Casarett and Doull's Essentials of Toxicology PDF

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Under no circumstances shall McGraw-Hill Education and/or its licensors be liable for any indirect, incidental, special, punitive, consequential or similar damages that result from the use of or inability to use the work, even if any of them has been advised of the possibility of such damages. This limitation of liability shall apply to any claim or cause whatsoever whether such claim or cause arises in contract, tort or otherwise. Contents Contributors Preface UNIT 1 GENERAL PRINCIPLES OF TOXICOLOGY 1. The Evolving Journey of Toxicology: A Historical Glimpse Philip Wexler and Antoinette N. Hayes 2. Principles of Toxicology Lauren M. Aleksunes and David L. Eaton 3. Mechanisms of Toxicity Lois D. Lehman-McKeeman 4. Risk Assessment Elaine M. Faustman UNIT 2 DISPOSITION OF TOXICANTS 5. Absorption, Distribution, and Excretion of Toxicants Angela L. Slitt 6. Biotransformation of Xenobiotics Andrew Parkinson, Brian W. Ogilvie, David B. Buckley, Faraz Kazmi, and Oliver Parkinson 7. Toxicokinetics Kannan Krishnan UNIT 3 NON-ORGAN-DIRECTED TOXICITY 8. Chemical Carcinogenesis James E. Klaunig and Zemin Wang 9. Genetic Toxicology Joanna Klapacz and B. Bhaskar Gollapudi 10. Developmental Toxicology John M. Rogers UNIT 4 TARGET ORGAN TOXICITY 11. Toxic Responses of the Blood Martyn T. Smith and Cliona M. McHale 12. Toxic Responses of the Immune System Barbara L.F. Kaplan, Courtney E.W. Sulentic, Helen G. Haggerty, Michael P. Holsapple, and Norbert E. Kaminski 13. Toxic Responses of the Liver Robert A. Roth, Hartmut Jaeschke, and James P. Luyendyk 14. Toxic Responses of the Kidney Rick G. Schnellmann 15. Toxic Responses of the Respiratory System George D. Leikauf 16. Toxic Responses of the Nervous System Virginia C. Moser, Michael Aschner, Jason R. Richardson, Aaron B. Bowman, and Rudy J. Richardson 17. Toxic Responses of the Cornea, Retina, and Central Visual System Donald A. Fox and William K. Boyes 18. Toxic Responses of the Heart and Vascular System Matthew J. Campen 19. Toxic Responses of the Skin Donald V. Belsito 20. Toxic Responses of the Endocrine System Patricia B. Hoyer and Jodi A. Flaws 21. Toxic Responses of the Reproductive System Paul M.D. Foster and L. Earl Gray Jr. UNIT 5 TOXIC AGENTS 22. Toxic Effects of Pesticides Lucio G. Costa 23. Toxic Effects of Metals Alexander C. Ufelle and Aaron Barchowsky 24. Toxic Effects of Solvents and Vapors James V. Bruckner, S. Satheesh Anand, and D. Alan Warren 25. Toxic Effects of Radiation and Radioactive Materials David G. Hoel 26. Toxic Effects of Plants and Animals John B. Watkins, III 27. Food Toxicology: Fundamental and Regulatory Aspects Supratim Choudhuri 28. Toxic Effects of Calories Martin J.J. Ronis, Kartik Shankar, and Thomas M. Badger 29. Nanoparticle Toxicology David B. Warheit, Günter Oberdörster, Agnes B. Kane, Scott C. Brown, Rebecca D. Klaper, and Robert H. Hurt UNIT 6 ENVIRONMENTAL TOXICOLOGY 30. Ecotoxicology Richard T. Di Giulio and Michael C. Newman 31. Air Pollution Daniel L. Costa and Terry Gordon UNIT 7 APPLICATIONS OF TOXICOLOGY 32. Analytical and Forensic Toxicology Bruce A. Goldberger, Dayong Lee, and Diana G. Wilkins 33. Clinical Toxicology Louis R. Cantilena Jr. 34. Occupational Toxicology Peter S. Thorne Answers to Chapter Questions Index Contributors Lauren M. Aleksunes, PharmD, PhD, DABT Associate Professor Department of Pharmacology and Toxicology Rutgers University Piscataway, New Jersey Chapter 2 S. Satheesh Anand, PhD, DABT Manager, Safety Assessment Life Science Research Battelle West Jefferson, Ohio Chapter 24 Michael Aschner, PhD Professor Department of Molecular Pharmacology Albert Einstein College of Medicine Bronx, New York Chapter 16 Thomas M. Badger, PhD Professor Emeritus Department of Pediatrics University of Arkansas for Medical Sciences Little Rock, Arkansas Chapter 28 Aaron Barchowsky, PhD Professor Department of Environmental and Occupational Health Graduate School of Public Health University of Pittsburgh Pittsburgh, Pennsylvania Chapter 23 Donald V. Belsito, MD Leonard C. Harber Professor Department of Dermatology Columbia University Medical Center New York, New York Chapter 19 Aaron B. Bowman, PhD Associate Professor Department of Pediatrics Vanderbilt University Medical Center Nashville, Tennessee Chapter 16 William K. Boyes, PhD Environmental Health Scientist Toxicity Assessment Division/Neurotoxicology Branch U.S. Environmental Protection Agency Research Triangle Park, North Carolina Chapter 17 Scott C. Brown, PhD Principal Scientist The Chemours Company Wilmington, Delaware Chapter 29 James V. Bruckner, PhD Professor Emeritus Department of Pharmaceutical and Biomedical Sciences College of Pharmacy University of Georgia Athens, Georgia Chapter 24 David B. Buckley, PhD, DABT Director, DMPK Roivant Sciences, Inc Durham, North Carolina Chapter 6 Matthew J. Campen, PhD, MSPH Regent’s Professor Department of Pharmaceutical Sciences  College of Pharmacy University of New Mexico Albuquerque, New Mexico Chapter 18 Louis R. Cantilena, Jr., MD, PhD1 Professor of Medicine and Pharmacology Director, Division of Clinical Pharmacology and Medical Toxicology Uniformed Services University Bethesda, Maryland Chapter 33 Supratim Choudhuri, PhD Toxicologist Division of Biotechnology and GRAS Notice Review, Office of Food Additive Safety Center for Food Safety and Applied Nutrition U.S. Food and Drug Administration College Park, Maryland Chapter 27 Daniel L. Costa, ScD, DABT Office of Research and Development (Emeritus) U.S. Environmental Protection Agency Adjunct Professor Chapel Hill, University of North Carolina Chapter 31 Lucio G. Costa, PhD, ATS Department of Environmental and Occupational Health Sciences University of Washington Seattle, Washington Chapter 22 Richard T. Di Giulio, PhD Sally Kleberg Professor of Environmental Toxicology Nicholas School of the Environment Duke University Durham, North Carolina Chapter 30 David L. Eaton, PhD, DABT, ATS, NASEM Dean and Vice Provost University of Washington Seattle, Washington  Chapter 2 Elaine M. Faustman, PhD, DABT Professor and Director Institute for Risk Analysis and Risk Communication Department of Environmental and Occupational Health Sciences University of Washington Seattle, Washington Chapter 4 Jodi A. Flaws, PhD Professor Department of Comparative Biosciences University of Illinois Urbana, Illinois Chapter 20 Paul M.D. Foster, PhD, ATS Senior Scientist; Chief, Toxicology Branch Division of the National Toxicology Program (NTP), retired National Institute of Environmental Health Sciences (NIEHS) Fuquay-Varina, North Carolina Chapter 21 Donald A. Fox, PhD, ATS, ARVO Fellow Toxicology and Pharmacology Expert Robson Forensic Austin, Texas Chapter 17 Bruce A. Goldberger, PhD, F-ABFT Chief, Director and Professor Departments of Pathology, Immunology, and Laboratory Medicine College of Medicine, University of Florida Gainesville, Florida Chapter 32 B. Bhaskar Gollapudi, PhD Senior Managing Scientist Health Sciences Center Exponent, Inc. Alexandria, Virginia Chapter 9 Terry Gordon, PhD  Professor Department of Environmental Medicine New York University School of Medicine New York, New York Chapter 31 L. Earl Gray, Jr., PhD Research Biologist Office of Research and Development National Health and Environment Effects Research Laboratory Reproductive Toxicology Branch Environmental Protection Agency Durham, North Carolina Chapter 21 Helen G. Haggerty, PhD Distinguished Research Fellow Immuno and Molecular Toxicology Drug Safety Evaluation Bristol-Myers Squibb New Brunswick, New Jersey Chapter 12 Antoinette N. Hayes, MS, DABT Lead Associate Scientist Nonclinical Drug Safety and Bioanalytical Alnylam Pharmaceuticals Cambridge, Massachusetts Chapter 1 David G. Hoel, PhD Distinguished University Professor Department of Public Health Sciences Medical University of South Carolina Charleston, South Carolina Chapter 25 Michael P. Holsapple, PhD, ATS Professor Michigan State University MPH Toxicology Consulting, LLC East Lansing, Michigan Chapter 12 Patricia B. Hoyer, PhD Professor Emeritus Department of Physiology College of Medicine The University of Arizona Tucson, Arizona Chapter 20 Robert H. Hurt, PhD Professor of Engineering Brown University Providence, Rhode Island Chapter 29 Hartmut Jaeschke, PhD, ATS Professor and Chair Department of Pharmacology, Toxicology, and Therapeutics University of Kansas Medical Center Kansas City, Kansas Chapter 13 Norbert E. Kaminski, PhD Professor, Pharmacology and Toxicology Director, Institute for Integrative Toxicology College of Human Medicine Michigan State University East Lansing, Michigan Chapter 12 Agnes B. Kane, MD, PhD Professor Department of Pathology and Laboratory Medicine Brown University Providence, Rhode Island Chapter 29 Barbara L.F. Kaplan, PhD Assistant Professor Center for Environmental Health Sciences Department of Basic Sciences College of Veterinary Medicine Mississippi State University Mississippi State, Mississippi Chapter 12 Faraz Kazmi, PhD Scientist Pharmacokinetics, Dynamics and Metabolism Janssen Research and Development Spring House, Pennsylvania Chapter 6 Joanna Klapacz, PhD Senior Toxicologist Toxicology and Environmental Research and Consulting The Dow Chemical Company Midland, Michigan Chapter 9 Rebecca D. Klaper, PhD Professor and Director of the Great Lakes Genomics Center School of Freshwater Sciences University of Wisconsin-Milwaukee Milwaukee, Wisconsin Chapter 29 James E. Klaunig, PhD Professor Department of Environmental Health Indiana University Bloomington, Indiana Chapter 8 Kannan Krishnan, PhD, DABT, FATS, FCAHS Chief Scientific Officer Institut de recherche Robert-Sauvé en santé et en sécurité du travail Montreal, Quebec, Canada Chapter 7 Dayong Lee, PhD, F-ABFT Houston Forensic Science Center Houston, Texas Chapter 32 Lois D. Lehman-McKeeman, PhD, ATS Vice President Pharmaceutical Candidate Optimization Bristol-Myers Squibb Company Princeton, New Jersey  Chapter 3 George D. Leikauf, PhD Professor Department of Environmental and Occupational Health Graduate School of Public Health University of Pittsburgh Pittsburgh, Pennsylvania Chapter 15 James P. Luyendyk, PhD Associate Professor Department of Pathobiology and Diagnostic Investigation Michigan State University East Lansing, Michigan Chapter 13 Cliona M. McHale, PhD Senior Researcher School of Public Health University of California, Berkeley Berkeley, California Chapter 11 Virginia C. Moser, PhD Retired Division of Toxicity Assessment National Health and Environmental Effects Research Laboratory U.S. Environmental Protection Agency Research Triangle Park, North Carolina Chapter 16 Michael C. Newman, PhD A. Marshall Acuff, Jr. Professor of Marine Science College of William & Mary Virginia Institute of Marine Science Gloucester Point, Virginia Chapter 30 Günter Oberdörster, PhD Professor of Toxicology University of Rochester Medical Center Rochester, New York Chapter 29 Brian W. Ogilvie, PhD Vice President of Scientific Consulting Sekisui XenoTech, LLC Kansas City, Kansas Chapter 6 Andrew Parkinson, PhD CEO XPD Consulting Shawnee, Kansas Chapter 6 Oliver Parkinson, PhD Consultant XPD Consulting Shawnee, Kansas Chapter 6 Jason R. Richardson, PhD Professor Pharmaceutical Sciences Northeast Ohio Medical University Rootstown, Ohio Chapter 16 Rudy J. Richardson, ScD Professor of Toxicology Environmental Health Sciences University of Michigan Ann Arbor, Michigan Chapter 16 John M. Rogers, PhD Director, Toxicity Assessment Division