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In vitro cell-based toxicity testing methods generate large amounts of data informative for risk-based evaluations. To allow extrapolation of the quantitative outputs from cell-based tests to the equivalent exposure levels in humans, reverse toxicokinetic (RTK) modeling is used to conduct in vitro-to-in vivo extrapolation (IVIVE) from in vitro effective concentrations to in vivo oral dose equivalents. IVIVE modeling approaches for individual chemicals are well-established; however, the potential implications of chemical-to-chemical interactions in mixture settings on IVIVE remains largely unexplored. We hypothesized that chemical co-exposures could modulate both protein binding efficiency and hepatocyte clearance of the chemicals in a mixture, which would in turn affect the quantitative IVIVE toxicokinetic parameters. To test this hypothesis, we used 20 pesticides from the Agency for Toxic Substances and Disease Registry (ATSDR) Substance Priority List, both individually and as equimolar mixtures, and investigated the concentration-dependent effects of chemical interactions on in vitro toxicokinetic parameters. Plasma protein binding efficiency was determined by using ultracentrifugation, and hepatocyte clearance was estimated in suspensions of cryopreserved primary human hepatocytes. We found that for single chemicals, the protein binding efficiencies were similar at different test concentrations. In a mixture, however, both protein binding efficiency and hepatocyte clearance were affected. When IVIVE was conducted using mixture-derived toxicokinetic data, more conservative estimates of Activity-to-Exposure Ratios (AERs) were produced as compared to using data from single chemical experiments. Because humans are exposed to mixtures of chemicals, this study is significant as it demonstrates the importance of incorporating mixture-derived parameters into IVIVE for in vitro bioactivity data in order to accurately prioritize risks and facilitate science-based decision-making.

Background: Both trichloroethylene (TCE) and tetrachloroethylene (PCE) are high-priority chemicals subject to numerous human health risk evaluations by a range of agencies. Metabolism of TCE and PCE determines their ultimate toxicity; important uncertainties exist in quantitative characterization of metabolism to genotoxic moieties through glutathione (GSH) conjugation and species differences therein. Objectives: This study aimed to address these uncertainties using novel in vitro liver models, interspecies comparison, and a sensitive assay for quantification of GSH conjugates of TCE and PCE, S -(1,2-dichlorovinyl)glutathione (DCVG) and S -(1,2,2-trichlorovinyl) glutathione (TCVG), respectively. Methods: Liver in vitro models used herein were suspension, 2-D culture, and micropatterned coculture (MPCC) with primary human, rat, and mouse hepatocytes, as well as human induced pluripotent stem cell (iPSC)-derived hepatocytes (iHep). Results: We found that, although efficiency of metabolism varied among models, consistent with known differences in their metabolic capacity, formation rates of DCVG and TCVG generally followed the patterns , and primary . Data derived from MPCC were most consistent with estimates from physiologically based pharmacokinetic models calibrated to in vivo data. Discussion: For TCE, the new data provided additional empirical support for inclusion of GSH conjugation-mediated kidney effects as critical for the derivation of noncancer toxicity values. For PCE, the data reduced previous uncertainties regarding the extent of TCVG formation in humans; this information was used to update several candidate kidney-specific noncancer toxicity values. Overall, MPCC-derived data provided physiologically relevant estimates of GSH-mediated metabolism of TCE and PCE to reduce uncertainties in interspecies extrapolation that constrained previous risk evaluations, thereby increasing the precision of risk characterizations of these high-priority toxicants. https://doi.org/10.1289/EHP12006

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