be about 45 g, but large interindividual differences in susceptibility occur in humans. Some studies on clinical use and on accidental human exposure have also been reported. The human estimated inhalation LOAEC is 249 mg m−3 and the oral LOAEL is 90%) of absorbed chloromethane is metabolized, approximately half of which is exhaled as CO2. The primary chloromethane metabolite is formaldehyde, which is formed by a glutathione (GSH)-mediated pathway. Formaldehyde is oxidized to formate by the GSH-dependent enzyme formaldehyde dehydrogenase. Formate enters the one-carbon pool, and can be incorporated into macromolecules (proteins, DNA, RNA) or converted to CO2 via folate-dependent single-carbon pathways. The primary urinary metabolite in humans is S-methylcysteine. The urinary levels of S-methylcysteine in humans who inhale chloromethane fall into two groups, due to polymorphisms in the glutathione-S-transferase (GST) isozyme GSTT1–1. The isozyme has been found in human erythrocytes, liver, and kidneys. Examination of the GST activity toward chloromethane in erythrocyte preparations from a group of 45 volunteers showed that 60% had variable GST activity (“conjugators” or “rapid metabolizers”), and the rest had no detectable activity (“nonconjugators” or “slow metabolizers”). Results were not sex dependent. It is unknown if GST genotype affects the toxicological risk from chloromethane.

Chloromethane (methyl chloride)

931

Mechanisms of toxicity The most marked effect of exposure to chloromethane in humans is central nervous system (CNS) toxicity, characterized by headache, nausea, vision disturbance, ataxia, muscle spasms, convulsions, and respiratory depression. Degenerative changes occur in the brain and spinal cord, and lesions are also seen in the liver, kidneys, lung, heart, and gastrointestinal (GI) tract. Similar toxic effects occurred in animal studies, including decrements in neurofunctional tests, weakness, cerebellar and testicular degeneration, and lesions in the liver, kidneys, and spleen. The mode by which chloromethane causes toxicity in the CNS and other organs has not been elucidated. It has been proposed that chloromethane may be metabolized to methanethiol, which causes neurotoxic effects similar to those of chloromethane. The acute toxicity of chloromethane in the liver, kidneys, and brain of rats and mice was shown to be dependent on the presence of GSH, as pretreatment with inhibitors of GSH synthesis prevented the toxic effects. Chloromethane inhalation was shown to deplete the nonprotein sulfhydryl content and/or GSH levels in the liver, kidneys, and lungs in rats. The chloromethane-induced GSH depletion was associated with lipid peroxidation paralleling hepatocellular toxicity. The GSH depletion also inhibited the GSH-dependent enzyme formaldehyde dehydrogenase, which was postulated to lead to the accumulation of formaldehyde in tissues. Co-treatment with the anti-inflammatory agent BW755C reduced brain, liver, and kidney toxicity caused by chloromethane, suggesting that chloromethane may disturb prostaglandin or leukotriene metabolism.

Acute and short-term toxicity Animal One study was located in which chloromethane was administered orally, in which rabbits were gavaged with chloromethane in olive oil for 60 doses over 83 or 85 days. The only treatment-related effect was toxicity of the spleen, characterized by congestion, phagocytosis, and hemosiderosis. In acute inhalation studies, cerebellar degeneration was the primary adverse effect in mice, rats, guinea pigs, dogs, and cats. Other effects included weakness and decrements in psychomotor performance. Lesions were seen in the testes and/or liver in some studies.

Human No studies of human exposure to chloromethane by the oral or dermal route were located. Volunteers exposed to 20–150 ppm chloromethane for up to 7.5 h per day over 2–5 consecutive days had no detrimental effects on physiological, clinical, neurological, or cognitive parameters. Volunteers exposed for 3 h to 200 ppm had marginally decreased performance in several psychomotor tasks. Acute inhalation exposure to unspecified concentrations of chloromethane resulted in multiple symptoms of CNS depression, including headache, nausea, dizziness, disturbed vision, slurred speech, incoordination, muscle spasms, convulsions, respiratory depression, unconsciousness, and coma, and possibly elevated mortality from cardiovascular disease. Death resulted from acute exposure to 30,000 ppm in a number of case reports. Severe CNS toxicity was accompanied by degeneration of the brain and spinal cord, and also caused toxic changes in the liver, kidneys, lung, heart, and GI tract.

Chronic toxicity Animal Chronic exposure led to cerebellar degenerative lesions, testicular lesions, renal toxicity, splenic atrophy, and hepatocellular lesions in rats and/or mice. In some cases, the effects occurred at concentrations that were lethal. The female C57BL/6 mouse appeared to be more susceptible to cerebellar lesions than the male, and both sexes of B6C3F1 mice.

Human No studies of human exposure to chloromethane by the oral or dermal route were located. Long-term inhalation of low levels of chloromethane ( 8000 mg L−1 for the protozoa Entosiphon sulcatum. The 48-h EC50 value for Daphnia magna exposed in a closed system was 200 mg L−1. Gas production was inhibited in methanogenic bacteria at chloromethane concentrations of 50 mg L−1 and in Nitrobacter at >2 g mL−1, and cell multiplication was inhibited at 500 mg L−1. Exposure to 5000 mg m−3 chloromethane was toxic to a number of plant species, including wheat, soybeans, sugar beets, tomatoes, and sunflowers.

Exposure standards and guidelines The U.S. Occupational Safety and Health Administration established a permissible exposure limit of 100 ppm as a time-weighted average (TWA) inhalation concentration for chloromethane, and a 200 ppm ceiling with a 5-min maximum peak of 300 ppm in any 3-h period (OSHA, 2018). The American Conference of Government Industrial Hygienists (ACGIH) determined a TWA threshold limit value (TLV-TWA) of 50 ppm and a short-term exposure limit of 100 ppm for occupational inhalation exposure (ACGIH, 2022). The current TLV document further listed a skin notation and a cancer category of A4 (Not Classifiable as a Human Carcinogen) for chloromethane. NIOSH (National Institute of Occupational and Health) lists an REL (Recommended Exposure Limit) up to 10-h TWA of the lowest feasible concentration (NIOSH, 2018).

Chloromethane (methyl chloride)

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The U.S. EPA Integrated Risk Information System (IRIS) program developed a reference concentration for chronic inhalation exposure of 9
10−2 mg m−3, which was based on cerebellar lesions seen in female C57BL/6 mice exposed by inhalation continuously (22–22.5 h per day) for 11 days. IRIS did not develop a reference dose for chronic oral exposure (U.S. EPA (Environmental Protection Agency), 2001). The 2018 edition of the U.S. EPA Drinking Water Standards and Health Advisories lists 1- and 10-day health advisories of 9 mg L−1 and 0.4 mg L−1, respectively, for a 10-kg child (U.S. EPA, 2018). The Agency for Toxic Substances Disease Registry (ATSDR) developed an acute inhalation Minimal Risk Level (MRL) of 0.5 ppm based on impaired motor coordination and cerebellar lesions in female mice exposed by inhalation continuously for 11 days (ATSDR, 2022, 2009, 1998). In the current ATSDR document (2022) there is no intermediate MRL due to lack of sufficient data. However, the ATSDR did base an intermediate inhalation MRL of 0.2 ppm on increased activities of liver enzymes in male mice after 6 months of intermittent chloromethane inhalation (ATSDR, 1998). A chronic inhalation MRL of 0.03 ppm is based on axonal swelling and slight degeneration of axons in the spinal cord seen after 2 years in male mice (CIIT, 1981). Additional regulations and guidelines applicable to chloromethane can be found in EPA AEGLs (Acute Exposure Guideline Levels, https://www.epa.gov/aegl/access-acute-exposure-guideline-levels-aegls-values), ACGIH TLVs and BEIs (Threshold Limit Values and Biological Exposure Indices), AIHA ERPGs (Emergency Response Planning Guidelines), and DOE PACs (Protective Action Criteria; https://edms3.energy.gov/pac/TeelDocs).

See also: Anesthetics; Neurotoxicity

Further reading American Conference of Governmental Industrial Hygienists (ACGIH) (2022) TLVs and BEIs Based on the Documentation of the Threshold Limit Values for Chemical Substances and Physical Agents & Biological Exposure Indices. Cincinnati, OH: American Conference of Governmental Industrial Hygienists. ATSDR (June 24, 2009) Addendum to the Toxicological Profile for Chloromethane. Atlanta, GA: Agency for Toxic Substances and Disease Registry (ATSDR), U.S. Department of Health and Human Services, Public Health Service. ATSDR (January 2022) Toxicological Profile for Chloromethane, Draft, Atlanta, GA. Available at https://www.atsdr.cdc.gov/toxprofiles/tp106.pdf. ATSDR (Agency for Toxic Substances and Disease Registry) (December 1998) Toxicological Profile for Chloromethane (Update). Atlanta, GA: Agency for Toxic Substances and Disease Registry (ATSDR), U.S. Department of Health and Human Services, Public Health Service. CIIT (Chemical Industry Institute of Toxicology) (1981) Final Report on a Chronic Inhalation Toxicology Study in Rats and Mice Exposed to Methyl Chloride. Report Prepared by Battelle Columbus Laboratories for the Chemical Industry Institute of Toxicology. EPA/OTS Doc #878212061, NTIS/OTS0205952. Dodd DE, Bus JS, and Barrow CS (1982) Nonprotein sulfhydryl alterations in F-344 rats following acute methyl chloride inhalation. Toxicology and Applied Pharmacology 62: 228–236. HSDB (Hazardous Substances Data Bank) (2022) Methyl chloride. National Library of Medicine. https://pubchem.ncbi.nlm.nih.gov/compound/6327. NIOSH (2018) CDC - NIOSH Pocket Guide to Chemical Hazards—Methyl Chloride. https://www.cdc.gov/niosh/npg/npgd0403.html. OSHA (2018) Subpart Z—Toxic and Hazardous Substances. Air Contaminants. Occupational Safety and Health Standards. Code of Federal Regulations 29 CFR 1915.1000. https:// www.govinfo.gov/app/details/CFR-2018-title29-vol7/CFR-2018-title29-vol7-sec1915-1000. SIDS (Screening Information Data Set for High Production Volume Chemicals) (2004) SIDS for Methyl Chloride. Online at http://www.inchem.org/documents/sids/sids/CLMETHANE.pdf. TOXNET Database (2021) Toxnet (Toxicology Data Network): Search Under Toxline for Chloromethane. USA: National Institutes of Health. Online at https://www.nlm.nih.gov/pubs/ techbull/nd19/nd19_toxnet_new_locations.html. U.S. EPA (1987) Health Effects Assessment for Chloromethane. Document no. EPA/600/8–88/024. Cincinnati, OH: U.S. EPA Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment, Office of Research and Development. Available at: https://nepis.epa.gov/Exe/ZyPDF.cgi/2000TGNR.PDF?Dockey¼2000TGNR.PDF. U.S. EPA (2018) 2018 Edition of the Drinking Water Standards and Health Advisories, EPA 822-F-18-001. Washington, DC: Office of Water U.S. Environmental Protection Agency. U.S. EPA (Environmental Protection Agency) (1986) Health and Environmental Effects Profile for Methyl Chloride. Document no. EPA/600/X-86/156. Cincinnati, OH: U.S. EPA Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment, Office of Research and Development. Available at: https://cfpub.epa.gov/ncea/iris/ iris_documents/documents/toxreviews/1003tr.pdf. U.S. EPA (Environmental Protection Agency) (2001) Methyl chloride. Integrated Risk Information System (IRIS). Assessment last revised 7/17/2001. Online at https://iris.epa.gov/ ChemicalLanding/&substance_nmbr¼1003. Accessed 27/01/2023.

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Chlorophenols Murali Badanthadka, NUCARE Nitte (Deemed to be University), NGSM Institute of Pharmaceutical Sciences (NGSMIPS), Department of Nitte University Centre for Animal Research and Experimentation (NUCARE), Deralakatte, Mangalore, India © 2024 Elsevier Inc. All rights reserved. This is an update of M. Badanthadka, H.M. Mehendale, Chlorophenols, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 896–899, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00281-5.

Chemical profile Introduction Background Uses Exposure and exposure monitoring Toxicokinetics (ADME) Mechanism of toxicity In vitro toxicity data Acute and short-term toxicity Animal Human Chronic toxicity (or exposure) Animal Human Immunotoxicity Reproductive toxicity Genotoxicity Carcinogenicity Organ toxicity Interactions Toxicogenomics Clinical management Environmental fate and behavior Ecotoxicology Toxicity to marine organisms Exposure standards and guidelines Conclusion References Further reading

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Abstract Chlorophenols (CPLs) are a group of chemicals produced by electrophilic halogenation of phenol with chlorine. There are five basic types and 19 different chlorophenols. Some chlorophenols are used as pesticides and herbicides. Others are used as antiseptics and disinfectants. Pentachlorophenol (PCP: CAS Registry Number: 87-86-5) is the most important compound in this group. It is a chlorinated aromatic compound and has been extensively used as a fungicide. First registered as a wood preservative in the United States in 1936, it has also been used in the preparation of ropes, paints, adhesives, canvas, insulation, and brick walls. In 1999, the International Agency for Research on Cancer classified it as a possible human carcinogen. CPLs can be damaging the liver, brain, gastrointestinal tract, upper respiratory tract, central nervous system (CNS). 4-Chlorophenol is considered very hazardous to human skin (a contact irritant; whitens and cauterizes the skin & mucous membranes). Undiluted compound can cause damage at a threshold concentration of 0.75% (w/v). Methemoglobinemia, Heinz body hemolytic anemia, hyperbilirubinemia and male reproductive toxicity in humans have also been reported with some of these phenols. EPA has classified pentachlorophenol as a Group B2, probable human carcinogen.

Keywords Carcinogen; Chlorophenols; 4-Chlorophenol; Fungicide; Herbicide; Pentachlorophenol (PCP); Pesticide; Trichlorophenol; Wood preservative

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00621-7

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Key points

• • •

Chlorophenols are used as pesticides, herbicides, antiseptics, disinfectants and as wood preservative. Pentachlorophenol is the most important compound in this group. PCP is categorized as a probable human carcinogen.

Chemical profile

• • • • • •

Representative compound: Pentachlorophenol (PCP) Name: Pentachlorophenol Synonym: 1-Hydroxy-2,3,4,5,6-pentachlorobenzene. Chemical Abstracts Service Registry Number: 87-86-5 Molecular Formula: C6HCl5O Chemical Structure:

Introduction Chlorophenols are chlorine-containing aromatic compounds and are widely used in industry as an essential raw chemical for synthesizing other chemicals. Apart from industrialization, agricultural activities also promote the production of chlorophenolic compounds. Uncontrolled chemical and pharmaceutical industry activities resulted in clorophenols ending up in the environmental (Czaplicka, 2004; Michałowicz and Duda, 2007; Jensen, 1996). The extensive presence of these compounds in the environment is related to the production, its use and low degradation. Because of carcinogenic and mutagenic properties and low environmental degradability, these chemicals became persistent organic pollutants (Olaniran and Igbinosa, 2011; Igbinosa et al., 2013). Their treatment, disposal, and general management have become a severe challenge to stakeholders in the health sector (Weber et al., 2008). Bioremediation using microorganisms has been suggested as a remedy to the effects of chlorophenols (Czaplicka, 2004; Pandey et al., 2003; Igbinosa et al., 2007). Several microbes have been harnessed and applied in the bioremediation of environmental contaminants (Pandey et al., 2003). However, contaminants have been shown to be refractory to microbial degradation, thereby either not metabolizable or transformed into other metabolites and thereby accumulate in the environment (Esteve-Núñez et al., 2001). Thus, study on the degradation of chlorophenols is of importance and value to the management of the ecological environment and human health (Liu et al., 2021; Gupta et al., 2002).

Background Pentachlorophenol (PCP) is a synthetic substance made from other chemicals and does not occur naturally in the environment. At one time, it was one of the most widely used biocides in the United States. Since 1984, the purchase and use of PCP have been restricted to certified applications. It is no longer available to the general public. Before restrictions, PCP was widely used as a wood preservative. It is now used industrially as a wood preservative for power line poles, cross arms, and fence posts. PCP can be found in two forms: PCP and the sodium salt of PCP. The sodium salt dissolves easily in water but PCP does not. The physical properties of these two forms are different but their toxic effects are expected to be similar. Humans are generally exposed to technical-grade PCP, which usually contains toxic impurities such as polychlorinated dibenzop-dioxins and dibenzofurans. In addition to workplace exposure, humans can be exposed to very low levels of PCP in indoor and outdoor air, food, soil, and drinking water. Exposure may also result from dermal contact with wood treated with preservatives that contain PCP.

Uses Chlorophenols are used in the synthesis of dyes, fungicides, herbicides, wood preservatives, and as ingredients in alcohol denaturants.

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Exposure and exposure monitoring Exposure to chlorophenols may occur through ingestion, inhalation, or dermal contact at workplaces where it is used or produced. The major occupational exposure is to workers in the wood products industry. Air and urine samples taken at 25 factories using PCP as a wood preservative showed that the average worker’s exposure in air was 0.013 mg m−3. Elevated levels were found in the urine and serum of workers.

Toxicokinetics (ADME) Absorption of PCP is rapid through oral, dermal, or inhalation exposure. The major tissue deposits vary somewhat between species. In humans, the liver, kidneys, brain, spleen, and fatty tissues are the major deposition sites. In the mouse, the gall bladder is a principal storage site. In the rat, it is the kidneys. The primary route of elimination is through the kidneys in unchanged form. Labeled PCP given to rats by injection or oral route yielded 41–43% unchanged PCP in the urine. One metabolite, tetrachlorohydroquinone (5–24%), was identified. The elimination half-life for PCP may be up to 20 days in chronically exposed individuals. In one study, a single dose of PCP (15 mg kg−1) was administered intravenously and orally to B6C3F1 mice. After intravenous administration, the clearance values and volume of distribution were 0.057
0.007 L h−1 kg−1 and 0.43
0.06 L h−1 kg−1, respectively. The elimination half-life was 5.2
0.6 h. After oral administration, the peak plasma concentration (28
7 mg mL−1) occurred at 1.5
0.5 h and bioavailability (1.06
0.09) was complete. The elimination half-life was 5.8
0.6 h. Only 8% of the PCP dose was excreted unchanged in the urine. PCP was primarily recovered in urine as glucuronide and sulfate conjugate metabolites. A portion of the dose was recovered in urine as tetrachlorohydroquinone (5%) and its conjugates (15%). For both PCP and tetrachlorohydroquinone, sulfates accounted for 90% or more of the total conjugates. There are marked gender differences in the biological half-life in non-human primates. The biological half-life for excretion in Rhesus monkeys was 41 and 92 h in males and females, respectively.

Mechanism of toxicity Chlorophenols block ATP production without blocking the electron transport chain. They inhibit mitochondrial oxidative phosphorylation, thereby causing accelerated aerobic metabolism. This increases the basal metabolic rate and body temperature, which leads to clinical hyperthermia. As body temperature rises, heat-dissipating mechanisms are activated to overcome hyperthermia by accelerated metabolism. ADP and other substrates accumulate and stimulate the electron transport chain further. This process demands more oxygen in a futile effort to produce ATP. This oxygen demand quickly surpasses the oxygen supply, and energy reserves of the body become depleted.

