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Toxicity Testing for Assessment of Environmental Agents: Interim Report (2006)

Chapter:2 Animal and In Vitro Toxicity Testing

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Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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2
Animal and In Vitro Toxicity Testing

Animals have been used as sentinels for early detection of potential risk to humans or as models to study the causes, pathogenesis, progression, and treatment of diseases. The latter use gave rise to the field of investigative toxicology, wherein animals are used as surrogates to predict possible adverse health effects to humans arising from chemical exposures. That approach is challenged by some people for scientific, ethical, and philosophic reasons, but the use of animal models to assess hazards and risks to humans continues to be the standard for protecting human health. Over the last several decades, scientists have developed standardized protocols for testing potentially hazardous chemicals to ensure sound scientific methods and generation of high-quality data that are critical for assessing human hazards and risks.

Toxicity testing in animals is conducted to identify possible adverse effects resulting from exposure to an agent and to develop dose-response relationships that allow evaluation of responses at other exposures. Toxicity tests are designed to minimize variance, bias, and the potential for false-positive and false-negative results. Those goals, however, are weighed in light of constraints on costs and other resources. The types and extent of human exposure are important considerations in designing toxicity studies for human health risk assessment. An understanding of duration, frequency, intensity, and routes of exposure and an understanding of chemical stability and possible chemical breakdown products are helpful in guiding the selection of the dosing regimen, the test medium, and the test material.

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

Animal toxicity studies conducted for regulatory submission typically are conducted in rats, mice, rabbits, and dogs with greater focus on rats. Testing guidelines generally require that common laboratory strains be used. At least three dose groups and a control group usually are required. For most toxicity tests, the U.S. Environmental Protection Agency (EPA) requires that the highest dose elicit signs of toxicity without compromising survival. EPA strongly recommends that the lowest dose not produce any evidence of toxicity. The numbers of animals required are defined in each study protocol and range from five rats per sex per dose in 28-day toxicity studies to 10 rats per sex per dose in subchronic studies to 50 rats per sex per dose in carcinogenicity assays. For developmental and reproductive studies, the litter is considered the experimental unit, and at least 20 litters per dose are required. The statistical power of a study is determined by the number of animals used and the sensitivity of the end point being evaluated.

This chapter provides an overview of consensus-study protocols developed or codified by several organizations, including EPA and the Organisation for Economic Co-operation and Development (OECD). EPA specifies the types and extent of toxicity data that it requires to make regulatory decisions regarding the risks and benefits associated with pesticide products in accordance with the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Federal Food, Drug, and Cosmetic Act (FFDCA). The specific data requirements are listed in the Code of Federal Regulations (CFR), Title 40, Subpart E, Part 158 (40CFR158). EPA also requires testing of industrial chemicals under the Toxic Substances Control Act (TSCA). EPA has harmonized the testing protocols that may be used in support of FIFRA registrations and TSCA test rules and has harmonized the guidelines with those of OECD. Appendix B of this report provides a list of EPA’s harmonized health-effects test guidelines.

OECD also develops test guidelines and guidance documents to help to characterize potential hazards associated with new and existing chemicals. The OECD document, Guidelines for the Testing of Chemicals (OECD Guidelines), is a collection of the most relevant internationally agreed-on testing methods used by government, industry, and independent laboratories (OECD 2004a). OECD publishes the guidelines to relieve some of the burden of chemical testing and assessment in multiple countries. Appendix B provides a list of OECD’s health-effects test guidelines.

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

In addition to its guidelines, OECD publishes a monograph series called guidance documents and detailed review documents that provide information on available testing methods and on how to use OECD guidelines in a testing strategy for classification of specific end points.1 They also discuss when such testing is useful or necessary, end points of concern, approaches for statistical analysis, and limitations of tests. The detailed review documents are prepared when it is necessary to assess the state of the art; they reflect a description of scientific progress, an inventory of gaps in the current set of testing guidelines, recommendations of guidelines that need updating, and proposals for developing or updating guidelines.

The specific testing requirements developed by EPA and OECD are assumed to have a sound scientific foundation and are generally accepted by interested stakeholders. As indicated, this chapter discusses the consensus protocols focusing primarily on EPA guidelines. It has been organized to present the more general toxicity tests first and then the tests designed to evaluate specific toxicity end points. Thus, the toxicity tests characterized by exposure duration—acute, subchronic, and chronic—are reviewed first; these tests are designed to gain an understanding of systemic effects, given various lengths of exposure, and can be used to guide human health risk assessment for those exposure durations. Toxicity tests designed to evaluate specific end points are discussed next and include tests for reproductive and developmental toxicity, neurotoxicity, immunotoxicity, and genotoxicity. It is important to note that some specialized end points are evaluated by various clinical measures or histopathology conducted in the exposure-duration tests. Results of general toxicity tests often indicate a need to conduct more specialized tests. The chapter concludes with a discussion of metabolism and pharmacokinetic studies. The intent of this chapter is to provide an overview of the rationale for conducting specific toxicity tests, the basic aspects of the study protocols, and the possible shortcomings of currently accepted tests. The descriptions are meant not to be exhaustive but simply to provide a context for evaluating toxicity-testing strategies. Detailed descriptions of study protocols can be found in the cited references.

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

TOXICITY TESTING CHARACTERIZED BY EXPOSURE DURATION

Acute Toxicity Testing

Acute toxicity tests evaluate the adverse effects of short-term exposure and are considered by EPA to be an “integral step in the assessment of [a chemical’s] toxic potential under the regulatory framework of its pesticide and toxic substances programs” (EPA 1998a). To be considered an acute exposure, dosing may be done once or may be done several times within or continuously throughout a 24-hour period, but use of a single dose is by far the most common method. The test animals, typically rodents (rats or mice) are observed for a period of several days to 2 weeks after dosing, and observations of deviant behavior, growth, or mortality are recorded. Historically, the primary focus of an acute toxicity test was to determine a chemical’s median lethal dose (LD50), the dose that causes death in 50% of the test animals. Today, acute toxicity tests are used also to determine dosing regimens for longer-term toxicity tests and to evaluate more fully the effects of acute exposure.

Acute testing protocols have evolved over the years to conserve animal use, to minimize the pain and discomfort of the test animals, and to obtain more information on the pathogenesis of toxicity. If a chemical is judged to have low toxicity, a limit test is first conducted. The limit test is a sequential test that uses a maximum of five animals with a starting test dose of 5,000 mg/kg (EPA 1998b). If three or more animals survive, the LD50 is considered to be greater than 5,000 mg/kg, and no further testing is conducted. If the substance proves to be more toxic than expected (that is, three or more animals die), the primary test recommended by EPA to assess acute oral toxicity is the up-down procedure (UDP) (EPA 1998b). The UDP uses one animal per exposure, and the animals are dosed sequentially at 48-hour intervals. The first animal is dosed a step below the best estimate of the LD50. If the animal survives, the second animal receives a dose that is higher by a factor of 3.2; if the first animal dies or appears moribund, the second animal receives a dose that is lower by a factor of 3.2. This process continues until death is observed or an upper bound is reached (usually 2,000 or 5,000 mg/kg). EPA has developed a software program that incorporates the data obtained from the UDP to calculate the LD50 and the confidence interval.

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

Although the preceding discussion focused on oral exposure, the route most relevant to potential human exposure (oral, inhalation, or ermal) is typically evaluated. Acute testing protocols are available for inhalation and dermal exposure (EPA 1998c,d). EPA has developed toxicity categories on the basis of LD50 or median lethal concentration (LC50) values (see Table 2-1). OECD (2001a) has a similar ranking system. EPA uses the categories to determine precautionary labeling requirements, personal protective equipment requirements, and restrictions on entry into pesticide-treated areas.

