A detailed report is presented on the performance of the embryonic stem cell test (EST) in a European Centre for the Validation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects in animals and/or humans, were tested in four laboratories. The outcome showed that the EST can be considered to be a scientifically validated test, which is ready for consideration for use in assessing the embryotoxic potentials of chemicals for regulatory purposes.

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Blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse can be induced to differentiate in culture into a variety of cell types, including cardiac muscle cells. The embryonic stem cell test that makes use of the differentiation of ES cells into cardiomyocytes in a standardized in vitro model was developed to offer an alternative method to comprehensive in vivo studies in reproductive toxicology about toxic effects of chemicals. ES cells of the mouse cell line D3 are investigated for their preserved capability to differentiate following drug exposure, and both ES cells and differentiated fibroblast cells of the mouse cell line 3T3 are comparatively analyzed for effects on viability. The following endpoints are used to classify the embryotoxic potential of chemicals into three classes of in vitro embryotoxicity (non-, weakly or strongly embryotoxic). These endpoints are: (1) the inhibition of differentiation of ES cells into cardiomyocytes after 10 days of treatment, and the decrease of viability (cytotoxicity) of (2) 3T3 cells and (3) ES cells after 10 days of treatment, determined by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) test. 50% inhibition concentrations for differentiation (ID₅₀) and cytotoxicity (IC₅₀D3 and IC₅₀3T3) are calculated from concentration-response curves. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed. This procedure identified three variables, the lg(IC₅₀D3), the lg(IC₅₀3T3) and the relative distance between IC₅₀3T3 and ID₅₀, that improved the separation of the three classes of embryotoxicity compared to the prediction model that was originally proposed after test development. Unlike the orginal PM, the improved PM incorporates as one variable the relative distance between IC₅₀3T3 and ID₅₀, instead of the ratio ID₅₀/IC₅₀D3 that was used previously.

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