Appendix DExperimental Animal and In Vitro Study Designs

Prenatal Developmental Toxicity

The prenatal developmental toxicity study provides information on the effects of repeated exposure to an agent during pregnancy (OECD 2000a; EPA 1998a; FDA 2000). It is normally conducted in two species, a rodent (usually rat) and a nonrodent (usually rabbit), although not all guidelines specify nonrodents. Animals are exposed to an agent, usually via ingestion or inhalation, during the period of major organogenesis. The protocols include exposure to the end of gestation in order to cover developmental events that occur later in gestation (e.g., central nervous system, skeletal growth, sexual differentiation). Offspring are delivered by cesarean section on the day before the expected day of parturition, and a maternal necropsy is conducted, including examination of the uterus for number of implantations, resorptions, fetal deaths, and live fetuses. Corpora lutea in the ovaries are also counted. Live fetuses are weighed and examined carefully for external, visceral, and skeletal malformations and variations. Although the terminology used for malformations and variations has been variable from laboratory to laboratory, attempts have been made at standardization (Wise et al. 1997).

Developmental Neurotoxicity

The objective of developmental neurotoxicity studies is to assess the potential of an agent to affect neurodevelopment (EPA 1998c). The protocol is designed to be used either as a separate study, usually as a follow up to other studies, or as part of a multigeneration reproduction study. A test agent is administered at a minimum of three dose levels to pregnant animals in groups that are large enough to produce 20 litters per dose group from day 6 of gestation through day 10 postnatally (the first half of lactation). (This is the minimum exposure period. Dosing can be continued throughout lactation or, in the context of a multigeneration study, dosing is done daily over two generations.) Pregnant and lactating dams are assessed for clinical signs of neurodevelopmental effects and for their performance in a functional observation battery. Litter sizes can be adjusted by random selection to provide equal numbers of male and female offspring (usually four of each). Offspring are randomly selected from litters for neurotoxicity evaluation, including gross neurological and behavioral disorders, motor activity, response to auditory startle, learning and memory, brain weight, and neuropathological examination. Motor activity is studied on postnatal days 13, 17, 21, and 60 ± 2. Auditory startle tests are conducted on postnatal days 22 and 60 ± 2. Learning and memory are evaluated in the offspring around the time of weaning (postnatal day 21) and again in adulthood (postnatal day 60 ± 2). Neuropathology is examined in the offspring on postnatal day 11 and at the termination of the study. The neuropathology analysis includes simple morphometric measurements of brain areas.

Although these studies are designed to specifically assess the effects of developmental exposures on nervous system structure and function, they are limited in the extent to which this complex system can be evaluated as part of routine testing. For example, assessment of social and reproductive behavior and condition (such as anxiety) are not included, different types of learning and memory–such as spatial and sequential learning, reference and working memory, or the effects of recall delay–are not assessed, and long-term effects of developmental exposures (beyond 60 days (d)) are not evaluated. Several efforts are under way to evaluate the utility of such protocols and to improve the methods used in rodent studies so that they are more comparable to those used in humans.

In Vitro Assays

Any developmental toxicity assay that uses a test subject other than a pregnant mammal falls under the general heading of an “in vitro assay.” Examples include isolated whole mammalian embryos in culture, nonmammalian embryo culture, and tissue, organ, and cell culture. Several manipulations are possible using in vitro assay systems that are not possible using pregnant mammals, such as the removal of the maternal environment, the removal or transplantation of specific tissues and cells, and the ability to track specific cells and molecules, to genetically alter cells, or to monitor embryo physiology.