In vitro toxicity data In vitro studies demonstrated inhibition of apoptosis induced by pentachlorophenol in liver and bladder cells that contributes to tumor promotion (Wang et al., 2000, 2001; Sai et al., 2001). Pentachlorophenol can induce direct necrosis and its metabolic product 4-chlorohydrocarbohydrate can break DNA chains, producing more severe toxicity than parent compound itself (Wang et al., 2001). Inhibition of apoptosis was also observed in human bladder cells T-24 and hepatoma cells HepG2 after treatment with PCP (Wang et al., 2001; Sai et al., 2001).

Acute and short-term toxicity Animal The lethal dose 50% (LD50) for PCP in laboratory animals ranges from 30 to 100 mg kg−1 (Ma et al., 2019).

Human The most prevalent signs and symptoms after ingestion of 30–250 mL of chlorophenols are corrosion of tissue, profuse sweating, intense thirst, nausea, vomiting, diarrhea, convulsions, pulmonary edema, cyanosis, and coma. If death from respiratory failure is not immediate, jaundice and oliguria or anuria may occur (Nnodu and Whalen, 2008).

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Chronic toxicity (or exposure) Animal The International Agency for Research on Cancers categorized chlorophenols into five groups belonging to the 2B group of potential human carcinogens (Seiler, 1991). This category includes chemical agents with sufficient evidence of animal carcinogenicity and inadequate evidence of carcinogenicity in humans.

Human Repeated exposure may cause symptoms of acute poisoning. Skin sensitivity reactions occur occasionally. Prolonged skin contact with chlorophenols may cause bladder tumors, hemolytic anemia, and lens opacities (Triebig et al., 1987). Pathologic findings after death due to chlorophenols include necrosis of mucous membranes, cerebral edema, and degenerative changes in the liver and kidneys. The WHO classified some chlorophenols (2,4,6-trichlorophenol, 2,4,5-trichlorophenol, and pentachlorophenol) as compounds suspected of having carcinogenic properties (IARC, 1991, 1999).

Immunotoxicity The immunogenic effects of PCP were evaluated by checking the frequency of lymphocyte phenotypes, functional responses, serum immunoglobulin levels, and autoantibodies in 38 individuals who were exposed to PCP in manufacturer-treated log houses. A comparison of subjects with controls revealed that the exposed individuals had activated T cells, autoimmunity, functional immunosuppression, and B-cell dysregulation. Autoimmunity was shown by elevation of TA1 phenotype frequencies and 21% incidence of anti-smooth muscle antibody. Functional immunosuppression was shown by the significantly reduced responses to all mitogens tested and to allogeneic lymphocytes in the mixed lymphocyte culture test. There was a significant elevation of CD10, and an 18% increase or decrease in serum immunoglobulins was noted. A striking anomaly was the enhanced natural killer activity found in exposed females but not in males. An in vitro experimental study showed a significant decrease in the tumor-killing (lytic) function of human natural killer cells indicating signs of immune alteration.

Reproductive toxicity Overexposure may cause reproductive disorder(s) based on tests with laboratory animals. PCP was administered to dams and their offspring via drinking water (6.6 mg L−1) during gestation and the lactation period. Tissue samples were obtained from dams, 3-week-old weanling pups, and 12-week-old pups. The results show that PCP exposure during development causes thyroid function vulnerability, testicular hypertrophy in adults, and aberrations in brain gene expression (Kawaguchi et al., 2008). Embryo–fetal toxicity and the teratogenic potential of PCP were studied following oral gavage to presumed pregnant female Sprague–Dawley rats. Doses of 0 (corn oil), 10, 30, and 80 mg kg−1 day−1 were administered to the rats at concentrations of 0, 2, 6, and 16 mg mL−1, respectively, from day 6 to day 15 of presumed gestation. The dose volume was 5 mL kg−1. Rats were sacrificed on day 20 of presumed gestation and necropsied. The no observed adverse effect level (NOAEL) for maternal toxicity and developmental NOAEL in rats were also found to be 30 mg kg−1 day−1. The lowest observed adverse effect level (LOAEL) for PCP developmental toxicity (80 mg kg−1 day−1) was associated with increased resorption, reduced live litter size, and fetal body weight, and caused increased malformations and variations. The potential of PCP to induce general and reproductive/developmental toxicity was evaluated in Crl Sprague–Dawley rats, using a two-generation reproduction toxicity study. PCP was administered by gavage at doses of 0, 10, 30, and 60 mg kg−1 day−1. It was concluded that 30 mg kg−1 day−1 is the LOAEL and 10 mg kg−1 day−1 is the NOAEL for both reproductive and general toxicity. In addition, the results did not indicate bioaccumulation and thereby PCP did not selectively affect reproduction or development of the offspring of rats at a dose of 10 mg kg−1 day1, a dose that is much higher than human exposure (Bernard and Hoberman, 2001; Bernard et al., 2002; Aoyama et al., 2005).

Genotoxicity PCP seems to be a weak inducer of DNA damage and has gentotoxic potential. It does not produce DNA strand breaks or clear differential toxicity to bacteria in the rec-assay in the absence of metabolic activation. Also, in the sister–chromatid exchange (SCE) induction assay, no increase can be observed in vivo, but PCP was found to be marginally active in in vitro experiments (Vlastos et al., 2016).

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An in vivo study on the genotoxic effects of PCP was carried out using freshwater fish (Channa punctatus). The fish were exposed to three sublethal doses of PCP (0.2, 0.4, and 0.6 ppm) by medium treatment. The results confirmed the genotoxicity of PCP in this organism (Farah et al., 2003). Chromosome analyses were carried out on peripheral lymphocytes from 22 male workers employed at a factory producing PCP. A small but significant increase in the frequency of dicentric and acentric was observed, suggesting that PCP has a genotoxic effect.

Carcinogenicity There is limited evidence of carcinogenicity of PCP in humans. The evidence is based on assays that utilized less than pure PCP. Contaminants of PCP include tri- or tetrachlorophenol, hexachlorobenzene, polychlorinated dibenzo-p-dioxins, or polychlorinated dibenzofurans. Indications are that the positive evidence for carcinogenicity is from the contaminant(s) and not the PCP. This product is or contains a component that has been reported to be possibly carcinogenic based on its International Agency for Research on Cancer (IARC), American Conference of Governmental Industrial Hygienists (ACGIH), National Toxicology Program (NTP), or Environmental Protection Agency (EPA) classification. Additionally, there is sufficient evidence in experimental animals for the carcinogenicity of PCP. Therefore, PCP is a probable human carcinogenic agent based on inadequate human data and sufficient evidence of carcinogenicity in animals.

Organ toxicity Liver: Both technical grade and pure pentachlorophenol appear to induce toxic effects in the liver of animals exposed sub-chronically to doses ranging from 1 to 30 mg kg−1/day. Cardiovascular System: Acute oral exposure to pentachlorophenol may induce tachycardia in humans and cause extensive vascular damage in animals.

Interactions Such studies are limited. A study demonstrated that modification in mixture composition with either a solvent and/or a surfactant could influence pentachlorophenol diffusion in the skin. Physicochemical interactions between the skin surface and stratum corneum contributed to pentachlorophenol transport. These interactions were identified by assessing chemical diffusion in biological and inert membrane systems (Baynes et al., 2002).

Toxicogenomics Details are available in the data base (http://ctdbase.org/detail.go?type¼chem&acc¼D010416).

Clinical management After exposure by ingestion, if corrosive injury is absent, decontamination to prevent further absorption may be achieved using activated charcoal. Emesis by syrup of ipecac may be considered but is not preferred. Next, milk should be given to drink. Gastric lavage and emesis are contraindicated in the presence of esophageal injury. In the case of dermal exposure, the poison should be removed by washing the affected skin or mucous membrane with copious amounts of water for at least 15 min.

Environmental fate and behavior Production of PCP may result in its release to the environment through various waste streams; its use as a wood preservative and surface disinfectant can result in its direct release to the environment. Vapor-phase PCP is degraded into hydroxyl radicals by photochemical reaction. The half-life for this reaction in air is estimated to be 29 days (Essam et al., 2007). If released into soil, PCP is expected to have low to no mobility based on measured values for the organic carbon partition coefficient (Koc) ranging from 1250 for the dissociated form to 25,000 for the undissociated form. The dissociation constant (pKa) of PCP is 4.70, indicating that this compound exists almost entirely in the anion form in the environment and anions generally do not adsorb more strongly to soils. Volatilization from moist soil is not expected because the acid exists as an anion and anions do not volatilize. PCP may not volatilize from dry soil surfaces based on its vapor pressure. If released into water, PCP is expected to adsorb to suspended solids and sediment based on its measured Koc values. Bioconcentration factor (BCF) AU2 values from approximately 5 to 5000 indicate that the bioconcentration of PCP in aquatic organisms is low to high; the value is greatly influenced by environmental pH. PCP is not expected to undergo hydrolysis in the

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environment due to the lack of functional groups that hydrolyze under environmental conditions (Jensen, 1996; Homolková et al., 2016). The environmental impact of PCP increases when its many degradation products are taken into consideration. Biodegradation has been extensively studied in Sphingobium chlorophenolicum [ATCC 39723]. In water and under ambient conditions of temperature and pressure, it is completely destroyed at catalyst/substrate ratios of 1:715 within minutes (Huang et al., 2008).

Ecotoxicology Classification and Koc values indicate that PCP is expected to adsorb to suspended solids and sediment in water. pKa values indicate that PCP exists almost entirely in the anion form at pH 5–9 and therefore, volatilization from water surfaces is not expected to be an important fate process. BCF values suggest that the bioconcentration in aquatic organisms is low to very high; this value is greatly influenced by the environmental pH. PCP rapidly photodegrades in surface water when exposed to direct sunlight; the half-life for photolysis in solution has been reported to be 0.86 h (McLellan et al., 2007). Effect of PCP on Aquatic Organisms. Organism

Effect

Golden orfe Rainbow trout Water flea Bacteria (div.) Algae

LC50 0.60 mg L–1 (96 h) LC50 0.12–0.26 mg L−1 (96 h) LC50 0.33–0.41 mg L−1 (96 h) NOEC 12.3 mg L−1 (30 min), growth EC50 10–7000 mg L−1 (96 h), growth

EC50, half maximal effective dose; LC50, lethal concentration 50%; NOEC, no observed effect concentration.

Toxicity to marine organisms Reported lethal (effect) concentration 50% L(E)C50 data ranging from 0.6 to 19.5, 2.55 to 29.7, and 5 to 7 mg L−1 for algae, crustaceans, and fish, respectively, indicate moderate to high acute toxicity. The toxicity of chlorophenols to aquatic organisms increases with an increase in the degree of chlorination and substitution away from the ortho position. The higher toxicity of the more highly chlorinated congeners can be ascribed to an increase in lipophilicity, which leads to a greater potential for uptake into the organism. Ortho-substituted congeners are generally of lower toxicity than the meta- and para-substituted compounds, as the close proximity of the ortho-substituted chlorine to the OH group on the molecule appears to shield the OH, which apparently interacts with the active site in aquatic organisms, causing the observed toxic effects. Toxicity also depends on the extent to which the chlorophenol molecules are dissociated in the exposure medium. No data could be located for sediment-dwelling organisms.

Exposure standards and guidelines The Occupational Safety and Health Administration permissible exposure limit is 0.5 mg m−3 for the 8-h time-weighted average (TWA). The threshold limit value is 0.5 mg m−3 for the 8-h TWA. The National Institute of Occupational Safety and Health recommended exposure limit is 0.5 mg m−3 for the 10-h TWA.

Conclusion Pentachlorophenol (PCP) was extensively used as a biocide in the United States, but it is now a restricted use pesticide. PCP is a man-made chemical that does not occur naturally. Humans are usually exposed to impure pentachlorophenol (also called technical grade pentachlorophenol). In the past, pentachlorophenol was extensively used as a pesticide and wood preservative. Since 1984, only certified applicators are allowed to use of this compound (unavailable to the general public). However, it is still used as a wood preservative for utility poles, railroad ties, and wharf pilings. EPA has classified pentachlorophenol as a Group B2, probable human carcinogen.

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Igbinosa EO, Odjadjare EE, Chigor VN, Igbinosa IH, Emoghene AO, Ekhaise FO, Igiehon NO, and Idemudia OG (2013) Toxicological profile of chlorophenols and their derivatives in the environment: The public health perspective. The Scientific World Journal 2013: 460215. Jensen J (1996) Chlorophenols in the terrestrial environment. Reviews of Environmental Contamination and Toxicology 146: 25–51. Kawaguchi M, Morohoshi K, Saita E, Yanagisawa R, Watanabe G, Takano H, and ⋯ Himi T (2008) Developmental exposure to pentachlorophenol affects the expression of thyroid hormone receptor b1 and synapsin I in brain, resulting in thyroid function vulnerability in rats. Endocrine 33(3): 277–284. Liu X, Deng W, and Yang Y (2021) Characterization of a novel laccase LAC-Yang1 from white-rot fungus Pleurotus ostreatus strain Yang1 with a strong ability to degrade and detoxify chlorophenols. Molecules 26(2): 473. Ma D, Wei J, Zhang H, Zhou Y, Shen J, Wang L, and Zhang P (2019) Acute toxicity evolution during ozonation of mono-chlorophenols and initial identification of highly toxic intermediates. Environmental Science: Processes & Impacts 21(9): 1509–1518. McLellan I, Carvalho M, Silva Pereira C, Hursthouse A, Morrison C, Tatner P, Martins I, San Romão MV, and Leitão M (2007) The environmental behaviour of polychlorinated phenols and its relevance to cork forest ecosystems: A review. Journal of Environmental Monitoring 9(10): 1055–1063. Michałowicz J and Duda W (2007) Phenols—Sources and toxicity. Polish Journal of Environmental Studies 16(3): 347–362. Nnodu U and Whalen MM (2008) Pentachlorophenol decreases ATP levels in human natural killer cells. Journal of Applied Toxicology 28(8): 1016–1020. Olaniran AO and Igbinosa EO (2011) Chlorophenols and other related derivatives of environmental concern: Properties, distribution, and microbial degradation processes. Chemosphere 83(10): 1297–1306. Pandey G, Paul D, and Jain RK (2003) Branching of o-nitrobenzoate degradation pathway in Arthrobacter protophormiae RKJ100: Identification of new intermediates. FEMS Microbiology Letters 229(2): 231–236. Sai K, Kang KS, Hirose A, Hasegawa R, Trosko JE, and Inoue T (2001) Inhibition of apoptosis by pentachlorophenol in v-myc-transfected rat liver epithelial cells: Relation to down-regulation of gap junctional intercellular communication. Cancer Letters 173(2): 163–174. Seiler JP (1991) Pentachlorophenol. Mutation Research 257(1): 27–47. Triebig G, Csuzda I, Krekeler HJ, and Schaller KH (1987) Pentachlorophenol and the peripheral nervous system: A longitudinal study in exposed workers. British Journal of Industrial Medicine 44(9): 638–641. Vlastos D, Antonopoulou M, and Konstantinou I (2016) Evaluation of toxicity and genotoxicity of 2-chlorophenol on bacteria, fish and human cells. Science of the Total Environment 551: 649–655. Wang YJ, Ho YS, Jeng JH, Su HJ, and Lee CC (2000) Different cell death mechanisms and gene expression in human cells induced by pentachlorophenol and its major metabolite, tetrachlorohydroquinone. Chemico-Biological Interactions 128(3): 173–188. Wang YJ, Lee CC, Chang WC, Liou HB, and Ho YS (2001) Oxidative stress and liver toxicity in rats and human hepatoma cell line induced by pentachlorophenol and its major metabolite tetrachlorohydroquinone. Toxicology Letters 122(2): 157–169. Weber R, Gaus C, Tysklind M, Johnston P, Forter M, Hollert H, and ⋯ Zennegg M (2008) Dioxin-and POP-contaminated sites—Contemporary and future relevance and challenges. Environmental Science and Pollution Research 15(5): 363–393.

Further reading CTD (Comp Toxicol. Database). http://ctdbase.org/detail.go?type¼chem&acc¼D010416. Hsu Y-C, et al. (2021) Emissions of PAHs, PCDD/Fs, dl-PCBs, chlorophenols and chlorobenzenes from municipal waste incinerator cofiring industrial waste. Chemosphere 280: 130645. https://pubmed.ncbi.nlm.nih.gov/33933998/. Liu X, et al. (2023) Free radical mechanism of toxic organic compound formations from o-chlorophenol. Journal of Hazardous Materials 443(Pt B), 130367. https://pubmed.ncbi.nlm. nih.gov/36444078/. Polouliakh N, et al. (2022) Toxicity analysis of pentachlorophenol data with a bioinformatics tool set. Methods in Molecular Biology 2486: 105–125. https://pubmed.ncbi.nlm.nih.gov/ 35437721/. Pubchem: 2-Chlorophenol. https://pubchem.ncbi.nlm.nih.gov/compound/2-Chlorophenol. Pubchem: 3-Chlorophenol. https://pubchem.ncbi.nlm.nih.gov/compound/3-Chlorophenol. Pubchem: 4-Chlorophenol. https://pubchem.ncbi.nlm.nih.gov/compound/4-Chlorophenol. Pubchem: Pentachlorophenol. https://pubchem.ncbi.nlm.nih.gov/compound/992.

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Chlorophenoxy herbicides Fred F Farris, School of Pharmacy, West Coast University, Los Angeles, CA, United States © 2024 Elsevier Inc. All rights reserved. This is an update of S. Karanth, Chlorophenoxy Herbicides, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, pp. 900–902, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00114-7.

Introduction Herbicidal action Production and use Human exposure Signs and symptoms of poisoning Mechanisms of toxicity Toxicokinetics Absorption Distribution Metabolism Excretion Environmental fate Atmospheric fate Aquatic fate Terrestrial fate Microbial degradation Abiotic degradation Carcinogenicity Antidote and emergency treatment Interactions References Further reading

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Abstract Chlorophenoxy herbicides are derivatives of phenoxyacetic acid, to which at least one chlorine atom has been attached to the phenyl ring. These agents are structurally similar to and share many of the growth regulatory characteristics of, the natural auxin, indoleacetic acid (IAA). The most frequently used member of this class of herbicides is 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D is one of the earliest of this class of herbicides to have been developed (1944), and also one of the best known, because of its use as one of the two herbicides in the manufacture of the military defoliating agent, Agent Orange; the other being 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). As herbicides, this class of agents stimulates uncontrollable growth in susceptible plants, leading ultimately to their death. The chlorophenoxy herbicides are one of the most commonly used groups of herbicides because of their low cost, effectiveness and good water solubility. They have been produced extensively since the 1950s and are used both agriculturally and recreationally for post-emergence control of annual and perennial broad leaved weeds in cereals, herbage seed crops, flax, rice, vines, peas, potatoes, asparagus, grassland, turf, orchards, roadsides, forestry, and aquatic areas. Exposure to chlorophenoxy herbicides can occur through inhalation of contaminated air, consumption of foods containing herbicide residues, dermal contact during their application, or by entering areas where the compounds have been recently applied. Signs and symptoms of exposure to chlorophenoxy herbicides typically include vomiting, diarrhea headache, confusion, bizarre or aggressive behavior, peculiar odor on breath, muscle weakness, peripheral neuropathy, and loss of reflexes. The mechanism of toxicity is unclear but may involve mitochondrial injury, cell membrane damage, uncoupling of oxidative phosphorylation, and disruption of acetyl coenzyme A metabolism. Widespread muscle damage occurs, and the cause of death is usually ventricular fibrillation.