National Health and Environmental Effects Research Laboratory Office of Research and Development United States Environmental Protection Agency Research Triangle Park, North Carolina Chapter 10 Martin J.J. Ronis, PhD Professor Department of Pharmacology and Experimental Therapeutics Louisiana State University Health Sciences Center  New Orleans, Louisiana Chapter 28 Robert A. Roth, PhD, DABT Department of Pharmacology and Toxicology Institute for Integrative Toxicology Michigan State University East Lansing, Michigan Chapter 13 Rick G. Schnellmann, PhD Professor and Dean Department of Pharmacology and Toxicology School of Pharmacy University of Arizona Tucson, Arizona Chapter 14 Kartik Shankar, PhD, DABT Associate Professor Department of Pediatrics University of Arkansas for Medical Sciences Little Rock, Arkansas Chapter 28 Angela L. Slitt, PhD Associate Professor Department of Biomedical and Pharmaceutical Sciences College of Pharmacy University of Rhode Island Kingston, Rhode Island Chapter 5 Martyn T. Smith, PhD Professor of Toxicology School of Public Health University of California, Berkeley Berkeley, California Chapter 11 Courtney E.W. Sulentic, PhD Associate Professor Department of Pharmacology and Toxicology Boonshoft School of Medicine  Wright State University Dayton, Ohio Chapter 12 Peter S. Thorne, MS, PhD Professor and Head Department of Occupational and Environmental Health Director Environmental Health Sciences Research Center University of Iowa College of Public Health Iowa City, Iowa Chapter 34 Alexander C. Ufelle, MBBS, MPH, PhD Assistant Professor Department of Public Health and Social Work Slippery Rock University Slippery Rock, Pennsylvania Chapter 23 Zemin Wang, MD, PhD, DABT Research Assistant Professor Department of Environmental Health Indiana University Bloomington, Indiana Chapter 8 David B. Warheit, PhD, DABT, ATS Technical Fellow Chemours Company Wilmington, Delaware Chapter 29 D. Alan Warren, PhD Academic Program Director Program in Environmental Health Sciences University of South Carolina Beaufort Bluffton, South Carolina Chapter 24 John B. Watkins, III, PhD, DABT Professor Emeritus of Pharmacology and Toxicology Indiana University School of Medicine Medical Sciences  Bloomington, Indiana Chapter 26 Philip Wexler, MLS Technical Information Specialist (retired) Toxicology and Environmental Health Information Program National Library of Medicine Bethesda, Maryland Chapter 1 Diana G. Wilkins, PhD, MT(ASCP) Professor and Division Chief of Medical Laboratory Sciences Department of Pathology School of Medicine University of Utah Salt Lake City, Utah Chapter 32 1Deceased Preface This fourth edition of Essentials of Toxicology condenses the principles and concepts of toxicology that were described in the ninth edition of Casarett & Doull’s Toxicology: The Basic Science of Poisons. Essentials of Toxicology succinctly defines the expansive science of toxicology, and includes important concepts from anatomy, physiology, and biochemistry to facilitate the understanding of the principles and mechanisms of toxicant action. We greatly appreciate the authors who contributed to the ninth edition of Casarett & Doull’s Toxicology: The Basic Science of Poisons because their chapters in the parent text provided the foundation for this edition of Essentials of Toxicology. The book is organized into seven units: (1) General Principles of Toxicology, (2) Disposition of Toxicants, (3) Non-organ-directed Toxicity, (4) Target Organ Toxicity, (5) Toxic Agents, (6) Environmental Toxicology, and (7) Applications of Toxicology. Key points and review questions are provided for each chapter. We trust that this book will assist students in undergraduate and graduate courses in toxicology, as well as students from other disciplines, to develop a strong foundation in the concepts and principles of toxicology. We invite readers to send us suggestions of ways to improve this text and we appreciate the thoughtful recommendations that we received on the last edition. We particularly give a heartfelt and sincere thanks to our families for their love, patience, and support during the preparation of this book. The capable guidance and assistance of the McGraw-Hill staff are gratefully acknowledged. Finally, we thank our students for their enthusiasm for learning and what they have taught us during their time with us. Curtis D. Klaassen John B. Watkins III UNIT 1 GENERAL PRINCIPLES OF TOXICOLOGY  CHAPTER 1 The Evolving Journey of Toxicology: A Historical Glimpse Philip Wexler and Antoinette N. Hayes INTRODUCTION ABOUT HISTORY TOXICOLOGY IN ANTIQUITY Ancient China Ancient India Ancient Egypt Pontus, Mithridates, and Theriacas Ancient Greece Ancient Rome THE MIDDLE AGES AND RENAISSANCE 18TH AND 19TH CENTURIES THE MODERN ERA Radiation Food and Drugs Pesticides Research and Chemical Warfare: A Surprising Alliance High Profile Poisonings Mass Environmental Exposures, the U.S. EPA, and Environmental Legislation INTERNATIONAL ENVIRONMENTAL CONVENTIONS AND OTHER GLOBAL EFFORTS  KEY POINTS Toxicology is the study of the adverse effects of xenobiotics on living systems. Toxicology assimilates knowledge and techniques from biochemistry, biology, chemistry, genetics, mathematics, medicine, pharmacology, physiology, and physics. Toxicology applies safety evaluation and risk assessment to the discipline. INTRODUCTION The word toxicology is derived from the Latinized form of the Greek word toxicon, meaning “arrow poison.” Poison, as a noun, dates back to the Old French poison or puison, meaning, originally a drink, especially a medical drink, but later signifying more of a magical potion or poisonous drink. The commonly misused term toxin formally should be used to refer to toxic substances produced biologically. Other terms, toxicant, toxic agent, and toxic substance, could be used to delineate the broader category of substances that are toxic, regardless of origin. Xenobiotics is a term referring to substances, whether toxic or not, foreign to a given organism. ABOUT HISTORY History is about the past; it is not the past. The past is passive, objective, all encompassing. History is active, subjective, and selective. The further back in time that we look, the more problematic it is for us to reach, in the present, conclusions about what happened in the past. Science begins with observation. In the distant past, our observational skills did not extend beyond our senses in assessing toxicity and safety. Our hominin ancestors used the process of trial and error making careful note of which substances, particularly potential food sources, were safe and which were hazardous. Although it might very well be after the damage was done, they and their tribe and descendants would quickly learn to differentiate between the safe and toxic. Toxic substances were to be avoided, unless used against enemies. TOXICOLOGY IN ANTIQUITY Ancient China Shen Nong, the legendary founder of Chinese Herbal Medicine, also known as the farmer god, and said to live circa 2800 B.C., was said to have tasted hundreds of herbs daily to differentiate the poisonous from the medicinal or just plain edible. He is considered the compiler of perhaps the world’s first pharmacological compendium, Divine Farmer’s Classic of Materia Medica. Du is the standard word for poison or toxicity in Chinese. It was understood by the ancient Chinese that herbals were potentially toxic and dose played a role. Aconite, derived from the plant wolfsbane and possessing extreme potential toxicity, was widely used medicinally in small doses in China over 2000 years ago—usually applied externally to treat various wounds or ingested as a tonic to restore qi (the vital energy defined by Chinese medicine) and extend life. Unadulterated aconite in larger doses was often used to murder. Today we know that the alkaloids in aconite have a narrow therapeutic index and their use is not generally recommended. Ancient India Ancient India was no stranger to the knowledge and uses of poisons. Indian surgeon Sushruta prepared Suśrutasam.hitā, a foundational medical/surgical compendium for Ayurveda (traditional Indian medicine). Volume 5 contains several chapters related to poisons and poisoning, including descriptions of vegetable and mineral poisons and animal poisons, as well as advice on medical treatment of snake bites and insect bites. Agada Tantra, one of the eight clinical specialties of Ayurvedic medicine, is specifically associated with toxicology. India has a long tradition of tales about the so-called “venomous virgin” who would, as a young girl, be fed “tolerably minute, but gradually increasing, amounts of poison or snake venom, so that by the time she was an attractive young woman, the level of toxin in her body would be so high that she could be sent to an enemy king as a gift. Upon kissing her, making love to her, or even just sharing glass of wine with her, he would instantly fall dead.” Ancient Egypt Venomous snakes and insects were well known and the focus of toxicology as it existed in ancient Egypt. One of the major documents examining snakebite is the Brooklyn Papyrus (held by the Brooklyn Museum), 525–600 b.c. For example, Paragraph 15 of the Papyrus describes the snake known by the Egyptians as Apophis which, mythologically, personified evil. Scholars believe this may be the Boomslang (Dyspholidus typhus) in the Colubridae family. Symptoms and signs of snake envenomation are presented in the Papyrus. The treatments offered could be general, for any snakebite, or specific. Bites by snakes known to be lethal generally received no treatment. Therapeutic measures, overall, were largely symptomatic. Toxicity is addressed to a lesser extent in the Berlin, Edwin Smith, and Ebers papyri. The death of Cleopatra VII, born in 69 b.c. and one of the most fascinating personalities in Egypt when Greece and Rome held sway, holds toxicological interest for us. After learning that Marc Anthony killed himself by a self-inflicted sword wound, Cleopatra decided to follow suit by supposedly holding an asp (Egyptian cobra) to her breast and succumbing to its venomous bite. However, she may have been murdered perhaps with a poisonous draught by Octavian, the victor in their battle, who then spread the rumor of her suicide to avoid retribution by her adoring subjects. Pontus, Mithridates, and Theriacas Mithridates VI, ruler of Pontus in northeastern Turkey beginning in 120 b.c., experimented with poisons and antidotes, even on himself. Son of a father who was murdered with poison and a mother who would have poisoned him in order to ascend to the throne, he went into hiding for a period of years. He returned to capture his rightful position by likewise using poison, probably arsenic. He feared assassination by poison and took precautions to avoid it.  