Acute toxicity data have benefits beyond toxicity ranking. Acute studies reveal whether frank toxicity is sudden, delayed, time-limited, or continuous. The time to onset and resolution of toxicity can provide insight into the time course of absorption, distribution, and clearance of a toxicant. Acute toxicity data can provide some idea of relative bioavailability by comparing data on various routes of exposure and can provide information on clinical signs potentially relevant for physicians who are treating patients and for scientists who are developing hypotheses about pathogenesis and target organs affected by acute exposures. That is especially important because toxic effects of acute exposure are often different from those of prolonged lower-level exposure. As discussed in greater detail in Chapter 6, acute toxicity tests can be redesigned to provide additional information on more subtle effects than lethality and gross clinical signs. One particular end point that has received increasing attention is cardiovascular toxicity, specifically adverse effects on ion channels in the myocardium that lead to abnormalities in the electrocardiogram, namely prolongation of the QT interval. Changes in the QT interval have been linked with cardiac arrhythmia that can progress to more serious cardiac events, including failure. However, the link has not yet been proven, and many believe that more research is needed on an alternative indicator of cardiac arrhythmia. The pharmaceutical industry

TABLE 2-1 EPA Acute-Toxicity Categories

Study

Category I

Category II

Category III

Category IV

Oral LD50

≤50 mg/kg

>50-500 mg/kg

>500-5,000 mg/kg

>5,000 mg/kg

Dermal LD50

≤200 mg/kg

>200-2,000 mg/kg

>2,000-5,000 mg/kg

>5,000 mg/kg

Inhalation (4-h) LC50

≤0.05 mg/L

>0.05-0.5 mg/L

>0.5-2 mg/L

>2 mg/L

 

Source: EPA 1998a.

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

does evaluate effects on cardiovascular function of potential drug candidates as part of its regulatory process; EPA does not have formal guidelines for evaluating cardiovascular toxicity.

Acute toxicity studies are based on the assumption that acute toxicity and lethality in animal models are relevant to humans. For many chemicals, the experience in humans is inadequate to confirm that assumption, but enough examples support it to continue this mode of hazard assessment. However, dose extrapolations from animals to humans are not simple: smaller rodents generally have a far greater rate of metabolism than do humans and therefore clear a chemical more rapidly, reducing total exposure relative to dose. Extrapolations therefore use plasma or tissue concentrations, an adjustment or uncertainty factor, or, as a surrogate for metabolic rate, doses calculated on the basis of body surface area or a quantity equal to body weight raised to the ¾ power. Moreover, metabolic and biologic differences sometimes lead to responses in animals or humans that are absent in the other species, termed species specificity. Knowledge of species differences in toxic responses is critical for extrapolating from animal data to human risk.

The scientific consensus remains that assessment of acute toxicity can help scientists to evaluate and manage the risks associated with potential exposure to noxious agents. Acute toxicity tests provide at least one relatively quick and inexpensive tool in testing schemes that screen large numbers of chemicals and identify chemicals that warrant further toxicity testing.

Subchronic or Repeated-Dose Toxicity Testing

Subchronic studies evaluate the adverse effects of continuous or repeated exposure over a portion of the average life span of experimental animals. They provide information on target-organ toxicity and bioaccumulation potential and are designed to determine no-observed-adverse-effect levels (NOAELs), which are used to establish standards or guidelines for human exposure. Subchronic studies are not designed to assess effects that have a long latency period, such as cancer, but do provide information that can be used in setting doses for chronic toxicity and carcinogenicity studies.

The exposure durations for subchronic studies are typically 28 or 90 days (see Appendix B for a list of EPA and OECD guidelines). Administration of the chemical (oral, inhalation, or dermal) is usually deter-

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

mined by the route of potential or actual human exposure. Depending on exposure duration, animals are often observed for 2 or 4 weeks after the end of treatment for reversibility, persistence, or delayed occurrence of adverse effects.

In 90-day studies, 20 rodents (10 of each sex) or eight nonrodents (four of each sex) are used for each dose group and the control group. Additional animals are included in the control and high-dose groups if satellite groups are used to evaluate effects after termination of treatment. In some cases, the shorter-term studies are conducted with fewer animals, such as five rats per sex per dose, and may evaluate fewer measures than the 90-day studies.

Typically, doses in subchronic studies are selected to define a dose-response relationship. The lowest dose should produce no adverse effects, the highest dose should induce toxic effects without compromising survival or inducing severe suffering, and the intermediate dose should produce a gradation of effects. A control group is also included. When a dose of 1,000 mg/kg per day in oral or dermal studies or 1 mg/L in inhalation studies is not toxic, further dosing above these quantities is not required. Oral dosing occurs daily if test material is incorporated in food or water or 5 days/week if the test material is administered by gavage (the method typically used for rodents) or capsule (typically used for dogs). In inhalation studies, exposure is usually conducted for a period of 6 hours/day for 5 or 7 days/week. Test guidelines require measurement and evaluation of a number of parameters, including clinical signs (such as changes in skin, fur, eyes, secretions, gait, posture, and response to handling), motor activity, grip strength, sensory reactivity to stimuli, body weight, food consumption, clinical pathology (clinical chemistry and hematology), and ophthalmology. At study termination, a gross necropsy is conducted on all animals, and selected organs are weighed. A full histopathologic analysis is conducted on all animals in the control and high-dose groups, on all animals that were killed or died during the study, and on all gross lesions. Target organs are examined in all animals. Statistical methods are used to evaluate the data.

Subchronic studies can provide initial or definitive data for risk-assessment purposes. However, the studies are sometimes limited by the smaller sample size, which reduces the sensitivity of the study to detect adverse effects. They often provide the basis of dose selections for longer-term studies, including chronic toxicity and carcinogenicity studies.

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

Chronic Toxicity and Carcinogenicity

The purpose of chronic toxicity testing is to determine the cumulative adverse effects of repeated daily oral, dermal, or inhalation exposures of test animals to various doses of a chemical for at least 12 months (EPA 1998e). The purpose of carcinogenicity testing is to determine the cumulative neoplastic effects of repeated daily oral, dermal, or inhalation exposures to various doses of test chemicals over most of the life span of the test species (EPA 1998f). EPA provides separate guidelines for chronic toxicity and carcinogenicity, but testing is most often combined for these two end points (EPA 1998g).

EPA guidelines (EPA 1998e) for chronic toxicity specify that “testing should be performed with two mammalian species, one a rodent and the other a nonrodent. The rat is the preferred rodent species and the dog is the preferred nonrodent species.” Other species can be used with adequate justification. Dose selection is generally based on results of a 90-day study; the highest dose should be the one that causes only mild signs of toxicity and does not alter the length of the study. The intermediate dose is chosen to produce a gradation of toxic effects, and the lowest dose should produce no evidence of adverse effect and thus should allow determination of a NOAEL. At least three dose groups and a control group should be included with 40 rats (20 of each sex) or eight dogs (four of each sex) in each group. EPA guidelines state that body weights and food consumption should be measured and that clinical pathology (hematology, clinical chemistry, and urinalysis) should be conducted at specified intervals during the study. At the end of the study, all animals should be subjected to gross necropsy, weights of major organs should be determined, and all gross lesions and tissues and organs of the digestive system, nervous system, glandular system, respiratory system, cardiovascular and hematopoietic system, and urogenital system should be preserved for histopathologic examination. Ophthalmologic examinations are also recommended. A full histopathologic analysis should be conducted on all controls and animals in the high-dose group and on gross lesions. If exposure-related changes are detected, the analysis is extended to all treatment groups (EPA 1998e).

Carcinogenicity bioassays are conducted with rodents, typically rats and mice, for a minimum of 24 months (rats) and 18 months (mice) and are designed to provide data for cancer-hazard identification and dose-response evaluation. Dose-selection guidelines are similar to those for

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

the chronic toxicity studies; however, group sizes are larger (50 rodents of each sex per group), and clinical pathology involves examination of blood smears. At the end of the study, gross necropsy and histopathology are extensive because the primary focus is on detecting neoplasms. The National Toxicology Program (NTP) has conducted over 600 lifetime cancer bioassays and has been at the forefront of developing definitive guidelines for detecting carcinogenic activity in rodents; the carcinogenicity data obtained reside in a public database.

