Reproductive toxicity includes the toxic effects of a substance on the reproductive ability of an organism and the development of its offspring. Reproductive toxicity is defined by the Globally Harmonized System (GHS; rev. 5) as “adverse effects [of chemicals] on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring” (UNECE, 2013, Part 3). Developmental toxicity means “adverse effects induced during pregnancy, or as a result of parental exposure…manifested at any point in the life span of the organism” (UNECE, 2013, Part 3).
The definitive animal test for evaluating potential developmental toxicity is the prenatal developmental toxicity test [e.g., OECD Test Guideline (TG) 414]. This test is usually performed with female rats and rabbits. The test substance can be administered by a variety of routes depending upon the routes of human exposure, usually beginning at implantation and continuing through the end of organogenesis or to full term. The pregnant animals are then killed and the fetuses are weighed, sexed, and examined in exquisite detail for external, visceral, and skeletal alterations.
For reproductive toxicity, a variety of tests are available. A so-called screening level test is the Reproductive/Developmental Toxicity Screening Assay (OECD TG 421), which uses male and female rats dosed with the test substance for two weeks prior to mating, during mating and gestation (3 weeks), and parturition up until postnatal day 4. This same core study design can be coupled with more extensive evaluation of repeated dose parental toxicity endpoints (OECD TG 422). Long considered the definitive test of reproductive toxicity is the two-generation reproduction toxicity study (OECD TG 416), which involves continuous dosing of parental rats and their offspring with the test substance (usually in the diet, but other routes are possible) for two entire generations, up through weaning of the F2 pups at 3 weeks of age. A large number of endpoints are evaluated to assess reproductive performance and fertility, growth and survival of offspring, achievement of developmental landmarks, and potential endocrine-mediated effects.
Recently, a number of large retrospective analyses have indicated that the data from the second generation has had little impact on the final outcome of the study in terms of regulation. Therefore, a new test design called the extended one-generation reproductive toxicity study (EOGRTS; OECD TG 443) was developed. This test omits the second mating (unless triggered by specific study findings), but in its place makes much more complete use of the F1 offspring by carrying them out for extended periods in order assess development of immune system function, developmental neurotoxicity, as well reproductive function and additional endocrine-sensitive end points. Whereas the two-generation study consumes well over 2000 animals, the EOGRTS actually provides more data but uses almost half as many animals. However, the EOGRTS is quite challenging to perform due to the logistical complications of collecting so much data in a compressed time frame.
Should a test focused on developmental neurotoxicity be needed, OECD TG 426 can be conducted. This 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.”
Several in vivo screens to detect potential endocrine activity are part of the US EPA’s Endocrine Disruptor Screening Program. These include the Uterotrophic assay (OCSPP Guideline 890.1600), which is a short-term screening test for oestrogenic properties conducted in immature or ovariectomized rodents typically exposed for three days followed by measurement of uterine weight (uterotrophic response). Its counterpart for the detection of potential anti-androgenic activity is the Hershberger assay (OCSPP Guideline 890.1400), which utilizes immature male animals. To evaluate other aspects of the hypothalamic-pituitary-gonadal axis, the male and female pubertal assays (OCSPP Guidelines 890.1500 and 890.1450, respectively) are used.[The above tests are all for agricultural and industrial chemicals. ICH Guidelines would be followed for the evaluation of drugs.]
Developmental toxicity is one of the most animal-intensive areas of regulatory toxicology, but also remains one of the most challenging for the application of alternative methods. Although there are a few in vitro assays which work reasonably well as pre-screens for teratogenicity, none cover the broader range of potential adverse developmental outcomes needed to assure safety. In addition, many of the assays focus on narrow windows of development or just a single embryonic lineage (e.g., cardiomyocyte lineage in the embryonic stem cell assay). The field is gradually moving toward integrated testing and assessment strategies in order to cover the majority of possible mechanisms driving a broader range of potential adverse outcomes.
The ECVAM Scientific Advisory Committee (ESAC) “endorsed three in vitro methods for embryotoxicity testing as scientifically validated” (ESAC Statements, May 1, 2002):
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.
|Embryonic stem cell test (EST)||Embryotoxicity/Teratogenicity|
|Whole rat embryo culture (WEC)||Embryotoxicity/Teratogenicity|
The EST uses two cell lines and three endpoints to predict embryotoxic chemicals (INVITTOX protocol). The cell lines are mouse embryonic stem cells (ES) and mouse 3T3 fibroblast cells. The assay endpoints are inhibition of differentiation of the ES cells, inhibition of ES and 3T3 cell viability, and inhibition of ES and 3T3 cell proliferation.
The micromass test evaluates effects of a test substance on the differentiation and growth of micromass cultures of rat limb bud (INVITTOX protocol). The limb bud cultures are a model that represents various developmental processes in cartilage histogenesis such as cell proliferation and differentiation, as well as cell-cell communication.
The whole rat embryo culture uses isolated and cultured early post-implantation rat embryos to study embryotoxic effects of chemicals (INVITTOX protocol). The morphology of 48-hour cultured embryos (controls and chemical exposed) are compared for delays in the development of certain organ systems or their malformation.
The micromass test and whole rat embryo culture entail the killing of animals for the tissue used in the assays, whereas the EST (which uses cell lines) does not.