Profiling assay performance in the DevTox germ layer reporter platform

Abstract

The U.S. Environmental Protection Agency (U.S. EPA) is mandated to develop new approach methods (NAMs) to detect chemicals risks to susceptible populations, including effects on pregnant women and their offspring. With limited hazard information available for current and new chemicals, NAMs can provide greater relevance to human biology, mechanistic insight, and higher testing capacity than traditional animal models. The DevTox Germ Layer Reporter (GLR) model platform was recently established for high-throughput screening and prioritization of potential developmental hazards. The model platform utilizes the RUES2-GLR pluripotent stem cell reporter line that expresses fluorescent fusion protein biomarkers SOX17 (endoderm), Brachyury (mesoderm), and SOX2 (ectoderm and pluripotency); enabling a multi-lineage readout of gastrulation lineages. The DevTox GLR-Endo assay used the model platform to evaluate chemical effects on differentiating endoderm, yielding a balanced accuracy (BA) of 72% against a training set of 43 developmental toxicants and 23 non-developmental toxicants. To assess the predictivity of additional early embryonic lineages, assays for pluripotency (DevTox GLR-Pluri), ectoderm (DevTox GLR-Ecto), and mesoderm (DevTox GLR-Meso) were developed. Chemical reference set (12 developmental toxicants and 4 non-developmental toxicants) activity for each assay revealed BAs of 92% for DevTox GLR-Endo and DevTox GLR-Pluri, 71% for DevTox GLR-Ecto, and 58% for DevTox GLR-Meso. Expanded testing of the DevTox GLR-Endo and DevTox GLR-Pluri with 63 developmental and non-developmental toxicants yielded BAs of 75% and 68%, respectively. Amongst the four DevTox GLR platform assays, the DevTox GLR-Endo assay maintained the highest degree of efficacy and overall predictive accuracy for the compound set evaluated in this study.