A comparison of the embryonic stem cell test and whole embryo culture assay combined with the BeWo placental passage model for predicting the embryotoxicity of azoles

•Addition of toxicokinetic data improves the predictivity of developmental toxicity.•Biomarkers of developmental toxicity advance the comparison of in vitro systems.•Retinoic acid genes identify early embryotoxic responses to azoles. In the present study, we show the value of combining toxico-dynamic and -kinetic in vitro approaches for embryotoxicity testing of azoles. Both the whole embryo culture (WEC) and the embryonic stem cells test (EST) predicted the in vivo potency ranking of twelve tested azoles with moderate accuracy. Combining these results with relative placental transfer rates (Papp values) as determined in the BeWo cell culture model, increased the predictability of both WEC and EST, with R2 values increasing from 0.51 to 0.87 and from 0.35 to 0.60, respectively. The comparison of these in vitro systems correlated well (R2 = 0.67), correctly identifying the in vivo strong and weak embryotoxicants. Evaluating also specific gene responses related with the retinoic acid and sterol biosynthesis pathways, which represent the toxicological and fungicidal mode of action of azoles respectively in the WEC and EST, we observed that the differential regulation of Dhrs3 and Msmo1 reached higher magnitudes in both systems compared to Cyp26a1 and Cyp51. Establishing sensitive biomarkers across the in vitro systems for studying the underlying mechanism of action of chemicals, such as azoles, is valuable for comparing alternative in vitro models and for improving insight in the mechanism of developmental toxicity of chemicals.