Keywords Agent orange; Auxin; Auxinic herbicides; Chlorophenoxy herbicides; Chlorophenoxyacetic acid; Defoliant; Herbicide; Indoleacetic acid; Phenoxyacetic acid

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00747-8

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Introduction Auxins are indole-based plant hormones (or growth regulators) derived from tryptophan that can control the division and elongation rate, as well as the identity or differentiation state of plant cells (Peris et al., 2010). Auxins are found in the tips of shoots and roots and promote cell division, and stem and root growth. They can also have a radical effect on plant orientation by promoting cell division on one side of the plant in response to sunlight and gravity, a process referred to as phototropism. Auxinic herbicides are organic compounds that are used as active ingredients in many herbicide formulations. They are the oldest of the synthetic herbicides, and are herbicidally active growth regulators or herbicides with growth regulating action. There are five groups of auxinic herbicides, including the chlorophenoxy herbicides. Chlorophenoxy herbicides are derivatives of phenoxyacetic acid, to which at least one chlorine atom is attached to the phenyl ring. The chlorophenoxy herbicides 4-chloro-2-methylphenoxyacetic acid (MCPA) (Fig. 1) and 2,4-dichlorophenoxyacetic acid (2,4-D) (Fig. 2) were discovered independently by British and American scientists, respectively, during World War II, and were instrumental in increasing cereal yields during this conflict (Sterling and Hall, 1997). These herbicides were designed to be structurally analogous to the natural plant auxin indoleacetic acid (IAA) (Fig. 3). When sprayed on broad-leaf plants these herbicides induce rapid, uncontrolled growth, leading to death, but leave monocotyledonous crops such as wheat or corn, relatively unaffected (Grossmann, 2010). The best known and most widely used chlorophenoxy herbicide is 2,4-D, but the class includes other members such as MCPA, and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Analogs of each of these three compounds, with an extra methyl group attached next to the carboxylic acid, were subsequently developed and marketed as dichlorprop (2,4-DP) (Fig. 4), mecoprop, and fenoprop, respectively. The addition of the methyl group creates a chiral center in these molecules and biological activity is found only in the (2R)-isomer (Wendeborn et al., 2012). A formulation of equal parts 2,4-D and 2,4,5-T is famously, or perhaps infamously, known as Agent Orange. The chlorophenoxy herbicides are white crystalline solids at room temperature, and their free acid and salt forms are stable. Their melting points and water solubility vary with structure. When heated to decomposition, they can release toxic gases including hydrogen chloride (HCl) and nitrogen oxides. In the free acid form, their pKa’s typically range from around 2–4 (PubChem, 2,4-D, n.d.; PubChem, MPCA, n.d.).

Fig. 1 MCPA.

Fig. 2 2,4-D.

Fig. 3 IAA.

Fig. 4 Dichlorprop.

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Herbicidal action Some researchers argue that the auxinic herbicides do not actually mimic the effects of the natural auxin IAA. However, the chlorophenoxyacetic acid herbicides share several important characteristics with IAA (Sterling and Hall, 1997), including: a. Dose-response patterns: At low doses they act as plant growth regulators, having a stimulatory effect on plant cell growth, but at high doses they act as phytotoxins b. When placed in plant cell cultures, some auxinic herbicides (e.g., 2,4-D) can replace IAA as the hormone for proper plant growth c. Different plant tissues, as well as tissues at different stages of development, display differential sensitivity to both natural auxins and chlorophenoxy herbicides d. Both auxins and chlorophenoxy herbicides induce growth by cell elongation, as opposed to cell division. e. Many studies characterizing IAA effects have replaced IAA with 2,4-D or similar herbicides In addition to the above, the effects of chlorophenoxy herbicides on growth and anatomy of susceptible plants are similar to those produced by IAA. As dosage is increased, leaves become stunted and cupped and terminal leaf growth ceases. Roots become thickened and stunted and numerous structural changes occur internally. Herbicides including 2,4-D, and 2,4,5-T have been reported to promote ethylene biosynthesis in many plants (Ashton and Crafts, 1981), and 2,4-D and IAA exhibit similar dose-dependent increases in ethylene production (Morgan and Hall, 1962). It is now known that ethylene is one of the most important hormones in leaf senescence regulation, and that it can trigger the senescence process, especially in sensitive species (Iqbal et al., 2017). The physiological changes associated with auxin-induced cell growth are also linked to stimulation of nucleic acid and protein biosynthesis (Ashton and Crafts, 1981: Sterling and Hall, 1997), increased proton extrusion through the plasma membrane of the plant, and an increased influx of Ca2+ into the cell cytoplasm (Sterling and Hall, 1997). Death of most plant tissues treated with 2,4-D is caused by the accumulation of abscisic acid (ABA) and ethylene induced oxidative stress (Grossmann, 2010).

Production and use The chlorophenoxy herbicides are one of the most commonly used groups of herbicides because of their low cost, effectiveness and good water solubility (Troyer, 2001). They have been produced extensively since the 1950s and are used both agriculturally and recreationally for post-emergence control of annual and perennial broad leaved weeds in cereals, herbage seed crops, flax, rice, vines, peas, potatoes, asparagus, grassland, turf, orchards, roadsides, forestry, and aquatic areas. In 2019, the US Geological Survey mapped use of herbicides in the US. By far the most highly used of the chlorophenoxy herbicides was 2,4-D, with 45 million pounds (20,000,000 kg) applied that year (ATSDR, 2020). Although use varies from yearto-year, production of this herbicide was similar in 2019 to that 3 decades ago; 52–67 and 47 million pounds in 1990 and 2001, respectively. Pasture and hay fields, wheat, soybeans, and corn crops receive the greatest applications (ATSDR, 2020). It has been suggested that development of genetically modified crops with increased tolerance to 2,4-D may result in an increase in the total amount applied annually to crops such as soybeans. The use of the next most commonly applied herbicide in the class, MCPA, corresponded to only 2 million pounds (or less than 5% of the 2,4-D application) in 2019. 2,4-D is produced by the reaction of 2,4-dichlorophenolate with monochloroacetic acid or by the reaction between 2,4-dichlorophenol and chloroacetic acid in aqueous sodium hydroxide. Although the free acid can be used as a herbicide, there are numerous commercially available formulations, including emulsifiable concentrates, wettable granules, wettable powders, emulsions (esters), and aqueous solutions (salts) (PubChem). Almost 90–95% of total 2,4-D global use is accounted for by the dimethyl amine salt and ethylhexyl ester (NPIC, 2008). 2,4-D and its different chemical forms are listed as the singular active ingredient or in combination with other ingredients, in about 600 agricultural and residential products. The use of 2,4-D ranks first among herbicides in frequency of home and garden applications and third in national herbicide use for agriculture (Gilliom et al., 1999). In the U.S., 2,4-D use is most extensive in the Midwest, Great Plains, and Northwestern regions (EPA, 2005).

Human exposure People can be exposed to chlorophenoxy herbicides through inhalation of contaminated air, exposure from soil and dust resuspension, consumption of foods containing herbicide residues, dermal contact during their application, or by entering areas where the compounds have been recently applied (ATSDR, 2020). Persons residing within or near areas of heavy herbicidal use (e.g., farms) have an increased risk of exposure. 2,4-D was detected in indoor air and on surfaces (floors, tabletops, and windowsills) inside single-story Midwestern residences following lawn applications (Nishioka et al., 2001). It was determined that the main transport routes of 2,4-D into the home were from the homeowner applicator and by pets (ATSDR, 2020). The primary mechanism for human exposure is through direct contact during use of the product. Dermal contact appears to be a major route of exposure for workers, although inhalation exposure and accidental ingestion via hand-to-mouth activity is possible. Forestry workers using backpack sprayers receive an estimated average exposure of 98 mg/kg/day. This is substantially lower than

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received by commercial pesticide applicators (2.75 mg/kg/day) and farm workers (5.78 mg/kg/day). However, forestry workers often did not comply with current product label requirements for personal protective equipment (PPE). In manufacturing plants, exposures occur during the handling of raw materials, intermediates, finished products and process wastes. Increased urinary levels of chlorophenoxy compounds and increased concentrations of some chlorinated dibenzodioxins in adipose tissue have been measured in highly exposed persons. The presence of dibenzodioxins and dibenzofurans has been demonstrated in the adipose tissue of nonoccupationally exposed people in many countries (ATSDR, 2020). Due to some human health concerns, 2,4-D was placed in pre-Special Review by the EPA in 1986. In 1988, it was proposed that Special Review not be initiated due to the lack of epidemiological data linking 2,4-D and carcinogenicity and the final decision was deferred until reregistration. Between 1988 and 2005, the EPA reviewed epidemiological data and found that no new links between 2,4-D and human cancer. In order to address safety concerns, the 2,4-D Industry Task Force agreed to make changes to labeled uses to reduce exposure. In 2005, the EPA drafted its Reregistration Eligibility Decision (RED); it was determined that 2,4-D was eligible for reregistration and it was decided not to initiate Special Review (EPA, 2005).

Signs and symptoms of poisoning Manifestations of systemic toxicity of chlorophenoxy compounds are known mainly from cases of deliberate suicidal ingestion of large quantities. While most clinical reports involve exposure to 2,4-D and mecoprop, it is assumed that all chlorophenoxy herbicides will share similar clinical profiles. Signs and symptoms of exposure to chlorophenoxy herbicides typically include vomiting, diarrhea headache, confusion, bizarre or aggressive behavior, peculiar odor on breath, muscle weakness, peripheral neuropathy, and loss of reflexes. Chlorophenoxy acids are weak uncouplers of oxidative phosphorylation, and large doses may produce hyperthermia from increased production of body heat. In addition, some of the chlorophenoxy acids, and their derivatives, can be irritating to skin, eyes and respiratory and gastrointestinal epithelium. In a few individuals, local cutaneous depigmentation may result from protracted dermal contact with chlorophenoxy compounds. Chloracne (a chronic, disfiguring skin condition) and other dermal effects have been reported, and appear to be specific for exposure to these compounds (Roberts and Reigart, 2013). One unusual effect that has been reported is that chlorophenoxy herbicides, particularly 2,4-D and 2,4-DP, inhibit the human (but not animal) taste receptor for sweets. This finding could potentially be of diagnostic use (Maillet et al., 2009). Ingestion of large amounts of chlorophenoxy acids can cause severe metabolic acidosis, electrocardiographic changes, myotonia, muscle weakness, myoglobinuria and elevated serum creatine phosphokinase in humans. These effects reflect injury to striated muscle. A few instances of peripheral neuropathy, some following dermal exposures to 2,4-D and another following ingestion, have also been reported. Myotonia and muscle weakness may persist for months after acute poisoning. Additional findings include loss of reflexes and fasciculation (Roberts and Reigart, 2013). Most reports of fatal outcomes involve renal failure, acidosis, and electrolyte imbalance (Flanagan et al., 1990: Keller et al., 1994). Mental status changes occur, with progression to coma and death in severe cases (Flanagan et al., 1990; Roberts et al., 2005). Moderate cerebral edema has also been reported (Roberts and Reigart, 2013). Respiration is not depressed, but hyperventilation is sometimes evident; probably secondary to the metabolic acidosis that occurs. Convulsions occur very rarely. Metabolic acidosis is manifest as a low arterial pH and bicarbonate content. The urine is usually acidic. Skeletal muscle injury, if it occurs, is reflected in elevated creatine phosphokinase and, sometimes, myoglobinuria. Moderate elevations of blood urea nitrogen and serum creatinine are commonly found as the toxicant is excreted. Renal failure have been accompanied by hyperkalemia or hypocalcemia, and were thought to result in the cardiovascular instability that led to death. Tachycardia is commonly observed and hypotension has also been reported. T-wave flattening has also been observed. Mild leukocytosis and biochemical changes indicative of liver cell injury have been reported (Roberts and Reigart, 2013).

Mechanisms of toxicity The precise mechanisms of toxicity of the chlorophenoxy herbicides is not known, but studies have revealed several cellular and biochemical alterations that occur in response to exposure. Based on their review of the literature, Bradberry et al. (2000) proposed three possible modes of action for chlorophenoxy herbicides. First, studies in model membrane systems show chlorophenoxy herbicide-induced alterations to membrane structure, disruption of cell membrane transport mechanisms, and inhibition of ion channels. Second, because chlorophenoxyacetic acids form analogs of acetyl coenzyme A (AcCoA) in vitro, it is possible that they disrupt cellular metabolic pathways involving AcCoA. Finally, in vitro studies have shown that phenoxy herbicides can uncouple oxidative phosphorylation, thereby disrupting cellular energy generation and utilization. Some effects reported in humans following poisoning with chlorophenoxy herbicide formulations and in animals following high doses of 2,4-D, such as damage to the blood-brain barrier, myotonia, and muscle twitching, are consistent with the above modes of action. Lerro et al. (2017) evaluated associations between urinary 2,4-D and selected urinary markers of oxidative stress (malondialdehyde [MDA], 8-hydroxy-20 -deoxyguanosine [8-OHdG], and 8-isoprostaglandin-F2a [8-isoPGF]) among 30 Iowa corn farmers who

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utilized pesticides and 10 controls. Exposure to 2,4-D was associated with elevated levels of 8-OHdG (b ¼ 0.066; 95% CI 0.008–0.124) and 8-isoPGF (b ¼ 0.088; 95% CI 0.004–0.172). The link between 2,4-D exposure and oxidative stress was also investigated in animals. Twenty-five-day-old offspring from female rats given 100 mg 2,4-D/kg/day on days 9–25 after birth of offspring, showed significant increases in reactive oxygen species in the midbrain, striatum, and prefrontal cortex (Ferri et al., 2007). Milder effects were observed in the hippocampus and no effects were seen in the hypothalamus. In other studies, oxidative stress increased and antioxidant enzyme levels decreased in livers from rats and their pups following maternal exposure to 126 mg of 2,4-D/kg/day from day 14 of gestation to day 14 after birth (Troudi et al., 2012a). In addition, increased oxidative stress, and decreased levels of both antioxidant and non-antioxidant enzyme activity were found in hemolysate and bone homogenates of the offspring (Troudi et al., 2012b). Exposure of rats to 100 mg 2,4-D/kg/day on days 1–19 of gestation, produced increased levels of malondialdehyde and reduced levels of antioxidant enzymes in the liver of dams and fetuses sacrificed on gestation day 20. Exposure to 2,4-D has also been reported to produce behavioral alterations in adult rats, through serotonergic and dopaminergic mechanisms. When given in combination with amphetamine, a ‘Serotonergic Syndrome’ was induced. Bortolozzi et al. (1998) reported that these amphetamine induced responses are due to increased serotonin and dopamine in the substantia nigra, ventral tegmental area, nucleus accumbens, striatum, midbrain, and cerebellum. In one study, dopamine D2 receptors appeared to be involved in the stimulant-induced behavioral sensitization (Bortolozzi et al., 2002), and it was shown that the D2 receptors were increased in density by about 40% in the striatum of rats exposed perinatally and then directly to 2,4-D. Increases were also reported in the prefrontal cortex and cerebellum (Bortolozzi et al., 2004). Exposure to 2,4-D in utero and through lactation produced a permanent increase in serotonergic neurons in all mesencephalic nuclei from offspring (Garcia et al., 2001). However, perinatal exposure followed by direct exposure produced an increase only in serotonergic neurons from the dorsal raphe nuclei. In other studies, dopamine and dopamine metabolite levels were decreased in the right side with respect to the left side in the striatum and nucleus accumbens in rats exposed perinatally and then directly. Rat pups exposed to 2,4-D via lactation, showed decreased tyrosine hydroxylase immunoreactivity in the substantia nigra and ventral segmental area in the midbrain and a significant drop in serotonin fiber density (Garcia et al., 2004, 2006). Behavioral alterations can be related to induction of reactive gliosis in the hippocampus and cerebellum of rat pups exposed to 2,4-D through maternal milk. Changes in myelin deposition and ganglioside patterns in brain were found in rat pups exposed to 2,4-D either directly or through maternal milk. 2,4-D can also disrupt microtubule assembly, and disorganize the Golgi apparatus in cultured cerebellar granule cells (ATSDR, 2020).

Toxicokinetics Absorption Studies conducted with human volunteers, indicate that oral absorption of 2,4-D is rapid and virtually complete. Oral administration of a single dose of 5 mg/kg 2,4-D to six male volunteers showed significant plasma levels at 1 h after dosing and peak plasma levels of approximately 30 mg/mL 7–24 h after dosing (Kohli et al., 1974); a plasma half-life of 33 h was estimated. In another study, five male volunteers received 5 mg/kg of 2,4-D. The estimated absorption ranged from 87.6% to 108.3%. Peak plasma levels of 10–30 mg/g occurred 6 h after dosing (Sauerhoff et al., 1977); a plasma half-life of 11.6 h was estimated. Dermal absorption of 2,4-D in humans is low compared to oral absorption. Male volunteers who received a topical application of 4 mg/cm2 of 2,4-D in acetone on the ventral forearm excreted only 5.8% of the applied dose in the urine over a 5-day period. In a similar study, male volunteers excreted 4.5% of a 10 mg dose of 2,4-D in acetone/water applied to a 9 cm2 area on the dorsum of the hand, over a 144-h period. Approximately 2% of 2,4-D in soil was absorbed through human skin in vitro. However, when using acetone as a vehicle, 19% of an applied dose of 2,4-D was absorbed (ATSDR, 2020).

Distribution Several investigators (ATSDR, 2020) have measured 2,4-D levels in humans who died following oral ingestion of 2,4-D and found it to be widely distributed to tissues including, brain, liver, kidney, spleen, muscle, body fat, pancreas, heart, and lungs. Studies in animals have also shown that 2,4-D is widely distributed in tissues after oral dosing. In a study in rats, some 2,4-D-derived radioactivity was detected in all 12 tissues examined as early as 1 h after gavage dosing (Khanna and Fang, 1966). Rats given 3 mg/kg 2,4-D had peak concentration in tissues 6–8 h after dosing, but no radioactivity was detectable 24 h after dosing. Aside from the stomach, the kidneys had the highest amount of radioactivity and the brain had the least. At doses of 240 mg/kg 2,4-D peak concentrations occurred 8 h after dosing and could still be detected after 41 h. Elimination half-lives ranged from 3 to 3.5 h; the brain had the lowest radioactivity at all times and the kidneys had the highest. In subcellular distribution studies, 2,4-D was found in the nuclear, mitochondrial, microsomal, and soluble fractions of the kidneys, liver, spleen, brain, heart, and lungs. A separate study in rats showed that postnatal dietary maternal exposure to 2,4-D can result in transfer of 2,4-D to the offspring via the milk (Sturtz et al., 2006). Saghir et al. (2013) also demonstrated excretion of 2,4-D in rat’s milk following perinatal exposure to 2,4-D in the diet. No information was located regarding distribution of 2,4-D following dermal exposure of humans or animals, but it is reasonable to assume that 2,4-D will distribute in a manner similar to that reported in oral animal studies.

