Abstract
The embryonic stem cell test (EST), an alternative model to animal studies, is a reliable and scientifically validated in vitro system for testing embryotoxicity. In contrast to most in vivo animal tests, two permanent cell lines, murine fibroblasts (BALB/c‑3T3 cells) and murine embryonic stem cells (mES‑D3 cells), are used in EST instead of animals in standard tests of toxicity. The embryotoxic potential of compounds (non, weak or strong embryotoxicity) may be obtained with a biostatistics‑based prediction model and calculated from three different experimental endpoint values: The potency to inhibit growth of i) BALB/c‑3T3 cells and ii) mES‑D3 cells (IC503T3 and IC50ES) as presented using a cell cytotoxicity assay, and iii) the potency to inhibit differentiation of mES‑D3 cells into contracting cardiomyocytes (ID50 D3) as demonstrated in a mES‑D3 cell differentiation assay. In the present study, a model of EST with mES‑D3 cells and BALB/c‑3T3 cells was established, according to the standard EST system of the EU Center for the Validation of Alternative Methods, and verified it with 5‑fluorouracil (strong embryotoxicity) as a positive control and penicillin G (non‑embryotoxic) as a negative control. In addition, the authors further assessed the embryotoxicity of four compounds (eugenol, carnosic acid, procyanidin and dioctyl phthalate) with this model. The embryotoxic potentials of the four compounds were successfully classified by the EST system. Eugenol exhibited strong embryotoxicity, carnosic acid and dioctyl phthalate exhibited weak embryotoxicity, while procyanidin exhibited non‑embryotoxicity.