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STANDARDIZATION AND VALIDATION OF PROPOSED PROTOCOLS FOR IN VITRO SCREENING ASSAYS AND QSAR FOR ESTROGEN RECEPTOR AND ANDROGEN RECEPTOR
Impact/Purpose:
As a result of concerns about the effects of endocrine disrupting chemicals (EDCs) on humans and wildlife, the Agency was charged with developing a screening and testing program to identify estrogens and other hormonally active substances, as deemed appropriate by the Administrator. The legislative mandate for this program exists under the FQPA and SDWA of 1996. As a consequence of this legislation the Agency commissioned a multi stakeholder committee under the Federal Advisory Committee Act (FACA), the Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC), to advise the Agency in this endeavor. The 1998 EDSTAC Final Report recommended several short-term in vitro assays as part of the screening battery. In addition to providing better estimates of estrogen/androgen/thyroid hormone activity, these alternative tests should ideally use fewer animals, or no animals at all. The objective of this research is to develop and standardize in vitro assays and QSAR approaches. Research will focus on developing protocols for in vitro assays that will detect estrogenic and androgenic activity (i.e., agonist and antagonist) and that are subsequently amenable to standardization and validation.
Description:
Screening EDCs for androgenic and antiandrogenic activities was recommended by the EDSTAC Committee in it Final Report. This research will develop in vitro approaches to assess estrogen receptor binding, develop cell lines that stably express estrogen receptor for screening EDCs for estrogenic and antiestrogenic activities in vitro, develop androgen receptor binding assays, develop androgenic-dependent gene expression assays with transiently transfected or transduced cells, and develop androgen-dependent gene expression assays that stably express androgen receptor and the luciferase reporter genes. In order to compare the assays in which development has been completed, we have run a suite of chemicals with known activity. Other assays are still under development, but upon completion, they will be evaluated with the same group of ligands. We will continue the development and distribution of stable cell lines so that equivalent results can be obtained in other laboratories. Other species ERs also will be stably transfected for reporter gene assays upon isolation, sequencing and expression. Assays will be evaluated for reproducibility, ease of use, inter and intra-assay variability, cost, specificity, sensitivity, and relevance to stated screening objectives.