Abstract
Introduction
While the seeds of toxicology were planted millennia ago, we have recently witnessed a dramatic growth of the science, data generation, and risk assessment paradigms that border on logarithmic. The seventh edition of this classic text examines and details the state of the science in 39 chapters. These chapters are designed for both students and professionals. The history, current status, and projected scientific advances in areas that are key to our understanding of the components of toxicology as well as the procedures in studying adverse effects. Importantly the role of regulatory agencies is integrated into how data are developed. People of faith have texts that include the Bible, Qur’an, and Torah; toxicologists have one text that should not be left on the shelf: Hayes’ Principles and Methods of Toxicology 7th Edition. Sir Colin Berry wrote the prefaces to both Volumes I and II. The preface clarifies the reasons and basis for developing a new edition to this text. Simply stated: principles, methodologies, and assessments that are “integral to toxicology.” New or upgraded topics include hormesis, medical devices, biotechnology products, cosmetics, and personal care products (PCPs).
The Seventh edition is presented in two volumes: Volume I (Chapters 1 to 20) addresses principles and agents; Volume II (Chapters 21 to 39) reviews methods and organ-specific toxicology.
The 39 chapters provide newly minted graduate students with the tools to grasp the varied landscape represented by toxicology as well as help guide him or her to the sub-areas that are of interest. In pursuing this specialty, the issues for consideration are clearly explained. The sequence of Editions reflects the rapid growth of the science, measured parameters, and ultimate risk assessments of new chemical entities as well as established drugs, pesticides, and industrial chemicals. Some Figures from the 6th Edition are carried over to the 7th. This seventh edition is timely and welcome. While many authors characterize their chapters as an overview only, they cite numerous references that provide details supporting their positions, risk assessments, and best practices.
Overall, these two volumes are well written and provide a definitive “state of affairs” regarding the individual topics. If a toxicologist needs a “go to” reference for a new project with which he or she is not fully up to date, the relevant chapter will provide both background and current understanding of the topics/subtopics. The comprehensive list of references provides further help if needed. Cursory summaries may be provided elsewhere, but not here.
The first chapter displays an amalgam of history and clear writing. The authors do an excellent job of putting toxicology in historical perspective. This high quality is continued with all the chapters that follow.
Applying appropriate statistics in the design and analysis of studies is critically important if data generated are to make sense and thus effect the development of drugs, the safety of pesticides, and the protection of the environment. The basis of various tests and identification of potential errors are set forth. Moving a new drug entity through the discovery, testing, and regulatory process is a major challenge, both in terms of time, money, and professional resources. Important steps and pitfalls are discussed. Accurate risk assessments must include the details of exposure; the approaches to obtaining these are provided.
Conducting sound risk assessments is not a slam dunk. Components that support meaningful risk assessment are examined. Exposure is one of the elements for developing risk assessments. The role of physiologically based pharmacokinetic (PBPK) models provides sets of equations that simulate the time courses of chemicals and their metabolites in various tissues throughout the body. Advances in computational power have increased the value of PBPK evaluations.
Omics (genomics, proteomics, lipidomics, and metabolomics – to note just a few) are components that can be integrated into an approach to establishing risk. These components are reviewed and explained. Epidemiology studies target cause and effect associations but are fraught with numerous pitfalls. Both students and professionals need to understand the roles of absolute risk, relative risk, odds ratio, hazard ratio, and standardized mortality ratio in tweezing out epidemiologic “truth”.
The chapter on plant and animal poisons is particularly well-written and comprehensive. The toxicology of cosmetics and PCPs is at the forefront of alternative methods for developing risk assessments; these are discussed in depth. The array and chemical physical properties of solvents, along with their potential toxicity and methods to reduce exposure, are examined. The toxicity of essential and non-essential metals is clarified, along with the regulatory guidance, where established. Radiation and its effects on organisms have been studied extensively; in fact, the bulk of toxicological studies focus on its effects. Roger McClellan’s review of radiation toxicity combines an in-depth review with the numerous studies with which he was intimately involved. While we are not judging one chapter versus another, this one certainly stands out.
The humane care and use of laboratory animals is an important aspect of toxicological research, particularly as public perception has driven some regulatory requirements. The “Three Rs” reflecting replacement, reduction, and refinement are reviewed. There are new “novel” and “alternative” approaches to toxicology testing. Chapter 22 reviews many of these approaches and the array of governmental and regulatory organizations that weigh in on such testing.
A deep dive into modern instrumental methods reviews the equipment and mathematics that support gas and liquid chromatography, as well as other approaches (e.g., NMR, ICP, ESR, SFE, and AAS). The use of both stable and radioactive isotopes is reviewed. Chapters on both acute (single dose) and subchronic (multiple short-term doses) provide the background and mechanics of obtaining reliable (regulatory-compliant) data. Genetic and epigenetic testing procedures and the interpretation of data from respective testing methods are reviewed.
