: Zacharewski, T.; Campbell, P.; Routledge, E.; Huet, M.-C.; Cooper, R.; Kime, D.; Wenzel, A.

Abstract
A number of in vitro test methods have been used to assess the ability of substances to interact with, and potentially modulate, components of the endocrine system. Many of these test methods have utility for elucidating mechanisms of action, and have been demonstrated to be useful for assessing potential hazards of single substances and complex mixtures. However, the utility of in vitro methods is limited by a number of important disadvantages. Taking into account the known advantages and disadvantages of in vitro assays, it is concluded that in vitro tests are complementary to, and not substitutes for, in vitro tests. There may be fundamental problems associated with interpreting synergistic interactions in vitro - which could account for the contradictory results obtained from different laboratories addressing this issue. Evidence from comparative endocrinology studies indicate that there is a sufficient degree of conservation between many aspects of the reproductive systems of vertebrate species, e.g., steroid receptors and steroidogenic enzymes, to allow reasonable species to species predictions to be made between certain vertebrate classes. However, it was recognised that species differences do exist and our present knowledge regarding the extent and significance of these differences is of concern, since this understanding is needed to provide a scientific database to justify species to species predictions. The development of internationally harmonised in vitro methods to detect endocrine activity will first require the identification of suitable assays. The nomination of two in vitro assays, designed to detect two different MOA; (1) receptor binding and (2) effects on steroidogenisis, is recommended. These assays should then be optimised and validated using the agreed criteria and process developed by ECVAM and ICCVAM. Based on the information and experience gained from this validation exercise, additional assays could then undergo the formal test optimisation and validation procedure in order to encompass a wider range of potential mechanisms of action. The mechanistic understanding contributed by in vitro tests will be an important factor for identifying EMs and, together with knowledge from comparative endocrinological studies, may be useful for extrapolating to or understanding of potential effects in different species.