His approach was to ingest small doses of toxicants to become tolerant to them. His lifelong pursuit was to create a universal antidote, which came to be known as a theriac. His Mithridatium was a concoction of tiny amounts of deadly poisons and antidotes. When Mithridates wanted to end his own life, consumption of poison weakened him, but he did not die. In one version of his actual death, he appealed to his bodyguard to impale him with a sword. Ancient Greece Nicander of Colophon (fl. 130 b.c.), a Greek poet and physician, is the author of two of the oldest extant works on poisons—Theriaka and Alexipharmaka. The Theriaka concerns venomous animals, including snakes, spiders, scorpions, insects, lizards, and fish. His Alexipharmaka deals with 21 poisons from the vegetable, mineral, and animal kingdoms. Among them are aconite, white lead, and hemlock. In both works, Nicander describes the poison, its symptoms, and antidotes. The Greek philosopher, Socrates (469–399 B.C.), became an iconic figure in the history of toxicology through his death. Convicted of corrupting the youth of Athens and disrespecting the gods, he was sentenced to death. His execution was supposedly carried out in suicidal fashion, with Socrates condemned to drink an extract of hemlock (Conium maculatum). This story has been questioned largely because the account provided in Plato’s Phaedo describes a clinical disorder not caused by hemlock poisoning, but rather a possible mixture of hemlock and opium. Alexander the Great (born in 356 b.c.) plays a role in the history of toxicology in that the cause of his death is an unsolved mystery. He is said to have drunk vast quantities of wine at a banquet in Babylon, after which he suffered severe abdominal pain. Over days, things went from bad to worse and he developed partial paralysis finally dying two weeks later. Rumors of poisoning began circulating in no time. Though speculation is widespread, the true cause of Alexander’s death has never been confirmed. The Oracle at Delphi, perhaps the most important and sacred shrine in ancient Greece, is associated with the Greek god Apollo. Pilgrims to Delphi would address their questions to the Pythia, a role filled by various women at different times. Plutarch, the celebrated Greek biographer and essayist, served as one of the priests at the temple of Apollo at Delphi. He noted that pneuma (a kind of gas or vapor) was emitted in the adyton, a small inner sanctum type area. The Pythia would inhale the pneuma and go into a trance, after which a priest would ask her the questions raised by the petitioners. It may be that the trancelike state of the Pythia was induced by inhaling ethylene gas or a mixture of ethylene and ethane from a naturally occurring vent of geological origin. Toxicology is also heir to a rich mythological tradition. After Hercules killed the nine-headed sea monster known as the Hydra, as part of his second labor, he cut it open and dipped his arrows in its venom, providing him with what may have been the first biological weapon for use in battle. Achilles, one of the prominent heroes in Homer’s Iliad was a victim of just such a poison. Immersed as an infant in the river Styx by his mother to make him immortal, she failed to realize that in holding him by the heel, that very part of the body would make him susceptible to future danger. In the final battle of the Trojan War, he was killed by a poisoned arrow shot into this heel. Ancient Rome The Romans of antiquity were also knowledgeable in the principles and practice of toxicology. Dioscorides (born in 40 a.d.), a native of Anazardus, Cilicia, Asia Minor, was a physician who traveled throughout the Roman Empire and collected samples of local medicinal herbs. The information he gleaned was compiled in the encyclopedic De materia medica in the first century a.d. In it he classified poisons as animal, plant, or mineral. More specifically, De Venenis and De venenosis animalibus, ascribed to Dioscorides but probably not written by him, covered poisons in general and animal venoms, respectively. Galen, born (129 a.d.) in Pergamon, was a firm subscriber to the theory of the humors (blood, yellow bile, black bile, and phlegm), the origins of which may go back to ancient Egypt but which were first articulated about medicine by Hippocrates. Galen formulated his own Galeni Theriaca and claimed it improved upon the one concocted by Mithridates. He wrote about assorted theriac compounds in his books De Antidotis I and II and De Theriaca ad Pisonem. The legal framework of toxicology is sometimes dated back to the age of the Roman military and political leader Sulla. Under the lex Cornelia de sicariis et veneficis (81 b.c.), punishment was imposed for anyone who prepared, sold, bought, kept, or administered a noxious poison (venenum malum). In 1983, Jerome Nriagu popularized the idea that the metal lead was responsible for the fall of the Roman Empire. It has been stated that lead contamination in water supplies, cooking, and the production of wine, ultimately decreased fertility and reproductive capacity. Recent archaeological investigations have found that the mean skeletal lead content of populations at the time was less than half that of present-day Europeans in the same regions. The assertion that lead was the primary culprit in Rome’s decline and fall has been largely refuted. THE MIDDLE AGES AND RENAISSANCE The Venetian Council of Ten was a governing body in Venice from around 1310 until 1797. They were known for conducting secret tribunals whereby figures perceived as a threat to the state were ordered to be executed. Many of these executions were carried out by poisoning. Poisoning as an assassination method was widespread during the fourteenth to sixteenth centuries in Europe. Letters to Grand Duke Cosimo I de’Medici affirm as much. Animal venoms, phytotoxins, and mineral poisons were all employed. Poisoning was clearly a family affair with the Medicis, and Cosimo’s sons Ferdinando and Francesco were equally complicit in it. Many legends surround Catherine de’Medici who moved to France to marry the future King Henry II. Despite multiple purported victims, there is no definitive evidence that she poisoned anyone. Developing and testing antidotes was also part of the Medicis’ stock-in-trade. Another powerful and infamous Italian family, originally from Spain, and on whom were pinned numerous heinous crimes, poisoning among them, were the Borgias. There were claims, for example, that Cesare murdered a servant who was a lover of his sister, Lucretia, in front of their father Pope Alexander. Cesare was also said to have poisoned Cardinal Juan Borgia. The reputation of Lucretia herself was stained with allegations that she was a poisoner. Documents uncovered recently in the Vatican archives refute these and other claims that the Borgias’ reputation for extensive poisonings and murders stems from rumors spread by their enemies. In seventeenth century France, during the reign of Louis XIV, a series of poisonings became known as L’affaire des poisons (the Affair of the Poisons). Madame de Brinvilliers was convicted of poisoning her father and two brothers and attempting to poison other family members. Prior to her execution she implicated, without specifically naming them, many others, who were subsequently prosecuted and sentenced to death. The notorious Catherine Deshayes, also known as La Voisin, an acknowledged sorceress, was burned at the stake in 1680 for her crimes. It is thought that two women in Palermo, Francesca la Sarda and Teofania di Adamo, jointly concocted and marked a poison known as “Acqua Tufania” for which they were executed. Under the leadership of Giulia Tofana, possibly Teofania’s daughter, they carried on the business. The poison became known as Aqua Tofana. Arsenic was likely a primary ingredient. An increasingly sophisticated understanding of toxicology developed during the Middle Ages and Renaissance. Moses Maimonides, the great Jewish philosopher, theologian, and scientist, wrote his Treatise on Poisons and their Antidotes, originally in Arabic, in 1198. Part I was concerned with bites from snakes and rabid dogs, and stings of scorpions and insects. Part II dealt with poisons in food and minerals, as well as remedies. He made a distinction between “hot” and “cold” poisons which, it has been claimed, may be equivalent to modern-day hemolysins and neurotoxins. Maimonides also emphasized preventive measures. The study of toxicants was widespread in Persian and Arabic countries. Known by his Latin name of Avicenna in the West, Abū ‘AlīAal-H.usayn ibn Abd Allāh ibn Sīnā was perhaps the most noteworthy physician/scientist/philosopher of the Islamic world. His celebrated “Canon of Medicine” remained the most popular medical textbook for some six centuries, and it included detailed descriptions of venoms and other poisons, as well as instructions related to antidotes. On a very practical level, it became clear to ordinary people that their very occupations could be harmful. Georgius Agricola (1494–1555), born in Saxony, currently part of Germany, is known as “the father of mineralogy” largely as a result of his best known monograph, De Re Metallica, published in 1556. The unorthodox medical revolutionary, Theophrastus von Hohenheim, called Paracelsus (1493/94–1541) was born in Einsiedeln, a municipality now in modern-day Switzerland. He theorized that there were four pillars of medicine: natural philosophy, astronomy, alchemy, and medical virtue. In addition to his medical works, he was a keen observer and investigator of toxic effects of various agents and wrote a treatise about their effects upon miners. He concludes this work with a discussion of metallic mercury and criticizes its use at the time as therapy for people afflicted with syphilis. The most famous toxicological adage associated with Paracelsus is “The dose makes the poison,” which is a distillation of what he wrote in his Seven Defenses: Wenn jhr jedes Gifft recht wolt außlegen/ Was ist das nit Gifft ist? alle ding sind Gifft/ vnd nichts ohn Gifft/ allein die Dosis macht/ dz ein ding kein Gifft ist. When you want to correctly evaluate a poison, what is there that is not poison? All things are poison and nothing is without poison; only the dose determines that something is not a poison. Paracelsus deserves the laurel crown and the oft-cited appellation, “Father of toxicology.” An understanding of the dose–response relationship is no less significant to our understanding of toxicology today than it was 500 years ago. 18TH AND 19TH CENTURIES The scientific method gained increasing prominence in the eighteenth and nineteenth centuries as a way of understanding our universe, and toxicology benefited from this more sophisticated and methodical approach. Richard Mead (1673–1754) authored the first book in English devoted solely to poisons, A Mechanical Account of Poisons in Several Essays. He described the signs and symptoms of snake envenomation, performed chemical tests on venom, and experimented on snakes (to study their venom delivery system) and other animals. Bernardino Ramazzini was a physician whose seminal achievements have earned him the moniker Father of Occupational Medicine. The first edition of his most famous book, De Morbis Artificum Diatriba (A Treatise on the Diseases of Workers), published in 1700, is the first comprehensive and systematic work on occupational diseases. It outlined the health hazards of chemicals and other substances, including repetitive motions, encountered by workers in over 50 occupations. Percivall Pott (1714–1788) published in 1774 an essay, Chirurgical Observations Relative to the Cataract, the Polypus of the Nose, the Cancer of the Scrotum in which he made the link between the profession of chimney sweeps (regarding soot lodging in the folds of scrotal skin) and scrotal cancer. This was the first occupational link to cancer and Pott’s investigations contributed to the science of epidemiology. It wasn’t until the 1920s that benzo[a]pyrene was identified as the actual chemical responsible. Four scientists who made remarkable advances in the chemical detection of poisons were Karl Wilhelm Scheele, Christian Friedrich Samuel Hahnemman, Johann Daniel Metzger, and Valentine