EPA guidelines for combined chronic toxicity and carcinogenicity testing (EPA 1998g) combine testing for chronic toxicity and carcinogenicity summarized above. In a combined test, the two species typically used are rats and mice—rats mainly for dosing by oral and inhalation routes and mice for the dermal route. Three dose groups and a control group are used, and at least 100 animals (50 of each sex) are used for each group. Additional animals—at least 20 (10 of each sex)—are included at each dose and in the control group as satellite groups for determination of chronic toxicity after 12 months; end points similar to those described for the chronic toxicity test are used. The minimal duration of daily exposure is 2 years for rats and 18 months for mice, and end points similar to those described for the carcinogenicity test are examined at the end of the study.

Considerable effort is being devoted to developing alternative transgenic and knockout animal models for carcinogenicity testing in Europe and the United States. The goal is to develop models that will increase the sensitivity of detection of carcinogenic lesions and shorten the time for their appearance; the latter would have the effect of conserving the resources required to test each agent and increase the number of agents that can be tested. Some of the efforts are being coordinated through the International Conference on Harmonization (ICH) Expert Working Group on Safety. That group, in collaboration with the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute (ILSI), conducted an evaluation of six animal models for their ability to detect the effects of a group of 21 chemicals, which included genotoxins and carcinogens. The results of those efforts were discussed at a workshop (Cohen et al. 2001) and presented in a special issue of Toxicologic Pathology (Vol. 29, supplement issue, 2001), which also presented detailed information on the models. The conclusion drawn from the evaluations was that some of the models might have use in hazard identification, providing information similar to that obtained from the 2-year combined chronic toxicity and carcinogenicity bioassay,

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

in conjunction with data from other sources in a weight-of-evidence approach to risk assessment. Determination of the usefulness of the models is still limited by the amount of comparative data available. Considerable effort is being devoted to broadening the comparison of tumor data from transgenic mouse strains and strains of mice traditionally used in lifetime bioassays.

TOXICITY TESTING CHARACTERIZED BY SPECIFIC END POINT

Toxicity testing of most chemicals begins with acute testing, progresses to subchronic testing, and, depending on the results, concludes with chronic testing. Evaluation in those studies may indicate the need to obtain more information on specific toxicity end points. The following sections discuss the tests used to evaluate reproductive and developmental toxicity, neurotoxicity, immunotoxicity, and genotoxicity. In vitro tests for cytotoxicity and other end points are also briefly discussed.

Reproductive and Developmental Toxicity

Reproductive and developmental toxicity testing includes a broader category of end points than other kinds of toxicity testing because of the multiple stages of exposure and the variability of possible effects. Exposures of sexually mature animals can result in sterility or decreased fertility by depleting or affecting ova or sperm or by affecting endocrine functions of organs involved in reproduction. If fertilization occurs, abnormalities of ova and sperm can result in embryonic death, failure of implantation, congenital malformations, embryonic growth retardation, genetic disease, or cancer in the offspring. Exposures during pregnancy can result in embryonic or fetal death, congenital malformations, reversible or irreversible growth retardation, or premature or delayed parturition; they may also have delayed postnatal effects, such as cancer, neurobehavioral effects, growth retardation, and death. Toxicant exposures of neonatal, immature, or adolescent organisms may result in growth retardation or stimulation, endocrine abnormalities, immunologic deficits, neurobehavioral effects, cancer, or death.

The general purpose of reproductive and developmental toxicity assays is to evaluate the competence of breeding pairs to produce pheno-

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

typically normal offspring. All or most of the reproductive cycle is evaluated. Four types of reproductive and developmental studies are discussed here—screening-level reproductive-toxicity assays, prenatal developmental-toxicity and teratology studies, generational tests, and reproductive assessment with continuous breeding. These assays are conducted because of their assumed relevance for predicting human hazard potential, but the data from such models may or may not be relevant for predicting human risk. Thus, the predictive power of the tests may be limited by differences in the underlying biology. A famous example of how species differences can be important is developmental exposure to thalidomide, to which rats are highly resistant and humans are exquisitely sensitive.

The assays described are apical tests—complex experiments that measure complicated end points, each of which is an integrated measure of multiple facets of the machinery necessary for successful reproduction and development. Apical tests provide little insight into the hundreds of molecular events, mechanisms, and targets responsible for toxicant action. Although they are useful for determining whether there is an overall effect, the lack of mechanistic insight is an important limitation. Future advances in testing will probably rely on our ability to discern the individual biologic underpinnings of toxicity, a complicated task in this setting.

Screening-Level Reproductive-Toxicity Assays

In these assays, animals are dosed with the test chemical for at least 2 weeks before mating and then for a maximum of 2 weeks of breeding. The females are dosed through gestation, and the test is terminated on postnatal day 4. The measurements made provide insight into gonadal function, fertility, pregnancy, parturition, and prenatal and postnatal developmental toxicity. OECD testing guidelines (TG) 421 and 422 (OECD 1995a, 1996) are reproductive and developmental screening tests; however, TG 422 (OECD 1996) is a combined repeated-dose toxicity study in combination with the reproductive and developmental screening test. These are screening-level assays used to make decisions about the need for further testing as part of the OECD screening information dataset (SIDS) program.

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×
Prenatal Developmental-Toxicity and Teratology Studies

The prenatal developmental-toxicity study (OECD 2001b) is used to examine embryonic and fetal toxicity as a consequence of exposure during pregnancy (for example, growth retardation, anatomic variations, teratogenicity, and lethality). Young mature virgin females are artificially inseminated or mated. The time of mating is noted, and groups of pregnant animals are either untreated or treated with three different doses of the test agent. In OECD TG 414 (OECD 2001b), the treatment is given from the time of implantation to scheduled cesarean section. If preliminary studies do not indicate a high potential for preimplantation loss, treatment may be extended to include the period from mating to the day before the scheduled cesarean section. The day before expected birth, the uterus is removed by cesarean section, and the uterus and fetuses are examined. If dosing is initiated before or at the time of implantation, preimplantation loss is evaluated.

Generational Tests

The prototypical reproductive-toxicity assay is the one-generation test (OECD 1983), although this test is not included by EPA in its test guidelines. The test chemical is administered to young adult rats of both sexes (generally to breeding pairs) during a prebreeding period covering one spermatogenic cycle and the last two stages of oocyte maturation and during mating. Dosing of females continues through pregnancy and nursing (3 weeks after birth). Pups are evaluated from birth through weaning, and birthweight, postnatal growth, survival, litter sizes, and sex distribution are recorded. Adult males typically are killed after the mating period, and sperm production and quality are assessed. Reproductive organs of both sexes of the parental generation are assessed grossly and histologically. The assay includes assessment of gonadal function, estrous cycling, mating behavior, fertility, parturition, and lactation in the parental animals and prenatal and postnatal development in the offspring. The assay has been modified over time, most recently to include end points that are sensitive to endocrine-disrupting chemicals.

The results of the one-generation reproductive-toxicity test in rodents are often used in risk assessment. In chemical regulation in Europe, it is part of a tiered testing system in which a two-generation test

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

may follow the one-generation test. The two-generation test is most often triggered when a specified production volume is reached. The ICH has published a guideline on reproductive-toxicity testing of medicinal products. The guideline describes a flexible design in which a reproduction and fertility test comparable with the one-generation reproductive-toxicity test can be run as a stand-alone assay or as part of the developmental-toxicity and perinatal and postnatal assessment of new drugs. Although there are differences from the OECD one-generation protocol, the general principle of the test is comparable.

The design of the two-generation test (OECD 2001c) is in principle similar to that of the one-generation test, but the first generation of offspring (F1) is followed through sexual maturation and the production of a second generation. The treatment of the parental generation is equivalent to that in the one-generation test. Data on sperm quality and estrous cycling in the offspring are collected, and the offspring are observed for developmental milestones, including some behavioral measures and histopathologic characteristics of sex organs, brain, and other potential target organs. Direct dosing of the F1 animals begins at weaning and is continuous through the end of the test. On reaching sexual maturity, these animals are bred. The F2 generation is evaluated through weaning. The data collected are similar to those in the one-generation study. The two-generation test is considered the appropriate test for reproductive toxicity, and the OECD test guideline has recently been updated to reflect the scientific state of the art (OECD 2005). EPA lists only the two-generation test as its reproductive toxicity assay (EPA 1998h).