Carcinogenicity bioassays have been the bedrock of early toxicity studies. The relevance of these studies is under scrutiny. Both DNA-reactive and epigenetic carcinogens are reviewed. The stepwise actions of mutagenic carcinogens that lead to tumors are addressed. Behavioral toxicity is reviewed in Chapter 28. The aspects of motor and sensory function, related to memory and operant conditioning, are examined. In addition, modifications of the brain that can result in the appearance of tremors, decreased strength and changes to the endpoints measured in functional observational battery tests are outlined.
Section IV deals with organ- and system-specific toxicology. Dermatotoxicology reviews the skin structure and function. Determinates of percutaneous absorption and the variety of tests to characterize toxicity of compounds when applied to the skin are reviewed. Aspects of inhalation toxicity including procedures in generating test atmospheres and the methods of measuring dosing are reviewed. As with other organ systems, the authors detail the anatomy of the lungs. In addition to noting the detailed anatomy of the gastrointestinal tract, Chapter 31 reviews the digestive function and the contribution of ancillary organs. Other target organs include the liver, kidneys, cardiovascular system, and nervous system. The endocrine system is reviewed in depth as well as toxicity of both the male and female reproductive systems. Finally, the immune system is addressed by the Burlesons (a family affair) and Victor Johnson.
Considering the depth and comprehensive content, no deficiencies were identified. The only error noted was in Figure 3.14, where Winslow’s birth date was 1977 instead of the correct date of 1877. We are all familiar with Paracelsus’s “Poison is in everything, and nothing is without poison. The dosage makes it either a poison or a remedy”. This two-volume text develops Paracelsus’s adage into a comprehensive, authoritative approach that will support the work of today’s toxicologist.
Individual Chapters
History, Understanding, and a Possible Future of Toxicology: Harming and Helping through Time
This chapter starts with a Preface that clearly describes the challenges before toxicologists today. The introduction is a reminder that data and public perceptions often differ. It is our job to put the challenges posed by chemicals into an understandable perspective. The review of poisoning throughout the ages is both historical and comprehensive. The authors note many of the issues facing toxicologists today.
Use of Toxicology in the Regulatory Process
The roles of hazard identification, dose-response, exposure assessment, and risk characterization, along with the uncertainty associated with each of these components, make the role of the toxicologist challenging. The roles various governmental agencies play in the risk assessment process along with the key data that are components to deriving these risks are explained both broadly and with specific examples. The challenges of interpreting data derived from high-dose studies to apply to humans with lower exposure levels are noted. This chapter is augmented with many Tables and Charts that help visualize the data described.
Dose-Response: A Fundamental Concept in Toxicology
There are an array of approaches to examining dose-response. Historical through current models are reviewed. Hormesis, linear non-threshold, biphasic, and dose-time responses are included in the dose-response options.
Metabolism: A Determinant of Toxicity
The knowledge of how chemicals are metabolized and the effects that these metabolic pathways have on toxicological endpoints has blossomed. This comprehensive chapter discusses and distinguishes different enzymes and their effects. Illustrations are noteworthy, clearly an advertisement for BioRender.com. For students targeting the pharmaceutical industry, this chapter must be targeted for study in depth.
Toxicokinetics
Pharmacokinetics (PK) and pharmacodynamics (PD) are key components of toxicokinetics. Leanne L. Bedard deals with the principles, mathematical approaches, dosing options, sample collection and analysis, interpretation of the data, and the regulatory role that these studies play. The risk assessment process integrates both toxicokinetics and toxicodynamics so that exposure is assessed and hazards are identified. This chapter is replete with mathematical models and equations that encompass the essence of toxicodynamics. Self-assessment exercises challenge students to implement TK procedures using (fake) data.
Physiologically Based Pharmacokinetic and Toxicokinetic Models
Physiologically based pharmacokinetic (PBPK) models provide sets of equations that simulate the time courses of chemicals and their metabolites in various tissues throughout the body. This chapter reviews the history of PBPK development and its application to a variety of exposure situations for which risk assessments are sought. Computer advances with their power to implement modeling programs have helped assign specific concentrations of chemicals to different tissue compartments. Models discussed include R/RStudio, MATLAB/Simulink, GastroPlus, PK-Sim PLETHEM, Simcyp, Magnolia, and MCSim. The importance of maintaining mass balance in PBPK analysis is stressed. Examples of different models with diagrammatic input/output parameters are provided. The goal of PBPK modeling “remains simple.” This is to predict the target tissue dose of compounds and their metabolites at target tissues and, in some cases, to describe interactions in target tissues. Today’s PBPK tools that are employed to achieve this end are provided. As with other chapters, this review can support graduate courses.