Rose. Scheele discovered oxygen before Joseph Priestley, although he published his results later. He is also credited with the discovery of hydrofluoric, hydrocyanic, and arsenic acids, and devised methods for detecting arsenic in body fluids and corpses. Hahnemman discovered a test for arsenic oxide. Rose and Metzger discovered the first methods for detecting elemental arsenic and arsenic oxides in fluids and tissues. In 1836, the English chemist James Marsh developed what came to be known as the Marsh test, a groundbreaking method for detecting arsenic. Mathieu Joseph Bonaventure Orfila (1787–1853) experimented widely with dogs, varying the amount of poison (such as arsenic) administered and the route of administration, and tested antidotes and treatments. He authored Traite des poisons in 1814/5. He subsequently extracted the sections on antidotes and treatments and published them in a compact free-standing volume designed for physicians and for lay audiences that may not have access to medical care but need to know what to do in the event of a poisoning emergency. As a medical expert, Orfila is best known for a case involving Marie Lafarge, charged with poisoning her husband. Eyewitnesses had seen her buying arsenic (used to exterminate rats) and stirring a white powder into her husband’s food. Upon his exhumation, Orfila found definite traces of arsenic in the body and demonstrated that it did not come from the surrounding soil. Marie Lafarge was found guilty of murder and received a death sentence, later commuted to life in prison. The case cemented Orfila’s reputation as the greatest toxicologist of the day. And like Paracelsus, Orfila has been called “Father of Toxicology,” but of course representing a different era, and for different reasons. In France, Francois Magendie (1783–1855) was best known for his pioneering contributions in neuroscience and neurosurgery, and experimental physiology. His studies on the effects of drugs on different parts of the body led to the introduction of compounds such as strychnine and morphine into medical practice. His research into the mechanisms of toxicity of these and other alkaloids furthered the science of toxicology. Claude Bernard (1813–1878), Magendie’s most celebrated pupil, discovered the role of the pancreas in digestion, the regulation of the blood supply by vasomotor nerves, and the glycogenic function of the liver. His work also led to an understanding of the self-regulating process of living organisms we now refer to as homeostasis. He won acclaim for his book Introduction à l’Etude de la Médecine Expérimentale (An Introduction to the Study of Experimental Medicine). His approach of starting with a hypothesis and having results, which are reproducible, furthered the paradigm of the modern scientific method. In the realm of toxicology, Bernard demonstrated that the mechanism of action of curare resulted from its interference in the conduction of nerve impulses from the motor nerve to skeletal muscle. In addition to curare, he studied the toxicological properties of other neuroactive compounds such as opium, atropine, strychnine, and nicotine. He was the first to describe the hypoxic effects of carbon monoxide. Greatly influenced by Orfila, Robert Christison (1797–1882), a Scottish physician, was interested in underpinning medical jurisprudence, especially toxicology, with a scientific foundation. Early on, he investigated the detection and treatment of oxalic acid poisoning and followed this up with investigations on arsenic, lead, opium, and hemlock. His celebrated book, Treatise on Poisons, first published in 1829, went through four editions. THE MODERN ERA Radiation The late nineteenth century is about the time when an understanding of radiation and its potentially hazardous effects began to surface. In 1895, Wilhelm Röntgen discovered that x-rays could penetrate human flesh. In 1896, Nikola Tesla intentionally exposed his fingers to x-rays and reported burns. In that same year, Henri Becquerel discovered that uranium salts naturally emitted similar rays. Marie Curie, a student of Becquerel, named the phenomenon “radioactivity.” She went on to discover thorium, polonium, and radium. Soon after radium’s discovery, it was manufactured synthetically and appeared in food products such as bread, chocolate, toys (because of its luminescence), toothpaste, cosmetics, suppositories, and products to treat impotence. One of the first revelations about the scope of its potential danger concerned the unfortunate girls who became radium watch dial painters in the early 1900s. These “radium girls” applied radium paint to watch and clock faces so they would glow in the dark. They were instructed to use their lips to shape the brushes to a fine point. By 1927, over 50 women died due to radium paint poisoning, and many survivors suffered significant health problems. The detonation of atomic bombs over the cities of Hiroshima and Nagasaki in World War II killed a couple hundred thousand almost immediately. Tens of thousands of people in both cities would later die of radiation exposure or otherwise suffer devastating injuries. Food and Drugs Toxicology has developed and continues, to some extent, to develop as a reactive (rather than proactive) field. Chemical laws and regulations often are enacted in reaction to major or widespread exposure incidents. Lewis Caleb Beck, an American physician and chemist, published in 1846 Adulterations of Various Substances Used in Medicine and the Arts with Means of Detecting Them: Intended as a Manual for the Physician, the Apothecary, and the Artisan. His publication helped promote the Drug Importation Act of 1848, which required the U.S. Customs Service to inspect and stop any adulterated drugs from entering the U.S. market. Inspectors could conduct qualitative tests, such as those detailed in Beck’s publication, to determine if a drug was adulterated. In 1902, Harvey W. Wiley administered the so-called “Poison Squad” experiments, asking healthy volunteers to consume measured amounts of preservatives routinely added to food items to determine whether they were safe for human consumption. In time, hundreds of patent medicines were identified as misleading, harmful, and sometimes deadly. In 1905, Samuel Hopkins Adams published, in Collier’s Weekly, “The Great American Fraud,” a sensational article exposing the hoax of patent medicines. Upton Sinclair’s 1906 book, The Jungle, detailed unsanitary conditions of workers in the meat packing industry. The Pure Food and Drugs Act and the Meat Inspection Act were passed on the very same day in 1906. England’s attention to the adulteration of food and drugs actually preceded that of the United States. Friedrich Accum published a book in the 1820s titled A Treatise on the Adulterations of Food, and Culinary Poisons with the subtitle There Is Death in the Pot. Accum wrote about hundreds of poisonous additives commonly used in food products to either sweeten, color, or bulk up foods. His and others’ campaign to prevent food adulteration eventually resulted in food and drug legislation in the United Kingdom. The 1906 Pure Food and Drug Act in the United States did not have the broad impact that was intended. Its main purpose was to ban foreign and interstate traffic of adulterated, falsely advertised, or mislabeled food and drug products. It empowered the U.S. Bureau of Chemistry to inspect products and refer offenders to prosecutors, but gave no prosecutorial power to the agency itself. The United States Pharmacopeia (USP) and the National Formulary served as a foundation for the Pure Food and Drugs Act. Although the law was popular, it was virtually impossible to enforce. The 1906 law prevented the manufacture, sale, or transportation of adulterated, misbranded, poisonous, or deleterious foods, drugs, medicines, and liquors. Prohibition in the United States ran from 1920 to 1933. During this time, there were very few legal means for obtaining alcohol. One infamous concoction was Jamaica Ginger, which contained between 70% and 80% alcohol by weight. The U.S. Treasury Department required changes to the ingredients of Jamaica Ginger to discourage its abuse. The minimum requirement of ginger solids per cubic centimeter of alcohol resulted in a bitter concoction that was not palatable. An alternative recipe that could pass the inspection and taste well enough to sell was prepared by adding tri-ortho-cresyl phosphate (TOCP) to the mixture. In early 1930, reports detailed a strange paralysis of the legs, arms, and wrists with little to no recovery in large numbers of people. By 1931, the disease, known colloquially as Ginger Jake paralysis, had reached epidemic proportions affecting an estimated 10,000 people and the adulteration with tri- ortho-cresyl phosphate was discovered. Sulfa drugs were a twentieth century miracle for the treatment of bacterial and fungal infections. The first sulfa drug, Protonsil, showed no effect in vitro with bacterial assays but was extremely effective in vivo. It was later discovered that Protonsil is metabolized to sulfanilamide in vivo and the science of the bioactivation of drugs was revealed. However, for a drug to be effective there needed to be an effective delivery system. Sulfanilamide is highly insoluble in an aqueous solution. Originally prepared as an elixir in ethanol, chemists discovered that the drug was more soluble in diethylene glycol. Many patients, most of whom were children, died of acute kidney failure resulting from metabolism of the glycol to metabolites that crystallized in the kidney tubules. This tragedy led to the passage of the 1938 Food, Drug, and Cosmetic (FD&C) Act. It contained provisions for both misbranding and adulteration. The law also required that a package’s ingredients and their amounts, as well as the name and address of the manufacturer, packer, or distributor, be clearly displayed on the label. To enforce the statute, the FDA was given search, seizure, and prosecutorial powers. In 1960, a new drug thalidomide (Kevadon), an anti-nausea medication also used to alleviate morning sickness in pregnant women, was distributed by the Merrell drug company to over 1200 U.S. doctors with the expectation that it would be approved quickly. By 1961, it became clear that thalidomide posed a serious safety risk. Infant deaths and deformities occurred at an alarming rate across Europe and the German manufacturer began pulling the drug from the market in the late 1961. By 1962, the application for approval in the United States was withdrawn completely. Though never licensed in the United States, physicians distributed drug samples to around 20,000 patients in the United States. By late 1962, there were at least 10,000 babies born with thalidomide-related defects and countless pregnancies that ended in miscarriage worldwide. The thalidomide tragedy led to the 1962 Kefauver-Harris Amendments, which gave the FDA the authority to require proof of efficacy (rather than just safety) before a new drug could gain approval. The amendments created the groundwork for the multi-phased approval process involving clinical trials, which is still very much in use today. Even with the current laws in place, occasionally a drug must be highly regulated, recalled, or removed from the open market for reasons such as toxicity, impurities, lack of efficacy, or abuse potential. Mylotarg (gemtuzumab ozogamicin), for example, was approved under an accelerated approval process in 2000 for the treatment of acute myelogenous leukemia. In 