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Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition.

Teratogenic effects caused by a new nitroimidazopyridazine were examined in Wistar (WU) rats after repeated oral administration of 0, 2.5, 10, and 40 mg/kg, given on days 6-17 post coitum (p.c.) (Day of mating = Day 0) in a regular study on embryo-fetal development according to ICH S5A. At day 20 p.c., fetuses were removed and carefully examined under a dissecting microscope for external, visceral and skeletal malformations. The exposure to the high dose of the test compound during the organogenesis and early histogenesis periods of prenatal development induced prominent CNS malformations (exencephaly, neural tube defects (NTD)) associated with external malformations (hyperflexion of the forelimbs). To support the data from this study additional histological evaluation of the brains was performed with the following results: disorganization of the cerebral cortex associated with ectopic subcommissural organs. Additionally, an in vitro test (whole embryo culture, WEC) showed alterations of the developing neural tube after the incubation of rat embryos with the test compound on gestation days 9.5-11.5. Our data demonstrated that nitroimidazopyridazine caused NTDs and limb malformations during organogenesis. Based on these data the further development of the test compound was stopped.