Toxicopanomics: Application of Genomics, Transcriptomics, Proteomics, Metabolomics, and Lipidomics in Predictive Mechanistic Toxicology
As the authors note, “Systems toxicology is a subdivision of systems biology that aims to provide a holistic view of biological processes by using systems wide molecular measurements, which are commonly termed “omics” such as genomics, proteomics, lipidomics and metabolomics (etc.).” Design of systems toxicology studies is critical for harvesting useful data. Inputs include enunciation of key assumptions, adequate measurement methods, and statistical tools to properly interpret the results. Components of studies that support these inputs are elucidated. High-throughput sequencing; the microbiome which has more genes that contained in the human genome; and the array of proteins (our proteome) all interact and are affected by exogenous chemical insults. Tweezing the ‘bottom line’ effects of these chemicals on the integrated “omics” reality is discussed, and approaches are outlined.
Toxicologic Assessment of Pharmaceutical, Medical Device, and Biotechnology Products
Drug development is not for the faint of heart. It is resource intensive and requires a team of professionals to oversee the process. The fact that so few potential drug candidates actually emerge as marketed products underscores the need for care when moving through the various stages of development. There is an 8- to 12-year path from drug discovery to successful launch of the drug. Included in this path are non-clinical development and clinical trials (phases 1 to 3). This chapter reviews the challenges and component parts to moving New Chemical Entities through the multiple steps for FDA registration. It takes a team with expertise in toxicokinetics, clinical trials, regulatory requirements, and post-marketing oversight to ensure each step needed for drug development is checked. Those critical steps are reviewed.
Statistics and Experimental Design for Toxicologists
Gad’s update of his statistical summaries is both comprehensive and robust. The many references support an array of statistical approaches that include bias, experimental design, statistical tests, types of errors, and the ability to tweeze out statistical significance from biological significance. This chapter, alone, could be the basis for an in-depth course for toxicologists who wish to incorporate appropriate statistical methods and analysis in their studies.
The Practice of Exposure Assessment
“The Dose makes the Poison”. Clearly toxicologists evaluate the dose-response in evaluating risk to individuals and the environment. This chapter reviews the approaches to establishing dose via routes that include skin, inhalation, and oral. The mechanics of estimating exposure values is dependent on initial dose levels, and the consideration of uncertainty embedded with these measurements. Improvements in the paradigm of hazard identification, dose-response assessment, exposure assessment, and risk characterization have been made but focus on hazard and dose-response; the need for better exposure assessment is essential to further refine the toxicological end product of risk characterization. Monte Carlo techniques have helped refine exposure assessments that are not overly conservative (and thus difficult to deal with). Both intake and uptake paradigms are discussed. Definitions are provided for bioavailability, relative bioavailability, relative absorption factors, and bioaccessibility. Exposure-related tools reviewed include time-weighted average (TWA); average daily dose (LADD); average daily dose (ADD); Total Exposure Assessment Methodology (TEAM); Dietary Exposure Evaluation Model (DEEM); Cumulative and Aggregate Risk Evaluation System (CARES); Stochastic Human Exposure and Dose Simulation Model (SHEDS); Child Health Outcomes (ECHO); Exposure and Fate Assessment Screening Tool (E-FAST); Consumer Exposure Model (CEM);Multi-chamber Concentration and Exposure Model (MCCEM); and Indirect Dietary Residential Exposure Assessment Model (IDREAM). As with the other chapters, here exposure assessment is brought up to date.
Epidemiology for Toxicologists
Epidemiology attempts to associate chemical insult with risk outcome. This cause-and-effect link, however, is fraught with challenges to the uninitiated. The roles of absolute risk, relative risk, also known as the risk ratio (RR), odds ratio (OR), hazard ratio (HR), and standardized mortality ratio (SMR) in tweezing out epidemiologic “truth” from available data are reviewed. The authors discuss topics to include measures of disease frequency; study designs; measures of exposure; measures of association; bias and confounding; and causal inference. Prevalence and incidence data are distinguished. Observational studies are designated cohort, case-control, cross-sectional, and ecological; each of these is explained. Bias with its many iterations and confounders must be accounted for and addressed. The Hill guidelines are one of the keys to epidemiology studies (strength of the association, consistency, specificity, temporal relationship, biological gradient, biological plausibility, coherence of the evidence, experiment, and reasoning by analogy); each is explained. The authors stress the importance of bias when developing conclusions to epidemiology studies.