Reproductive Assessment by Continuous Breeding

The protocol for reproductive assessment by continuous breeding has been conducted almost exclusively by the NTP. Breeding pairs co-habit for an extended period (14 weeks), during which they are continuously exposed to the test agent. Each litter produced is examined and then discarded. This study design allows the determination of the maximal number of litters that can be produced. If effects on fertility are noted, additional study legs can be run to determine which sex is affected and to generate hypotheses regarding the mechanism of toxicity. The test can also be extended into a two-generation-like protocol. The assumptions and uncertainties are similar to those of the one-generation

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

test. This study design has not been required as part of the regulatory assessment process for pesticides, but the data are useful.

Neurotoxicity

Neurotoxic effects in animals and humans can be assessed with a wide array of methods, including neurochemical, anatomic, physiologic, and behavioral. For example, neurochemical effects of an agent can include selective effects on synthesis, reuptake, release, or metabolism of specific neurotransmitters. Anatomic changes can include alterations of the cell body, the axon, or the myelin sheath of neurons or of the thickness of cell layers in specific brain regions. At the physiologic level, a chemical might reduce the speed of neurotransmission or change the thresholds for neural activation. Behavioral alterations can include changes in sensations of sight, hearing, or touch; alterations in simple or complex reflexes and motor functions; alterations in cognitive functions, such as learning, memory, and attention; and changes in a wide array of psychologic and social behaviors (WHO 2001).

Regulatory agencies consider data from required animal toxicology studies and effects reported in the published literature when evaluating the neurotoxic potential of chemicals. This section briefly summarizes the types of neurotoxicity evaluations that are conducted in animals and recommended in toxicity-test guidelines by EPA and OECD (see Appendix B for a summary of the guidelines). More comprehensive reviews of the available neurotoxicity tests have been published by the National Research Council (NRC 1992), EPA (1998i), the World Health Organization (WHO 2001), and OECD (2004c).

Neurotoxicity Testing in Standard Toxicity Studies

Neurotoxicity evaluations required by EPA and OECD guidelines for standard acute, subchronic, and chronic toxicity tests include detailed clinical observations, functional tests, and histopathology. Detailed clinical observations are made outside the home cage, preferably in a standard arena, and at similar times on each occasion. Observations should include evaluation of skin and fur, eyes, and mucous membranes; respiratory and circulatory effects; autonomic effects, such as salivation;

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

central nervous system effects, including tremors and convulsions; level of activity; gait and posture; reactivity to handling or sensory stimuli; altered strength; stereotypies; and bizarre behavior, such as self-mutilation and walking backward. Observations should be detailed and carefully recorded, preferably with scoring systems explicitly defined by the laboratory. Toward the end of the repeated-dose standard adult toxicity studies, functional tests are required, including an assessment of motor activity, grip strength, and sensory reactivity to stimuli of different types, such as visual, auditory, and proprioceptive stimuli. Brain weight and histopathologic characteristics are required. Specifically, multiple sections of the brain should be examined (including cerebrum, cerebellum, medulla, pons, and pituitary), as should sciatic and tibial nerves close to muscle, specimens of three levels of the spinal cord (cervical, midthoracic, and lumbar), and eyes, including the retina and optic nerve. The clinical and functional end points mentioned here can be altered by specific and nonspecific effects on the nervous system, especially at maximum tolerated doses that can cause substantial systemic toxicity.

The standard developmental study (EPA 1998j) includes gross pathologic assessment of the nervous system. Two-generation reproduction studies (EPA 1998h) are used to evaluate clinical signs of toxicity and brain weight in offspring. Those evaluations can provide an initial indication of potential neurotoxic effects after postnatal exposures but are much more limited than those required in the standard adult toxicity studies.

Adult Neurotoxicity Studies

OECD TG 424 (OECD 1997) and the EPA neurotoxicity screening battery (EPA 1998k) are similar. Both include detailed clinical observations or a functional observational battery in the home cage and open field; functional tests, including assessments of motor activity, grip strength, and reactivity to sensory stimuli; and neuropathologic examination of perfusion-fixed tissues. Adult neurotoxicity studies require functional tests and clinical observations similar to those in standard toxicity studies but require perfusion-fixed tissues, more frequent measurement of functional tests, and observations to be conducted without knowledge of treatment level.

In general, OECD and EPA neurotoxicity tests may be required when there are structure-activity concerns or when neurotoxic effects

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

have been revealed in standard toxicity, reproductive, or developmental studies. The OECD neurotoxicity TG recognizes the redundancy of standard toxicity studies, and TG 424 encourages flexibility to minimize the number of end points that merely duplicate those of standard repeated-dose toxicity studies.

Developmental Neurotoxicity Studies

The developmental-neurotoxicity study protocol (EPA 1998l) is designed to develop data on the potential functional and morphologic hazards to the nervous system in offspring of mothers exposed during pregnancy and lactation. OECD (2004b) has developed similar draft guidelines for a developmental-neurotoxicity test. The EPA guidelines require that pregnant females be dosed from gestational day (GD) 6 through postnatal day (PND) 10. Recently, EPA extended the dosing period from GD 6 through PND 21 (that is, until weaning). Motor activity is measured repeatedly on PND 13, 17, 21, and 60. Auditory-startle habituation is measured around weaning and on PND 60. Auditory-startle habituation, as conducted in this study, is primarily a measure of reactivity to repeated loud bursts of noise (for example, 120 dB for 10 msec). A test of learning and memory is also required around weaning and on PND 60. The EPA developmental-neurotoxicity test guidelines (EPA 1998l) require periodic clinical observations of the dams and pups with standardized procedures by trained technicians who are unaware of the animals’ treatment. Interobserver reliability is required if more than one observer is used in a given study. The EPA guidelines (EPA 1998l) require detailed neuropathologic evaluation, including measurements of immersion-fixed brain taken on PND 11. EPA allows neuropathologic evaluation at PND 22 because the exposure period was extended through PND 22, but the pups should be perfusion-fixed. At the termination of the study (usually on PND 60), the pups are perfusion-fixed, and the central and peripheral nervous systems are evaluated according to the EPA neurotoxicity-screening battery guideline with the additional requirement of measurements of the brain.

An important limitation in using the rat model for developmental-neurotoxicity testing is that there are important species differences in brain development relative to birth. In general, brain development in rats from birth to about PND 11 is roughly equivalent to brain development in human fetuses during the third trimester of gestation (Rice and Barone

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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2000). Thus, it could be difficult to experimentally reproduce potential environmental exposure to the human fetus in rat developmental-neurotoxicity studies. For example, exposure through maternal rat milk may be substantially different from human in utero exposures in terms of relative amounts of parent compound and metabolite. But direct dosing of pups during early lactation may not necessarily reflect human in utero exposure after dietary, dermal, or inhalation exposure to pregnant females. Measurement of parent compound and toxic metabolite in milk and evalution of biomarkers of exposure or effect after exposure can potentially be used in physiologically based pharmacokinetic and pharmacodynamic models to characterize dose-response relationships and improve extrapolation of results from animal studies to humans (Dorman et al. 2001). Although studies of lactational or placental transfer could prove valuable for interpreting toxicity-study results, such study designs are less well established under conditions in which human data will not be available, the mode of action is not well characterized, and the toxic component has not been identified. Therefore, an ILSI-HESI committee evaluating use of pharmacokinetic and metabolism data for developmental and developmental-neurotoxicity testing recently concluded that more experience is needed on how best to carry out these studies before pharmacokinetic data on fetus, offspring, and maternal milk are routinely required (Barton et al. 2005).