2010, the drug was voluntarily withdrawn from the market because a phase 3 comparative controlled clinical trial demonstrated an increase in mortality. The nonsteroidal, anti-inflammatory medication for arthritis, Vioxx (refecoxib), was responsible for perhaps over 27,000 heart attacks and cardiac deaths. These effects did not emerge in the original clinical trials. From around 1938 to 1971, millions of pregnant women were prescribed diethylstilbestrol (DES) as a hormone-replacement therapy and to prevent miscarriages and premature births. It was discovered that DES caused a rare vaginal cancer (clear cell adenocarcinoma) in girls and young women who had been exposed to DES in the womb. It was recalled from the market in 1971. In some cases, a drug may be removed from the market temporarily to protect consumers. In 1982, there were several deaths eventually linked to Tylenol brand acetaminophen capsules. The capsules were laced with potassium cyanide. Several copycat crimes followed this incident. In 1987, Stella Nickell laced Excedrin capsules with cyanide, killing both her husband and a woman who purchased the tampered product. Crimes such as these led to the passage of the Federal Anti-Tampering Act of 1983. Pesticides Research and Chemical Warfare: A Surprising Alliance Naturally derived pesticides have been used to protect crops for thousands of years. Some 4500 years ago, the Sumerians dusted their crops with elemental sulfur. Around 3200 years ago, the Chinese used mercury and arsenic compounds to control body lice. Synthetic pesticide development and use is a product of the twentieth century. Germany was responsible for much of the large-scale production of pesticides and warfare gases used in the early to mid-1900s. Fritz Haber, a German scientist, with contributions from Carl Bosch, sought a way to capture nitrogen in the air for use in large-scale fertilizer production. The Haber-Bosch process was instrumental in the manufacture of nitrogen-based explosives for the German Army during World War I. Bosch also researched the weaponization of toxic substances such as chlorine, phosgene, and mustard gas, leading to the largest deployment of chemical weapons in modern history. During World War I, the Germans launched a chemical attack using chlorine gas in Ypres, Belgium in 1915. Phosgene employment accounted for nearly 85% of all gas-related fatalities during that war. The human toxicity of tabun, a new organophosphate insecticide synthesized in 1937 by Gerhard Schrader, results from inhibition of acetylcholinesterase in the peripheral and central nervous systems. DDT was recognized as an insecticide by Paul Hermann Müller in 1939. DDT was extremely effective in preventing the spread of malaria in developing countries. It was the chemical of choice for controlling insect populations in the United States as well. Not long after its introduction, DDT was discovered to cause eggshell thinning. Many birds didn’t reproduce effectively, and their populations diminished over time. The work and research of Rachel Carson brought this to everyone’s attention by publishing her book Silent Spring in 1962. The grassroots effort of environmental advocacy movements were instrumental in influencing the government to create the Environmental Protection Agency (EPA) in 1970. Another critically important book is Theo Colburn’s Our Stolen Future published in 1996, which brought the concept of endocrine disruption to the public and scientific forefront. The book reported that endocrine-active (or estrogen mimicking) compounds may be eliciting effects at doses considerably lower than toxicities caused by other mechanisms, and that reproductive and developmental risks can be significant. High Profile Poisonings Poisonings have continued unabated from ancient times forward. In 1978, Jim Jones, founder of the Peoples Temple, led over 900 of his followers, one-third of them children, to their deaths, by ordering them to drink a cyanide-laced punch drink in Jonestown, Guyana. An umbrella outfitted with a firing mechanism was used to administer the poison ricin into the leg of Georgi Markov, a Bulgarian dissident and writer. He died several days later. Nazi leaders such as Hitler, Himmler, and Goering committed suicide with cyanide. In 1995, five plastic bags of liquid sarin were punctured with metal-tipped umbrellas in Tokyo subway cars during rush hour releasing the deadly nerve gas. In 2004, Viktor Yuschchenko was poisoned with dioxin, resulting in severe facial disfigurement due to chloracne. In 2006, Alexander Litvinenko, a former officer of the Russian state security organization FSB died from radioactive polonium-210 poisoning. In February 2017, Kim Jong Nam, the half-brother of North Korean Leader Kim Jong Un, was assassinated at a Malaysian airport when two women rubbed his face with the lethal nerve agent VX. It is clear that poisons continue to be a weapon of choice in politics and in society. Mass Environmental Exposures, the U.S. EPA, and Environmental Legislation Years prior to the advent of a full-fledged grassroots environmental movement, various events made clear the fragility of our environment. The Donora Smog was a historic air inversion in Pennsylvania that killed 20 people and sickened 7000 more in 1948. In 1952, during the so- called Great Smog of London, over five days, more than normal coal emissions mixed with fog in a temperature inversion resulted in thousands of deaths and tens of thousands of hospitalizations. It was the Great Smog that led to passage of the 1956 Clean Air Act in the United Kingdom. In Cleveland, the Cuyahoga River is remembered as the body of water polluted from decades of industrial waste, which caught fire in 1969 (and, in fact, on earlier occasions as well). More direct cause–effect incidents involving chemicals began surfacing. Most companies created landfills for dumping chemical byproducts that accumulated from the manufacturing process. The increase in the manufacture of chemicals translated to both an increase in direct human exposure via ingestion of products kept in the home, and an increase in indirect human exposure via leaching of dumped chemicals into the ground water, air, and food supply. Love Canal in Niagara Falls, New York, was used as a dump site by the Hooker Chemical Company for over a decade. In the 1970s, long after it was capped and an entire community built on top of it, weather patterns forced chemical waste into the groundwater and at surface. The entire area was found to be contaminated with a variety of toxic chemicals, which led to a cluster of illnesses among the residents living in the area. The activism around the contamination and subsequent cleanup led to legislation that would ensure that other chemically contaminated sites would receive government funding for cleanup. The Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), commonly known as Superfund, was enacted on December 11, 1980. It authorizes the cleanup of uncontrolled or abandoned hazardous-waste sites as well as accidents, spills, and other emergency releases of pollutants and contaminants into the environment. Superfund was amended owing to the release of methyl isocyanate from a Union Carbide insecticide plant in Bhopal, India, in 1984. With an immediate death toll of some 4000, a final death toll of many thousands more, and even more victims who suffered and are still suffering lingering effects, the Bhopal disaster remains probably the worst industrial accident in history. An important law authorized by Title III of the 1986 Superfund Amendments and Reauthorization Act (SARA) is the Emergency Planning and Community Right-to-Know Act (EPCRA). It requires public records of chemicals managed at facilities and provides the EPA with the authority to work with states and localities to prevent accidents and develop emergency plans in case of dangerous releases of chemicals. The EPA’s Toxics Release Inventory (TRI), a publicly accessible online database, is an outgrowth of EPCRA. For decades in the early part of the twentieth century, one of Japan’s Chisso Corporation plants began releasing methylmercury in industrial wastewater to Minamata Bay. It bioaccumulated in the aquatic life in the Bay and was eaten by the local populace, as well as animals. With the situation not discovered until 1956, it took a severe toll on the population. Over 2000 victims suffered from severe nervous system symptoms, and many of those died. Itai- itai, another disease outbreak in Japan, was caused by cadmium poisoning from the release of large quantities of this chemical into the Jinz River from mining operations. Weak and brittle bones are among the main effects. In Seveso, Italy, an industrial accident resulted in the exposure of thousands of people to dioxin. Chloracne was among the main sequelae, and there was an excess risk of lymphatic and hematopoietic tissue neoplasms in the most exposed zones. On August 15, 1984, Lake Monoun in West Province, Cameroon, exploded in a limnic eruption, in which dissolved carbon dioxide suddenly erupted from deep lake waters, forming a gas cloud with suffocating potential. The gas killed 37 people. On August 21, 1986, an even more deadly eruption occurred at Lake Nyos killing approximately 1746 people and more than 3000 livestock. Lake Monoun, Lake Nyos, and Lake Kivu are the only known volcanic lakes in the world to have high concentrations of gas dissolved deep below the surface. The Toxic Substances Control Act (TSCA), the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the Clean Air Act, the Clean Water Act, the Safe Drinking Water Act, and the Resource Conservation and Recovery Act (RCRA) give the EPA the authority to control hazardous waste from “cradle to grave.” All have been strengthened in various ways with amendments since their initial implementation. The Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) is a European Union regulation that requires all companies manufacturing or importing chemical substances into the European Union in quantities of one ton or more per year to register these substances with the European Chemicals Agency (ECHA) in Helsinki, Finland. INTERNATIONAL ENVIRONMENTAL CONVENTIONS AND OTHER GLOBAL EFFORTS Given that toxic agents do not respect national borders, some Multilateral Environmental Agreements (MEAs) are designed to manage potentially hazardous chemicals. Three MEAs are the Basel Convention on the Control of Transboundary Movements of Hazardous Wastes and their Disposal (adopted 1989; entered into force in 1992), the Rotterdam Convention on the Prior Informed Consent Procedure for Certain Hazardous Chemicals and Pesticides in International Trade (adopted 1998; entered into force in 2004), and the Stockholm Convention on Persistent Organic Pollutants (adopted 2001; entered into force in 2004). Though most research focuses on single chemicals, we are exposed to many chemicals at a time and over time. Learning how they interact with each other in causing their effects upon organisms is a critical question. Related to this is the issue of the effects of chemicals or combinations thereof in common household products including furniture, cars, electronics, and baby products. The upward trajectory of toxicology continues unabated. Its scientific foundation is becoming more assured, precise, and relevant. Challenges will remain and include intermittent funding and political constraints. Toxicology will continue to build upon its history and build a trail of new history. A better understanding of toxicant exposures, individual and combined, and their effects upon living organisms will lead to an era when the global environment will be significantly safer and the world’s populace healthier. BIBLIOGRAPHY Hays HW. Society of Toxicology History, 1961–1986. Washington, DC: Society of Toxicology; 1986. Nunn JF. Ancient Egyptian Medicine. Norman, OK: University of Oklahoma Press; 1996:144. Wexler P, ed. History of Toxicology and Environmental Health; Toxicology in Antiquity I and II, Academic Press; 2014. Wexler P, ed. History of Toxicology and Environmental Health; 2nd Edition. Academic Press; 2018. Wexler P, ed. Toxicology in the Middle Ages and Renaissance. Academic Press; 2017. Wujastyk D. The Roots of Ayurveda: Selections from Sanskrit Medical Writings. London: Penguin; 2003:144. Yang S, ed. The Divine Farmer’s Materia Medica: A Translation of the Shen Nong Ben Cao. Boulder, CO: Blue Poppy Press; 1998. QUESTIONS Choose the one best answer. 