This tier involves testing chemical candidates on a range of physiological and behavioral effects in a rat or mouse model. This tier has several unique qualities that make it critical for assessing chemicals for potential EDC effects. While a chemist may wish to use any of other tiers as an entry point for initial screens, the Mammalian tier should usually be reserved only for the final level of testing due to the greater complexity of the experimental design, the need for a developmental model that spans several months, and our respect for minimizing the use of mammals for testing unless absolutely necessary.
If a chemical passes screening in earlier Tiers, it is important to determine its effects in a mammalian model. Rodents are recommended due to their high conservation of endocrine and neurobiological systems with other species, including humans. In addition, both rat and mouse models for EDCs are well studied and validated in the literature. They undergo relatively rapid postnatal development, they reach maturity in approximately 2 months or less, and they have short gestations (18-22 days). Other tiers use cells and cells lines, as well as non-mammalian testing protocols, which are highly relevant to human health. However, no other tier has the unique features of internal pregnancy in a uterine environment, followed by lactation and maternal care. Considering that most researchers believe the greatest cause for concern for EDC exposures is to developing fetuses and in infancy, which are critical developmental periods, such a mammalian model is needed.
The principal goal of the Mammalian tier is to assess the long-term health effects of early life exposure to the chemical of interest. Tests are designed to maximize the likelihood of identifying any effects on overall growth and development, pubertal timing, behavior, sexual dimorphisms, immune function, and other life history endpoints that are not possible to comprehensively assess in silico, in vitro, or in non-mammalian animal models.
An advantage of the Mammalian Tier is its malleability and flexibility, depending on the information needed. For example, if a compound displayed no EDC activity in the other tiers, this tier could be used to obtain confirmatory data from a mammalian model. Alternatively, this tier could be used to more comprehensively assess the endocrine disrupting potential of a compound for which results in other tiers were difficult to interpret. The endpoints chosen will thus necessarily differ depending on the type of information needed and the performance of the compound in the other tiers.
For some compounds, a guideline study using a rat model may be required as part of routine risk assessment and toxicity testing. Unfortunately most of the government guideline studies as currently proposed contain an insufficient number of endocrine sensitive endpoints and do not include doses that are considered to be “environmentally relevant” and, due to their government-standard “Good Laboratory Practices” requirements, are very expensive.
This Tier, as designed, has the advantage that it can provide information on more human disease-focused data than governmental guideline studies at a fraction of the cost, and can be adapted to enhance a guideline study to include EDC-sensitive endpoints, additional doses, and account for critical animal care issues that can have an impact outcomes, such as caging materials, light cycle, diet, handling, and strain differences. Note That mammalian studies can and should be done with a TiPED collaborator experienced with both mammalian screening and risk assessment requirements.
Rodent testing is not high-throughput, requires labor-intensive monitoring, demands specialized skills to do properly, and is expensive.
A potential pitfall of this Tier is that rodents are sensitive to the laboratory environment, with husbandry, food, temperature, social environment, and other factors changing basic developmental and physiological processes. These tests must be conducted in partnership with an experienced TiPED collaborator once the compound of interest has been screened in at least one of the other tiers to obtain basic information about its potential EDC activity. It is absolutely essential that the person conducting the assays be trained and a confirmed expert on each aspect of the work.
Because we respect the importance of minimizing animal testing, appropriate statistical analyses such as power analysis must be conducted prior to performing any work. Additionally, teams of experts working together are the ideal scenario to collect tissues to minimize use of animals. These caveats underscore the point that experienced TiPED partners are needed to design, conduct, and interpret results.