The developmental-neurotoxicity test is one of the most logistically difficult EPA-guideline tests to conduct and requires specialized expertise in neurobehavioral testing and morphometric analysis. Historical control data from laboratories conducting EPA developmental-neurotoxicity guideline studies can be variable, particularly at the earlier times of PND 13 and 17 (Raffaele et al. 2003, 2004; Sette et al. 2004). Some of the variability could be reduced by improving environmental experimental conditions and methods, as discussed by Garman et al. (2001) and Cory-Slechta et al. (2001). However, the variability may also be due, in part, to normal variability during a period of rapid development, to the practical definition of birth date that could span 23 hours and does not take into account gestational age, and to the practical necessity of testing large numbers of animals over several hours during the day and across multiple days of testing depending on when the pups were born (Li 2005).

Initial analysis of the relative sensitivity of developmental-neurotoxicity testing compared with other standard end points and studies indicates that, in general, the developmental-neurotoxicity study is

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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not more sensitive than chronic bioassays and other reproductive-developmental end points but can provide additional characterization of potential neurotoxic effects after developmental exposures (Makris et al. 1998; EPA 1999; Middaugh et al. 2003).

Specialized Studies for Neurotoxicity Testing

EPA has developed six test guidelines for neurotoxicity testing (see Appendix B). The neurotoxicity-screening battery (EPA 1998k) and developmental-neurotoxicity study (EPA 1998l) were discussed above. The delayed-neurotoxicity test is typically required for organophosphorus substances and includes behavioral, histopathologic, and neurochemical assessments in the hen (EPA 1998m). The remaining guidelines for schedule-controlled operant behavior (EPA 1998n), peripheral-nerve function (EPA 1998o), and sensory-evoked potential (EPA 1998p) are outlined briefly in this section. A more comprehensive review of neurotoxicity end points can be found in EPA’s neurotoxicity risk-assessment guidelines (EPA 1998i).

The test guideline for schedule-controlled operant behavior requires that subjects be trained until they display demonstrable stability in performance before exposure. This guideline is designed to evaluate performance of a learned behavior and not learning or memory itself. EPA states that substances that have been observed to produce neurotoxic signs in other toxicity studies (such as central nervous system depression or stimulation) and substances that are structurally similar to neurotoxicants that affect performance, learning, or memory may be appropriate to evaluate with this test. Although schedule-controlled operant behavior testing may be useful to more fully characterize the potential neurotoxicity of a chemical, it has not been found to be more sensitive than a functional observational battery or an assessment of motor activity in several independent laboratories for different classes of chemicals (Moser et al. 2000).

The peripheral-nerve test function is used to evaluate peripheral-nerve conduction velocity and amplitude in anesthetized animals with electrophysiologic techniques. EPA indicates that substances that have been shown to produce related effects in other studies (such as neuropathologic changes in peripheral nerves) and substances with a structural similarity to those causing peripheral neuropathy may be appropriate to evaluate with this test.

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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The test for sensory evoked potentials is also an electrophysiologic test that evaluates the effects of chemicals on brain electric potentials after stimulation of the visual, auditory, or somatosensory system. The test is recommended if there is reason to believe that particular sensory functions are specifically sensitive to the test compound.

In contrast with EPA, OECD did not develop specific test guidelines for more specialized neurotoxicity evaluations. Instead, OECD developed a guidance document for neurotoxicity testing that provides general descriptions, references, and commentary for a wide variety of behavioral, neurologic, neurochemical, neurophysiologic, and morphologic techniques (OECD 2004c). The OECD neurotoxicity guidance document emphasizes an “iterative” testing strategy that includes an evaluation of the degree of concern about the neurotoxic effects and the possible concentrations to which people may be exposed to determine the adequacy of the existing data to evaluate potential risk.

OECD indicates that initial animal data for neurotoxicity assessment are most often provided by standard single-dose (OECD 1981, 1987, 2001d,e,f) or repeated-dose toxicity studies (OECD 1995b, 1998) in which functional histopathologic information is gathered on all major organ systems, including the nervous system. If information indicates possible neurotoxic effects, OECD recommends that additional end points be included in the initial standard tests to obtain in-depth information about specific neurotoxic effects. However, OECD cautions that the decision to add specific end points to the initial study should take into consideration the potential for confounding toxicologic effects of the higher doses usually required in the initial studies. For example, excessive systemic toxicity at doses near the maximum tolerated dose can cause indirect effects on the nervous system that confound interpretation of tests of learning and memory.

In summary, a tiered or iterative approach to neurotoxicity testing is being used by OECD and EPA. Initial evaluation of the nervous system can be obtained with standard toxicity studies, which are typically conducted over a range of doses that include the maximum tolerated dose. OECD encourages an iterative approach that includes a determination of whether the neurotoxic effects and exposure assessment provide sufficient data for an assessment of risk (OECD 2004c). The available information on the test chemical and on structurally related chemicals can be used to guide selection of additional neurotoxicity tests.

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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Immunotoxicity

The immune system is responsible for defending the body against infection by viruses, bacteria, and other disease-producing microorganisms. It also plays a role in cancer surveillance, destroying cells that have become transformed and might otherwise develop into tumors. For the immune system to recognize the wide array of pathogens that are present in the environment, it relies on many cell types that play mutually supporting roles in generating an immune response. Those cells arise from stem cells in the bone marrow and thymus and are found throughout the body in lymphoid tissues and in the blood as white blood cells. Because of the complexity of the immune system, assessing the influence of chemical exposure is a complicated and difficult task.

Two main types of immunotoxic responses may result from xenobiotic exposure: immunosuppression (in which one or several specific functions of the immune system are inhibited, and the inhibition results in increased susceptibility to infections or tumors) and antigenicity (in which the immune system recognizes the xenobiotic as foreign and mounts an immune response to it). Antigenicity is more commonly known as allergy, which is often referred to as hypersensitivity even though it constitutes a normal immune response to a foreign substance. Contact hypersensitivity is a relatively common cutaneous immunotoxic response that depends on the sensitization of T lymphocytes and leads to the development of inflammatory lesions in the skin on re-exposure to the sensitizing chemical. Alternatively, IgE-antibody-mediated allergic responses occur immediately on re-exposure to a sensitizing chemical, and symptoms depend on the route of exposure to the xenobiotic. If the exposure occurs by inhalation, asthmatic conditions, such as wheezing or pulmonary congestion, may arise; if the exposure is oral, there may be an intestinal reaction of diarrhea or vomiting; and systemic effects may include decrease in blood pressure, vessel leakage, or shock.

A third, less studied immunotoxic effect of xenobiotic exposure is autoimmunity. In this situation, the xenobiotic may act as a partial antigen (a hapten) and create a new antigen by binding to tissue protein. If the immune system recognizes the new tissue-associated protein as foreign, an immune response to the tissue is generated and results in an autoimmune response and, if it is persistent, autoimmune disease.

Potential immunosuppressive effects are often identified during subchronic toxicity testing. Whole and differential blood-cell counts,

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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lymphoid organ weights, and histopathologic examination of the spleen, thymus, lymph nodes, and bone marrow can indicate effects on the immune system. However, those end points alone may not always be predictive of immunotoxicity (Luster et al. 1992, 1993). Because test animals are routinely maintained under very clean housing conditions, the immune system can essentially be considered to be “resting” unless specifically challenged with antigen. Therefore, a specific immune response must be induced to assess the ability of a xenobiotic to cause immunosuppression.

The functional immune test recommended by EPA is the in vivo antibody response to sheep red blood cells (SRBCs) (EPA 1998q). The test is done in mice and rats unless the pharmacokinetic data are similar in both species, in which case either species may be used. The animals are exposed to test and control compounds for at least 28 days and are then immunized with SRBCs. At the end of the exposure period (4-5 days after SRBC injection), either a plaque-forming cell assay to measure the number of B cells making anti-SRBC antibody (immunoglobin M, IgM) or an enzyme-linked immunosorbent assay (ELISA) to measure serum anti-SRBC IgM is performed. Additional or followup tests may include flow cytometry to evaluate subpopulations of T and B cells if there is substantial suppression of the anti-SRBC response or a functional test for natural killer cells to assess the chemical’s effect on nonspecific immunity (EPA 1998q). The Food and Drug Administration (FDA) also recommends the SRBC assay as a test for immunosuppression if pharmacokinetic studies indicate that an investigational new drug or its metabolites concentrate in immune tissues (FDA 2002).