1. Which one of the following statements regarding toxicology is true? a. Modern toxicology is concerned with the study of the adverse effects of chemicals on ancient forms of life. b. Modern toxicology studies embrace principles from such disciplines as biochemistry, botany, chemistry, physiology, and physics. c. Modern toxicology has its roots in the knowledge of plant and animal poisons, which predates recorded history and has been used to promote peace. d. Modern toxicology studies the mechanisms by which inorganic chemicals produce advantageous as well as deleterious effects. e. Modern toxicology is concerned with the study of chemicals in mammalian species. 2. Knowledge of the toxicology of poisonous agents was published earliest in the: a. Ebers papyrus. b. De Historia Plantarum. c. De Maateria Medica. d. Lex Cornelia. e. Poisons and their Antidotes. 3. Paracelsus, a physician-alchemist, formulated many revolutionary views that remain integral to the structure of toxicology, pharmacology, and therapeutics today. He focused on the primary toxic agent as a chemical entity and articulated the dose–response relation. Which one of the following statements is NOT attributable to Paracelsus? a. Natural poisons are quick in their onset of actions. b. Experimentation is essential in the examination of responses to chemicals. c. One should make a distinction between the therapeutic and toxic properties of chemicals.  d. These properties are sometimes but not always indistinguishable except by dose. e. One can ascertain a degree of specificity of chemicals and their therapeutic or toxic effects. 4. The art of toxicology requires years of experience to acquire, even though the knowledge base of facts may be learned more quickly. Which modern toxicologist is credited with saying that “you can be a toxicologist in two easy lesions, each of 10 years?” a. Claude Bernard. b. Rachel Carson. c. Upton Sinclair. d. Arnold Lehman. e. Oswald Schmiedeberg. 5. Which of the following statements is correct? a. Claude Bernard was a prolific scientist who trained over 120 students and published numerous contributions to the scientific literature. b. Ginger Jake paralysis was caused by tri-ortho-cresyl phosphate. c. An Introduction to the Study of Experimental Medicine was written by the Spanish physician Orfila. d. Magendie used autopsy material and chemical analysis systematically as legal proof of poisoning. e. Percival Potts was instrumental in demonstrating the chemical complexity of snake venoms.  CHAPTER 2 Principles of Toxicology Lauren M. Aleksunes and David L. Eaton INTRODUCTION SUBDISCIPLINES OF TOXICOLOGY SPECTRUM OF UNDESIRED EFFECTS Allergic Reactions Idiosyncratic Reactions Immediate versus Delayed Toxicity Reversible versus Irreversible Toxic Effects Local versus Systemic Toxicity Interactions of Chemicals Tolerance CHARACTERISTICS OF EXPOSURE Route and Site of Exposure Duration and Frequency of Exposure DOSE–RESPONSE RELATIONSHIPS Individual, or Graded, Dose–Response Relationships Quantal Dose–Response Relationships Dose Extrapolation Across Species Shapes of Dose–Response Curves Threshold and Linear, Nonthreshold Models Nonmonotonic Dose–Response Curves Essential Nutrients  Hormesis Endocrine Active Chemicals Assumptions in Deriving the Dose–Response Relationship Evaluating the Dose–Response Relationship Therapeutic Index Margins of Safety and Exposure Potency versus Efficacy ASSESSING TOXICOLOGICAL RESPONSES Causation in Toxicology Mechanisms and Modes of Action Adverse Outcome Pathways VARIATION IN TOXIC RESPONSES Selective Toxicity Species Differences Modifying Factors Genetics Age Sex Circadian Rhythm Microbiome TOXICITY TESTING Acute Toxicity Testing Subacute (Repeated-Dose) Toxicity Testing Subchronic Toxicity Testing Chronic Toxicity Testing Developmental and Reproductive Toxicity Mutagenicity Carcinogenicity Neurotoxicity Assessment Immunotoxicity Assessment  Sensitization Eye and Skin Irritation and Corrosion Other Toxicity Tests SYSTEMS TOXICOLOGY Transcriptome Epigenome Proteome Metabonomics/Metabolomics Exposome High-Content Screening Computational Toxicology Innovative Testing Models KEY POINTS A poison is any agent capable of producing a deleterious response in a biological system. A mechanistic toxicologist identifies the cellular, biochemical, and molecular mechanisms by which chemicals exert toxic effects on living organisms. Toxicogenomics permits identification and protection of genetically susceptible individuals from harmful environmental exposures, and customizes drug therapies based on their individual genetic makeup. A descriptive toxicologist is concerned directly with toxicity testing, which provides information for safety evaluation and regulatory requirements. A regulatory toxicologist both determines from available data whether a chemical poses a sufficiently low risk to be marketed for a stated purpose and establishes standards for the amount of chemicals permitted in ambient air, industrial atmospheres, and drinking water. Selective toxicity means that a chemical produces injury to one kind of living matter without harming another form of life even though the two may exist in intimate contact. The individual or “graded” dose–response relationship describes the response of an individual organism to varying doses of a chemical. A quantal dose–response relationship characterizes the distribution of responses to different doses in a population of individual organisms. Hormesis, a “U-shaped” dose–response curve, results with some xenobiotics that impart beneficial or stimulatory effects at low doses but adverse effects at higher doses. Descriptive animal toxicity testing assumes that the effects produced by a compound in  laboratory animals, when properly qualified, are applicable to humans, and that exposure of experimental animals to toxic agents in high doses is a necessary and valid method of discovering possible hazards in humans. INTRODUCTION Toxicology is the study of the adverse effects of chemical, biological, or physical agents on living organisms and the environment. These toxic substances include naturally occurring harmful chemicals, or toxins, as well as foreign substances called xenobiotics. Toxins are poisons that originate from plants and microbial organisms and include venoms released by animals in order to injure predators. By comparison, xenobiotics include a variety of synthetic chemicals with different intended purposes. Generally, such toxic chemicals are referred to as toxicants, rather than toxins, because they are not produced by biological systems. Toxic chemicals may also be classified in terms of physical state (gas, dust, liquid, size); chemical stability or reactivity (explosive, flammable, corrosive); general chemical structure (aromatic amine, halogenated hydrocarbon, etc.); or ability to cause significant toxicity (extremely toxic, very toxic, slightly toxic, etc.). Classification of toxic chemicals based on their biochemical mechanisms of action (e.g., alkylating agent, cholinesterase inhibitor, and endocrine disruptor) is usually more informative than classification by general terms such as irritants and oxidizers. However, more descriptive categories such as air pollutants, occupation-related exposures, and acute and chronic poisons may be useful to associate toxic chemicals that result in similar adverse events or are encountered under particular conditions. Virtually every known chemical has the potential to produce injury or death if it is present in a sufficient quantity. Table 2–1 shows the dose of chemicals needed to produce death in 50% of treated animals (lethal dose 50 [LD50]). It should be noted that measures of acute lethality such as LD50 do not accurately reflect the full spectrum of toxic responses, or hazards, associated with exposure to a chemical. For example, some chemicals may have carcinogenic, teratogenic, or neurobehavioral effects at doses that produce no evidence of acute or immediate injury. In addition, there is a growing recognition that various factors such as age, genetics, diet, underlying diseases, and concomitant exposures can account for an individual’s susceptibility to a range of responses. For a particular chemical, multiple different effects can occur in a given organism, each with its own dose–response relationship. TABLE 2–1 Approximate Acute LD50 Values of Some Representative Chemicals  A toxicologist is an individual trained to examine and communicate the nature of a toxicant’s properties and identify approaches to prevent or mitigate harm done to human, animal, and environmental health. Toxicological research identifies the cellular, biochemical, and molecular mechanisms of action of toxic chemicals and determines the extent to which these actions cause functional perturbations in critical organ systems. Using these data, a toxicologist then assesses the relationship between toxicant exposure (or dose) to the response (or outcome) and in turn the probability of an adverse event to occur. This determination requires an assessment of risk which is the quantitative estimate of the potential effects of a chemical on human and environmental health at particular exposure levels. Toxicology is a broad applied science that draws upon multiple disciplines including chemistry, biology, physiology, pathology, pharmacology, molecular biology, physics, statistics, and more. SUBDISCIPLINES OF TOXICOLOGY The professional activities of toxicologists fall into three main categories: mechanistic, hazard assessment, and regulatory. Although all three categories have distinctive characteristics, each contributes to the others, and all are vitally important to chemical risk assessment (see Chapter 4). A mechanistic toxicologist identifies the cellular, biochemical, and molecular mechanisms by which chemicals exert toxic effects on living organisms (see Chapter 3). The results of mechanistic studies have implications in many areas of toxicology. In risk assessment, mechanistic data may be useful in determining whether an adverse outcome (e.g., cancer and birth defects) observed in laboratory animals may occur in humans or that may not be relevant to humans. Mechanistic data are also useful in the design and production of safer alternative chemicals and in therapies for chemical poisoning and treatment of disease. A hazard assessment toxicologist conducts toxicity testing that provides comprehensive information for the evaluation of a chemical’s safety and to meet important regulatory requirements. A hazard is a chemical or action that causes harm, whereas