There are two potential applications for in vitro assays: screening for developmental toxicity and analyzing mechanisms of normal and abnormal development. In vitro assays to screen chemicals for potential developmental toxicity have been under development for approximately 15 years with the idea that they could be used to assess larger numbers of chemicals than can be evaluated with in vivo developmental toxicity tests in mammals, could reduce the number of experimental animals used in those tests, and could be used to reduce the costs of testing large numbers of chemicals. A number of attempts have been made to validate in vitro assays for screening chemicals, and efforts are under way to validate the rodent embryo culture, micromass, and stem cell assays in a European-sponsored trial (Spielmann et al. 1998), and the frog embryo teratogenicity assay (Xenopus) in an interlaboratory comparison (Fort et al. 1998). Validation requires certain considerations in study design, including defined endpoints for toxicity, an understanding of the procedure's ability to respond to chemicals that require metabolic activation, and the accuracy of the test's response to chemicals that cause developmental toxicity or no effect in whole animal studies (Kimmel et al. 1982; Kimmel and Kochhar 1990; Schwetz et al. 1991). Since most in vitro systems involve an interruption in normal metabolism and the biological interrelationships found in the intact system, the range of developmental effects that can be produced and the power of the study to detect an effect are compromised as compared to those obtained using standard study designs in whole animal systems (Kimmel 1990). For these and other reasons, in vitro developmental toxicity assays are unlikely to be used alone to screen chemicals for risk assessment purposes when there is no prior knowledge about the potential for developmental toxicity. In the case of priority-setting in early drug or chemical development, such assays may be useful for eliminating those with toxicity that can be detected in these systems, leading to further development of those with little or no toxicity, with the expectation that standard in vivo assays would be conducted before actual marketing. In vitro screens also may be useful for assessing the developmental toxicity of chemicals or chemical classes for which there is already some information about toxicity from in vivo studies for the purpose of describing the relative toxicity (potency) of members of chemical families. If chemicals are likely to act through a common mechanism, a Single in vitro screen that is sensitive to a particular mechanism may predict the relative potencies of a class of chemicals. For example, an in vitro mouse limb bud cell screen has been used successfully to rank the relative teratogenic potential of a large series of synthetic retinoids (Kistler 1987). In addition, in vitro assays may be useful for studying complex mixtures for synergism or antagonism, and for evaluating the cumulative risk of two or more chemicals that have similar mechanisms or effects.

In vitro assays have become widely used for mechanistic studies in developmental toxicology (Harris 1997). An advantage to using in vitro assays for such studies is that they utilize decreasing levels of biological complexity to isolate specific developmental processes. In vitro assays are useful for identification of tissue sites of accumulation, initial biochemical insults, gene expression changes, structure-activity relationships, and disrupted developmental pathways. It is important to link the information developed in these assays to the whole tissue and organism events that are seen as a result of developmental toxicity in order to be most useful for risk assessment purposes. Such information can be employed in developing biologically based dose-response models for developmental toxicity (e.g., Shuey et al. 1994).

Male Reproductive Toxicity

Expressions of male reproductive toxicity can involve alterations in the male reproductive organs or in related endocrine systems. Such alterations can include changes in sexual behavior (mating behavior, libido, erection, intromission, ejaculation), onset of puberty (delayed physical and behavioral development), fertility (achieving conception within a defined period), pregnancy outcome (production of normal quality and number of offspring), reproductive organ structure and morphology, reproductive endocrine parameters (including peptide and steroid hormone control), or other functions that compromise the integrity of the male reproductive system.

Female Reproductive Toxicity

Female reproductive toxicity includes adverse effects on reproductive organs and related endocrine systems. Endpoints that reflect toxicity include sexual behavior (receptivity to the male at appropriate times in the cycle), age at onset of puberty, fertility (the ability to produce offspring in normal number), gestation length, parturition, lactation, loss of primordial follicles, and age at reproductive senescence.

Single-Generation Reproduction Study

The single-generation test can provide useful information on basic reproductive function (OECD 1983). It also provides information on the effects of subchronic exposure of peripubertal and adult animals.

In a single-generation reproduction study, males and females can be exposed during the same or separate trials to determine whether one or both sexes are affected. Daily dosing of male laboratory animals should begin when they are 5 to 9 weeks (wk) old and continue for 10 wk (for rats) or 8 wk (for mice) before the mating period. This schedule exposes the animals to an agent for the duration of one complete cycle of spermatogenesis (approximately 70 d in rats and 56 d in mice). Daily dosing of females should begin when they are 5 to 9 wk old and continue for at least 2 wk (OECD 1983) before mating. Females should continue to receive daily doses of the test agent throughout the 3-wk mating period, during pregnancy, and until offspring are weaned. At least three dose groups and one control group are usually included. Either one male to one female or one male to two female matings can be used, resulting in group sizes of at least 20 females and 10 or 20 males. The goal is to produce a minimum of 20 pregnant females per treatment group. Animals that have not mated or that remain infertile should be separated and studied for the cause of their infertility.