Immunotoxicity testing for contact hypersensitivity has been conducted for many years; guinea pigs have been the model of choice. The guinea pig maximization test (GPMT) and the Buehler test (BT) have been validated for use in screening for skin sensitizers. The BT and GPMT involve delivery of a test substance or vehicle to the skin of guinea pigs either topically (BT) or by intradermal injection with and without Freund’s adjuvant (GPMT). In both tests, skin inflammatory reactions are graded and recorded after challenge doses of the test substance.

The expense and technical difficulties associated with guinea pig tests led to a concerted effort among interested parties to develop and validate a mouse model that could replace the guinea pig tests. The mouse local lymph node assay (LLNA) has now obtained regulatory acceptance for screening for skin sensitizers The LLNA detects proliferation of lymphocytes in lymph nodes on application of potential skin sensitizers to the ears of mice. The test affords a quantitative measure of

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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cell proliferation as a function of incorporation of radioisotope into DNA of dividing lymphocytes. The LLNA is considered a refinement of traditional guinea pig skin sensitization assays because it minimizes pain and distress of animals and uses fewer animals (EPA 2003; FDA 2002; NTP 1999). Alternatively, the mouse ear swelling test may be used to detect moderate to strong sensitizers; if an agent is positive, it may be designated a potential sensitizer without further testing in guinea pigs (EPA 2003).

Tests for immunotoxicity are also outlined in guidelines for microbial-pesticide toxicity studies (EPA 1996). For those agents, EPA considers reporting of observed allergic responses of humans to microbial pest-control agents (MPCAs) sufficient to address potential health concerns about allergy or hypersensitivity. If there is a potential for a virus-containing MPCA to cause immunodeficiency in mammals, specific followup tests are prescribed case by case.

Genotoxicity

Genotoxicity refers to adverse effects on DNA, genes, and chromosomes, and genetic toxicology is the branch of toxicology that studies those effects. There is concern about such effects because many human diseases are of mutational origin. Down, Klinefelter, and Turner syndromes are the most frequently encountered chromosomal aberrations in humans, and retinoblastoma (a gene mutation) occurs in one of 20,000 births (Flamm et al. 1977). Brusick (2001) provides information on the effects of mutagens on the human gene pool.

The purpose of conducting genotoxicity studies is to identify agents that have the potential to alter DNA. Originally, the focus of genetic toxicology was on whether the effects could be transmitted to future generations. To evaluate that possibility, methods were developed to determine the transmissibility of genetic damage to progeny. Those methods used whole animals, primarily rats or mice. Over the years, the concern about what the effects mean for potential carcinogenicity has increased. One could argue that most studies as now practiced are directed toward evaluating carcinogenic potential (FDA 2000a). As a result, more in vitro testing for genotoxicity is done. Animal studies, however, remain a part of test guidelines of EPA, FDA, and other government agencies and organizations.

Test methods in genetic toxicology are categorized according to their ability to detect gene or point mutations; chromosomal effects, such as breaks, gaps, translocations, and aneuploidy (loss or gain of one or

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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more chromosomes); or other DNA damage, as can be indicated by unscheduled DNA synthesis (Herbold et al. 2001). There are over 50 specific tests of genetic toxicity; they are conducted in bacteria, yeast, fruit flies, mammalian cells, and whole animals. Table 2-2 lists representative genotoxicity tests, which include both in vitro and whole-animal tests, particularly in the case of chromosomal abnormalities. Only the tests that are used most often for regulatory purposes are discussed here.

With the exception of the heritable-translocation assay and the dominant-lethal assay, the tests discussed here have a common limitation. They provide information only on carcinogenic or mutagenic potential (that is, hazard identification) and are less useful for providing quantitative information needed for risk assessment. That glaring deficiency is most apparent when one considers how to assess the risks to humans from chemicals that cause gene mutations. The only assay that can be used is the specific locus assay conducted in mice. However, that assay is not typically used, because it is costly and requires the use of large numbers of animals.

Gene or Point Mutations

A variety of methods that use bacteria or mammalian cells have been developed. Because of costs, few studies are conducted in vivo. Most gene-mutation tests involve in vitro single-cell systems, and only in vitro tests that are used most frequently are discussed here.

Gene mutations are usually single-base or base-pair alterations in DNA. Tests measuring those effects may be subdivided into tests that detect reverse or forward mutations. In general, the most sensitive tests for detecting gene mutations are the reverse-mutation methods, such as the bacterial reverse-mutation tests that use Salmonella typhimurium and Escherichia coli (EPA 1998r). The sensitivity of those organisms has been increased by modifications that allow chemicals to penetrate their cell walls more easily or prevent the DNA-repair process. The modifications have increased the ability of tests to detect mutagens. Some scientists view them as less relevant to the human situation and thus not very useful for providing quantitative information needed for risk assessment. However, these tests are extremely useful for hazard identification, and most scientists do not doubt the utility of single-cell gene-mutation systems for screening chemicals as the first steps in a tiered-testing approach. Positive test results are thought to indicate the potential muta-genicity of a chemical in both animals and humans. The S. typhi-

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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TABLE 2-2 Representative Genetic Toxicology Tests

Point or Gene Mutation

Chromosomal Aberration

DNA Damage

Salmonella microsome assaya

Human lymphocyte cellse

E. coli pol A (W3110/P3478)i

E. coli WP2uvrApKM101b

Chinese hamster ovary cellsd

Bacillus subtilis rec (H17/M45)i

Mouse lymphoma L5178Yc

Mouse lymphoma L5178Yc

Rat liver primary cellsj

Chinese hamster ovary cellsd

Rodent bone marrow cellsf

 

 

Dominant-lethal assayg

Heritable-translocation assayh

aMaron and Ames 1983.

bEPA 1998r.

cClive et al. 1979.

dPreston et al. 1981.

eDean and Danford 1984.

fKilian et al. 1977.

gGreen et al. 1985.

hGeneroso et al. 1980.

iLeifer et al. 1981.

jWilliams 1977.

murium assay has also been shown to be useful for detecting mutagenic carcinogens.

Other in vitro systems that can be used to detect gene or point mutations are mouse lymphoma, Chinese hamster ovary, and Chinese hamster V79 cells (EPA 1998s). The mouse lymphoma test is also capable of detecting chromosomal aberrations. With most in vitro methods, metabolic-activation capabilities should be added to allow detection of chemicals that require activation to produce mutagenicity.

Chromosomal Aberrations

Chromosomal aberrations may be thought of as structural or numerical alterations of chromosomes. After exposure to a clastogen (an agent that causes structural alterations), breaks or gaps in the continuous structure of the chromosome can be viewed with a microscope. Clasto-

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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gens are thought to cause major harm to DNA. Mammalian cells in culture—such as Chinese hamster fibroblasts, human or animal peripheral lymphocytes, and cells from whole animals—can be used (EPA 1998t). In animals, structural alterations can be observed in somatic cells, such as peripheral blood cells, and in germ (spermatogonial) cells. The value of assessing structural chromosomal damage is in the corroboration of an agent as a potential mutagen, that is, producing not only gene mutations but also chromosomal alterations. In addition, results of in vitro testing can be verified in vivo with the same end point (chromosomal aberrations), and this is not easily done in the case of gene mutations. It must be recognized, however, that not all chemicals that produce gene mutations produce structural chromosomal alterations (Zeiger 1998), and some human carcinogens, such as asbestos (Jaurand 1997), do not produce gene mutations but do cause chromosomal mutations.

Another type of structural alteration is formation of micronuclei. Micronuclei can be defined as cytoplasmic chromatin-containing bodies that are formed when acentric chromosomal fragments or whole chromosomes lag during anaphase and fail to become incorporated into daughter-cell nuclei during cell division (FDA 2000b). The mammalian red-blood-cell micronucleus test detects chromosomal fragments in bone marrow and other tissues. It can also detect numerical alterations (aneuploidy) (FDA 2000b; Hayashi et al. 2000).