the risk is the likelihood for a hazard to result in harm. Risk is determined by the extent of the exposure to the hazard. Toxicologists must be concerned with the risk posed by chemicals (drugs, food additives, insecticides, herbicides, solvents, etc.) to humans, fish, birds, and plants, as well as other factors that might disturb the balance of the ecosystem. A regulatory toxicologist has the responsibility for deciding, on the basis of data provided by descriptive and mechanistic toxicologists, whether a drug or other chemical poses a sufficiently low risk (or, in the case of drugs, a favorable risk/benefit profile) to be marketed for a stated purpose. Computational toxicologists are needed to develop and implement computer-based models to predict adverse health effects resulting from the interaction of chemicals with biological organisms. Occupational toxicologists are responsible for conducting research and making recommendations for the prevention of work-related injury and illness. Regulatory toxicologists are also involved in the establishment of standards for the amount of chemicals permitted in ambient air, industrial atmospheres, and drinking water, often integrating scientific information from basic descriptive and mechanistic toxicology studies with the principles and approaches used for risk assessment. Other specialized areas of toxicology are forensic, clinical, and environmental toxicology. Forensic toxicology is the application of analytical chemistry to toxicology. This field covers the medicolegal aspects of the deleterious effects of chemicals on animals and humans. The expertise of forensic toxicologists is used to aid in establishing the cause of death and determining its circumstances in a postmortem investigation (see Chapter 32). Clinical toxicology is the realm of medical science concerned with disease caused by or uniquely associated with toxic substances (see Chapter 33). Generally, clinical toxicologists are physicians who receive specialized training in emergency medicine and poison management. Efforts are directed at treating patients poisoned with drugs or other chemicals and at the development of new techniques to treat intoxications. Environmental toxicology focuses on the impact of chemical pollutants in the environment on biological organisms. Ecotoxicology is a specialized area within environmental toxicology that focuses on the impacts of toxic substances on population dynamics in an ecosystem. The transport, fate, and interactions of chemicals in the environment constitute a critical component of both environmental toxicology and ecotoxicology. Information from the toxicological sciences, gained by experience or research, has a growing influence on our personal lives as well as on human and environmental health across the globe. Complementary fields such as exposure science advance toxicology by studying the magnitude and duration of contact between toxic chemicals and biological organisms. Knowledge about the toxicological effects of a compound and the extent of exposure has important implications for drugs, consumer products, waste cleanup, manufacturing processes, regulatory action, civil disputes, and broad policy decisions. The growing influence of toxicology on societal issues carries with it substantial ethical, legal, and societal implications for toxicological research and testing. Recognizing the many critical roles that toxicologists play throughout society, the Society of Toxicology, a professional organization for toxicologists, has developed the Code of Ethics that frames the expected behaviors and attitudes of its members (Table 2–2). Adherence to these professional behaviors and ideals is critical for ensuring the public’s trust of the data and assessments conducted by toxicologists. TABLE 2–2 Toxicology Code of Ethics SPECTRUM OF UNDESIRED EFFECTS The spectrum of undesired effects elicited by chemicals can be broad. Some effects are harmful, whereas others are not. Prescription drugs produce a number of effects, but typically only one of these actions is intended to be therapeutic; all of the other responses are referred to as side effects. However, some of these side effects may be preferred for another therapeutic indication. Drug effects that are never desirable and are always harmful to the well-being of animals and humans are referred to as the adverse, deleterious, or toxic effects of the drug. Allergic Reactions A chemical allergy is an adverse reaction of the immune system to a chemical in response to a previous exposure to that chemical or to a structurally similar one. The term hypersensitivity is most often used to define this allergic response, but allergic reaction and sensitization reaction may also describe the situation when a prior exposure (sensitization) to the chemical is required to produce its subsequent toxic effect. Once sensitization has occurred, allergic reactions may result from exposure to relatively very low doses of chemicals. For a given individual, allergic reactions can be dose-related. For example, it is well known that the allergic response to poison ivy or poison sumac in sensitized individuals is related to the extent of the skin’s exposure to urushiol oils found in the leaves. Some sensitization reactions, such as allergic reactions to nuts, shellfish, and other foods as well as certain antibiotics and xenobiotics, may be severe and occasionally fatal. Hypersensitivity reactions are discussed in more detail in Chapter 12. Idiosyncratic Reactions Chemical idiosyncrasy refers to the abnormal reactivity of an individual to a chemical based on its genetics or other individual sensitivity factors. Idiosyncratic drug responses involve a combination of individual differences in the ability to (1) form a reactive intermediate (usually through oxidation to an electrophilic intermediate), (2) detoxify the reactive intermediate (usually through hydrolysis or conjugation), (3) mount an immune response through human leukocyte antigens (HLAs) as well as T cells, and/or (4) cause cell death. Patient-specific factors that extend beyond genetics include inflammatory stress, infection, mitochondrial dysfunction, and environmental factors. Typically, there is a delay between the time drug therapy is initiated and the clinical presentation of symptoms. Idiosyncratic reactions can occur in any organ system; however, the skin, liver, hematopoietic, and immune systems are the most often affected. Because of the life-threatening nature of idiosyncratic drug reactions, it is critical that researchers continue to identify modifying factors that initiate and/or heighten these responses. Immediate versus Delayed Toxicity The toxic effects of a chemical can develop rapidly after a single exposure or may be delayed for some time. Immediate toxicity can be observed for most chemicals, whereas delayed toxicities of chemicals may take months or years to be recognized. Because of the long latency period, decades may pass after exposure to a carcinogen before tumors are observed in humans. Reversible versus Irreversible Toxic Effects Some toxic effects of chemicals are reversible, whereas others are irreversible. The likelihood of a toxic response to be reversed largely depends on the ability of an injured tissue to adapt, repair, and regenerate. For tissues such as the liver and gastrointestinal tract that have a high ability to regenerate, many injuries are reversible. By comparison, the CNS has a much more limited ability to divide and replace damaged neurons making damage largely irreversible. Cancers and birth defects caused by chemical exposures, once they occur, are often also considered irreversible toxic effects. Therefore, it is important to understand the regenerative and reparative capacity of a target organ in order to counteract a chemical’s toxic effects. Local versus Systemic Toxicity Toxic responses are also characterized according to the proximity between the site of chemical exposure and the site(s) of molecular action. Local effects are those that occur where contact is first made by the toxicant and the biological system. Such effects are produced by the ingestion of toxic substances or the inhalation of irritant materials. By comparison, systemic effects require the absorption and distribution of a toxicant from its entry point to a distant site where the deleterious effects are produced. Most chemicals usually elicit their major toxicity in only one or two organs, the target organs of toxicity. While it may be true in some cases, the target organ of toxicity is not always the site of the highest concentration of the chemical. Interactions of Chemicals Throughout the day, an individual may contact many chemicals at any given time (in the workplace, cosmetics, medications, diet, hobbies, etc.). As a result, it is necessary to consider how various chemicals may interact with each other. Interactions may impact absorption, protein binding, receptor signaling, and the biotransformation and excretion of one or both of the interacting toxicants. Additive—An additive effect occurs when the combined responses of two chemicals are equal to the sum of the responses to each chemical given alone (e.g., 2 + 3 = 5). Synergistic—A synergistic effect is observed when the combined responses of two chemicals are much greater than the sum of the response to each chemical when given alone (e.g., 2 + 2 = 20). Potentiation—Potentiation occurs when one substance does not produce any toxicity on a particular tissue or system but when added to another chemical makes that chemical much more toxic (e.g., 0 + 2 = 10). Antagonism—Antagonism occurs when two chemicals administered together interfere with each other’s actions or one interferes with the action of the other (e.g., 4 + 6 = 8; 4 + (−4) = 0; 4 + 0 = 1). There are four major types of antagonism: receptor, chemical, dispositional, and functional. Receptor Antagonism—Receptor antagonism occurs when two chemicals that bind to the same receptor produce less of an effect when given together relative to the addition of their separate effects (e.g., 4 + 6 = 8) or when one chemical antagonizes the effect of the second chemical (e.g., 0 + 4 = 1). Receptor antagonists are often termed blockers. Chemical Antagonism—Chemical antagonism or inactivation is simply a direct chemical reaction between two compounds that produces a less toxic product. For example, chelators bind to metal ions, such as arsenic, mercury, and lead, decreasing their toxicity. Dispositional Antagonism—Dispositional antagonism occurs when the absorption, distribution, biotransformation, or excretion of a chemical is altered so that the concentration and/or duration of the chemical at the target organ is reduced. Functional Antagonism—Functional antagonism occurs when two chemicals counterbalance each other by producing opposing effects on the same physiological function, often through different signaling pathways. For example, the marked fall in blood pressure during severe intoxication with a barbiturate can be effectively antagonized by the intravenous administration of a vasopressor such as norepinephrine. In this case, the barbiturate works through GABAA receptors and norepinephrine activates α-adrenergic receptors to produce opposing effects on vascular tone. Tolerance Repeated exposure to a chemical can reduce its pharmacologic and/or toxicologic actions, a process called tolerance. Cross-tolerance occurs when structurally related chemicals cause diminished responses. Typically, days or weeks of