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In a study that only evaluated brain distribution, subcutaneous administration of 300 mg/kg 2,4-D (half the LD50) followed by IV radioactive 2,4-D resulted in widespread distribution of activity (cerebral cortex, striatum, medulla oblongata, cerebellum, and midbrain brain, including hippocampus, hypothalamus, and thalamus) without any one area showing preferential accumulation (Tyynela et al., 1990). A study in pregnant rabbits followed 2,4-D distribution after IV injection of 14C-labeled 2,4-D on days 28–30 of gestation. Radioactivity was rapidly transferred to fetal plasma and brain (Sandberg et al., 1996), peaking in fetal plasma approximately 30 min after injection and remaining relatively constant for the 2-h sampling period. Except for plasma, maternal kidneys and uterus showed the highest tissue activity area under the curves (AUCs). In maternal brain, lateral and ventricular choroid plexus had the highest concentration of radioactivity (about 10 times higher than any other brain region). Fetal brain had the lowest activity of any maternal or fetal organ sampled. The concentration in fetal brain tissue was 7% of that in fetal plasma compared to 2% of that in maternal plasma. In general, maternal and fetal tissue AUCs increased proportionally to the dose of 2,4-D, from 1 to 10 mg/kg. However, in fetal tissues, activity also increased 10-fold when the maternal dose was raised from 10 to 40 mg/kg. The investigators suggested that because only unbound compound was available for placental transfer, the greater increase in fetal AUCs suggested saturation of maternal 2,4-D plasma protein binding (Sandberg et al., 1996).

Metabolism In a group of six male volunteers, only unchanged 2,4-D was detected in urine samples over a 1-week period after receiving a single oral dose of 5 mg/kg 2,4-D (Kohli et al., 1974). Urine samples collected from five volunteers following ingestion of 5 mg/kg 2,4-D showed mostly parent 2,4-D (mean 82.3% of the administered dose), with smaller amounts of conjugates over a 6-day period (Sauerhoff et al., 1977). Griffin et al. (1997) compared 2,4-D metabolism in rats, mice, and hamsters following oral dosing of 14C-labeled 2,4-D. The parent molecule was the major urinary metabolite in all three species. A glycine conjugate was found in the urine of mice and hamsters, a taurine conjugate in the urine of mice and male hamsters, and a glucuronide was detected only in urine from hamsters. In a comparative study in rats and dogs receiving a single oral dose of 14C-labeled 2,4-D, only the parent molecule was excreted in the urine of rats (van Ravenzwaay et al., 2003). The urine of dogs, however, contained taurine, serine, glycine, glutamic acid cysteine, sulfate, and glucuronide conjugates of 2,4-D. Interestingly, it was reported that dog plasma contained only unchanged 2,4-D.

Excretion The chlorophenoxy herbicides are excreted almost exclusively in the urine. In some animals including humans, metabolism is minimal and the majority of the compound is excreted as the parent molecule. In six healthy male volunteers dosed orally with 5 mg/kg 2,4-D, unchanged compound was detected in the urine within 2 h after ingestion, and over 75% of the parent compound was excreted within 4 days (Kohli et al., 1974). In a separate study, in which volunteers also received a single oral dose of 5 mg/kg of 2,4-D, the parent was excreted in the urine within 3 days of dosing (Sauerhoff et al., 1977). By the sixth day after dosing, urinary excretion was complete. The elimination half-life was estimated to range from 10.2 to 28.5 h, and the estimated fraction of the dose eliminated in the urine ranged from 47.8 to 96.5%. Under normal conditions, the average half-life of 2,4-D in humans is between 13 and 39 h, that of 2,4,5-T about 24 h, and that of MCPP about 17 h. However, half-life varies markedly with urinary pH, with excretion being greatly enhanced in an alkaline urine. A half-life as long as 70–90 h may occur with acidic urine. Half-life is also longer with large doses and prolonged exposure (Roberts and Reigart, 2013). In volunteers receiving 4 mg/cm2 of 2,4-D in acetone, applied dermally, most of the absorbed dose was eliminated in the urine within 72 h (Feldmann and Maibach, 1974). In a similar study, subjects given 10 mg of 2,4-D in acetone, applied dermally, excreted most (84.8%) of the absorbed dose in the urine in 96 h. The approximate mean urinary excretion half-life was 39.5 h (Harris and Solomon, 1992).

Environmental fate Atmospheric fate Atmospheric levels of chlorophenoxy herbicides are generally very low, but detectable levels may be present in agricultural areas where they have been recently applied (WHO, 2003). According to a gas/particle partitioning model of semivolatile organic compounds in the atmosphere, both 2,4-D and MCPA, will exist in both the vapor and particulate phases in the ambient atmosphere. Both 2,4-D and MCPA in the vapor phase are degraded by reaction with photochemically-produced hydroxyl radicals. The half-lives for this process are estimated at 19 h and 30 h for 2,4-D and MCPA, respectively. Particulate-phase may be removed from the air by wet and dry deposition. Aerial drift from spraying operations can transport the herbicides to nearby ponds and streams. A multi-year study of MCPA in surface water of a small prairie watershed concluded that the aqueous levels of MCPA were related to elevated precipitation and air levels rather than to runoff losses. MCPA may undergo direct photolysis since it is photochemically reactive in water. Exposure of aqueous solutions of MCPA in distilled water to Oct sunlight in Davis, CA resulted in a 14% decomposition after 245 h (PubChem, MPCA, n.d.).

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Aquatic fate Chlorophenoxy herbicides may enter rivers, lakes, and ponds from spray drift following its aerial application or from runoff and erosion of soils. Some of these herbicides, such as 2,4-D, may also be directly applied to water surfaces in order to eradicate nuisance aquatic plants. The aerobic aquatic metabolism half-life of 2,4-D was reported to be about 15 d; however, it was more persistent in anaerobic aquatic metabolism studies, with a half-life ranging from about 41 to 333 d (EPA, 2005). Degradation studies of MCPA suggest its complete degradation will take 96 wks. Photolysis in sunlit surface waters may also be an important degradation process for these herbicides, and the direct photolysis half-life of MCPA in water (at surface conditions under summer sunlight) is about 19–20 d. Loss of chlorophenoxy herbicides via hydrolysis and evaporation from the water surface is negligible. A bioconcentration factor (BCF) of 1, measured in carp, suggests that bioconcentration in aquatic species is low (PubChem, 2,4-D, n.d.; PubChem, MPCA, n.d.).

Terrestrial fate Once deposited into soils, the chlorophenoxy herbicides typically undergo biodegradation rapidly. Photolysis and hydrolysis can contribute to their removal, but volatilization is a negligible factor. In a series of field dissipation studies conducted over a two year period, 2,4-D typically showed degradation half-lives ranging from a few days to a few weeks depending upon the soil properties, water content of the soil, and whether 2,4-D was applied as a liquid or granular formulation (Wilson et al., 1997). A literature review of available laboratory persistence studies of MCPA in soil found half-lives ranging from liver > whole blood. Conjugation with glutathione constitutes the primary route of metabolism of chlorothalonil. Liver represents the major site for chlorothalonil conjugation. In the enzymatic reaction, 4-(glutathione-S-yl)2,5,6-trichloroisophthalonitril is formed initially. This is also a substrate for glutathione-S transferases (GSTs), resulting in the substitution of a second chlorine atom to give 4,6-bis(glutathione-S-yl)-2,5-dichloroisophthalonitril. Hydrolysis studies indicated that the metabolism of chlorothalonil is pH dependent. Thus, 4-hydroxy-2,5,6-trichloroisophthalonitrile and 3-cyano-2,4,5,6tetrachlorobenzamide are formed at pH 9 but not at pH 7 (Garron et al., 2012). The metabolism of chlorothalonil was recently investigated in liver and gill cytosolic and microsomal fractions from channel catfish using high performance liquid chromatography. The reports indicate that chlorothalonil is detoxified in vitro by GST-catalyzed glutathione conjugation. However, no human data are currently available for the biotransformation of chlorothalonil. Chlorothalonil is primarily eliminated via the kidneys (Garron et al., 2012). Evaluation of exposure of Chlorothalonil on an estuarine polychaete Laeonereis acuta biochemical biomarkers of oxidative metabolism and cholinesterases indicated an increase in the levels of antioxidant capacity against peroxyl radicals (ACAP) in the animals treated with higher concentrations of chlorothalonil along with induced GST activity and reduced GSH content of these animals. This indicates increased oxidative stress and finally proving that chlorothalonil exposure causes increase in the AChE activity possibly related to membrane lipids (Dellal et al., 2019). Following administration of 1 mg kg−1 chlorothalonil endotracheally, orally, or dermally to rats, less than 6% was recovered in blood or urine within 48 h. The major route of elimination following oral administration to rats is in the feces (>80%), with 5.4–11.5% being excreted in the urine as the dose increases from 5 to 200 mg kg−1. Marked species differences exist in the pharmacokinetic behavior of chlorothalonil. Thus, following oral administration of 50 mg kg−1 chlorothalonil, dogs and rhesus monkeys excrete up to 98 and 92% of the dose, respectively, in the feces compared with 82% in rats (PubChem, 2022).

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Mechanism of toxicity Glutathione conjugation represents a bioactivation reaction for chlorothalonil resulting in the formation of S-conjugates toxic to the kidney. Chlorothalonil acts as an alkylating agent and reacts with cellular sulfhydryl compounds. Alkylation of biological molecules results in effects on cellular function and viability. Chronic damage to the proximal tubular epithelium may be involved in the mechanism of chlorothalonil tumorigenicity to the kidneys (WHO, 1999). Assessment of effect of chlorothalonil (60 mg/kg/per day and 180 mg/kg/per day) on Wistar rats by gavage not only indicated increase in the absolute weights of their kidneys but also demonstrated increased urea and creatinine as compared to untreated rats. The kidney microphotographs of the treated rats revealed alterations as peritubular inflammatory reaction, necrosis, cell desquamation and degenerated distal tubules (Dellal et al., 2019).

Animal toxicity Forestomach and the renal proximal tubule are the primary target tissues of chlorothalonil toxicity in Sprague–Dawley rats. Toxicity is characterized by hypertrophy, hyperplasia, vacuolization, and degeneration of renal tubular epithelium and acanthosis, hyperkeratosis, and hyperplasia of the squamous epithelium of the forestomach. Chlorothalonil is a well-known skin and eye irritant. Exposure effects of chlorothalonil on human colon adenocarcinoma cell line (Caco-2) monolayer depicted an increase in the paracellular permeability paired with downregulation of the tight junction genes while showing an upregulation of apoptotic genes combined with phosphorylation of mitogen activated protein kinase (MAPK). This strongly validates that chlorothalonil exposure leads to intestinal barrier dysfunction brought by the activation of the MAPK pathway (Tao et al., 2021). It is also known to cause severe ocular lesions in rabbits that are irreversible (Gupta, 2018). Sustained contact with the squamous epithelium of the forestomach can lead to an inflammatory response. The earliest observation following chlorothalonil administration at 175 mg kg−1 day−1 to rats for varying periods of time for up to 91 days has been characterized by multifocal ulceration and erosion of the mucosa, subsequently progressing to hyperplasia and hyperkeratosis. These lesions have been observed in subchronic and chronic studies in rats and mice (no observed effect level (NOEL)  2 mg kg−1 day−1) and in chronic studies appear to be closely related with incidence of neoplasia (NOEL 4–21 mg kg−1 day−1) (USEPA, 1989). Undiluted chlorothalonil is a strong irritant and produces irreversible corneal, iridal, and conjunctival effects in rabbits. Weakness and sedation precede death in animals given acute toxic doses intraperitoneally. Chronic oral administration to rat results in ataxia. Chlorothalonil may cause reproductive or developmental toxicity, however, a significant increase in the occurrence of post-implantational loss due to early embryonic death is observed in mice (Gupta, 2018). Hematuria, vaginal bleeding, and epistaxis are seen in rats following chronic oral exposure. In chronic dermal exposures to chlorothalonil dissolved in acetone, the no-effect level for irritation is 0.001%. The 0.01% concentration is a mild irritant and 0.1% a moderate irritant. Prolonged exposure of rodents to chlorothalonil results in nephrotoxicity and renal tubular hyperplasia, and these effects, if sustained, can lead to a tumorigenic response. Chlorothalonil produced a dose-related increased incidence of renal tubular adenomas and adenocarcinomas in rats. The oral LD50 in rats is greater than 10 g kg−1. Chlorothalonil is predicted to be a rodent carcinogen via a nongenotoxic mechanism (WHO, 1999; USEPA, 1989). LD50 values for a variety of experimental animals are as follows: Animal Model

Route of administration

LC50/LD50

Source

Mouse Mouse Rat Rat Rat Rat Rabbit

Intraperitoneal Oral Intraperitoneal Dermal Oral Inhalation Percutaneous

2500 mg kg−1 3700 mg/ kg 2500 mg kg−1 10,000 mg kg−1 10,000 mg kg−1 0.1 mg/L 5000 mg kg−1

Cayman Chemicals Cayman Chemicals Quali-Pro TM/C WDG Turf & Ornamental Fungicide Quali-Pro TM/C WDG Turf & Ornamental Fungicide Quali-Pro TM/C WDG Turf & Ornamental Fungicide Quali-Pro TM/C WDG Turf & Ornamental Fungicide Zhejiang Rayfull Chemicals., Ltd. (Zhejiang Rayfull Chemicals, n.d.)

Human toxicity Facial dermatitis has been reported in occupational exposures and can occur in the absence of direct skin contact, due to the high volatility of chlorothalonil. Chlorothalonil is a strong primary skin irritant and may also cause allergic contact urticaria and anaphylaxis. Patch testing with concentrations greater than 0.01% may produce primary irritant reactions. Hypersensitivity reactions characterized by facial erythema, periorbital erythema and edema, eczema, and pruritus have been observed following chlorothalonil exposure. Photosensitivity reactions were seen in some individuals. High concentrations of chlorothalonil produce delayed irritant reactions. Delayed dermal irritant effects have also been noted 48–72 h after cessation of exposure. Immediate respiratory reactions such as tightness of chest and throat may occur following inhalation exposure to chlorothalonil (Mozzachio et al., 2008). A recent review of the potential cancer risks of chlorothalonil to operators and consumers conducted in the United

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Kingdom for the Pesticide Advisory Committee provided evidence that chlorothalonil is not genotoxic. The primary potential for human exposure to chlorothalonil is to forest service applicators applying the fungicide. Chlorothalonil does not have any adverse reproductive or teratogenic effects at expected exposure levels. Data suggesting carcinogenic potential of chlorothalonil remain inconclusive at the present time (Organization, 1994; Commission, 2002). Investigation of impending genotoxic effects of different concentrations of Chlorothalonil in human lymphocytes in vitro demonstrated increased frequency of chromosomal aberrations and sister chromatid exchanges as compared to the negative control to diminished mitotic value at all concentrations supporting the idea that chlorothalonil is clastogenic and potentially increases DNA damage in human lymphocytes (Dellal et al., 2019; Lopes et al., 2020).

Reproductive effects Long-term administration of chlorothalonil at high doses to both male and female rats did not affect reproduction or the litters that were produced. However, weight gains in both sexes decreased generation after generation. Administration of high doses of chlorothalonil to pregnant rabbits through the stomach during the sensitive period of gestation resulted in abortion. These studies suggest that chlorothalonil will not affect human reproduction except at very high doses. In second- and third-generation reproductive toxicity studies, rats showed decreased food consumption, lower body weights, kidney lesions, and forestomach lesions after exposure to chlorothalonil in the diet. The only effect in the pups was a reduction in body weight exposed to chlorothalonil or SDS-3701 in utero (Arena et al., 2018). Although several studies have focused on the toxic effects of chlorothalonil on the male reproductive system, interrogation of Cholorthalonil toxicity on spermatogenesis has been limited. A recent study demonstrated that low dose chlorothalonil impairs mouse spermatogenesis through the intertwining of estrogen receptor pathway coupled with DNA and histone methylation (Zhang et al., 2019).

Developmental toxicity Developmental toxicity with Chlorothalonil has not been reported in rats or rabbits. However, dams treated with chlorothalonil have shown increased mortality accompanied with excess lacrimation, vaginal and nose discharges, anogenital stains, decreased food consumption, and reduced body weight (de Castro et al., 2000). Risk assessment of chlorothalonil on a native amphibian Rhinella arenarum by means of continuous treatments from larval to embryo stages presented substantial toxicity with increase in time. The larval stages showed relatively constant toxicity throughout time while the embryos depicted various sublethal effects with time (Acquaroni et al., 2021).

Teratogenic effects Administration of high doses of Chlorothalonil to rats led to reduced weights in both male and females. Elevated doses of chlorothalonil given to the female rats through the stomach during the gestational period had no notable effect on fetuses, although the dose was toxic to mothers. Limited study disclosed rabbits had no effects (Oregon State University, 1996). Based on these observations, chlorothalonil is expected to produce no birth defects in humans. (de Castro and Chiorato, 2007).

Mutagenic effects Mutagenicity studies conducted on various animals, bacteria, and plants indicate that chlorothalonil does not cause any chromosomal changes. The compound is therefore not expected to pose significant mutagenic risks to humans. The effects of chlorothalonil injected intraperitoneally in an estuarine fish Micropogonias furnieri, frequently consumed by humans was assessed. Chlorothalonil presented to be genotoxic by bringing about DNA damage while also indicated mutagenic effects on micronuclei, nuclear buds etc. (Lopes et al., 2020). Given that genomic instability contributes to carcinogenesis, it would be safe to say that chlorothalonil may affect the health of aquatic biosystem as well as humans (Castro et al., 2022). Chlorothalonil (>97% pure; up to 10 mg plate−1) was not mutagenic in Ames assays with strains TA98, TA100, TA1537, or TA1538 with and without rat liver S-9 homogenate pretreated with Aroclor 1254. Chlorothalonil (up to 50 mg plate−1) was also not mutagenic in the above strains with and without rat kidney S-9 homogenate. Chlorothalonil (97.3% pure) was not mutagenic when tested at 0.3 mg ml−1 in Chinese hamster lung fibroblasts (V79) cells, at 0.3 mg ml−1 with activation (rat liver S-9 homogenate pretreated with Aroclor 1254) in mouse BALB/3 T3 fibroblasts, and at 0.03 mg ml−1 without activation in BALB/3 T3 cells. However, chlorothalonil was positive in some chromosomal aberration assays and a DNA damage study. An epigenetic mechanism has been suggested for chlorothalonil-induced DNA damage in isolated human lymphocytes (EPA, 1997; Shi et al., 2018).