Pathology Principles and Practices for Toxicity Studies
Pathology is a specialized branch of toxicology that provides critical data for the evaluation and determination of cause and effect of exogenous chemicals on organisms. A table that presents a list of abbreviations for toxicologic pathology concepts and terms is included. Common clinical pathology analytes evaluated during routine toxicity studies are presented as well as a recommended tissue list for anatomic pathology evaluation during repeat-dose toxicity studies. Accurate necropsies are key to evaluating and interpreting pathological data. The technical keys and pitfalls of obtaining necropsy data and subsequent clinical and pathological data are outlined. The options available for staining histologic slides, depending on the target to be evaluated, are discussed. Adverse versus non-adverse changes in morphology, physiology, growth, development, reproduction, or life span that results in an impairment to maintain functional homeostasis are distinguished. Cited references (375) will fill any gaps in the author’s “broad introduction to the discipline of toxicologic pathology.”
Chemical Toxicology Information Resources
The resources for toxicologists that need to cover various issues related to physicians, governmental regulators, and workers in industry are covered here. Standardized animal testing is on the path to being replaced by the array of -omics that are emerging. In part, this is due to ethical considerations regarding the treatment of animals and – in part – due to the time and cost of such testing. Risk assessments invariably need reputable references which aid in the processes of hazard identification, dose-response, and exposure assessment; this chapter provides a path to those references (focusing on non-pharmaceutical chemicals). An array of books that may be useful to toxicologists are provided. Listed books cover Principles, Toxic Agents, Methods to Assess Toxicity, and Organ- and System-Specific Toxicology. Toxicology-related journals are listed, with those offering open access noted. Organizations, such as the Agency for Toxic Substances and Disease Registry (ATSDR) that produce review articles focused on specific areas of toxicology provide summaries of adverse effects, exposure scenarios, and other factors that shape risk assessments. ATSDR is just one of many (e.g., Concise International Chemical Assessment Document (CICAD); Australian Industrial Chemicals Introduction Scheme Public Report; Environmental Health Criteria (EHCs); European Union Risk Assessment Reports (RARs); IARC Monographs on the Evaluation of Carcinogenic Risks to Humans; Nordic Expert Group for Criteria Documentation of Health Risks from Chemicals; National Toxicology Program (NTP) Monographs; NTP Report on Carcinogens (RoC) Monographs; OECD Chemical Assessments; US EPA Integrated Risk Information System (IRIS); US EPA National Air Toxics Assessment (NATA); US EPA Office of Water Drinking Water Standards, Advisory Tables and Chemical Health Effect Documents; US EPA Office of Pesticide Programs (OPP; US EPA Office of Pollution Prevention and Toxics Chemicals Undergoing Risk Evaluations under Toxic Substances Control Act (TSCA); US EPA Provisional Peer-Reviewed Toxicity Values (PPRTV); and US EPA Risk Assessment Guidance). Resources that provide ‘bottom line’ summaries for both public and professional consumption are provided. Methods of conducting successful literature searches as well as bibliographic resources are reviewed. Finally, the advent of social media and push technology adds a finishing touch. This chapter is an essential reference for toxicologists.
Food Safety and Foodborne Toxicants
Areas reviewed for food safety include additives, supplements, pesticide residues, heavy metals, bacteria and viruses, and naturally occurring toxins. Standards for food additives, color additives, Generally Recognized as Safe (GRAS) ingredients, and New Dietary Ingredients (NDIs) are discussed. An approach to testing additives for potential use in foods is provided. Food allergies are an important aspect of foods, whether adulterated or not; a review of the mechanisms of allergies is discussed. The array of bacterial and viral-related toxins that cause sickness and, in extreme cases, death are listed. The text is augmented with a number of examples of the structures of offending toxins. The topics covered are, in short, an overview of risk assessment tasks that many toxicologists are faced with.
Plant and Animal Toxins
One aspect of interacting with the environment often focuses on the toxins present and “ready to pounce” on the unsuspecting. This chapter discusses toxins present in plants and animals and delves into the intricacies of their composure and how they trigger adverse reactions. Plant toxins include alkaloids, glycosides, proteinaceous compounds, non-protein amino acids, organic acid toxins, resinous and phenolic compounds, and mineral toxins. Toxins from cyanobacteria, along with an in-depth review of the various toxins associated with mushrooms, are surveyed. Animal toxins that are reviewed include snakes, insects, fish, and amphibia (to note a few). After reading this chapter, one might be reluctant to step outside lest they be ‘attacked’ by the many poisonous species.