Animals are allowed to litter normally and rear their progeny until weaning. Optionally, by the removal of some pups, the litters can be standardized (normally on day 4 postpartum) to include an equal number of pups of each sex (Agnish and Keller 1997). It is considered inappropriate to remove only runts or any other deviant animals. Adjustment of litter size is not possible when there are fewer than eight animals per litter. The major advantage of the standardization of litter size is the diminished variability of pup and litter data, because dams have equal lactational challenges and pups have similar possibilities for growth. The disadvantages of standardization have been documented extensively elsewhere (Palmer 1986; Palmer and Ulbrich 1997) and include the disruption of the normal distribution of litter sizes; standardized litter sizes that are below the natural mean, median, and modal values normally observed for most rat and mouse strains; the elimination of large numbers of offspring that normally would survive; the introduction of human bias in selection; and the raising of mean body weight of pups and the lowering of the challenge to the lactating ability of the dam.

The animals are observed daily throughout testing. Toxic effects, mortality, neurobehavioral changes, altered sexual behavior, and problems in parturition and lactation are recorded. Food consumption and weight of animals are measured at least weekly, and after parturition on the same days litters are weighed. Individual records of each parent test animal and litter are maintained. The time after pairing to achieve a sperm-positive smear (the precoital interval) and duration of pregnancy are recorded, and soon after delivery the number and sex of pups, stillbirths, live births, and the presence of gross anomalies in each litter are recorded. The pups are weighed at a minimum on the morning after birth, on days 4 and 7, and weekly thereafter. Dead pups and the excess pups killed at day 4, if the litter is standardized, should be studied for any defects. All abnormalities in the dams or offspring should be recorded.

At necropsy, the offspring are examined for structural abnormalities, particularly those of reproductive organs, that also can be preserved for histopathological study. At a minimum, all parental animals and offspring that die during the test, those in the highest dose group, and the controls should be examined. Whenever there are gross abnormalities in an organ, the animals also must be examined microscopically.

The data should be treated with appropriate statistical methods. If one male is mated to two females, then nested statistical analysis must be performed based on the number of males used. A well-conducted, single-generation reproductive toxicity study should provide an estimate of a no-observed-adverse-effect level (NOAEL) and an assessment of adverse effects on fertility, parturition, lactation, and postnatal growth. Significant detrimental effects on any endpoint should be considered adverse.

The primary limitation of a single-generation toxicity study is that it provides no information on the breeding capacity of offspring. Other limitations are noted in Table D-1. An EPA workshop (Francis and Kimmel 1988) examined the value of the single-generation reproductive study and concluded that it is “insufficient to identify all potential reproductive toxicants, because it would exclude detection of effects caused by prenatal and postnatal exposures (including the prepubertal period) as well as effects on germ cells that could be transmitted to and expressed in the next generation” (EPA 1996a).

Multigeneration Reproduction Study

Several authorities have published guidelines on multigeneration reproductive assays (OECD 2000b; FDA 2000; EPA 1998b). For a discussion of multigeneration tests, see articles by Lamb (1988, 1989) and Christian (1986).

Multigeneration reproduction studies determine the potential of an agent to produce adverse effects on the male and female reproductive systems, in the embryo and fetus, and in the neonate. The bioassay examines a wide variety of endpoints related to reproduction, including effects on libido; germ cells; gametogenesis; fertilization; implantation; embryonic, fetal, and neonatal growth; development; parturition; lactation; and postweaning growth and maturity. The direct toxic effects of an agent on the pregnant dam can be evaluated. In addition, the recently revised guidelines include measures of estrous cyclicity and ovarian primordial follicle counts in parental and first filial ( F₁) females, and sperm parameters (number, motility, and morphology) in parental and F₁ males. Development of the reproductive system and measures of sexual maturation (vaginal opening and preputial separation) are also included. Finally, organ weights of the reproductive organs, target organs, and brain, spleen, and thymus are included. Because of its study design, a multigeneration reproduction study can provide data that cannot be developed from other standard testing protocols. The observed effects can be different from those seen in other (e.g., subchronic) studies.

