Reproductive toxicity includes the toxic effects of a substance on the reproductive ability of an organism and the development of its offspring. Reproductive toxicity has been defined as "any effect of chemicals that would interfere with reproductive ability or capacity," including effects on lactation (UNECE, 2004). The definition of developmental toxicity is very broad, so the Globally Harmonized System (GHS) considers the following definition sufficient for classification purposes: "adverse effects induced during pregnancy, or as a result of parental exposure," that "can be manifested at any point in the life span of the organism" (UNECE, 2004).
The Animal Test(s)
Animal tests include evaluating the effects of prenatal exposure on pregnant animals and their offspring [OECD Test Guideline (TG) 414]. This test is usually performed with female rats and rabbits. The test substance is administered orally, the pregnant animals are killed just prior to delivery, and the fetuses are examined for toxic effects. A one-generation reproduction toxicity study (OECD TG 415) in rats or mice is used to evaluate toxic effects on male and female reproduction. Males and females are dosed orally before mating, and females during pregnancy. A two-generation reproduction toxicity study (OECD TG 416) continues dosing with the test substance to the first generation offspring. OECD TG 421 (Reproductive/Developmental Toxicity Screening Assay) uses male and female rats with the test substance administered orally for 4-9 weeks. Pathological effects are determined by daily observation, necropsy, and microscopic histopathology.
The Organisation for Economic Cooperation and Development (OECD) adopted two draft proposals for new reproductive/developmental toxicity TGs in October 2007. Draft Proposal 426, Developmental Neurotoxicity Study, involves the daily oral feeding of a test substance to female rats (preferred species) from the time of mating through lactation. The purpose is to determine in utero and early postnatal developmental neurological effects of the test substance. At least 20 litters per dose for three dose levels are tested. Offspring are evaluated for neurotoxic effects including "gross neurologic and behavioural abnormalities, and the evaluation of brain weights and neuropathology during postnatal development and adulthood." Draft Proposal for a New Guideline 440: Uterotrophic Bioassay in Rodents: A short-term screening test for oestrogenic properties is an in vivo assay to evaluate potential estrogen-disruptor effects of a test substance. The test evaluates increased uterine weight (uterotrophic response) in female animals with a nonfunctional hypothalamic-pituitary-ovarian axis after short-term, repeated exposure to the test substance. The European Centre for the Validation of Alternative Methods (ECVAM) has noted its opposition to the OECD "validation" of the Uterotrophic Bioassay, stating that previously agreed validation principles were not met (ESAC Statement, February 15, 2005).
Non-animal Alternative Methods
The challenge in replacing animal tests for reproductive and developmental toxicity testing is the many possible organs and physiological processes that could be affected by a chemical. In vitro models for all the potential mechanisms of toxicity need to be identified, developed, and organized into a rational testing scheme.
Many in vitro methods have been developed for reproductive/developmental toxicity testing (Bremer, et al., 2005), and they will be reviewed here in the future.
Validation and Acceptance of Non-animal Alternative Methods
An ICCVAM-NICEATM workshop reviewed the Frog Embryo Teratogenesis Assay: Xenopus (FETAX) as a potential alternative for assessing developmental toxicants. The method was deemed not ready for validation, so recommendations were made for its continued development.
The ECVAM Scientific Advisory Committee (ESAC) "endorsed three in vitro methods for embryotoxicity testing as scientifically validated" (ESAC Statements, May 1, 2002):
- Embryonic stem cell test for embryotoxicity
- Micromass embryotoxicity assay
- Whole rat embryo embryotoxicity assay
The ESAC recommended these in vitro methods as ready for regulatory acceptance but acknowledged they cannot replace the animal tests. However, when used as part of a testing strategy, they could contribute to reducing animal use.