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The cost of testing chemicals as reproductive toxicants precludes the possibility of evaluating large chemical inventories without a robust strategyfor prioritizing chemicals to test. The use of quantitative structure-activity relationships in early hazard identification is a cost-effective prioritization tool, but in the absence of systematic collection of interpretable test data upon which models are formulated, these techniques fall short of their intended use. An approach is presented for narrowing the focus of candidate ED chemicals using two in vitro assays: one optimized to measure the potential of chemicals to bind rainbow trout estrogen receptors (rtER), and a second to enhance interpretation of receptor binding data in a relevant biological system (i.e., fish liver tissue). Results of rtER competitive binding assays for 16 chemicals yielded calculable relative binding affinities (RBA) from 179 to 0.0006% for 13 chemicals and partial or no binding for an additional 3 chemicals. Eleven lower to no affinity chemicals (RBA < 0.1%) were further tested in trout liver slices to measure induction of rtER-dependent vitellogenin (VTG) mRNA in the presence of chemical passive partitioning (from media to multiple hepatocyte layers in the slice) and liver xenobiotic metabolism. VTG induction in slices was observed in a concentration-dependent manner for eight chemicals tested that had produced complete displacement curves in binding assays, including the lowest affinity binder with an RBA of 0.0006%. Two chemicals with only partial binding curves up to their solubility limit did not induce VTG. The monohydroxy metabolite of methoxychlor was the only chemical tested that apparently bound rtER but did not induce VTG mRNA. Data are presented illustrating the utility of the two assays in combination for interpreting the role of metabolism in VTG induction, as well as the sensitivity of the assays for measuring enantiomer selective binding and ER-mediated induction. The combined approach appears particularly useful in interpreting the potential relevance of extremely low affinity chemical binding to fish receptors (RBA = 0.01-0.0001%) within a defined toxicity pathway as a basis for prioritizing within large chemical inventories of environmental concern.

Legislation and prospective legislative proposals in for instance the USA, Europe, and Japan require, or may require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Chemicals found to test positive are considered to be endocrine active substances (EAS) and may be putative endocrine disruptors (EDs). To date, there is still little or no experience with incorporating metabolic and toxicokinetic aspects into in vitro tests for EAS. This is a situation in sharp contrast to genotoxicity testing, where in vitro tests are routinely conducted with and without metabolic capacity. Originally prepared for the Organisation of Economic Cooperation and Development (OECD), this detailed review paper reviews why in vitro assays for EAS should incorporate mammalian systems of metabolism and metabolic enzyme systems, and indicates how this could be done. The background to ED testing, the available test methods, and the role of mammalian metabolism in the activation and the inactivation of both endogenous and exogenous steroids are described. The available types of systems are compared, and the potential problems in incorporating systems in in vitro tests for EAS, and how these might be overcome, are discussed. Lastly, some recommendations for future activities are made.

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