Advanced developmental toxicity test method based on embryoid body's area

•Cytotoxicity in ESCs/3T3 cells and embryotoxicity in embryoid body were evaluated.•Embryoid body's area is an important endpoint to assess developmental toxicity.•Embryoid body's area was regulated by Dnmt3b, Cbx5, Hdac2 and Cdkn2a.•Embryoid bodies test is the useful and advanced developmental toxicity test method.

Embryonic stem cell test (EST) evaluates the embryotoxic potential of substances and measures the half inhibition in viability of mouse embryonic stem cells (ESCs), fibroblasts (3T3 cells) and in cardiac differentiation of ESC. In this study, we suggest the developmental toxicity test method (termed EBT) applying area of embryoid bodies (EBs) instead of cardiac differentiation of EST. In the assessment of 21 substances, EB area was logarithmically decreased in dose-dependent manner. Decline in EB area resulted in decrease of beating ratio during differentiation of ESCs. In classification by the EBT-based prediction model reflecting decline in cell viability and EB area, toxicity for 21 chemicals showed 90.5% accuracy. In the results of next generation sequencing, reduction in EB area resulted from cell cycle arrest mediated by HDAC2 and CDKN2A. Conclusively, EBT is advanced and is a useful tool to assess and classify various embryotoxicants in a short time with less effort.