Safety Assessment of Cosmetics and Personal Care Products
The authors note PCPs are expansive and diverse. They are important since they include broad population exposure, direct and intended contact, and multiple applications and uses. In addition, the issues of ethics and legal issues surrounding animal testing impact current methods for risk assessments. The bodies involved with the toxicology of cosmetics and PCPs include the Cosmetic Ingredient Review (CIR) Expert Panel, the Scientific Committee on Consumer Safety (SCCS), the Research Institute for Fragrance Materials (RIFM) and the Expert Panel for Fragrance Safety, and the Flavor Extract Manufacturers Association (FEMA) Expert Panel. An array of alternative tests are noted by the Alternative Methods International Cooperation on Alternative Test Methods (ICATM) and the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM). Approaches to obtaining adverse effects of cosmetics and PCPs due to oral, dermal, inhalation, eye, and skin exposure are reviewed. In addition, repeat dose, chronic, developmental, reproductive, cardiovascular, and carcinogenicity approaches are noted. Computational resources that are helpful alternative methods are discussed.
Solvents and Industrial Hygiene
The chemical properties, workplace exposure limits and controls, sampling methods, exposure and engineering controls, and toxicology of selected solvents are discussed. Organic solvents are used for degreasing agents, paint thinners, dry-cleaning agents, extractants, and carrier vehicles for paints, varnishes, and industrial coatings. Exposure controls are critical to minimize the adverse effects of these solvents. The chemical formulas for cyclic hydrocarbons, esters, alcohols, ketones, aromatic hydrocarbons, cyclic hydrocarbons, halogenated hydrocarbons, aldehydes, nitro-hydrocarbons, and ethers are presented and are a quick chemical reminder of these structures. The role of organizations and workplace guidance documents are discussed, including Occupational Safety and Health (NIOSH); Occupational Exposure Limits (OELs); Permissible Exposure Limits (PEL); Mine Safety and Health Administration (MSHA); National Institute for Occupational Safety and Health (NIOSH); Recommended Exposure Limits (RELs); New Chemical Exposure Limits (NCELs); Biological Exposure Indices (BEIs); Workplace Environmental Exposure Limits (WEELs); NIOSH Pocket Guide to Chemical Hazards (NPG); personal protective equipment (PPE); and Workplace Environment Exposure Limits (WEELs).
Crop Protection Chemicals: Mechanism of Action and Hazard Profiles
The hazards of pesticides are often on the minds of people. James Bus reviews the toxicity of fungicides, insecticides, and herbicides and describes their mechanism of action (when known). The Food Quality Protection Act of 1996 (FQPA) required that cumulative effect of pesticides having a common mechanism of toxicity be considered. The test guidelines for Office of Pollution Preventions and Toxics (OPPT), OPP, and OECD are enumerated. An array of fungicides (23), insecticides (17), and herbicides (30) are reviewed. The regulatory history of how EPA emerged as today’s governing authority over pesticide registrations was detailed. This chapter is required reading for pesticide toxicologists.
Metals
Metals are both dietary essentials and can be deleterious, depending on the chemical attributes and exposure levels. Tchounwou details the chemical/physical attributes of metals and their respective toxicities. Topics include essential versus nonessential metals; EPA/FDA’s risk management paradigm for metals; Minimal Risk Levels (MRLs); Regional Screening Levels (RSLs); Recommended Exposure Limits (RELs); Permissible Exposure Limits; and Maximum Contaminant Levels. Essential metals discussed include calcium, chromium, cobalt, copper, iron, magnesium, manganese, molybdenum, potassium, sodium, and zinc. Highly toxic metals: arsenic, cadmium, lead, and mercury. Toxic metals with RfDs: aluminum, antimony, barium, beryllium, boron, nickel, silver, strontium, uranium, and vanadium. Finally, metals for which RfDs have not been established: bismuth, cerium, gallium, germanium, gold, hafnium, indium, lithium, niobium, osmium, platinum, rhodium, tantalum, tellurium, thallium, tin, titanium, tungsten, yttrium, and zirconium. Generally, sources of each metal are noted, along with their adverse health effects and advisory, screening, and regulatory levels.
Radiation Toxicity
We live in “a sea of ionizing radiation” and as well-educated as toxicologists, we need to understand the sources, effects, and radiation components. This education includes the effects of the different forms of radiation (alpha, beta, and gamma); the effects of past nuclear detonations; the effects of radiation exposure; and the development of radiation countermeasures. Roger McClellan notes that writing this chapter was a pleasure as his career has been intertwined with many radiation-related projects. His personal take on many of these projects transformed this review into a very pleasurable read. The effects of radiation have been extensively investigated. As a result, there are many references noted that provide insight into the half-lives, exposure, effects, and ways to minimize adverse events. I would have liked to have a reference by Cyril Comar, professor at Cornell, included in the discussion 1 .