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Carcinogenic effects There exists sufficient evidence in experimental animals supporting carcinogenicity of chlorothalonil. Chlorothalonil is a potential human carcinogen, known to affect the kidney, ureter, and bladder in experimental animals. Both sexes of rats fed chlorothalonil daily over a lifetime developed carcinogenic and benign kidney tumors at the higher doses. In an unsettled study (REF), mouse fed daily high doses of chlorothalonil for 2 years were found to develop tumors in the forestomach area in females and carcinogenic and benign kidney tumors in males. A long-term National Toxicology Program study concluded that under the conditions of this bioassay, chlorothalonil is carcinogenic to Osborne–Mendel rats, but not to B6C3F1 mice (ORST, 1996; Catalog, 1975).

Clinical management One of the primary forms of treatment following Cholorthalonil exposure is to support respiratory and cardiovascular function. Dilution and dermal or eye decontamination are primary considerations. Following oral exposure, immediate dilution with 4–8 oz of milk or water is recommended. Vomiting must be induced if victim is conscious. If inhaled, victim must be immediately moved to fresh air; if the exposed individual fails to breathe, artificial respiration should be provided. In case of dermal exposure to chlorothalonil, the exposed area should be thoroughly washed with soap and water. Allergic contact dermatitis may be treated with antihistamines, topical steroids, or systemic steroids. Following an eye exposure, the affected eyes should be irrigated with copious amounts of tepid water for at least 15 min. Immediate medical attention for the eyes is also recommended. Exposure may cause temporary allergic side effects. Symptoms include redness of the eyes, mild bronchial irritation, and redness or rash on exposed skin. Temporary allergic reactions can be treated with antihistamines or steroid creams and/or systemic steroids upon consultation with the physician (King and Balogh, 2013; PubChem, 2022).

Ecotoxicology Chlorothalonil and its metabolites are highly toxic to fish, aquatic invertebrates, and marine organisms. Chlorothalonil levels of less than 1 ppm have been reported to produce noticeable toxicity in fish such as the rainbow trout (LC50 of 0.25mgl−1), bluegills (LC50 of 0.3mgl−1), and channel catfish (LC50 of 0.43mgl−1). However, it is relatively nontoxic to birds, mammals, and bees (PubChem, no date). Chlorothalonil has been known to impact the sperm quality in guppy Poecilia vivipara by affecting the RBC physiology and fertility in male P. vivipara (Lopes et al., 2020). In a study of 30-day exposure of relevant concentrations of chlorothalonil in the blood of economically important olive flounder Paralichthys olivaceus showed that prolonged exposure to chlorothalonil can affect susceptibility to pathogens via immunosuppression, hepatic toxicity and oxidative stress in Olive Flounder (Bacmaga et al., 2018; Shi et al., 2018; Lee et al., 2022).

Exposure standards and guidelines Owing to its potential for causing eye irritation, chlorothalonil is also classified as a toxicity class II, moderately toxic chemical. Based on the increased incidence of renal tumor in female rats, EPA currently lists chlorothalonil as a Group B2 (probable human) carcinogen, the Q∗ value being 0.007 66 mg kg−1 day−1 (United States Environmental Protection, 1999). Chlorothalonil has an acceptable daily intake and a reference dose value of 0.03 and 0.015 mg kg−1 day−1, respectively (Dellal et al., 2019; PubChem, 2022).

Conclusion Chlorothalonil is an important broad-spectrum, non-systemic, organochlorine fungicide that has been widely used for decades as an effective disease management tool for potatoes, peanuts, turf, a variety of vegetables and fruit crops (Phillip and Unive, 2019). It is also used to control fruit rots in cranberry bogs and is used in paints. Chlorothalonil is classified as a general use pesticide by the USEPA. This compound is considered Group B2, probable human carcinogen. In overexposure situations, GI decontamination and administration of activated charcoal along with cathartic sorbitol should be considered.

See also: Nematicides; Pesticides and its toxicity

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References Acquaroni M, Svartz G, and Pérez Coll C (2021) Developmental toxicity assessment of a chlorothalonil-based fungicide in a native amphibian species. Archives of Environmental Contamination and Toxicology 80(4): 680–690. Arena M, et al. (2018) Peer review of the pesticide risk assessment of the active substance copper compounds copper (I), copper (II) variants namely copper hydroxide, copper oxychloride, tribasic copper sulfate, copper (I) oxide, Bordeaux mixture. EFSA Journal 16(1): e05152. https://doi.org/10.2903/j.efsa.2018.5152. Bacmaga M, Wyszkowska J, and Kucharski J (2018) The influence of chlorothalonil on the activity of soil microorganisms and enzymes. Ecotoxicology 27(9): 1188–1202. Castro MS, et al. (2022) Genotoxic and mutagenic effects of chlorothalonil on the estuarine fish Micropogonias furnieri (Desmarest, 1823). Environmental Science and Pollution Research 29(16): 23504–23511. de Castro VLS and Chiorato SH (2007) Effects of separate and combined exposure to the pesticides methamidophos and chlorothalonil on the development of suckling rats. International Journal of Hygiene and Environmental Health 210(2): 169–176. de Castro VL, Chiorato SH, and Pinto NF (2000) Biological monitoring of embrio-fetal exposure to methamidophos or chlorothalonil on rat development. Veterinary and Human Toxicology 42(6): 361–365. Dellal I, Mallem L, and Abdennour C (2019) Effect of chlorothalonil on renal activity of the male rat. Ecology, Environment and Conservation 25(1): 252–256. Gao S, et al. (2019) Physicochemical properties and fungicidal activity of inclusion complexes of fungicide chlorothalonil with b-cyclodextrin and hydroxypropyl-b-cyclodextrin. Journal of Molecular Liquids 293: 111513. Garron C, et al. (2012) Assessing the genotoxic potential of chlorothalonil drift from potato fields in Prince Edward Island, Canada. Archives of Environmental Contamination and Toxicology 62(2): 222–232. Gupta PK (2018) Toxicity of fungicides. In: Veterinary Toxicology, pp. 569–580. Academic Press. King KW and Balogh JC (2013) Event based analysis of chlorothalonil concentrations following application to managed turf. Environmental Toxicology and Chemistry 32(3): 684–691. Lee S, et al. (2022) Long-term exposure to antifouling biocide chlorothalonil modulates immunity and biochemical and antioxidant parameters in the blood of olive flounder. Comparative Biochemistry and Physiology, Part C: Toxicology & Pharmacology 257: 109337. Lopes FC, et al. (2020) Impacts of the biocide chlorothalonil on biomarkers of oxidative stress, genotoxicity, and sperm quality in guppy Poecilia vivipara. Ecotoxicology and Environmental Safety 188: 109847. Lopes FC, Varela Junior AS, Corcini CD, et al. (2020) Impacts of the biocide chlorothalonil on biomarkers of oxidative stress, genotoxicity, and sperm quality in guppy Poecilia vivipara. Ecotoxicology and Environmental Safety 188: 109847. Meng C, He Q, Huang JW, et al. (2015) Degradation of chlorothalonil through a hydrolytic dehalogenase secreted from Bacillus subtilis WB800. International Biodeterioration & Biodegradation 104: 97–104. Mozzachio AM, et al. (2008) Chlorothalonil exposure and cancer incidence among pesticide applicator participants in the agricultural health study. Environmental Research 108(3): 400–403. Potter TL, Don Wauchope R, and Culbreath AK (2001) Accumulation and decay of chlorothalonil and selected metabolites in surface soil following foliar application to peanuts. Environmental Science and Technology 35(13): 2634–2639. https://doi.org/10.1021/es002054e. Pubchem (2022) https://pubchem.ncbi.nlm.nih.gov/compound/Chlorothalonil. Shi T, et al. (2018) Inventories of heavy metal inputs and outputs to and from agricultural soils: A review. Ecotoxicology and Environmental Safety 164: 118–124. Tao H, et al. (2021) Chlorothalonil induces the intestinal epithelial barrier dysfunction in Caco-2 cell-based in vitro monolayer model by activating MAPK pathway. Acta Biochimica et Biophysica Sinica 53(11): 1459–1468. USEPA (1989) Risk assessment: Guidance for superfund. Human Health Evaluation Manual (Part A), Interim Final. Washington, DC: Office of Emergency and Remedial Response, U.S. Environmental Protection Agency. Zhang P, et al. (2019) Low dose chlorothalonil impairs mouse spermatogenesis through the intertwining of estrogen receptor pathways with histone and DNA methylation. Chemosphere 230: 384–395.

Relevant websites http://extoxnet.orst.edu/pips/reflist6.htm :Catalog, E. N. L. (1975) Brief Records: EPA National Library Catalog. 1–16. https://www.mass.gov/doc/pesticide-reduction-resource-guide-for-citizens-municipalities/download :Commission, W. N. R (2002) Pesticide Reduction Resource Guide. Wellesley Natural Resources Commission. https://www3.epa.gov/pesticides/chem_search/cleared_reviews/csr_PC-081901_15-Dec-97_449.pdf :EPA (1997) HED Chlorothalonil RED. https://inchem.org/documents/hsg/hsg/hsg098.htm :IPCS: INCHEM (n.d.) Chlorothalonil. http://extoxnet.orst.edu/pips/chloroth.htm#::text¼Trade%20and%20Other%20Names%3A%20Trade,with%20many%20other%20pesticide%20compounds :EXTOXNET. Pesticide Information Profiles. https://pubchem.ncbi.nlm.nih.gov/compound/Chlorothalonil :National Center for Biotechnology Information (2022) PubChem Compound Summary for CID 15910, Chlorothalonil. https://www.fao.org/3/cb2762en/cb2762en.pdf :Organization, W. H. (1994) Pesticide Residues in Food 1993. http://extoxnet.orst.edu/pips/chloroth.htm :ORST (1996) EXTOXNET Chlorothalonil. https://www.canr.msu.edu/news/broad_spectrum_fungicides_for_vegetables :Phillip B and Unive MS (2019) Broad-Spectrum Fungicides for Vegetables. https://inchem.org/documents/ehc/ehc/ehc183.htm#SubSectionNumber:1.1.1 :United Nations Environment programme (1996) Chlorothalonil. https://efaidnbmnnnibpcajpcglclefindmkaj/https://www3.epa.gov/pesticides/chem_search/reg_actions/reregistration/fs_PC-081901_1-Apr-99.pdf :United States Environmental Protection (1999) Chlorothalonil Facts. In: Prevention, Pesticides and Toxic Substances. http://efaidnbmnnnibpcajpcglclefindmkaj/https://www.ncbi.nlm.nih.gov/books/NBK402050/pdf/Bookshelf_NBK402050.pdf :WHO (1999) International Agency for Research on Cancer IARC Monographs on the Evaluation of Carcinogenic Risks to Humans Tumours of the Kidney. http://www.rayfull.com/Productshows.asp?ID¼264#.YniNTdrMK3A :Zhejiang Rayfull Chemicals (n.d.), L. Chlorothalonil.

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Chlorpheniramine Samaneh Nakhaeea and Omid Mehrpourb, aMedical Toxicology and Drug Abuse Research Center (MTDRC), Birjand University of Medical Sciences, Birjand, Iran; bData Science Institute, Southern Methodist University, Dallas, TX, United States © 2024 Elsevier Inc. All rights reserved. This is an update of E.J. Hall, G.J. Hall, Chlorpheniramine, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 923–924, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00711-9.

Chemical profile Background and uses Exposure routes and pathways Pharmacokinetics Mechanisms of toxicity Acute and short-term toxicity or exposure Animal Human Chronic toxicity or exposure Animal Human Genotoxicity Clinical management Prognosis Patient education Antihistamine toxicity key facts References

989 990 990 990 990 991 991 991 991 991 991 992 992 992 992 993 993

Abstract Chlorpheniramine (CAS 132-22-9) is a first-generation antihistamine (H1-receptor antagonist) commonly used to alleviate the symptoms of allergic reactions. It has sedative and antimuscarinic properties. An overdose of sedating antihistamines can have anticholinergic effects. Chlorpheniramine poisoning is managed by providing both symptomatic and supportive care. The prognosis may vary depending on the amount of drug consumed, underlying medical conditions, and any co-ingestant(s). Children and the elderly are particularly vulnerable to adverse side effects.

Keywords Allergy medication; Anticholinergic; Antihistamine; Antipruritic; Chlorpheniramine

Chemical profile

• • • • • • •

Name: Chlorpheniramine. Chemical Abstracts Service Registry Number: 132-22-9. Synonyms: chlorpheniramine, Chlorphenamine, Teldrin, Chlor-trimeton,3-(4-chlorophenyl)-N,N-dimethyl-3-pyridin-2-yl-propan1-amine (IUPAC); Chlor-Trimeton® Chemical Formula: C16H19ClN2 Molecular Weight: 274.793. Solubility: Ethanol, chloroform, water, methanol. Slightly soluble in benzene and ether. Target receptors(s): o Histamine receptors (H1). o Cholinergic muscarinic receptors (Minigh, 2008). Cl

•

CH3 N

Chemical Structure:

CH3

N

Encyclopedia of Toxicology 4th Edition

https://doi.org/10.1016/B978-0-12-824315-2.00360-2

989

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Background and uses Allergies and allergic rhinitis are generally treated with antihistamines that bind to H-1 receptors. Based on their effects on the central nervous system, H-1 antihistamines are typically divided into sedative (first generation, lipid-soluble drugs) and non-sedative (second generation) forms, with non-sedative agents being more likely to cross the blood-brain barrier. The first generation of antihistamines binds both central and peripheral histamine-1 receptors, whereas the second generation of antihistamines only binds peripheral histamine-1 receptors. These differences can have different therapeutic and adverse effects. In addition to their antihistamine properties, some antihistamines possess additional anticholinergic, antimuscarinic or other effects (Olasi nska-Wisniewska et al., 2014; Monczor and Fernandez, 2016; Thomas, 2016; Farzam et al., 2019). Antihistamines that act on the H-1 receptor have anticholinergic properties, causing adverse effects; this is mainly associated with the first generation of antihistamines (Manning, 2012). Antihistamines of the first generation are commonly associated with sleepiness as a side effect. Due to this reason, they are sometimes used as a sleep aid for people who have difficulty sleeping (insomnia). Chlorpheniramine is an over-the-counter medicine and is classified as a first-generation antihistamine, which works by blocking the H-1 receptors responsible for alleviating allergic reactions caused by the release of histamine. It has sedative and antimuscarinic activity Aronson (2016). Chlorpheniramine reduces vasodilation and capillary permeability by inhibiting histamine’s effects on smooth muscle. This compound is used to treat pollen allergy and allergic rhinitis symptomatically (Minigh, 2008). In addition, it is used to treat food allergies, drug reactions, hay fever, urticaria, relief of itching associated with chickenpox, and anaphylactic reactions in an emergency situation (Waller and Sampson, 2018). Initial responses to chlorpheniramine are observed within 30 min of administration, with peak responses occurring 1–2 h later (Minigh, 2008). Though chlorpheniramine is included in many multi-symptom over-the-counter cold relief medications, the Food and Drug Administration (FDA) issued a safety alert in March 2011 detailing some risks associated with this medication. Chlorpheniramine is contraindicated in patients known to be hypersensitive to it. It should be used with caution in patients with prostatic hypertrophy, bladder neck obstruction, asthma, hepatic insufficiency, glaucoma, or ulcers (Minigh, 2008). Since the mechanism of bronchial asthma and systemic anaphylactic reactions involves many other mechanisms than just histamine release, histamine antagonists are used only as adjunctive therapy to epinephrine after the acute reactions have been controlled. Antihistamines may also be used to help ameliorate allergic reactions during blood or plasma transfusions. Chlorpheniramine is commonly used in small-animal veterinary medicine for its antihistaminic/antipruritic effects, especially for the treatment of pruritus in cats, and occasionally as a mild sedative. Studies have shown that chlorpheniramine has antidotal effects against dichlorvos (a synthetic organophosphate acetylcholinesterase inhibitor) similar to those of atropine (Mousa, 2009; Owunari and Chika, 2021b; Owunari and Chika, 2021a). Some preclinical data showed that chlorpheniramine can produce antinociception effects (Tamadonfard and Mojtahedein, 2005; Hasanein, 2009; Tzeng et al., 2015; Chiu et al., 2020).

Exposure routes and pathways Chlorpheniramine is available in oral and injectable formulations; thus, ingestion and injection are routes of intentional and unintentional exposure. Antihistamine toxicity is almost exclusively associated with oral intake. Household exposures to antihistamines are very limited (Borowy and Mukherji, 2019).

Pharmacokinetics Chlorpheniramine is well absorbed after oral administration and has a serum half-life of approximately 20 h in adults. The consumption of food slows the peak blood concentration of the drug but does not affect its absorption. In addition to being widely distributed throughout the body, chlorpheniramine also affects the central nervous system (Minigh, 2008). It undergoes a relatively high degree of first-pass metabolism in the gastrointestinal (GI) mucosa and liver; therefore, only about 25–60% of the drug is available systemically. It is metabolized by the liver using the P450 cytochrome system. The drug and its metabolites (desmethylchlorpheniramine and didesmethylchlorpheniramine) are excreted almost exclusively through the kidneys. The elimination half-life is more rapid in children, 9.5–13 h vs. 14–24 h in adults.

Mechanisms of toxicity Except for non-sedating antihistamines, H-1 blockers have anticholinergic effects. Non-sedating antihistamine drugs are generally less toxic than sedating antihistamines (Manning, 2012). The toxicity of antihistamines is generally apparent after ingestion of three to five times the usual daily dosage (Manning, 2012). Toxicity of antihistamines is usually related to their anticholinergic effects and may include loss of appetite, nausea, vomiting, diarrhea or constipation, and other GI effects, dry mouth, hot dry skin, dilated pupils, tachycardia, hypertension or hypotension, as well as dizziness, tinnitus, lassitude, incoordination, fatigue, blurred vision, diplopia, euphoria, nervousness, insomnia, and tremors. Acetylcholine is competitively blocked at muscarinic receptors, resulting in

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symptoms of anticholinergic poisoning. Concurrent use of alcohol, tricyclic antidepressants, monoamine oxidase inhibitors, or other central nervous system (CNS) depressants along with antihistamines may exaggerate and extend the anticholinergic and CNS depressant effects of antihistamines; concurrent use is not recommended. Products that were marketed prior to the FDA safety alert but not approved by the FDA included multi-symptom cold medications comprised of drug combinations of chlorpheniramine with decongestants, antitussives, and analgesics. Risks associated with the use of these products included improper use in children and infants, potentially risky combination of ingredients, and patients receiving too much or too little medication because of problems with the way some ‘extended-release’ products were made. Newborn and premature infants are even more prone to anticholinergic side effects and an increased susceptibility to convulsions; thus, this drug is not recommended at all for this age group. Geriatric patients are also more prone to anticholinergic effects, and a paradoxical reaction characterized by hyperexcitability may occur in some elders taking antihistamines. Overdosage may also produce central excitation resulting in convulsions. Further, overdose with chlorphenamine may result in significant serotonin toxicity due to cumulative effects (Walsh, 2012). Several studies have demonstrated that chlorphenamine inhibits serotonin re-uptake significantly. Overdosing with chlorphenamine and dextromethorphan-containing cough medications may result in significant serotonin toxicity, due to their combined serotonergic effects (Monte et al., 2010).

