Enlargement of the database of the validated embryonic stem cell test (EST), testing of eight substances
The embryonic stem cell test (EST) is a scientifically validated in vitro assay that was established to classify compounds with respect to their embryotoxic potential. This assay assesses the embryotoxic properties of chemicals by the evaluation of inhibitory effects on differentiation of contracting myocardial cells compared to cytotoxic effects on undifferentiated murine embryonic stem cells and differentiated 3T3 fibroblasts. On the basis of test data of 27 compounds a biostatistical prediction model (PM) was developed which enables the EST to predict the embryotoxic potential of test chemicals with an accuracy of 78%. In order to continually improve the predictivity of the EST, we intended to enlarge the data base of the validated EST by testing additional chemicals including pharmaceutical substances. In this study, we have tested eight different substances: benzoic acid, d-penicillamine, methyl azoxymethanol acetate, Y-27632, mometasone furoate, and three pharmaceutical inhouse substances. Different molecular and functional endpoints were assessed: detection of myosin heavy chain expression by flow cytometry analysis, microscopic evaluation of contracting cardiomyocytes and analysis of cytotoxic effects on embryonic stem cells and fibroblasts. On the basis of these in vitro endpoints we were able to correctly predict the embryotoxic potential in vivo.