Teratogenicity of valproic acid derivatives: Evaluation of structure-activity relationships using the embryonic stem cell test

allemand. Testing for reproductive and developmental toxicity of drugs and other chemical compounds in vitro is an attractive alternative procedure to time-consuming and expensive in vivo or ex vivoexperiments. The embryonic stem cell test (EST) represents a scientifically validated method for the detection and classification of compounds according to their teratogenic potency. However,more work is required to assess its applicability domain and to improve its predictive capacity before gaining full regulatory acceptance.We picked valproic acid (VPA) as a model compound to evaluate the suitability of the EST for distinguishing between developmental toxicity potencies of substances with closelyrelated structures. VPA is among of the most frequently used anti-epileptic drugs worldwide. Further, it is used for migraine prophylaxis and in the treatment of bipolar psychotic disorders.Two severe side effects of VPA, hepatotoxicity and teratogenicity have prompted research into derivatives of VPA. Here we investigate six closely related analogues of VPA whose teratogenicpotential has been previously determined in the NMRI exencephaly mouse model.The validated EST employs the murine embryonic stem cell line D3, which spontaneously differentiates into contracting cardiomyocytes.This morphological feature is used as an endpoint of differentiation along with the measurement of cytotoxicity on both D3 stem cells and 3T3 fibroblasts. Recently we reportedon an abbreviated protocol of the EST using flow cytometry,termed FACS-EST. This protocol reduces the assay length from ten to seven days and replaces the morphological assessment with the immunofluorescence detection of structural proteins ofthe sarcomere apparatus.Determining the concentration of half maximal inhibition of differentiation of VPA and its six congeners in the EST revealed a ranking similar to that found previously. Distinct embryotoxicitiesin vivo of stereoisomers, which differ only by their spatial configuration, were reproduced by the EST. Similarly, an increased in vivo potency correlating with longer chain lengthof the congener was evident as higher cytotoxicity in the EST.Comparing the validated EST with the FACS-EST demonstrated a good correlation, with the ranking of the substances being identical between methods.Our data demonstrate that the EST is capable of differentiating among closely related molecules according to their embryotoxic potency. Both in vitro differentiation and cytotoxicity haveto be considered to assess teratogenicity comparable to in vivo results. The FACS-EST is equally potent to the validated EST, and all substances were ranked in the same order and in full accordancewith data obtained in the NMRI exencephaly mouse model