The parental animals (P generation) are treated with an agent, usually via ingestion, for at least 10 wk before mating. Females continue to be exposed during gestation and lactation. Each dam can produce one to three litters, depending on whether the outcome in the first litter is unequivocal or confirmation is required of findings. This gives some flexibility in the protocol (multiple litters are needed only when initial results are equivocal), and it applies to P and F₁ generations. If the effects in the F₂ (second filial) generation are more marked than in the F₁ generation, additional generations can be examined to clarify potential transgenerational effects.

Dosing of all generations is continuous throughout the study. During lactation, pups receive the test substance through the dam's milk and later from the treated food or drinking-water. If inhalation exposure is used, grooming of the fur can lead to additional exposure to the test material. Coprophagia by the pups is another possible route of exposure. Upon reaching sexual maturity, at least one male and one female from the F₁ generation are selected from each litter for mating with another pup from a different litter but exposed to the same dose. F₁ generation rats are treated for at least 13 wk and F₁ mice are treated for at least 11 wk before mating.

The study report must include the following data:

• Species and strain.
• Toxic response data by sex and dose, including indices of mating, fertility, gestation, birth, viability, and lactation; offspring sex ratio; time-to-mating (including the number of days until mating and the number of estrous cycles until mating); duration of gestation.
• Day of death if that occurs during the study.
• Toxic or other effects on reproduction and pre- or postnatal growth of the offspring.
• Developmental data, such as anogenital distance (triggered in F₂ pups if positive findings are noted in the F₁ animals), age of vaginal opening, and preputial separation.
• Number of P and F₁ females with normal cycles and cycle length.
• Day of recording an abnormal effect and its subsequent course.
• Body weight data by sex for each generation.
• Dietary intake and food efficiency (body weight gain per food consumed), and test substance consumption for P and F₁ animals, except for the period of cohabitation.
• Sperm evaluation on data including total cauda epididymal sperm counts, percentage of progressively motile sperm, percentage of morphologically normal sperm, and percentage of sperm with each identified abnormality.
• Stage of estrous cycle at the time of death for P and F₁ females.
• Necropsy findings.
• Implantation data and postimplantation loss calculations for P and F₁ females.
• Absolute and relative organ weight data.
• Detailed descriptions of all histopathological findings.
• Adequate statistical analyses.
• A copy of the study protocol.

The multigeneration study is probably the most complex type undertaken for regulatory purposes and provides information on toxicity that follows treatment throughout the entire reproductive cycle, except that it does not evaluate reproductive senescence other than the evaluation of primordial follicles in females. Other limitations are noted in Table D-1. In general, significant detrimental effects on any endpoints or on indices derived from the data should be considered adverse. EPA (1996a) provides a detailed discussion on adverse effects.

Reproductive Assessment by Continuous Breeding Study

The U.S. National Toxicology Program (NTP) has developed a test protocol for evaluating toxicity through a reproductive assessment by continuous breeding (RACB) study design (Lamb 1985; Gulati et al. 1991). The protocol was originally developed for mice as a faster and more cost-effective alternative to the conventional regulatory reproductive toxicity studies, but it also has been used successfully with rats. After a 1-wk pretreatment period, males and females are housed as breeding pairs in individual cages and allowed to mate continuously for 14-wk. Exposure to the test substance (usually in feed or drinking-water) is continued throughout the study, and the offspring are removed from the cage immediately after parturition. After the cohabitation breeding period, the pair is separated and the last litter is raised to weaning. Pups from these litters can then be selected, and treatment is continued. The pups are used in a mating trial to evaluate effects in the second generation in a manner comparable to that described previously for the multigeneration design.