Acute and short-term toxicity or exposure Animal CNS stimulation (excitement to seizures) or depression (lethargy to coma) may follow overdosage, as well as previously mentioned anticholinergic effects, respiratory depression, or death. The normal dosage range for veterinary use in small animals is 0.2–0.8 mg kg−1; one report of a puppy ingesting 25 mg kg−1 described signs of ataxia, tremors, bradycardia, coma, and cardiac arrest, eventually resulting in death within 11 h of ingestion.

Human Most suicide attempts occur in two categories: pediatric patients and the elderly. Because of their therapeutic sedative properties and global availability, antihistamines can be commonly excessively administered by the former group. Older people are more susceptible to the sedative and anticholinergic effects of these medications (Borowy and Mukherji, 2019). As antihistamines have a broad range of toxic and therapeutic effects, the pathophysiology of antihistamine toxicity varies. The most common effect of H-1 antihistamines is hallucinations or antimuscarinic effects. An antihistaminic medication administered in a rapid manner intravenously produces a hallucinogenic effect. As a mnemonic, one can recall the presenting symptoms of anticholinergic toxicity as “red as a beet, dry as a bone, hot as a hare, blind as a bat, and mad as a hatter.” It results in vasodilation and reddening of the skin, anhidrosis and decreased sweat production, and hyperthermia because of inadequate sweat production. Besides, mydriasis can cause blurred vision. Also delirium, urinary retention and hallucination might occur due to reduced detrusor contraction (Rollstin and Seifert, 2013; Borowy and Mukherji, 2019). Symptoms of neurotoxicity, particularly with first-generation H-1 antihistamines, can occur 2 h after ingestion and appear as hallucinations and drowsiness. Also, in pediatric populations, ataxia and irritability might be seen. Seizures may occur at any time following ingestion, but they generally occur within the first hour or two after ingestion (Borowy and Mukherji, 2019). Last but not least, agitation and seizures and irritability may accompany rhabdomyolysis. Symptoms of visual disturbances include mydriasis, diplopia and blurred vision (Borowy and Mukherji, 2019). CNS stimulation following overdosage is more common in children than adults. Overdosage in adults usually causes CNS depression (lethargy to coma) followed by excitement, seizures, and postictal depression, as well as previously mentioned anticholinergic effects. Cardiovascular effects may include tachycardia with prolonged QTc and QRS intervals and nonspecific ST and T-wave changes, hypo- or hypertension, dysrhythmias, and cardiac arrest; in cases of anticholinergic toxicity, Brugada-like ECG patterns may be seen (Borowy and Mukherji, 2019), an extreme overdose may result in cerebral edema, coma, cardiovascular or respiratory arrest, or death. Symptoms may appear 30 min to 2 h after exposure, and death may occur several days after the onset of initial symptoms.

Chronic toxicity or exposure Animal Animal feeding models designed to test for carcinogenicity and mutagenicity following chronic exposure have so far proved negative.

Human Studies of chronic dosing in adults and children have shown the expected side effects of drowsiness and sedation in therapeutic doses. The FDA categorizes this drug as category B for use during pregnancy. Preferably, all medications, including antihistamines, should be postponed in the first trimester. If antihistamines should be prescribed, first-generation drugs should be preferred, with

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chlorpheniramine, hydroxyzine, and dexchlorpheniramine being the first choice (Kar et al., 2012). If second-generation antihistamines are preferred, loratadine is the first choice and cetirizine is the second choice (Stefaniak et al., 2022).

Genotoxicity Ames Salmonella and mouse lymphoma tests for mutagenicity have been negative.

Clinical management A typical anticholinergic syndrome may confirm the diagnosis of toxicity based on the history of ingestion. The most common antihistamines can be detected in a comprehensive urine toxicology test. There are no specific levels available for such tests (Manning, 2012). In urine drug tests, some over-the-counter antihistamines can lead to false-negative results for amphetamines, phencyclidine, and methadone, misleading practitioners. Antihistamine concentrations may be determined through extensive tests, such as liquid chromatography/mass spectroscopy or gas chromatography/mass spectroscopy. Nevertheless, these tests are rarely recommended due to the lengthy diagnostic process (Borowy and Mukherji, 2019). Initial evaluations should always include the following: performing an ECG, measuring the temperature, heart rate, respiration rate, and blood pressure (Borowy and Mukherji, 2019). In the case of sedating antihistamines, the patient’s level of consciousness and respiration should be monitored (Thomas, 2016). Symptomatic and supportive care including advanced life-support measures should be implemented as necessary. Induction of emesis and gastric lavage is indicated if the time of ingestion was recent, and the patient is conscious with stable CNS signs. Activated charcoal can be administered orally if the conditions are suitable. Due to slowed gastrointestinal motility, gut decontamination procedures may prove beneficial even in patients who arrive late for treatment, (Manning, 2012). Antihistamines are not effectively removed by hemodialysis, hemoperfusion, peritoneal dialysis, or repeat dosage activated charcoal (Manning, 2012). Agitated patients with tachycardia may benefit from sedation with benzodiazepines. Patients who have taken large overdoses should have electrocardiogram monitoring performed, though the use of antidysrhythmic associated with prolongation of the QT interval should be avoided (class 1a agents such as quinidine, disopyramide, procainamide, and aprindine, and most class III agents such as N-acetyl procainamide and sotalol). Intravenous benzodiazepines and phenytoin have been used to treat seizures caused by antihistamine toxicity in humans. In the event of seizures or severe agitation, creatinine kinase should be tested routinely. If hypotension develops, isotonic fluids should also be administered (Borowy and Mukherji, 2019). Overdoses caused by antihistamines do not have a specific antidote. A patient experiencing delirium or tachycardia may require the administration of physostigmine if the anticholinergic effects of the antihistamine are causing severe anticholinergic symptoms which are not responsive to conventional treatment. In spite of this, because antihistamine overdoses are associated with a greater risk of seizures and wide-complex tachycardia, physostigmine is not routinely recommended (Manning, 2012). A number of contraindications are associated with physostigmine, including widened QRS and pulmonary diseases such as asthma (Borowy and Mukherji, 2019). Known anticholinergic drugs such as atropine, tricyclic antidepressants, and scopolamine should not be co-administrated due to their potential for antimuscarinic toxicity (Borowy and Mukherji, 2019).

Prognosis Ingestion of toxic antihistamines can have varying outcomes depending on the amount of drug consumed, the underlying medical condition, and any co-ingestants, but usually, the prognosis is good if the patient is promptly admitted to the hospital emergency department and monitored closely. A high dose can result in multiorgan failure in the elderly and children (Borowy and Mukherji, 2019). Those patients who do not develop clinical symptoms within 6 h of antihistamine overdose may be discharged (Thomas, 2016).

Patient education Even though these medications can be purchased over the counter, they can still cause significant adverse effects and toxicity. Healthcare providers should educate patients about the signs and symptoms of antihistamine overdose. Medication should be kept in a secure location at home, away from children. Furthermore, these agents should not be used in conjunction with sedatives or hypnotics. Also, before being discharged from the hospital, patients who have intentionally consumed these medications need to see a psychiatrist (Borowy and Mukherji, 2019).

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Antihistamine toxicity key facts

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Remember: “red as a beet, dry as a bone, hot as a hare, blind as a bat, mad as a hatter, and full as a flask.” In anticholinergic toxicity, Brugada-like ECG patterns can be seen. The antihistaminic effects peak 2 h after ingestion. The first sign and symptom may be severe sedation. Some common over-the-counter antihistamines cause false-negative results in urine drug screenings for amphetamines, methadone, and phencyclidine.

References Chlorphenamine maleate. Aronson JK (ed.) (2016) Meyler’s Side Effects of Drugs, 16th edn, p. 270. Oxford: Elsevier. doi: 1016/B1978-1010-1444-53717-53711.00481-53719. Borowy CS and Mukherji P (2019) Antihistamine Toxicity. [Updated 2021 March 12] StatPearls. Treasure Island, FL: StatPearls Publishing. Available from: https://www.ncbi.nlm.nih. gov/books/NBK482318/. Chiu C-C, Liu K-S, Chen Y-W, et al. (2020) Chlorpheniramine produces cutaneous analgesia in rats. Pharmacological Reports 72: 827–832. Farzam K, Sabir S, and O’Rourke MC (2019) Antihistamines. [Updated 2019 March 28] In: StatPearls. Treasure Island, FL: StatPearls Publishing. Available from: https://www.ncbi.nlm. nih.gov/books/NBK538188. Hasanein P (2009) Effects of chlorpheniramine and hydroxyzine administration, as histamine H1-receptor antagonists, on the nociception threshold of cholestatic rats. Hormozgan Medical Journal 13: 173–181. Kar S, Krishnan A, Preetha K, et al. (2012) A review of antihistamines used during pregnancy. The Journal of Pharmacy and Pharmacology 3: 105. Manning B (2012) Chapter 18: Antihistamines. In: Olson KR (ed.) Poisoning & Drug Overdose, 6th edn. New York, NY: The McGraw-Hill Companies. https://accessmedicine. mhmedical.com/Content.aspx?bookid¼391§ionid¼42069832 (Accessed April, 2022). Minigh J (2008) Chlorpheniramine. In: Enna SJ and Bylund DB (eds.) xPharm: The Comprehensive Pharmacology Reference, pp. 1–6. New York: Elsevier. Monczor F and Fernandez N (2016) Current knowledge and perspectives on histamine H1 and H2 receptor pharmacology: Functional selectivity, receptor crosstalk, and repositioning of classic histaminergic ligands. Molecular Pharmacology 90: 640–648. Monte AA, Chuang R, and Bodmer M (2010) Dextromethorphan, chlorphenamine and serotonin toxicity: Case report and systematic literature review. British Journal of Clinical Pharmacology 70: 794–798. Mousa JY (2009) Effect of chlorpheniramine on acute dichlorvos poisoning in chicks. Iraqi Journal of Veterinary Sciences 23: 35–43. Olasinska-Wisniewska A, Olasinski J, and Grajek S (2014) Cardiovascular safety of antihistamines. Advances in Dermatology and Allergology/Poste¸ py Dermatologii i Alergologii 31: 182. Owunari GU and Chika IJ (2021a) Acute toxicity profile of chlorpheniramine: Potential use as antidote to dichlorvos poisoning. GSC Biological and Pharmaceutical Sciences 14: 149–153. Owunari GU and Chika IJ (2021b) Effect of chlorpheniramine on acute dichlorvos poisoning in wistar rats. GSC Biological and Pharmaceutical Sciences 14: 154–160. Rollstin A and Seifert S (2013) Acetaminophen/diphenhydramine overdose in profound hypothermia. Clinical Toxicology 51: 50–53. Stefaniak AA, Pereira MP, Zeidler C, et al. (2022) Pruritus in pregnancy. American Journal of Clinical Dermatology 1–16. Tamadonfard E and Mojtahedein A (2005) Effect of chlorpheniramine on formalin-indnced pain in mice: Its relation with opioid system. Journal of Veterinary Research 60: 363–368. Thomas SH (2016) Antihistamine poisoning. Medicine 44: 141–142. Tzeng J-I, Lin H-T, Chen Y-W, et al. (2015) Chlorpheniramine produces spinal motor, proprioceptive and nociceptive blockades in rats. European Journal of Pharmacology 752: 55–60. Waller DG and Sampson AP (2018) 39: Antihistamines and allergic disease. In: Waller DG and Sampson AP (eds.) Medical Pharmacology and Therapeutics, 5th edn, pp. 451–456. Elsevier. 1016/B1978-1010-7020-7167-1016.00039-00037. Walsh GM (2012) 15: Antihistamines (H1 receptor antagonists). In: Aronson JK (ed.) Side Effects of Drugs Annual, pp. 271–276. Elsevier. 1016/B1978-1010-1444-5949959490.00015-59495.

Relevant websites http://www.nlm.nih.gov/medlineplus/druginfo/meds/a682543.html :MedlinePlus. http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid¼2725.

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Chlorpromazine Sophia Anagnostisa, Nimrat Khehrab, and Mayur S Parmarc, aDr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Fort Lauderdale, FL, United States; bSaint James School of Medicine, Arnos Vale, Saint Vincent and the Grenadines; c Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Clearwater, FL, United States © 2024 Elsevier Inc. All rights reserved. This is an update of R.D. Beckett, Chlorpromazine, Editor(s): Philip Wexler, Encyclopedia of Toxicology (Third Edition), Academic Press, 2014, Pages 925–929, ISBN 9780123864550, https://doi.org/10.1016/B978-0-12-386454-3.00284-0.

Chemical profile Background Uses Mechanism of toxicity Acute and short-term toxicity Animal Human Chronic toxicity Human Environmental fate and behavior Exposure and exposure monitoring Toxicokinetics Exposure standards and guidelines Immunotoxicology Reproductive toxicity and teratogenicity Genotoxicity Carcinogenicity Clinical management Ecotoxicology Other hazards Exposure standards and guidelines Conclusion References

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Abstract Chlorpromazine is a first-generation low-potency typical antipsychotic agent. It exerts its antipsychotic effect by blocking dopaminergic receptors (D1–D4 but especially D2) in mesolimbic and mesocortical pathways. It is approved for use in humans as an antipsychotic agent to manage psychotic disorders, such as mania, schizophrenia, and bipolar disorder. Miscellaneous other uses are in the treatment of intractable hiccup, nausea, vomiting, preoperative anxiety, and severe behavioral problems in children. It is also useful as a treatment of psychogenic pruritus. Chlorpromazine’s use is associated with major adverse effects, such as extrapyramidal side effects, orthostatic hypotension, sedation, and ocular complications. Chlorpromazine has been associated with a greater risk of seizures among first-generation antipsychotics. Long-term use of chlorpromazine has been associated with corneal deposits and hepatoxicity, which includes abnormal liver function tests (increased serum aminotransferase levels), acute cholestatic liver injury, and jaundice. Chlorpromazine can cross the blood-brain barrier. With the development of second-generation antipsychotics that have a lower side-effect profile, chlorpromazine is now rarely used. It is extensively metabolized in the liver and kidneys (by isozymes CYP2D6, CYP1A2 and CYP3A4), yielding to many metabolites. This drug chapter aims to illustrate the uses (FDA-approved and Off-label), acute and chronic toxicity, and clinical management of the adverse and toxic effects associated with chlorpromazine. It also highlights the exposure routes, toxicokinetics, immunotoxicity, reproductive toxicity, genotoxicity, carcinogenicity, and ecotoxicity associated with chlorpromazine. There is no known antidote for this compound, and poisoned patients should receive prompt supportive care.

Keywords Adverse effects; Bipolar disorder; Chlorpromazine; Chlorpromazine toxicity; Dopamine antagonist; Nausea and vomiting; Neuroleptic malignant syndrome; Psychotropic disorder; Schezophrenia

Key points

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Chlorpromazine is a first-generation typical antipsychotic agent (a phenothiazine) and blocks dopaminergic receptors, especially D2 receptors, in mesolimbic and mesocortical pathways. Chlorpromazine is FDA approved for use in humans to manage psychotic disorders, such as mania and schizophrenia. Also useful as an antiemetic and antipruritic.

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Major adverse effects of chlorpromazine are extrapyramidal reactions (such as acute dystonia, akathisia, parkinsonism, and tardive dyskinesia), orthostatic hypertension, sedation, and ocular complications. Chlorpromazine-associated risk of arrhythmias and sudden cardiac death is attributed to QT prolongation. Chlorpromazine can be administered: orally, deep intramuscular (IM) injection, intravenous (IV) injection, or IV infusions.

Chemical profile

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Name: Chlorpromazine Chemical Abstracts Service Registry Number: 50–53-3 Synonyms: 2-Chloro-10-[3-(dimethylamino)propyl]phenothiazine; Chlorpromazine hydrochloride; CPZ; Thorazine Molecular Formula: C17H19ClN2S Chemical Structure:

Background First-generation antipsychotics were the most prevalently administered class of medications until the 1990s for treating and managing schizophrenia (Ban, 2007). In 1957, chlorpromazine, a typical first-generation antipsychotic, was approved by the US Food and Drug Administration (FDA). Chlorpromazine is also administered to treat and manage bipolar disorder, acute psychosis, and other medical conditions. In the 1990s, atypical second-generation antipsychotics such as olanzapine, clozapine, quetiapine, and risperidone gained significant popularity due to their decreased risk for extrapyramidal adverse effects (Gajwani et al., 2006).

Uses Chlorpromazine is FDA-approved for use in humans for the treatment and management of psychotic disorders such as schizophrenia and bipolar I acute manic type of manic-depressive illness. It is also useful in treating nausea/vomiting (migraine and intraoperative related), serotonin syndrome (as adjunctive therapy), tetanus (as adjunctive therapy), combativeness or explosive hyperexcitability behavior (acute agitation in children aged 1–12 years old), and short-term treatment of hyperactivity in children (Kohse et al., 2017; Valdovinos et al., 2020). Treatment of bipolar disorder with chlorpromazine was administered to control symptoms associated with manic episodes, such as abnormally elevated energy, decreased need for sleep, impulsive behavior, excitable behavior, and grandiose beliefs (McElroy and Keck, 2000). Chlorpromazine is also FDA-approved for treating persistent hiccups (singultus) lasting 48 h or greater (Kohse et al., 2017). Chlorpromazine’s off-label use includes the management of agitation in terminally ill cancer patients, cancer-associated pain, autonomic dysreflexia, cholera (as adjunctive therapy), opioid withdrawal, ocular pain, paralytic ileus, and phantom limb syndrome. It is also helpful for treating agitation and delirium in hospitalized patients without underlying psychiatric illnesses.

Mechanism of toxicity Acute and chronic toxicity due to chlorpromazine generally manifests as an extension of its normal pharmacological activity. The mechanism of action of chlorpromazine primarily involves the antagonism of dopaminergic (D2) neurotransmission at synaptic sites and blockade of postsynaptic dopamine (D2) receptor sites at the subcortical levels of the reticular formation, limbic system,

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and hypothalamus (Li et al., 2016). High occupancy of D2 receptor sites likely leads to adverse effects such as extrapyramidal symptoms (EPS), hyperprolactinemia, and cognitive dulling (Li et al., 2016). Chlorpromazine also has robust central and peripheral activity directed against adrenergic receptors and weak activity against histaminic (H1) and muscarinic receptors (Johnson et al., 2005). While chlorpromazine has serotoninergic receptor inhibition in vitro, it loses this inhibitory action after it has been metabolized in vivo (Suzuki et al., 2013). Chlorpromazine is known to depress vasomotor reflexes mediated by the hypothalamus and/or brain stem, inhibit the release of growth hormone, antagonize secretion of prolactin release-inhibiting hormone, and reduce the secretion of corticotropin-regulatory hormone (Hino et al., 1986). Chlorpromazine causes significant prolongation of the QTc interval by inducing early after-depolarizations and blocking the depolarizing sodium channels in cardiac myocytes. Metabolism of chlorpromazine occurs via the CYP3A4 substrate: CYP450 enzymes A12 and 2D6. Metabolism occurs via the gastrointestinal system (GI) and the kidneys, resulting in excretion in urine, bile, and feces. Chlorpromazine may be irritating to the eyes, mucous membranes, and skin. Contact and inhalation should be avoided.