Humane Care and Use of Laboratory Animals in Toxicology Research
Public perception of research that uses animals is often negative; the authors deal with the key aspects of animal care, facility requirements; regulatory requirements; caging; and the many animal models that are currently in use. Traditional and nontraditional species are reviewed. A list of professional organizations that support public communications on the use of animals in research is valuable. The “three Rs” (Replacement, Reduction, Refinement) are explained and reviewed. Zoonotic diseases impact the occupational health and safety aspects of laboratory management. It turns out that “dogs” are not dogs; that is, there are more than 15 breeds to choose from when designing studies. Some endpoints are best examined with particular breeds. Similarly, various species of nonhuman primate models and their respective research targets are noted. The differences between the old world (from Africa and Asia) and new world (Central and South America) monkeys are discussed and how the respective models are used. Environmental enrichment options need to be incorporated into each study design. The authors note that there needs to be a balance between scientific needs of each study and the need to ensure humane treatment of the test animals. The 434 references cited attest to the depth of this discussion.
Novel Approaches and Alternative Models: Validation and Regulatory Acceptance of Toxicological Testing Methods and Strategies
In addition to reviewing the key elements of Chapter 21 (care and use of lab animals), this chapter delves deeper into alternative models for toxicology testing. Discussions include Institutional Animal Care and Use Committees (IACUC); the International Cooperation on Alternative Test Methods (ICATM); FAIR data (Findable, Accessible, Interoperable, and Reusable); NAMs new approach and methodologies, integrated approaches for testing and assessment (IATA), the International Agency for Research on Cancer (IARC), Quantitative Structure Activity Relationships (QSAR) tools, and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). Alternative models are applied to Skin Sensitization, Acute Skin Irritation and Corrosion, Acute Eye Irritation and Corrosion, Acute Dermal Toxicity, Acute Inhalation Toxicity, Acute Systemic Toxicity, and Acute Oral Toxicity. Epigenetic mechanisms (e.g., DNA methylation and post-transcriptional modification of histones (TPMs)) elevate these discussions to the latest research.
Modern Instrumental Methods for Studying Mechanisms of Toxicology
The “latest research into the mechanisms of toxicology” is reviewed. These include gas chromatography (GC), liquid chromatography (LC), GC with mass spectrometry, LC with mass spectrometry (LC/MS), LC with mass spectrometry/mass spectrometry (LC/MS/MS), supercritical fluid chromatography (SFC), infrared (IR) and Raman spectroscopy, ultraviolet-visible spectroscopy (US-VIS), nuclear magnetic resonance spectrometry (NMR), atomic absorption spectroscopy (AAS), inductively coupled plasma (ICP), and sample preparation technologies. The mathematics associated with analyzing detector data from the respective instruments are provided. This review is essential for developing a hands-on approach to chromatography. An example of the results of a chromatogram for silylated green tea shows the exquisite detail that emerges. The authors for various sections are noted. Other resources include electron spin resonance (ESR), the use of stable and radioactive isotopes, atomic absorption spectroscopy (AAS) and inductively coupled plasma (ICP), and supercritical fluid extraction (SFE). The reference sections are separated by different spectroscopic approaches.
Acute Toxicity
After a review of the definitions and dose-relationship for acute toxicity, the standard approaches are explained (oral, dermal, and inhalation). Eye irritation and associated tests and procedures are noted. The regulatory overview of acute studies is reviewed (classification schemes, GHS labeling, test guidelines). Mathematics used for estimating LD50s, etc. are provided. The anatomy of the eye is detailed.
Short-Term Subchronic Toxicity
Regulatory requirements for repeat-dose studies are outlined, including the requirements for Good Laboratory Practices. Study designs, data analysis, and generation of adequate reports are detailed. The objectives and types of data that can (or should) be collected for sub-chronic studies are provided. It is critical that in-life evaluations be obtained accurately; physical exams, body weight measurements, feed consumption, ophthalmologic exams, and clinical pathology are reviewed. Postmortem procedures are also included (e.g., necropsy, organ weights, and microscopic pathology).
Genetic and Epigenetic Toxicology
First came the double helix; then, we learned how the genes collectively act. Today, we know more about the role of genes and how alterations to their structure (mutations) are linked to adverse effects. The basic genetic concepts, the mechanisms of genotoxicity, and the regulations governing this research area are reviewed. Both genetic and epigenetic mechanisms are noted. These include DNA methylation, histone modifications, chromatin structure assessment, and assessment of non-coding RMAs.
