Evaluating Chemical and Other Agent Exposures for Reproductive and Developmental Toxicity (2001) / Chapter Skim
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Appendix D: Experimental Animal and In Vitro Study Designs
Appendix D: Experimental Animal and In Vitro Study Designs
Pages 206-235
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From page 206... ...
By themselves, however, in vitro tests are insufficient for defining the potential reproductive or developmental toxicity of an agent. The primary information on experimental animal testing for reproductive and developmental toxicity potential is likely to be derived from standard studies used by regulatory agencies.
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From page 207... ...
This appendix describes experimental animal and in vitro studies that are used to assess developmental toxicity and male and female reproductive toxicity from exposures to pesticides, industrial chem~cals, and food ingredients. The testing of pharmaceutical agents is not described in detail here, but can be found in FDA (1994~.
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From page 211... ...
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From page 212... ...
Several other types of studies, although not solely designed to assess developmental toxicity, can be used for that purpose. They include single- and multigeneration reproduction studies, reproductive assessment by continuous-breeding studies, and serial mating (dominant lethal)
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From page 213... ...
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.
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From page 214... ...
In Vitro Assays Any developmental toxicity assay that uses a test subject other than a pregnant mammal fails 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.
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From page 215... ...
Tn 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.
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From page 216... ...
EPA (1991) published guidelines for developmental toxicity risk assessment that provide more detailed discussion of study result interpretation.
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From page 217... ...
Neurobehavioral development and function (actual enpoints measured depend on the function or organ system being studied) Reflex development Locomotor development Motor activity Sensory function Social-reproductive behavior Cognitive function Neuropathology and brain weight Reproductive system development and function Vaginal opening Onset of estrus Balano-preputial separation Ovarian cyclicity Quantitation of ovarian primordial follicles Sperm measures (e.g., morphology, motility, number)
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From page 218... ...
In these situations, developmental effects should tee attributed to developmental toxicity and should not be considered secondary effects of maternal toxicity. It is possible that the adult and the developing offspring are sensitive to the same dose of an agent.
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From page 219... ...
. Female reproductive toxicity includes adverse effects on reproductive organs and related endocrine systems.
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From page 220... ...
The animals are observed daily throughout testing. Toxic effects, mortality, neurobehavioral changes, altered sexual behavior, and problems in parturition and lactation are recorded.
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From page 221... ...
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)
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From page 222... ...
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.
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Dietary intake and food efficiency (body weight gain per food consumed) , and test substance consumption for P and Fat animals, except for the period of cohabitation.
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From page 224... ...
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.
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From page 225... ...
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.
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From page 226... ...
Although such studies are the most typical way to evaluate the reproductive toxicity of an agent, most provide insufficient evidence of whether the agent causes male or female reproductive toxicity in animals. There is, therefore, a need for additional data, which, in fact, can come from the same study.
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From page 227... ...
Preweaning index Mean pups born per litter minus mean pups weaned per litter Mean pups born per litter weight or altered morphology can provide sufficient evidence that an agent is a male reproductive toxicant or add weight to evidence that it is not a male reproductive toxicant. Likewise for females, evidence of ovarian toxicity measured by weight changes and altered morphology can provide sufficient evidence for female reproductive toxicity.
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From page 228... ...
Decreases in the weight of the seminal vesicles or ventral prostate can be sufficient evidence of male reproductive toxicity, but are more useful if supplemented by data on endocrine effects. Changes in pituitary weight alone would typically be insufficient evidence of male reproductive toxicity, because pituitary weight is an inaccurate indicator of changes in pituitary function, which are best measured by other parameters, such as hormone concentrations.
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From page 229... ...
Low-quality morphological techniques, such as formalin fixation and paraffin embedding, are never sufficient to show that an exposure did not produce testicular toxicity. Morphological changes in accessory sex organs are less common, but clear treatment-related effects also can provide sufficient evidence of male effects.
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From page 230... ...
Endocrine Evaluations If adequately designed studies detect changes in concentrations of gonadal steroid or gonadotropic pituitary hormones, these endocrine parameters do provide sufficient evidence of reproductive toxicity. Typically, adequate studies that show toxicity will have multiple samples obtained in a well-defined context that includes sex, age, reproductive state, day of cycle, and so on.
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From page 231... ...
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.
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From page 232... ...
By themselves, these alterations would be insufficient to identify an agent as a reproductive toxicant. Weight and Morphology Changes A statistically significant decrement in ovarian or uterine weight in a study properly controlled for cyclic variation is worthy of consideration and should signal the need for additional studies.
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From page 233... ...
The disadvantage of this method is the possibility that the injection of the releasing agent will cause an atypical physiological situation, so that one cannot extrapolate the effect it "unmasks" to unmanipulated animals. if changes in concentrations of gonadal steroid or gonadotropic pituitary hormones are detected in adequately designed studies, these endocrine parameters do provide sufficient evidence of reproductive , .
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From page 234... ...
in Vitro and Perfusion Systems Tissue culture methods have been used to study ovary slices in vitro, and cell culture methods have been used for studying granulosa cells and myometrial cells. In culturing ovary slices or granulosa ceils, investigators often use the release of sex steroids into the medium as an outcome parameter.
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From page 235... ...
Conversely, if an agent is not transferred into the milk in rodent studies, but it is clear that exposure to critical organ systems continues in utero at the same developmental stages in humans, it may be appropriate to conduct direct dosing studies in rodents to determine any potential effects on the structural and functional development of these systems.
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