Acute and short-term toxicity Animal Single and repeated exposures to oral chlorpromazine have been conducted in several species, including cats, dogs, horses, and rats, at doses ranging from 2 to 30 mg kg−1 day−1. The primary effects of chlorpromazine were cardiac arrhythmia, decreased activity, stimulation of hepatic microsomal enzyme activity, hormonal changes, hypotension, impaired motor activity, ocular lesions, photosensitization, tachycardia, reduced bile flow, reduced red blood cell count and hemoglobin. The oral 50% lethal dose (LD50) for rats is 210 mg kg−1, and the IV LD50 for rats is 23 mg kg−1 (Irwin et al., 1959). When administered parenterally at doses ranging from 1.5 to 100 mg kg−1 in dogs, mice, and rats, chlorpromazine was associated with ataxia, CNS depression, decreased hemoglobin, hypotension, and stimulation of hepatic microsomal enzyme activity. When administered to pregnant rats, chlorpromazine IV doses ranging from 5 to 45 mg kg−1 day−1 were associated with delays in bone ossification, decreased body weight, increased fetal and maternal mortality, and skeletal malformations at higher doses (Kunimatsu et al., 2010).

Human Chlorpromazine has the propensity to cause non-neurologic adverse effects due to its low-potency nature. Chlorpromazine is stored in body fats due to its high lipid solubility resulting in slower drug elimination. Antimuscarinic adverse effects have been noted in patients administered chlorpromazine and exhibit symptoms such as dry mouth, blurred vision, constipation, dizziness, and urinary retention. Antihistamine (H1) effects, such as sedation, may be noted in patients administered chlorpromazine. Additionally, there is a significantly increased risk of exhibiting angle closure glaucoma in the elderly (Solmi et al., 2017). Endocrine adverse effects such as hyperprolactinemia can occur in both males and females due to chlorpromazine’s blockade of D2 receptors in the tuberoinfundibular pathway (Li et al., 2016). Males may exhibit erectile dysfunction, gynecomastia, and galactorrhea in the setting of hyperprolactinemia. In rare instances, priapism can also occur. Females may exhibit irregular menstruation, oligomenorrhea, amenorrhea, and galactorrhea. Intramuscular or intravenous administration of chlorpromazine may cause hypotension and headache. Patients with hypersensitivity and allergies to phenothiazines should not be administered chlorpromazine. Patients taking antihypertensive medications should be administered chlorpromazine with caution due to the increased risk of severe hypotension (Gugger, 2011). Concurrent administration of central nervous system (CNS) depressing drugs is an absolute contraindication as chlorpromazine prolongs and intensifies the action of CNS depressants (Landoni and Martin, 2022). Additionally, chlorpromazine is not recommended for patients with a past medical history of poorly controlled seizure disorders (Hedges et al., 2003). Chlorpromazine has not been approved for its use in dementia-related psychosis (Yunusa et al., 2021). It is reported to increase mortality in elderly patients with dementia-related psychosis (US Boxed Warning). Since chlorpromazine is a D2 blocker, it may interfere with the effectiveness of dopamine agonist drugs such as levodopa and cabergoline. Antidepressants and selective serotonin reuptake inhibitors (SSRIs), such as citalopram and escitalopram, are absolute contraindications. Clinicians administering chlorpromazine in pregnant women should assess the risk and benefits; extra caution and medical supervision are recommended, especially during the third trimester (Einarson and Boskovic, 2009). It is recommended physicians and patients follow a risk-benefit analysis when determining treatment with low-dose chlorpromazine in breastfeeding mothers (Klinger et al., 2013). Acute toxicity and/or overdosage of phenothiazines, such as chlorpromazine, is expected to present as an extension of normal adverse effects observed at therapeutic doses. The most commonly reported manifestations of acute toxicity are severe extrapyramidal reactions, hypotension, and sedation. Patients with early or mild intoxication may present with confusion, disorientation, drowsiness, excitement, or restlessness. Patients in the late stage of toxicity may present with CNS stimulation and convulsions followed by respiratory and/or CNS depression (Landoni and Martin, 2022). Other signs of toxicity include cardiovascular shock, cardiovascular conduction abnormalities, dysrhythmias, agitation, anticholinergic effects, alterations in body temperature, vomiting, difficulty breathing, and pulmonary edema.

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The severity of toxic effects is dose-related. For phenothiazines, doses at least 10 times the defined daily dose are considered potentially lethal. Regarding chlorpromazine, this value is approximately 3000 mg day−1. Due to the varied, dose-related nature of chlorpromazine toxicity, typical therapeutic blood concentrations (i.e., 0.02–3.0 mg l−1) overlap with reported lethal blood concentrations (i.e., 1–44 mg l−1). Fatalities have been reported in children, youths, adults, and elderly patients following the ingestion of chlorpromazine (Cancro and Wilder, 1970; Dorson and Crismon, 1988; Landoni and Martin, 2022). Common mechanisms of sudden death reports include cardiovascular events (Cancro and Wilder, 1970; Ye et al., 2018; Landoni and Martin, 2022).

Chronic toxicity Human Due to the potential for adverse reactions associated with long-term therapy, patients with a history of long-term treatment of any antipsychotic should be periodically reevaluated to determine whether continued therapy is warranted. The following additional syndromes may occur at normal or toxic doses of chlorpromazine. Up to 60% of patients receiving antipsychotics, including chlorpromazine, experience extrapyramidal reactions such as dystonic reactions and feelings of motor restlessness. Parkinsonian manifestations such as hypersalivation, tremors, shuffling gait, slowed speech, dysphagia, akinesia, or bradykinesia may also occur due to chlorpromazine toxicity. Patients may also experience tardive dyskinesia as potentially irreversible, involuntary, dyskinetic movements. Tardive dyskinesia may partially or completely remit if the causative agent is discontinued; however, there is no established, reliable treatment. Chlorpromazine toxicity can cause neuroleptic malignant syndrome (NMS) (Berman, 2011), manifesting as hyperpyrexia, muscle rigidity, altered mental status, and autonomic instability (i.e., cardiac dysrhythmias, diaphoresis, irregular heart rate or blood pressure, and tachycardia). Patients receiving antipsychotics may experience an encephalopathic syndrome manifesting as confusion, extrapyramidal symptoms, fever, and lethargy. Additional potential adverse reactions associated with chlorpromazine include anticholinergic adverse effects, cardiac arrest, cerebral edema, convulsive seizures, drowsiness, fever following large IM doses, jaundice, hematological disorders including agranulocytosis, lactation, peripheral edema, postural hypotension, psychotic symptoms, systemic lupus erythematosus-like syndrome, and tachycardia. The following adverse reactions are particularly related to long-term therapy: skin hyperpigmentation, deposition of particulate matter in the lens and cornea, subsequent development of opacities, and the development of multifocal tics and vocalizations following long-term treatment. In a case study following 7 years of chlorpromazine treatment, the development of blue-gray pigmentation of the skin and corneal and lens opacities was observed in a psychiatric patient. However, 10 months after discontinuation of chlorpromazine, partial improvement was observed in the skin discoloration and anterior lens deposits. No change was observed in the corneal deposits post-discontinuation (Huff et al., 2014). Chlorpromazine may also cause cholestatic jaundice due to impaired flow of bile (LiverTox, 2012). Chlorpromazine may also cause inflammation and injury of the liver resulting in drug-induced hepatotoxicity (Björnsson, 2016). To prevent the occurrence of hepatotoxicity, routine monitoring of liver function tests (LFTs) is essential. In the event of hepatotoxicity, cessation of chlorpromazine is required with subsequent initiation of symptomatic management.

Environmental fate and behavior Chlorpromazine occurs as a white, crystalline solid that is nearly water-insoluble but freely soluble in alcohol. The melting point of chlorpromazine is approximately 60  C. Chlorpromazine is commercially available as a hydrochloride salt, a white, crystalline powder that is soluble in water (1 g ml−1) and alcohol (667 mg ml−1) at 25  C. The commercially available product has a pH of 3–5. Additionally, it may contain inactive ingredients such as benzyl alcohol, sulfites, and others. Chlorpromazine is photosensitive. Therefore, it should be protected from light, moisture, and temperatures outside of 20–25  C. Chlorpromazine has the propensity to adsorb to plastic, such as tubing significantly. Thus, it should be avoided clinically. In ambient atmospheric conditions, chlorpromazine exists as a vapor and particulate. Photochemically produced hydroxyl radicals degrade chlorpromazine vapor with an estimated half-life of 1.6 h. Chlorpromazine particulates are removed by wet or dry deposition. Chlorpromazine is likely to be immobile in soil (Koc 9900, pKa 9.3); also, chlorpromazine adsorbs sediment if released into the water. It is not expected to volatilize from soil or water. Lastly, there is a high potential for bioconcentration.

Exposure and exposure monitoring Chlorpromazine can be administered in several ways: orally, deep intramuscular (IM) injection, intravenous (IV) injection, or IV infusion. Importantly, chlorpromazine is not administered subcutaneously due to the risk of dermal irritation. Exposure to

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chlorpromazine can occur through dermal or inhalational contact, especially among pharmacists, physicians, and nurses dispensing or administering the drug. Although not a common clinical practice, therapeutic drug concentrations can be monitored. Among patients with schizophrenia, there is significant variation in the plasma level in the occurrence of clinical response.

Toxicokinetics Absorption: Chlorpromazine is rapidly absorbed orally and from parenteral injection sites, with peak plasma levels occurring at 2–4 h after oral administration and 15–30 min after IM injection. Following oral administration, pharmacological action occurs within 30–60 min; IM injection has an onset time of 15 min. Due to significant first-pass hepatic metabolism and intestinal wall metabolism that vary among individual patients, interindividual oral absorption is erratic. Drug information references cite 20–32% bioavailability; absorption following IM administration is expected to be 10-fold higher. Distribution: Chlorpromazine distributes to most fluids and body tissues with a volume of distribution ranging from 8 to 160 L kg−1. Chlorpromazine efficiently crosses the blood-brain barrier. Thus, concentrations in the central nervous system (CNS) are five times higher than in plasma. Chlorpromazine binds significantly to protein, mainly albumin; the plasma protein binding rate of 90–99% protein binding in the CNS ranges from 19% to 72%. After administering intramuscular doses of chlorpromazine to pregnant women before delivery, chlorpromazine and its metabolites were notably distributed to fetal plasma, amniotic fluid, and neonatal urine. Rapid placental transfer, with fetal levels approaching 50% of maternal values within 10 min, has also been observed in animal studies. Chlorpromazine is also distributed to breast milk of mothers during therapy. These levels are found not to correlate well with maternal dose or serum level (LactMed, 2006). Metabolism: Chlorpromazine undergoes extensive hepatic metabolism to 10–12 metabolites, both active and inactive. The N-dimethylaminopropyl side chain of chlorpromazine is demethylated and metabolized to an N-oxide while positions 3 and 7 of the phenothiazine nucleus undergo hydroxylation. The quantitatively important metabolites of chlorpromazine are nor2-chlorpromazine, chlorophenothiazine, methoxy and hydroxyl products, and glucuronide conjugates of hydroxylated compounds. Several of these metabolites are active, including nor2-chlorpromazine, nor2-chlorpromazine sulfoxide, and 3-hydroxy chlorpromazine. Chlorpromazine is known to be metabolized into at least 20 different metabolites through multiple metabolic pathways, including hydroxylation, demethylation, and sulfation (Usdin, 1971). Elimination: Less than 1% of a chlorpromazine dose is excreted in the urine as an unchanged drug; most elimination of a single 120 mg m−2 dose occurred within 6 h. Following treatment, chlorpromazine and its metabolites may be detected up to 6–18 months. 23–37% of a chlorpromazine dose is excreted as an unchanged drug or metabolite. The two major metabolites of chlorpromazine found in the urine are N-dedimethylchlorpromazine and 7-hydroxychlorpromazine. 20% of the chlorpromazine dose excreted in the urine consists of an unconjugated unchanged drug, demonomethylchlorpromazine, dedimethylchlorpromazine, sulfoxide metabolites, and chlorpromazine-N-oxide. 80% of the chlorpromazine dose excreted in the urine consists of conjugated O-glucuronides and ethereal sulfates of mono- and dihydroxychlorpromazine derivatives. The half-life of chlorpromazine is cited as 6 h; however, values ranging from 2 to 119 h have been reported. Elimination of chlorpromazine occurs faster in children than in adults. Chlorpromazine is also eliminated in the feces. Chlorpromazine is not dialyzable.

Exposure standards and guidelines Chlorpromazine can be administered orally, intramuscularly, or intravenously. Oral chlorpromazine HCl is available in 10 mg, 25 mg, 50 mg, 100 mg, and 200 mg dosages. Clinically, doses should be titrated to patient response using the lowest dose possible. For most adult psychotic patients, oral doses of 500 mg day−1 are sufficient for chronic treatment. However, the maximum recommended total daily IM dose during acute psychosis is 2400 mg. For pediatric patients (at least 6 months of age) exhibiting psychosis, the maximum recommended oral dose for chronic treatment is 0.55 mg kg−1 every 4 h. During acute episodes, IM doses up to 0.55 mg kg−1 every 6 h may be administered. The maximum dose for patients younger than 5 years of age or less than 22.7 kg is 40 mg IM day−1; the maximum dose for patients aged 5–12 years and weighing 22.7–45.5 kg is 75 mg IM day−1. There are no occupational exposure standards for chlorpromazine. Initial treatment of schizophrenia with chlorpromazine is initiated with 25–75 mg day−1 orally twice a day, and subsequent maintenance therapy requires a dosage of 200 mg day−1; the maximum oral dose is 800 mg day−1. Intravenous or intramuscular dosages are initiated at 25 mg. Following IM/IV initiation, intake is altered to an as needed basis to 25–50 mg after 1 to 4 h. The typical dose ranges from 300 to 800 mg day−1. To treat nausea and vomiting, oral chlorpromazine dose ranges from 10 to 25 mg every 4–6 h as needed. Persistent hiccups (singultus) can be treated orally with the administration of dosages ranging from 25 to 50 mg every 6–8 h of chlorpromazine. If symptoms persist for 2–3 days following oral administration of chlorpromazine, parenteral therapy is indicated. Regarding preoperative apprehension, oral chlorpromazine doses range from 25 to 50 mg, 2 to 3 h before the operation. Despite chlorpromazine metabolism via kidneys, renal dose adjustments are not required. However, it is important to proceed with caution in hepatically impaired patients.

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Immunotoxicology Chlorpromazine is contraindicated for patients with hypersensitivity to any phenothiazine due to the potential for crossover allergic reactions. Additionally, the parenteral product may cause anaphylaxis, life-threatening asthmatic episodes, or less severe asthmatic episodes in susceptible patients due to sulfites present in the formulation. Asthmatics are at heightened risk for this effect. Chlorpromazine hydrochloride injection may cause contact dermatitis, and direct contact with the skin should be avoided. In rare, reported cases, this can progress to persistent photosensitivity.

Reproductive toxicity and teratogenicity Chlorpromazine is pregnancy category C. Rigorous animal studies have not been performed to evaluate the safety of chlorpromazine during pregnancy or breastfeeding. Two previously conducted observational studies observed the relationship between chlorpromazine and perinatal mortality, defects, or malformations. However, studies did not reveal a significant correlation between chlorpromazine exposure and fetal mortality (Iqbal et al., 2005). A four-year follow-up study in 14 children exposed to chlorpromazine (50 to 150 mg) daily throughout the gestation found no congenital anomalies or behavioral or emotional problems (Kris and Carmichael, 1957). Reported congenital malformations attributed to chlorpromazine include syndactyly, microcephaly, clubfoot/hand, muscular abdominal aplasia, endocardial fibroelastosis, and brachymesophalangy. Microcephaly due to phenothiazines has also been reported independent of the aforementioned observational studies. During the third trimester of pregnancy, exposure to any phenothiazine is associated with increased neonatal risk for extrapyramidal reactions and/or withdrawal symptoms. Symptoms include agitation, feeding disorder, hypertonia, hypotonia, respiratory distress, somnolence, tardive dyskinetic-like symptoms, and tremors. It is important to note that in many reported cases of neonatal phenothiazine withdrawal, the mother was receiving multiple medications; however, some cases suggested risk with chlorpromazine alone. Overall, most studies showed chlorpromazine is not teratogenic and should be used cautiously during pregnancy when its benefits outweigh the potential risks involved (Iqbal et al., 2005). In an animal study, oral chlorpromazine was associated with ovarian toxicity and reduced fertility at doses of at least 10 mg kg−1 (Zamani et al., 2015). In male rats, testicular dysfunction, which includes the deregulation of reproductive hormones, is observed with chlorpromazine treatment (Oyovwi et al., 2021). Inhibition of testicular dehydrogenase enzymes, transmembrane ionic pumps, and sperm capacitation was noted in the animal with chlorpromazine treatment (Oyovwi et al., 2021).

Genotoxicity In animal studies, certain phenothiazines have been associated with chromosomal aberrations in spermatocytes and abnormal sperm. In an in vitro study utilizing a combination of the single-cell gel electrophoresis (COMET) assay, the photomutagenicity of chlorpromazine and its N-demethylated metabolites were tested in human skin fibroblast cell lines (Agúndez et al., 2020). The study results showed that upon UVA radiation exposure, chlorpromazine photosensitized DNA damage, and its metabolites demethylchlorpromazine (DMCPZ) and didemethylchlorpromazine (DDMCPZ) promoted extensive DNA-photodamage. The next-generation sequencing showed similar results with minor discrepancies for DDMCPZ (Agúndez et al., 2020).

Carcinogenicity Animal studies have shown an increased risk for the development of mammary neoplasms following long-term administration of prolactin-stimulating antipsychotics. Phenothiazines, including chlorpromazine, should be used with caution in patients with a history of breast cancer due to this theoretical risk. Current evidence is inconclusive as to the absolute risk associated with this issue.