Carcinogenicity Bioassays and Related Assays: Human Relevance
Rodent carcinogenicity bioassays (RCB) are reviewed, and their evolution to today’s approach to carcinogenicity assays is discussed. Topics include DNA-reactive carcinogens and epigenetic carcinogens. Examples of epigenetic carcinogens include endocrine modulators, immunomodulators, and cytotoxins. Details of how mutagenicity evolves into tumors are provided. They review the use of historical control data (HCD) and the relevance of rodent bioassays to humans. Emerging assays and alternative bioassays that employ transgenic mice are reviewed. The details of chromosomal alterations due to chemical insult are relevant to understanding the emergence of tumors.
Assessment of Behavioral Toxicity
The approaches to assessing behavioral toxicity are making significant advancement. This chapter includes reviews of motor functions, sensory function, learning, memory, schedule-controlled behavior, stimulus properties of chemicals, social behavior, and human behavioral testing. Respondent conditioning, operant conditioning, reinforcement, and punishment and extinction are reviewed. Modifications of the brain due to behavioral experience as well as observations regarding tremor, strength, gait, balance, and other components of functional observational batteries (FOB) are reviewed. Cambridge Neuropsychological Testing Automated Battery (CANTAB), a computer-automated battery of neuropsychological tests designed for accurate, sensitive cognitive assessment, is reviewed.
Dermatotoxicology
The authors (Hayes and Maibach) detail the skin structure and function. They review determinates of percutaneous absorption and responses of the skin such as neoplasticity, allergy, sensitization, irritation, and corrosion. The tests to determine adverse reactions to skin are reviewed (e.g., Draize, open epicutaneous, Buehler, FCA, optimization, split adjuvant, guinea pig maximization, local lymph node assay, and mouse ear swelling test, among others). Topics also include human irritation tests (e.g., single-application patch tests, repeat application patch tests, and exaggerated exposure irritation tests).
Inhalation Toxicology
This chapter reviews the anatomy and function of the respiratory tract, the procedures in generating and characterizing exposures to particles and gases, the design and operation of inhalation studies, and evolving approaches to assessing pulmonary responses. Omics methods are reviewed. Here, the internal and external environments interact efficiently (due to the large area represented by alveoli and the small air-blood barrier thickness). Test guidelines from EPA, OECD, and MAFF (Japan) are set forth. Aerodynamic models for pulmonary, bronchiolar, and nasopharyngeal deposition are described. The devices and facilities needed to expose animals to inhaled particles and gases are reviewed. The authors note inhalation studies are both “an art and science”. The format for listing references is not consistent with other chapters; here they are listed by name, not number.
Gastrointestinal Toxicology
The authors review the gastrointestinal immune system, digestive health, histological organization of the GI tract, control of digestive system function, enteric endocrine, and nervous functions. Details of the structures for the stomach, mouth, and digestive tract, as well as supporting organs (e.g., pancreas, gall bladder, and liver), are provided.
Hepatotoxicology
The structure and function of liver cells (hepatocytes, Kupffer cells, hepatic stellate cells, and cholangiocytes) as well as the biliary tract are outlined. The broad spectrum of hepatocyte toxicity and the models and methods for assessing toxicity are reviewed. Both non-alcohol and alcohol-induced fatty liver diseases are discussed. Cancer of the liver, the evaluation via the Bradford Hill criteria, and carcinogen modes of action are reviewed. The key role reactive oxygen species (ROS) plays in toxicity to the liver is noted. Clinical measures of liver function (via our standard blood tests) are reviewed. MiRNAs are noncoding RNAs that are endogenously expressed; their role in liver function and toxicity is reviewed. Because liver toxicity presents in a variety of ways, analysis of serum enzymes (e.g., ALP, AST, and GGT) along with other biomarkers (e.g., bilirubin, albumin, and ammonia) is useful in identifying adverse effects.
Principles and Methods for Renal Toxicology
Anatomy, physiology, and biochemistry of the kidney are introduced. Since the kidneys receive approximately 25% of the cardiac output, they are easy targets for systemic poisons. The kidneys maintain the body’s salt, water, and acid-base balance and do this through three mechanisms: glomerular filtration, tubular reabsorption, and tubular secretion. Damage to different sections of the kidneys will present differently. The presentation of urinalysis parameters, the purpose of measurement, and their link to clinical outcomes are noted. A number of experimental model systems along with their advantages and limitations are shown in Table 33.3. Advances include “kidneys on a chip” as well as creating organoids. For someone (the reviewer) who was on dialysis for 6 months and successfully got off (with a low but adequate kidney function), this chapter is a window into how lucky one can be.
Cardiovascular Toxicology Methods
A review of cardiac and vascular function and toxicity and the methodologies used in assessing adverse effects is provided. Cardiotoxicants (drugs, environmental, occupational, and endogenous) and examples of cardiovascular disease (CVD) are examined. While all organs are important for good health, a healthy heart is essential.
