A higher throughput method to the Embryonic Stem cell Test (EST), to detect embryotoxicity in early development

The in vitro embryonic stem cell test (EST) allows for categorisation of the embryotoxic potential of chemicals and drug candidates. For classification, a validated prediction model was developed based on the inhibition of differentiation of murine embryonic stem cells (D3 cells) into cardiomyocytes, and the cytotoxicity data of D3 cells and murine fibroblasts (3T3 cells). Alterations were made in order to simplify the experimental procedures of the EST; a low-cell-binding 96-well plate was used to obtain the embryonic bodies instead of the original hanging drop culture. Furthermore, we assessed the need to include the 3T3 cells and developed a new ranking system. D3 cells were exposed to test compounds either from day 0 or day 3 onwards and the compounds were ranked by their Relative Embryotoxic Potency (REP), relative to the positive control 6-Aminonicotinamide. This resulted in the following REP order; 6-Aminonicotinamide > Hydroxy urea > Valproic acid > Methoxyacetic acid > Penicillin G. A similar outcome was obtained when the validated prediction model was used. Exposure of cells from day 0 or day 3 onwards did not have any effect on the outcome as calculated with both methods. We propose a simplification of the in vitro EST procedure and REP values to rank compounds.