Clinical management The recommended treatment for chlorpromazine overdose, and overdose of phenothiazines in general, is supportive and symptomatic care. In the event of suspected chlorpromazine toxicity, patients should be immediately transferred to a healthcare facility. Careful patient history is recommended to characterize exposure to chlorpromazine and other neuroleptics. Concurrent ingestion of TCAs should be ruled out. Following the acquisition of precise patient medical history and subsequent suspected chlorpromazine toxicity, termination of chlorpromazine is recommended. An electrocardiogram (ECG), electrolytes, and arterial blood gases should be measured on admission. Continuous or repeat ECG is often necessary to rule out dysrhythmia development. If an ECG is abnormal, monitoring should continue for at least 24 h. Regular measurement of electrolytes, particularly potassium and magnesium, is intended to identify additional dysrhythmia risk. Arterial blood gases help rule out the development of respiratory acidosis, which can also increase the risk of dysrhythmia. Plasma chlorpromazine concentrations are recommended in the clinical management of suspected overdose. Abnormalities in

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electrolytes and acid-base balance should be corrected. Gastrointestinal decontamination with activated charcoal may be used for patients who present within 1 h of ingesting a potentially lethal dose. Repeat use of activated charcoal may enhance elimination; however, continued use increases the risk for paralytic ileus. Additionally, gastric lavage may be used. Since phenothiazines can decrease gastrointestinal motility, gastric lavage may be effective up to several hours following ingestion. Forced emesis, cathartics, and whole bowel irrigation are not recommended due to lack of benefit and increased risk for decreased consciousness, aspiration, seizures, dystonia, and paralytic ileus. The development of extrapyramidal reactions due to chlorpromazine overdose warrants the use of anticholinergic antiparkinsonian drugs. The preferred agent for acute treatment is benztropine 1–2 mg IV or IM. For persistent dystonias, oral benztropine 2–8 mg day−1 in divided doses or trihexyphenidyl 5–15 mg day−1 in divided doses can be administered. Limited data is available regarding the preferred treatment for chlorpromazine- or phenothiazine-induced dysrhythmias; however, sodium bicarbonate has been suggested due to its effectiveness in similar cardiac conduction defects in TCA toxicity. If a patient develops ventricular or supraventricular dysrhythmia, heart block, or a QRS interval wider than 120 ms, sodium bicarbonate may be given at doses of 1–3 mEq kg−1 IV bolus administered at 3 to 5-min intervals to a maximum blood pH of 7.55. Depending on the clinical scenario, depending on lidocaine, phenytoin, isoproterenol, ventricular pacing, and defibrillation may also be warranted for the second-line treatment. If sodium bicarbonate is ineffective in reducing a case of torsades de pointes, magnesium sulfate may be used. Medications that prolong the QTc interval (e.g., procainamide, quinidine) should be avoided due to the risk of dysrhythmia development during concomitant use with chlorpromazine. If patients develop hypotension, IV fluids and pressors should be administered to maintain perfusion. Norepinephrine or phenylephrine are preferred pressors, as chlorpromazine reverses the effects of epinephrine and dopamine. Stimulants (i.e., amphetamine, dextroamphetamine, caffeine with sodium benzoate) that do not increase seizure threshold may be used for patients with severe CNS depression. Benzodiazepines (e.g., Diazepam 5–20 mg IV) are preferred for treating seizures. Barbiturates should be relegated to second-line treatment due to their potential to induce respiratory depression. Development of NMS will require management in an intensive care setting with similar supportive care as described. Additionally, oral bromocriptine 2.5–10 mg three to four times daily or dantrolene 1–3 mg kg−1 day−1 IV in four divided doses is recommended for the treatment of severe cases and consideration for non-severe cases. Concomitant development of acute renal failure should be addressed per normal guidelines. Following several weeks of termination of chlorpromazine, low-dose chlorpromazine can be initiated again under medical supervision.

Ecotoxicology Chlorpromazine may be released into the environment through its production and clinical use; its theoretical risk for bioconcentration in fish is high, assuming the organism does not metabolize the drug.

Other hazards When chlorpromazine is heated to decomposition, fumes of hydrogen chloride, nitroxides, and sulfoxides are emitted.

Exposure standards and guidelines There are no occupational exposure standards for chlorpromazine. Clinically, doses should be titrated to patient response using the lowest dose possible. For most adult psychotic patients, oral doses of 500 mg day−1 are sufficient for chronic treatment; however, the maximum recommended total daily IM dose during acute psychosis is 2400 mg. For psychotic pediatrics at least 6 months of age, the maximum recommended oral dose for chronic treatment is 0.55 mg kg−1 every 4 h. During acute episodes, IM doses up to 0.55 mg kg−1 every 6 h may be used. The maximum dose for patients younger than 5 years of age or less than 22.7 kg is 40 mg IM day−1; the maximum dose for patients aged 5–12 years and weighing 22.7–45.5 kg is 75 mg IM day−1.

Conclusion Chlorpromazine is a first-generation low-potency typical antipsychotic agent that blocks dopaminergic receptors, especially D2 receptors, in mesolimbic and mesocortical pathways. It is approved to manage and treat schizophrenia, bipolar disorder, and acute psychosis. Other therapeutic uses are in the treatment of intractable hiccups, nausea, vomiting, preoperative anxiety, and severe behavioral problems in children. It is also useful to treat psychogenic pruritus. Chlorpromazine is metabolized in the liver and kidneys by CYP2D6 (major pathway), CYP1A2 and CYP3A4 yielding several metabolites. Less than 40% of the administered dose of chlorpromazine is excreted in the urine. Toxic signs and symptoms may include agitation, coma, convulsions, difficulty breathing & swallowing, dry mouth, extreme sleepiness, fever, intestinal blockage, irregular heart rate, low blood pressure, and restlessness. There is no known antidote for this compound, and poisoned patients should receive supportive medical care.

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Journal of Psychiatric Practice 15(3): 183–192. Gajwani P, Kemp DE, Muzina DJ, Xia G, Gao K, and Calabrese JR (2006) Acute treatment of mania: an update on new medications. Current Psychiatry Reports 8(6): 504–509. Gugger JJ (2011) Antipsychotic pharmacotherapy and orthostatic hypotension: identification and management. CNS Drugs 25(8): 659–671. Hedges D, Jeppson K, and Whitehead P (2003) Antipsychotic medication and seizures: a review. Drugs Today (Barc) 39(7): 551–557. Hino M, Ono H, and Fukuda H (1986) Brain stem involvement in the effects of chlorpromazine on the monosynaptic reflex of the rat lumbar spinal cord. General Pharmacology 17(4): 379–383. Huff LS, Prado R, Pederson JF, Dunnick CA, and Lucas LM (2014) Chlorpromazine-induced skin pigmentation with corneal and lens opacities. Cutis 93(5): 247–250. Iqbal MM, Aneja A, Rahman A, Megna J, Freemont W, Shiplo M, Nihilani N, and Lee K (2005) The potential risks of commonly prescribed antipsychotics: during pregnancy and lactation. Psychiatry (Edgmont) 2(8): 36–44. Irwin S, Slabok M, Debiase PL, and Govier WM (1959) Perphenazine (trilafon), a new potent tranquilzer and antiemetic. I. Behavioral mode of action. Archives Internationales de Pharmacodynamie et de Thérapie 118(3–4): 358–374. Johnson DE, Nedza FM, Spracklin DK, et al. (2005) The role of muscarinic receptor antagonism in antipsychotic-induced hippocampal acetylcholine release. European Journal of Pharmacology 506(3): 209–219. Klinger G, Stahl B, Fusar-Poli P, and Merlob P (2013) Antipsychotic drugs and breastfeeding. Pediatric Endocrinology Reviews 10(3): 308–317. Kohse EK, Hollmann MW, Bardenheuer HJ, and Kessler J (2017) Chronic hiccups: An underestimated problem. Anesthesia and Analgesia 125(4): 1169–1183. Kris EB and Carmichael DM (1957) Chlorpromazine maintenance therapy during pregnancy and confinement. The Psychiatric Quarterly 31(4): 690–695. Kunimatsu T, Kimura J, Funabashi H, Seki T, et al. (2010) The antipsychotics haloperidol and chlorpromazine increase bone metabolism and induce osteopenia in female rats. Regulatory Toxicology and Pharmacology 58(3): 360–368. LactMed (2006) Drugs and Lactation Database (LactMed). National Library of Medicine. Landoni JHD and Martin JDS (2022) Chlorpromazine (PIM 125). Internationally Peer Reviewed Chemical Safety Information, International Programme on Chemical Safety (IPCS). WHO. Li P, Snyder GL, and Vanover KE (2016) Dopamine Targeting Drugs for the Treatment of Schizophrenia: Past, Present and Future. Current Topics in Medicinal Chemistry 16(29): 3385–3403. LiverTox (2012) LiverTox: Clinical and Research Information on Drug-Induced Liver Injury. McElroy SL and Keck PE (2000) Pharmacologic agents for the treatment of acute bipolar mania. Biological Psychiatry 48(6): 539–557. Oyovwi MO, Nwangwa EK, Ben-Azu B, Rotue RA, et al. (2021) Prevention and reversal of chlorpromazine induced testicular dysfunction in rats by synergistic testicle-active flavonoids, taurine and coenzyme-10. Reproductive Toxicology 101: 50–62. Solmi M, Murru A, Pacchiarotti I, et al. (2017) Safety, tolerability, and risks associated with first- and second-generation antipsychotics: A state-of-the-art clinical review. Therapeutics and Clinical Risk Management 13: 757–777. Suzuki H, Gen K, and Inoue Y (2013) Comparison of the anti-dopamine D₂ and anti-serotonin 5-HT(2A) activities of chlorpromazine, bromperidol, haloperidol and second-generation antipsychotics parent compounds and metabolites thereof. Journal of Psychopharmacology 27(4): 396–400. Usdin E (1971) The assay of chlorpromazine and metabolites in blood, urine, and other tissues. Critical Reviews in Clinical Laboratory Sciences 2(3): 347–391. Valdovinos EM, Frazee BW, Hailozian C, Haro DA, and Herring AA (2020) A nonopioid, nonbenzodiazepine treatment approach for intractable nausea and vomiting in the emergency department. Journal of Clinical Gastroenterology 54(4): 327–332. Ye X, Shi C, Shen YW, Zhao ZQ, Jiang Y, and Li LL (2018) Forensic analysis of 24 cases of long-term antipsychotics use-induced sudden unexpected deaths. Fa Yi Xue Za Zhi 34(6): 644–647. Yunusa I, Rashid N, Abler V, and Rajagopalan K (2021) Comparative efficacy, safety, tolerability, and effectiveness of antipsychotics in the treatment of dementia-related psychosis (DRP): A systematic literature review. The Journal of Prevention of Alzheimer’s Disease 8(4): 520–533. Zamani ZS, Sadrkhanlou ZR, Ahmadi A, and Movahed E (2015) The effects of chlorpromazine on reproductive system and function in female rats. International Journal of Fertility and Sterility 9(3): 371–379.

Relevant websites https://pubchem.ncbi.nlm.nih.gov/compound/Chlorpromazine :PubChem https://go.drugbank.com/drugs/DB00477 :Drug bank

ENCYCLOPEDIA OF TOXICOLOGY

DEDICATION Dedicated to the remediation of planet earth’s largely human-generated centuries of environmental degradation and its resultant climate crisis, and also to the toxicologists who, via scientific research, application, and communication play a critical role in helping ameliorate and forestall further damage to the natural world and to the health of populations and individuals. Equally dedicated to the elimination of strife, injustice, conflict, and divisiveness among the world’s citizens and to the brave people and movements striving to create a peaceful and lawful realm where freedom is presumed, diversity is valued, and equal opportunity is affirmed. And to my mom, Yetty, who celebrated her 95th birthday in 2023.

ENCYCLOPEDIA OF TOXICOLOGY FOURTH EDITION

EDITOR IN CHIEF Philip Wexler Independent Toxicology Information Specialist and U.S. National Library of Medicine (retired)

VOLUME 3

ASSOCIATE EDITORS Mohammad Abdollahi Tehran University of Medical Sciences (TUMS), Tehran, Iran

Shayne Gad Gad Consulting Services, Raleigh, NC, USA

Helmut Greim Technical University of Munich, Freising-Weihenstephan, Germany

Mary Gulumian North West University, Water Research Unit, South Africa

Evangelia I. Iatrou Laboratory of Toxicology and Forensic Chemistry, Medical School, University of Crete, Heraklion, Crete, Greece

Diana Miguez Latitud - LATU Foundation, Technological Laboratory of Uruguay (LATU), Montevideo, Uruguay

Asish Mohapatra Health Risk Assessment and Toxicology Specialist, Environmental Health Program, Health Canada, Calgary, Alberta, Canada

Sidhartha D. Ray Department of Pharmaceutical & Biomedical Sciences, Touro University College of Pharmacy, NY, USA

Jose Tarazona European Food Safety Authority, Parma, Italy and Spanish National Environmental Health Centre (CNSA)Instituto de Salud Carlos III. Ministry of Science and Innovation. Madrid, Spain

Aristidis Tsatsakis Laboratory of Toxicology and Forensic Chemistry, Medical School, University of Crete, Heraklion, Crete, Greece

Timothy Wiegand University of Rochester Medical Center and Strong Memorial Hospital, Rochester, NY, USA

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Publisher: Oliver Walter Acquisitions Editors: Clodagh Holland-Borosh and Blerina Osmanaj Content Project Managers: Pamela Sadhukhan and Greetal Carolyn Associate Content Project Managers: Nandhini Mahendran and N. Kiruthigadevi Designer: Miles Hitchen

CONTENTS OF VOLUME 3 List of Contributors for Volume 3

xiii

Editor Biographies

xxiii

Foreword

xxix

Preface

xxxi

Chlorpyrifos

1

Shelley DuTeaux and Svetlana E Koshlukova

Chlorsulfuron

15

Sarina Paridehpour and Zahra Bayrami

Chlorzoxazone

23

Kashyap N Thakore

Choline

27

Effat Davoudi-Monfared

Cholinergics

31

Michael Liu

Cholinesterase inhibition

35

BN Szafran, GA Casillas, and BL Alman

Chromium

53

Shayne C Gad

Chromosome aberrations

59

E Renieri, E Vakonaki, and P Fragkiadaki

Chrysene

65

Adrienne T Black

Ciguatoxin

73

Michael A Darracq

Ciprofloxacin

83

Whitney Prochownik and Madan K Kharel

Circadian clock effects/chronotoxicology

89

Shayne C Gad

Cisplatin

95

Pollobi Akther, Azhar Hussain, and Sidhartha D Ray

Clean Air Act (CAA), US

105

Robert W Kapp Jr

Clean Water Act (CWA), US

113

Robert W Kapp Jr.

v

vi

Contents of Volume 3

Clinical chemistry

121

Shayne C Gad

Clofibrate

123

Satabdi Ray and Sidhartha D Ray

CN gas

131

Asieh Karimani, Bruno Mégarbane, Kaveh Tabrizian, Mahmoud Hashemzaei, and Ramin Rezaee

Coal tar

135

Lenny Kamelia and Linda G Roberts

Cobalt

145

Shayne C Gad

Cocaine

149

M Siegrist and Timothy J Wiegand

Coke oven emissions

157

Adrienne T Black

Colchicine

165

Swetaleena Dash

Combustion toxicology

169

Shayne C Gad

Comet assay

183

Solange Costa, Joana Pires, and Armanda Teixeira-Gomes

Common mechanisms of toxicity in pesticides

191

Antonio F Hernández

Comparative regulatory testing requirements

205

Taraneh Mousavi and Mohammad Abdollahi

Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA); Revised as the Superfund Amendments Reauthorization Act (SARA); Superfund, US

213

Robert W Kapp Jr

CompTox Chemicals Dashboard

221

Coniine

225

Ifra Rehman and Sidhartha D Ray

Consumer Product Safety Commission (CPSC)

231

Madiha Khalid and Mohammad Abdollahi

Contract Research Organizations

235

Shayne C Gad

Copper

241

Shayne C Gad

Corrosives

247

Jeanna M Marraffa

Corticosteroids

251

Inderbir Padda and Mayur S Parmar

Cosmetics and personal care products

259

Sophia Hatziantoniou, Ioannis Sotirios Kapetanstratakis, and Nikolaos Drakoulis

Cosmetics, endocrine disrupting ingredients

271

Marí a-Elena Fernández-Martí n and Jose V Tarazona

Cotinine Joshua P Gray

287

Contents of Volume 3

Coumarins

vii 293

W Eggleston

Creosote

299

Jaclyn Tetenbaum-Novatt

Cresols

303

Murali Badanthadka

Criminal Enforcement of Environmental Law in the European Union and the United States

311

Carlos de Miguel Perales and George Wilkinson

CRISPR in toxicology research

317

Amin Sobh, Rola S Zeidan, and Christopher D Vulpe

Cromolyn

325

Timothy J Wiegand

Crotonaldehyde

329

Gerard G Dumancas, Lakshmi Viswanath, Rosa Wang, Emily Gondek, Sathish Kumar Lageshetty, Beulah Solivio, Arnold A Lubguban, and Roberto M Malaluan

Cumene

337

Gelareh Abdolmaleki and Zahra Bayrami

Cumulative (combined exposures) risk assessment

345

Samaneh Nakhaee and Omid Mehrpour

Curare (D-Tubocurarine)

353

Ann E Rigby-Jones and Steven A Burr

Cuyahoga River

359

Karen L Mumy

Cyanamide

361

Daniela L Baconi, Miriana Stan, and Ana-Maria Vlasceanu

Cyanide

369

Steven A Burr

Cyanogen chloride

375

Mona Navaei-Nigjeh and Zahra Bayrami

Cyclodienes

385

Pete N Lohstroh and Svetlana E Koshlukova

Cyclohexane

399

Samantha E Gad

Cyclohexene

405

Sara Mostafalou and Perham Mohammadi

Cycloheximide

411

Alicia P DeFalco

Cyclophosphamide

417

Christine M Stork and Susan M Schreffler

Cyclosarin (GF)

423

Omid Mehrpour, Samaneh Nakhaee, and Farshad M Shirazi

Cyclosporine

433

Teresa Dodd-Butera and Molly Broderick Pritty

Cyfluthrin

439

N Assar, M Noruzi, and M Sharifzadeh

Cypermethrin Maysa M Falah and Steven A Burr

445

viii

Contents of Volume 3

Cytochrome P450

449

Karanpreet Singh Bhatia, Aarthi Nivasini Mahesh, and Shruti Bhatt

Dacarbazine

457

Nidhi Patel, Rabin Neupane, Swapnaa Balaji, Amit K Tiwari, and Sidhartha D Ray

Dalapon

465

David R Wallace

Danthron (1,8-dihydroxyanthraquinone)

471

Ningning Yang

Data fusion applications in toxicology

477

Gerard G Dumancas, Mary Krichbaum, Beulah Solivio, Arnold A Lubguban, and Roberto M Malaluan

DDT (dichlorodiphenyltrichloroethane)

487

Luisina D Demonte and Melina P Michlig

Decane

493

SR Clough

DEET (N,N-Diethyl-m-toluamide)

497

Luciana Griffero, Germán Azcune, and Andrés Pérez Parada

DEF (butyl phosphorotrithioate)

511

Priya Raman and Neha Bhavnani

Deferoxamine and other iron chelators

519

Niloofar Deravi, MD, Narges Norouzkhani, Kimia Keylani, Seyedeh Mona Haghi, and Seyyed Kiarash Sadat Rafiei

Delaney Clause

533

Robin C Guy

Deltamethrin

535

Maysa M Falah and Steven A Burr

Dental settings and toxic agents

539

Mahshid Hodjat, Nastaran Sharifi, and Madiha Khalid

Department of Energy, US

551

Omid Mehrpour, Farhad Saeedi, and Samaneh Nakhaee

Derived minimal effect level (DMEL)

557

Robert Roy, Robert Skoglund, and John Hutchinson

Derived no-effect level (DNEL)

561

Robert Roy, Robert Skoglund, and John Hutchinson

Detergent

565

I Cattaneo and S Levorato

Dextromethorphan

571

Jeanna M Marraffa

Diaminotoluenes

575

Ayesha Rahman Ahmed

Diazinon

585

Yuvashree Muralidaran, Neelakanta Sarvashiva Kiran, Yashaswini Chandrashekar, and Prabhakar Mishra

Diazoaminobenzene

591

Gabriel López-Berenguer, Emma Martí nez-López, and Antonio Juan Garcí a-Fernández

Diazoxide

597