The embryoid body test (EBT) is a developmental toxicity test method that assesses the half inhibitory concentrations of substances in the area of embryoid bodies (EBs), and in the viability of mouse embryonic stem cells (ESCs) and fibroblasts (3T3 cells). In the previous pre-validation study evaluated the predictive accuracy of the EBT using 26 coded test substances and highly accurate (above 80%) when substances were classified using the predictive model. EBT used two same endpoints as EST, the half inhibition concentrations for cell viability of mouse ESCs (IC50 E14) and 3T3 fibroblasts (IC50 3T3), but replaced the half inhibition concentration for cardiac differentiation (ID50 CM) with the half inhibition concentration for EB area (ID50 EB). We used the hanging drop method to form an embryoid bodies. In order to verify the proposed EBT method in this study, inter-laboratory reproducibility (5 substances in common) and predictive capacity (10 substances in each laboratory) tests were performed. To ensure reliability of the study results, the tests were conducted using identity-coded test substances. The results of statistical analysis of the inter-laboratory reproducibility test indicated that reproducibility accuracy 87%, sensitivity 78%, and specificity 100%. The results of statistical analysis of the predictive capacity test indicated that the lead laboratory had reproducibility accuracy 80%, sensitivity 86%, and specificity 67%. Participatory laboratory 1 had reproducibility accuracy 80%, sensitivity 71%, and specificity 100% and participatory laboratory 2 had reproducibility accuracy 80%, sensitivity 86%, and specificity 67%. The results of the intra- and inter-laboratory 2tests were highly accuracy 83%, sensitivity 80%, and specificity 89% when substances were classified using the predictive model. EBT can accurately classify various embryotoxicants in a short time with less effort and greater validation.
This research was supported by a grant (17182MFDS487) from Ministry of Food and Drug Safety in 2017.