Chromosomal aberrations in somatic cells have implications for carcinogenicity, whereas aberrations in germ cells imply transmission of effects to future generations. A number of whole-animal tests are used to detect chromosomal aberrations. The more prominent are the rat or mouse bone marrow cytogenetic assay (EPA 1998u), the rat or mouse dominant-lethal test (EPA 1998v), and the mouse heritable-translocation test (EPA 1998w). The dominant-lethal and heritable-translocation tests detect only chromosomal abnormalities that are produced in male germ cells. The bone marrow and dominant-lethal tests are usually used in the second tier of toxicity testing, and the heritable-translocation test in the third tier.

DNA Damage

Tests that detect DNA damage do not detect actual mutations. They yield indirect evidence of mutagenicity in that they detect an interaction with DNA that has affected the repair process. E. coli and B. subtilis (EPA 1998x) and such mammalian cells as liver cells are usually

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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used. In bacteria, cell survival differs between a strain that is capable of DNA repair and one that is not. The mutagen should cause more cell killing in the strain devoid of repair. In mammalian cells, a process called unscheduled DNA synthesis measures the repair (synthesis) of DNA at a period other than the usual S phase of the cell cycle (Herbold et al. 2001). The S phase is the stage of the cell cycle in which normal DNA synthesis occurs. Non-S-phase synthesis is thought to occur when the cell is undergoing the excision repair that has been induced by the interaction of a chemical with DNA. Tritiated thymidine is incorporated into the DNA of cells undergoing unscheduled DNA synthesis, and the resulting radiolabeled nuclear grains are counted. A substantial increase in the number of grains in the treated cells signifies an effect. The effect is indirect (that is, it does not detect a mutagenic event) and is usually viewed as indicating potential mutagenicity in mammalian cells. The method is used primarily as a screening assay at the first level of a tiered approach.

In Vitro Tests for Cellular Toxicity and Other End Points

Thousands of chemicals are synthesized or brought to market each year, and conventional toxicity testing has not kept up with the pace of development. The cost of animal testing is high; it may exceed several million dollars per substance. In addition, there are concerns about animal welfare because testing uses a large number of animals. The development of in vitro model systems to evaluate the toxicity of chemicals and drugs and potentially to reduce overall costs and animal use is receiving increased attention.

In vitro model systems, in general, have been used for two primary purposes: to gain a better (perhaps mechanistic) understanding of chemical-induced toxicity in animals and possibly humans and to serve as rapid screening systems for the toxicologic evaluation of chemicals, which may complement in vivo toxicity testing or may replace some in vivo models if scientifically validated and accepted by regulatory agencies. Commonly used in vitro models for assessing chemical toxicity include perfused organ preparations, isolated tissue preparations, single-cell suspensions, and cell-culture systems, such as primary cell cultures and mammalian cell lines. Of these in vitro models, cell-culture systems have been used more often by investigators because they are reliable, reproducible, and relatively inexpensive experimental systems to assess chemical toxicity at the cellular level. In vitro tests that assess cellular

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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toxicity may be characterized as tests that measure cellular functions and tests that measure cell death. Functional assays typically measure reversible events that reflect a state of impairment or reversible cell injury or toxicity, and cell-death assays (often referred to as permeability assays) estimate the failure of the permeability barrier of the plasma membrane, which represents irreversible cell injury or loss of cell viability.

As described previously (Table 2-2), cultured human and other mammalian cells, such as lymphocytes and fibroblasts, are an integral part of EPA test guidelines for the determination of gene mutation, sister-chromatid exchange, chromosomal aberrations, and unscheduled DNA synthesis. EPA has accepted a battery of in vitro tests to evaluate the high-production-volume chemicals. In that battery, in vivo cytogenetics can be replaced by in vitro cytogenetics. In addition, nonanimal methods for assessing cellular toxicity have been used for many years by manufacturers to support inhouse decisions about product development before conducting conventional animal testing (Bruner et al. 1996). The recommendation of nonanimal methods for use in regulatory toxicology has occurred case by case since the development of the field of validation and the establishment of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and its European counterpart, the European Centre for the Validation of Alternative Methods (ECVAM) in the 1990s. In recent years, ICCVAM and ECVAM have validated (or accepted as validated) and recommended for regulatory acceptance a number of in vitro tests. As seen in Table 2-3, the validated assays have primarily been assays that evaluate cytotoxicity or cell viability. The process for validating these methods has established their strengths and weaknesses and in some cases limited their applicability to specific chemical classes.

In addition to tests that have been validated and recommended for regulatory use, there are many examples in the literature of a wide variety of mammalian cells that are used to evaluate the toxicity and efficacy of chemical agents and to investigate mechanisms of toxicity. Several examples are discussed briefly below.

The Developmental Therapeutics Program at the National Cancer Institute (DTP 2005) developed an in vitro cell-line screen to support its drug-discovery program. The system, designed to screen up to 20,000 compounds per year for potential anticancer activity, uses 60 human tumor cell lines, representing leukemia, melanoma, and cancers of the

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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TABLE 2-3 Representative Validated In Vitro Methods for Cellular-Toxicity Testinga

Method

Purpose

Cells

End Points

Validated and Recommended for Regulatory Use by

Embryonic stem-cell test

Screening assay to identify potentially embryotoxic substances and to classify chemicals into three categories (strong, weak, and not embryotoxic)

Mouse embryonic stem (ES) cells and 3T3 adult mouse cell line

Inhibition of ES cell differentiation and inhibition of ES and 3T3 cell growth

ECVAM

Micromass test

Assay to evaluate ability of substances to inhibit differentiation

Micromass cultures of rat limb bud cells

Inhibition of differentiation, viability, and growth

ECVAM

Embryotoxicity testing in postimplantation rat whole-embryo culture

Assay to identify substances that induce malformation that results in embryo toxicity

Cultured rat embryos containing 1-5 somites

Embryo morphology after 48 hours of culture

ECVAM

In vitro 3T3 NRU phototoxicity testb

Assay to detect the phototoxicity induced by the combined action of a chemical and light

3T3 adult mouse fibroblast cell line

Neutral red uptake to indicate cytotoxicity

ECVAM

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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Method

Purpose

Cells

End Points

Validated and Recommended for Regulatory Use by

EPIDERM skin-corrosivity testc

Assay to detect ability of a substance to cause skin corrosivity

EPIDERM human epidermal model system

Cell viability as determined by reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT

ECVAM (replacement); ICCVAM (screen in tiered-testing strategy)

EPISKIN corrosivity testc

Assay to evaluate skin corrosivity

EPISKIN reconstructed human epidermis

Cell viability as determined by reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT

ECVAM (replacement); ICCVAM (screen in tiered-testing strategy)

Rat skin transcutaneous testd

Assay to evaluate skin corrositivity

Skin disks prepared from rats 28-30 days old

Ability to produce a loss of normal stratum corneum integrity and barrier function, which is measured as a reduction of the inherent TER below a predetermined threshold level

ECVAM (replacement); ICCVAM (screen in tiered-testing strategy)

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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Corrositexe

Screening assay to predict skin corrosivity for classification and labeling

Reconstituted collagen membranes

Time required for a test material to pass through a biobarrier membrane (a reconstituted collagen matrix, constructed to have physiochemical properties similar to those of rat skin), and produce a visually detectable change

ECVAM (replacement) ICCVAM (screen in tired-testing strategy)

In vitro cytotoxicity test for assessing acute systemic toxicityf

Assay to select starting doses for acute oral toxi city in rodents

BALB/c3T3, normal human keratinocytes

Cell survival or viability as determined by the cell’s ability to uptake and bind neutral red dye

ICCVAM (reduction)

aInformation from the ECVAM Web site (ECVAM2005) accessed September 2005 and personal communication with ICCVAM. The validations of the assays have established their strengths and weaknesses and in some cases limited their applicability to particular chemical classes or to levels within tiered-testing strategies. The Corrositex assay has been accepted for regulatory purposes and is approved for use by the Department of Transportation.

bOECD 2004d.

cOECD 2004e.

dOECD 2004f.

eOECD 2004g.

fInformation from ICCVAM Web site (ICCVAM/NICEATM 2001) accessed October 2005.

