Risk Characterization and Probabilistic Concentration–Response Modeling of Complex Environmental Mixtures Using New Approach Methodologies (NAMs) Data from Organotypic in Vitro Human Stem Cell Assays

BACKGROUND: Risk assessment of chemical mixtures or complex substances remains a major methodological challenge due to lack of available hazard or exposure data. Therefore, risk assessors usually infer hazard or risk from data on the subset of constituents with available toxicity values. OBJECTIVES: We evaluated the validity of the widely used traditional mixtures risk assessment paradigms, Independent Action (IA) and Concentration Addition (CA), with new approach methodologies (NAMs) data from human cell-based in vitro assays. METHODS: A diverse set of 42 chemicals was tested both individually and as mixtures for functional and cytotoxic effects in vitro. A panel of induced pluripotent stem cell (iPSCs)-derived models (hepatocytes, cardiomyocytes, endothelial, and neurons) and one primary cell type (HUVEC) were used. Bayesian concentration–response modeling of individual chemicals or their mixtures was performed for a total of 47 phenotypes to derive point-of-departure (POD) values. Probabilistic IA or CA was conducted to estimate the mixture effects based on the bioactivity profiles from the individual chemicals and compared with mixture bioactivity. RESULTS: All mixtures showed significant bioactivity, even though some were constructed using individual chemical concentrations considered “low” or “safe.” Even though CA is much more accurate as a predictor of mixture effects in comparison with IA, with CA-based POD typically within an order of magnitude of the actual mixture, in some cases, the bioactivity of the mixtures appeared to be much greater than that of their components under either additivity assumption. DISCUSSION: These results suggest that CA is a preferred first approximation for predicting mixture toxicity when data for all constituents are available. However, because the accuracy of additivity assumptions varies greatly across phenotypes, we posit that mixtures and complex substances need to be directly tested for their hazard potential. NAMs provide a practical solution that rapidly yields highly informative data for mixtures risk assessment. https://doi.org/10.1289/EHP7600