The same endpoints (fertility, pups per litter, pup weight, sex, survival) are studied in the RACB protocol as in the standard multigeneration protocol. It is the time between Litters and the progressive effects on fertility and reproduction that are specific to the RACB study design. The difference is that the RACB study design allows more than one litter to be examined per generation and can give an indication of subfertility and infertility. Adverse effects that might not be noted in the first mating may become evident later due to longer exposure time; such findings would not normally be detected in the conventional studies.

The RACB does not give information on specific male and female reproductive effects unless cross-breeding of control and treated males and females is done following the 14-week mating trial. It also does not provide information on effects in the second generation unless F₁ pups are raised to breeding age and mated to produce a second generation as in the multigeneration study design. Other limitations are noted in Table D-1.

Serial Mating Study (Dominant Lethal Study)

If a single-mating trial results in an adverse effect attributable to the male, it is difficult to determine the developmental stage in which the disruption occurs. It is well known that different stages of spermatogenesis are variably sensitive to toxic effects and that each toxic substance can affect different sperm cell populations (Parvinen 1982). Spermatogonia, for example, are sensitive to cyclophosphamide in experiments conducted in mice (Toppari et al. 1990), whereas spermatocytes are disrupted by ethylene glycol monomethyl ether in experiments conducted in rats (Chapin et al. 1985). The action of a compound that primarily affects the somatic Sertoli cells of the testis, for example, m-dinitrobenzene (Foster 1989), will produce an extensive period of infertility because of adverse effects on the function of these cells at various stages of germ cell differentiation.

Serial mating makes it possible to assess the sensitive stages of spermatogenesis and susceptible cell types. This information can be obtained from a specific serial-mating trial or from a similar protocol used for dominant lethal testing (OECD 1984; EPA 1998d). Adult males (usually rats) are exposed before mating, typically for 1-5 d, with 20 males per dose group, where after they are mated to one to three females weekly for the next 8-10 wk. Adverse effects on male reproduction are manifested as decreased numbers of implantation sites in uteri (indicative of failure of fertilization or preimplantation loss) and increased early fetal mortality (indicative of postimplantation loss or dominant lethality). To examine the uterine contents, dams are killed before parturition (e.g., on days 13-18 of pregnancy).

Any adverse effects can then be attributed to specific cell populations by back-calculation on the basis of the well-known kinetics of spermatogenesis (Chapin et al. 1985). The test was originally designed for detection of germ cell mutagenicity, and it requires a large number of female animals (e.g., an 8-wk trial would use 160-480 females), which is a disadvantage.

The limitations of serial-mating trials are similar to those shown in Table D-1 for other reproduction studies, except for the identification of stage of spermatogenesis affected. Additional endpoints of male reproductive toxicity and effects other than death of the offspring are not evaluated unless included in the protocol.

Total Reproductive Capacity

The total reproductive capacity study, a variant of the continuousbreeding study, is designed to assess ovarian toxicity. Female fetuses are particularly susceptible to agents that can adversely affect germ cells because development of the oocyte occurs prenatally; no new germ cells develop after birth. Female animals are exposed to a test substance for a short period in utero (i.e., days 9-16 of gestation) (McLachlan et al. 1981) or postnatally (Generoso et al. 1971) and allowed to mate with a single male as long as the females remain fertile. The numbers of litters and offspring are compared with those of control animals to estimate the loss of oocytes resulting from the exposure.

Total reproductive capacity studies have been designed with the specific purpose of evaluating female reproductive capacity and are not tests of general reproduction function.

Male Indices

Female Indices

Several endpoints listed in Table D-2 can provide evidence for female reproductive toxicity. For example, when a continuous-breeding study shows an adverse effect, it is desirable that the study also mate each member of a breeding pair to an untreated control to identify which member is affected by the agent. If a study has not taken this step, it cannot be said with certainty that the observed effect is the result of female reproductive toxicity; it can be equally likely that a male effect or a couple effect is involved.

Because most standard animal reproduction studies do not observe mating, they do not contain evaluations of an agent's effect on sexual behavior. If a study does report observations of mating, the failure of female rodents to assume a lordotic position and to accept mounting is evidence of abnormal sexual behavior. Additional signs include running from or fighting with the male (Uphouse and Williams 1989; Uphouse 1985).