The embryonic stem cell test (EST), an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells

The embryonic stem cell test (EST) was developed as an in vitro embryotoxicity test which does not use embryonic tissues from pregnant animals, but only two permanent mouse cell lines, 3T3 fibroblasts and embryonic stem (ES) cells of the D3 line. In the EST, cytotoxicity is determined in the two cell-lines after 10 days of culture, together with the differentiation of ES cells into contracting myocardial cells. The embryotoxic properties of 16 carefully selected chemicals were tested in the EST. Out of 12 endpoints and ratios of endpoints, stepwise discriminant analysis selected three endpoints characterized by the best correlation to the embryotoxic properties of the test chemicals. By using linear discriminant functions, a classification scheme was developed for the EST, in which test chemicals are assigned to three classes of in vivo embryotoxicity: not embryotoxic, moderate embryotoxic and strong embryotoxic. In the classification model of the EST, all the test chemicals were correctly assigned to their in vivo classes of embryotoxicity.