Abbreviations: MTT, 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; TER, transcutaneous electric resistance.

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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lung, colon, brain, ovary, breast, prostate, and kidney. The aim is to rank synthetic compounds or natural-product samples for selective growth inhibition or killing of particular tumor cell lines for further evaluation.

An engineered cell line, the MCL-5 human lymphoblast line, was designed to express five human cytochrome P450s (CYPs) (Crespi et al. 1991). The human CYPs incorporated into the MCL-5 cell line are the major P450s implicated in creating toxic metabolites and include CYPs 1A1, 1A2, 3A4, 2A6, and 2E1 and epoxide hydrolase. The MCL-5 assay an differentiate between parent and metabolite toxicity in one assay by comparing the concentrations of chemicals that produce 50% inhibition of growth (IC50) in the MCL-5 cells with the IC50 values obtained in a control cell line cH2, which does not express the P450s.

Considerable effort has been devoted to the development of human skin or surface epithelium equivalents to replace animals in toxicity testing and in mechanism studies (Ponec 2002). The human tissue equivalents mimic native tissue to a high degree. They originate in human keratinocytes that are cultured on a variety of matrices and can be mixed with other cell types found in skin, including dendritic cells, melanocytes, and fibroblasts. Human three-dimensional tissue models for various epithelial tissues—including epidermal, corneal, esophageal, oral, tracheal and bronchial, ectocervical, and vaginal—have been developed and are commercially available. Those tissue equivalents form a typical multilayer epithelium and express markers of epithelial differentiation (Ponec 2002). Two such models are used in corrosivity assays that have been validated and approved for regulatory use by ECVAM and ICCVAM (see Table 2-3).

The greatest level of xenobiotic biotransformation is attributed to the liver, and considerable effort has been devoted to the development of various liver cell and tissue-culture systems for use in studies of xenobiotic metabolism and toxicity. A number of recent reviews (Guillouzo 1998; Lerche-Langrand and Toutain 2000; de Kanter et al. 2002; Groneberg et al. 2002; Gebhardt et al. 2003; Brandon et al. 2003) have covered the advantages and limitations of various in vitro liver cell systems, including isolated perfused liver, isolated hepatocytes in short-term suspension and primary monolayer culture, various liver cell lines, and liver precision-cut slice cultures. The isolated liver-perfusion system most closely resembles the in vivo liver in maintenance of tissue architecture and cell-cell interaction. Its use can reduce but not eliminate animal use. Those in vitro systems were initially developed with rodent liver in research laboratories investigating liver biochemistry, mechanisms of

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

hepatocyte growth control, the role of cell-cell interactions, xenobiotic phase I and phase II biotransformation processes, and mechanisms of toxicity. They are widely used in drug development, where the objective is to obtain information that can be reliably extrapolated to the in vivo situation with regard to metabolism, drug-drug interactions, and mechanisms of toxicity. The increased availability of human liver (liver that is not usable for transplantation but is suitable for research studies) has provided data directly relevant to human liver biotransformation capacity and allowed comparison with data generated with rodent liver. Other organs and tissues also have biotransformation capacity that may contribute to tissue-specific toxicity, and the preparation of precision-cut tissue slices from various tissues (such as lung, kidney, and intestine) for use in studying tissue-specific biotransformation and toxicity has recently been reviewed by de Kanter et al. (2002).

Although in vitro systems are being used to assess the effects of chemicals on a number of cell types, only a few of the methods have been validated and recommended for acceptance by European and U.S. groups for regulatory purposes (see Table 2-3). Future research should be directed toward the refinement of existing methods and the development of new alternatives. As the level of technologic sophistication advances, an increased understanding of cellular mechanisms of toxicity will be achieved. Although in vitro model systems are now recognized as powerful tools in toxicity testing, their full potential remains largely undeveloped.

MECHANISTIC TESTING: METABOLISM AND PHARMACOKINETIC STUDIES2

Metabolism studies are used to identify potential biotransformation products after administration of test chemicals to experimental animals and to examine the potential for accumulation of compound or metabolites in tissues with repeated dosing. Pharmacokinetic (PK) studies can evaluate the time course of changes in concentrations in the body, persistence of radioactivity after dosing with radiolabeled compounds, or accumulation with multiple dosing. The EPA test guidelines for metabolism and pharmacokinetic studies (EPA 1998y) require a suite of studies

2  

Together, metabolism and pharmacokinetic studies have traditionally been referred to as ADME (absorption, distribution, metabolism, and excretion) studies.

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

to be submitted for review under FIFRA for pesticides. EPA requires development of data on metabolite formation, absorption, distribution, biotransformation, and excretion. The data are intended to aid in understanding mechanisms of toxicity and in determining whether animal toxicity studies are adequate for testing toxicity concerns associated with plant metabolites of the pesticide that might be present on raw agricultural products. The guidelines discuss two tiers of tests: a required core group of studies (tier 1) and a followup group (tier 2).

The tier 1 studies are more closely linked to specific requirements in the guidelines. In general, the guidelines expect tier 1 studies to satisfy regulatory requirements for biotransformation and PK data. Tier 1 tests use a radiolabeled test chemical to evaluate material balance, identify metabolites, and assess distribution and persistence of the radiolabel in various tissues. A single low-dose study is necessary with at least four young adult male animals, typically rats. The rationale for selection of dose route, specific dose rate, and animal sex needs to be described in the final report. By using radioactivity, the studies assess net behavior of the administered radiolabel rather than any specific characteristics of the parent chemical or important metabolites. The metabolism portion requires identification of all metabolites that constitute more than 5% of the original dose, from which the study report should propose a schematic of the pathways of metabolism.

Tier 2 studies should be designed to answer questions about chemical disposition on the basis of tier 1 results or other toxicity-test results. Any tier 2 study would be conducted according to agreement between a registrant and EPA, and considerable flexibility is allowable in tier 2 studies. Possible tier 2 studies identified in the test guidelines are evaluation of the extent of absorption, tissue-distribution time courses, plasma kinetics, and enzyme induction. The latter two still rely on radioactivity rather than speciation of the label or tracking of the parent chemical by chemical-specific analytic methods. The guidelines for tier 2 studies encourage physiologically based pharmacokinetic (PBPK) modeling and development of data needed for structured PK models.3

3  

PK models describe the time course in relation to model parameters that account for chemical movement and reactions within the body and elimination. In PBPK models, time-course behaviors are directly related to blood flows, tissue solubility, metabolic constants, and routes and processes of elimination. PBPK models have been developed to facilitate extrapolations required in human health risk assessments by predicting tissue doses of toxic compounds under a variety of exposure conditions in test animals and people, thereby allowing pre

Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

The guidelines also note that PBPK studies with parent chemicals might be submitted in lieu of other tier 1 studies if it is determined that PBPK studies would satisfy the metabolism and pharmacokinetics guidelines. Despite the encouragement, the guidelines do not define the studies required to generate datasets to support development of PK or PBPK models for any specific compound or class of compounds (that is, there are no formal guidelines for PK or PBPK model development). Current test guidelines for metabolism and PK studies will need to be revised to produce data more useful for PBPK model development. For example, the collection of data on radioactivity does not characterize the kinetics of specific forms of the compound (parent compound and major metabolites), and studies of individual compounds are needed for PBPK model development.

In general, the EPA guideline specifies a minimal dataset without providing a strong basis for evaluating alternative test strategies that might provide results more pertinent to human health risk assessment. The emphasis on measurement of radioactivity without speciation into specific compounds limits the value of the kinetic data collected in accordance with the guideline. Nevertheless, the studies are useful for assessing biotransformation products, for inferring the role of the parent chemical and metabolites in specific toxic responses, and for getting a sense of the overall pharmacokinetics of a compound in a test animal.

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Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

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Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
×

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×

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×

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Suggested Citation:"2 Animal and In Vitro Toxicity Testing." National Research Council. 2006. Toxicity Testing for Assessment of Environmental Agents: Interim Report. Washington, DC: The National Academies Press. doi: 10.17226/11523.
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