Neurotoxicology
The nervous system is complex; this chapter reviews the anatomy (central and peripheral nervous system), physiology (blood brain barriers), development, and the various cells of the nervous system. Illustrations of the chemoelectric transmission, descriptions of the major neurotransmitters and their receptors, and the variety of markers of neurotoxicity are discussed. The role of amyloid plaques in Alzheimer’s disease and Lewy bodies in Parkinson’s disease are reviewed. Nervous system toxicants reviewed included lead, methylmercury, cadmium, thallium, and quinine. The use of tetraethyl lead in gasoline and its eventual banning reflect the enthusiastic welcoming of new chemical entities (think: DDT) only to find adverse effects. The authors stress that the complexity of the nervous system and the array of unique vulnerability factors result that no two exposures will always result in similar outcomes.
Toxicology of the Endocrine System
The components of the endocrine system, along with their contribution to good health, are reviewed. These include the hypothalamus, pituitary, pineal, thyroid, thymus, adrenal, kidney, and pancreas glands. The key sexually related endocrine glands (ovaries in females; testes in males) are detailed. Detailed anatomy of key endocrine glands is presented. The illustrations could be “cleaner.” It was surprising to learn that the gut is the largest endocrine organ in the body. This is due to the enteroendocrine cells (EECs) that constitute approximately 1% of the GI intestinal epithelium. The presence and effects of synthetic endocrine-disrupting chemicals (compared with natural endocrine-disrupting chemicals – both exogenous and endogenous) are discussed (synthetic compounds play a minor role). A number of “pesticide active ingredients” (PAIs) have been tested for endocrine activity by EPA direction. HPV (high production volume) chemicals were also targets for endocrine-disrupting abilities. The Agency’s Endocrine Disruptor Screening Program (EDSP) resulted in many agrochemicals tested specifically for their endocrine-related effects. Both in vitro and in vivo assays for endocrine activity are reviewed. The tragedy of diethylstilbestrol (DES) resulting in clear cell adenocarcinomas of the vagina in the cervix after prenatal exposure noted how the medical establishment was slow to change directions, even in the presence of data. We live in a sea of endocrine-active chemicals; inaction in the face of data showing adverse effects of exposure to specific chemicals is unsupported.
Assessment of Male Reproductive Toxicity
Toxicity that has adverse effects on the ability to perform sexual functions has an effect that is not easily ignored. Sexual performance, in fact, takes center stage in today’s society. This chapter reviews the biology and toxicology of the male reproductive tract, the endpoints evaluated when evaluating male reproductive toxicity, testing protocols, and “tools for follow-up mechanistic assessments”. The stepwise development leading to puberty is detailed.
Female Reproductive and Developmental Toxicology
As with the male reproductive system, discussions include anatomy, physiology, and testing guidelines. The thalidomide disaster resulted in renewed interest and prevention of birth defects. Gametes contact one-half the normal number of chromosomes and, when united (one from the male; one from the female), they form a zygote that can develop into a new organism. The stages of ovulation and the hormones governing them are explained. Events during early development (from the fertilized ovum to development of the embryo) are described. The stages that are sensitive to malformation generation are examined. Of particular interest is the difference between humans and non-primates such as rats, mice, and rabbits. Study conduct and report generation are detailed, particularly as they apply to key aspects of the report (appropriate species, purity of the test article, dosing formulations, vehicle dosage selection, exposure, route, and documentation of the study).
Immunotoxicology: The Immune System Response to Toxic Insult
The immune system is complicated; the authors dissect this topic into little bites that are more easily understood. Topics include immune cells and tissues, immune mechanisms, evaluation of immunologic changes in humans, risk assessment, regulatory guidance, and future challenges. The differences between innate immune response and adaptive immune response are discussed. The complement system is a key component of innate immunity. A table of cytokines and chemokines are included. Details of test procedures are particularly helpful. The classification of the immune assessment test for humans includes CBC and differential blood count, along with IgG, IgA, and IgM levels. Acute-phase proteins (CRP), complement, and CMI (delayed-type skin test) may also be evaluated, along with cell-mediated immunity and humoral immunity. The immunoglobrulin classes (e.g., IgG, M, A, D, and E) and their biological functions are discussed.
Conclusion
This book is a “must have” for toxicology students as well as those in medical schools. While it is not a “quick read,” it is a two-volume text that should be relied upon for an excellent review of the 39 topics. Practicing toxicologists will appreciate the in-depth discussions and extensive reference lists provided. Toxicologists who have proven their expertise in the past have again proven it in the text.