repeated exposure are required for tolerance to occur. Dispositional tolerance occurs when the amount of chemical reaching the site of action decreases over time, leading to the reduced responsiveness of the tissue to stimulation. Chemical or cellular tolerance may result from a lower availability of receptors and/or mediators (e.g., neurotransmitters). CHARACTERISTICS OF EXPOSURE Toxicity to a biological system requires that sufficient concentrations of the “active” form of a chemical accumulate at the site of action for a requisite period of time. Whether a toxic response occurs is dependent on multiple factors: chemical and physical properties of the chemical, the exposure scenario, how the chemical is metabolized by the system, the concentration of the active form at the particular target site(s), and the overall susceptibility of the biological system to injury. To characterize fully the potential hazard of a specific chemical, one needs to know not only the type of effect it produces, and the dose required to produce that effect, but also the information about the chemical, route of exposure, and disposition. Route and Site of Exposure Toxic chemicals enter the body via the gastrointestinal tract (ingestion), the lungs (inhalation), and the skin (topical, percutaneous, or dermal). Chemicals generally produce the greatest effect and the most rapid response when given directly into the bloodstream (the intravenous route). Chemicals can also enter the body to varying degrees through other routes. An approximate descending order of effectiveness for the other routes would be inhalation, intraperitoneal, subcutaneous, intramuscular, intradermal, oral, and dermal. The vehicle, or the inert material in which the toxicant is dissolved, and other formulation ingredients can markedly alter chemical absorption after ingestion, inhalation, or topical exposure. In addition, the route of administration can influence the toxicity of chemicals. For example, a chemical that acts on the CNS, but is efficiently detoxified in the liver, would be expected to be less toxic when given orally than when given via inhalation, because the oral route requires that nearly all the doses pass through the liver before reaching the systemic circulation and then the CNS. Typically, different routes of toxicant entry into the body have been associated with certain types of exposures. Occupational exposure to chemicals most frequently results from breathing contaminated air (inhalation) and/or direct and prolonged contact of the skin with the substance (dermal exposure), whereas accidental and suicidal poisonings occur most frequently by oral ingestion. Duration and Frequency of Exposure The duration and frequency for exposure of experimental animals to chemicals is classified according to four categories: acute, subacute, subchronic, and chronic. Acute exposure refers to exposure to a chemical for less than 24 hours. While acute exposure usually refers to a single administration, repeated exposures may be given within a 24-hour period for some slightly toxic or practically nontoxic chemicals. Acute exposure by inhalation refers to continuous exposure for less than 24 hours, most frequently for 4 hours. Repeated exposure is divided into three categories: subacute, subchronic, and chronic. Subacute exposure refers to repeated exposure to a chemical for 1 month or less, subchronic for 1 to 3 months, and chronic for more than 3 months, although usually this refers to studies with at least 1 year of repeated dosing. These three categories of repeated exposure can be by any route, but most often they occur by the oral route. In situations of human exposure to chemicals, the frequency and duration of exposure are not well-defined compared to controlled animal studies; nonetheless, many of the same terms are used to describe general exposure situations. Thus, workplace or environmental exposures may be described as acute (occurring from a single incident or episode), subchronic (occurring repeatedly over several weeks or months), or chronic (occurring repeatedly for many months or years). For many chemicals, the toxic effects that follow a single exposure are quite different from those produced by repeated exposure. For example, the primary, acute toxic manifestation of benzene is CNS depression, but repeated exposures can result in bone marrow toxicity and an increased risk for leukemia. Chronic exposure to a toxic chemical may produce some immediate (acute) effects after each administration in addition to the long-term, low-level, or chronic effects of the toxic substance. Toxicokinetic studies are performed by sampling the blood or tissue at various times after exposure to determine the concentration of a chemical and better understand the influence of exposures on toxicity endpoints. Concentration-time profiles for a given chemical are influenced by the frequency of exposures. The relationship between elimination rate and frequency of chemical exposure is shown in Fig. 2–1. A chemical that produces severe effects with a single dose may have no effect if the same total dose is given in several intervals. For the chemical depicted by line B in Fig. 2–1, in which the half-life for elimination (time necessary for 50% of the chemical to be removed from the bloodstream) is approximately equal to the dosing frequency, a theoretical toxic concentration (shown conceptually as two concentration units in Fig. 2–1) is not reached until the fourth dose, whereas that concentration is reached with only two doses for chemical A, which has an elimination rate much slower than the dosing interval (time between each repeated dose). Conversely, for chemical C, where the elimination rate is much shorter than the dosing interval, a toxic concentration at the site of toxic effect will never be reached regardless of how many doses are administered. Of course, it is possible that residual cell or tissue damage occurs with each dose even though the chemical itself is not accumulating. The important consideration, then, is whether the interval between doses is sufficient to allow for complete repair of tissue damage. It is evident that with any type of repeated exposure, the production of a toxic effect not only is influenced by the frequency of exposure but also may be entirely dependent on the frequency rather than the duration of exposure. Chronic toxic effects may occur, therefore, if the chemical accumulates in the biological system (rate of absorption exceeds the rate of biotransformation and/or excretion), if it produces irreversible toxic effects, or if there is insufficient time for the system to recover from the toxic damage within the exposure frequency interval. For additional discussion of these relationships, see Chapters 5 and 7. FIGURE 2–1 Diagrammatic view of the relationship between dose and concentration at the target site under different conditions of dose frequency and elimination rate. (Line A) A chemical with very slow elimination (e.g., half-life of 1 year). (Line B) A chemical with a half-life approximately equal to frequency of dosing (e.g., 1 day). (Line C) Rate of elimination faster than the dosing frequency (e.g., 5 hours). Blue-shaded area is representative of the concentration of chemical at the target site necessary to elicit a toxic response. DOSE–RESPONSE RELATIONSHIPS Dose–response relationships are defined as the association between the amount of a toxicant administered and the extent to which changes are observed in a biological system. When describing chemical exposures, it is important to consider both the dose of the chemical administered or measured in the environment (external dose) and the amount of chemical absorbed and found at the site of biological activity (internal dose). The dose of a chemical or toxic agent can be expressed as a mass or concentration. The units of the concentration will depend upon whether the toxic chemical is found in the solid, liquid, or gaseous state. When considering the administration of chemicals to humans and animals, doses are conventionally expressed as milligrams per kilogram. Dose–response relationships are routinely divided into two types: (1) the individual dose– response relationship, which describes the response of an individual organism to increasing doses of a chemical, often referred to as a “graded” response because the measured effect is continuous over a range of doses, and (2) a quantal dose–response relationship, which characterizes the distribution of individual responses to different doses in a population of organisms. Individual, or Graded, Dose–Response Relationships Individual dose–response relationships are characterized by a continuous scale of doses that lead to an increase in the magnitude of a specific response. The graded dose–response relationship requires careful selection of a range of doses for evaluation and identification of a specific biochemical process. For example, Fig. 2–2 shows the dose–response relationship between different dietary doses of the organophosphorous insecticide chlorpyrifos and the extent of inhibition of two different enzymes acetylcholinesterase and carboxylesterase in the rat brain and liver, respectively. In the brain, the degree of inhibition of both enzymes is clearly dose-related, although the degree of inhibition per unit dose is different for the two enzymes. From the shapes of these two dose–response curves, it is evident that, in the brain, cholinesterase will be inhibited at a chlorpyrifos dose of 3 mg/kg that does not alter the activity of carboxylesterase. At higher doses of chlorpyrifos (5 mg/kg and higher), both enzymes will be inhibited, but the extent of inhibition will be greater for cholinesterase than carboxylesterase. The primary toxicological response that results from chlorpyrifos exposure is directly related to the degree of cholinesterase enzyme inhibition in the brain. Thus, clinical signs and symptoms for chlorpyrifos would follow a dose–response relationship like that for the brain cholinesterase enzyme. However, the observed response to varying doses of a chemical in the whole organism is often complicated by the fact that most chemicals have multiple sites or mechanisms of toxicity, each with their own “dose–response” relationship and subsequent adverse effect.  FIGURE 2–2 Dose–response relationship between different doses of the organophosphate insecticide chlorpyrifos and esterase enzyme inhibition in the brain. Open circles and blue lines represent acetylcholinesterase activity and closed circles represent carboxylesterase activity in the brains of pregnant female Long–Evans rats given five daily doses of chlorpyrifos. (A) Dose–response curve plotted on an arithmetic scale. (B) Same data plotted on a semi-log scale. (Data from Lassiter TL et al: Gestational exposure to chlorpyrifos: dose response profiles for cholinesterase and carboxylesterase activity, Toxicol Sci 1999: 52:92- 100.) Quantal Dose–Response Relationships In contrast to the “graded” or continuous-scale dose–response relationship that occurs in individuals, the dose–response relationships in a population are defined as quantal—or “all or none”—in nature, that is, at any given dose, an individual in the population is classified as either a “responder” or a “nonresponder.” Although these distinctions of “quantal population” and “graded individual” dose–response relationships are useful, the two types of responses are conceptually identical. The ordinate in both cases is simply labeled the response, which may be t