Hazard evaluation of substances of “unknown or variable composition, complex reaction products and biological materials” (UVCBs) remains a major challenge in regulatory science because their chemical composition is difficult to ascertain. Petroleum substances are representative UVCBs and human cell-based data has been previously used to substantiate their groupings for regulatory submissions. We hypothesized that a combination of phenotypic and transcriptomic data could be integrated to make decisions as to selection of group-representative worst-case petroleum UVCBs for subsequent toxicity evaluation in vivo. We used data obtained from 141 substances from 16 manufacturing categories previously tested in six human cell types (induced pluripotent stem cell (iPSC)-derived hepatocytes, cardiomyocytes, neurons, and endothelial cells, and MCF7 and A375 cell lines). Benchmark doses for gene-substance combinations were calculated, and both transcriptomic and phenotype-derived points of departure (PODs) were obtained. Correlation analysis and machine learning were used to assess associations between phenotypic and transcriptional PODs and to determine the most informative cell types and assays, thus representing a cost-effective integrated testing strategy. We found that two cell types—iPSC-derived-hepatocytes and -cardiomyocytes—contributed the most informative and protective PODs and may be used to inform selection of representative petroleum UVCBs for further toxicity evaluation in vivo. Overall, while the use of new approach methodologies to prioritize UVCBs has not been widely adopted, our study proposes a tiered testing strategy based on iPSC-derived hepatocytes and cardiomyocytes to inform selection of representative worst-case petroleum UVCBs from each manufacturing category for further toxicity evaluation in vivo.

Microphysiological systems are an emerging area of in vitro drug development, and their independent evaluation is important for wide adoption and use. The primary goal of this study was to test reproducibility and robustness of a renal proximal tubule microphysiological system, OrganoPlate® 3-lane 40, as an in vitro model for drug transport and toxicity studies. This microfluidic model was compared to static multi-well cultures and tested using several human renal proximal tubule epithelial cell (RPTEC) types. The model was characterized in terms of the functional transport for various tubule-specific proteins, epithelial permeability of small molecules (cisplatin, tenofovir and perfluorooctanoic acid) versus to large-molecules (fluorescent dextrans, 60-150 kDa), and gene expression response to a nephrotoxic xenobiotic. The advantages offered by OrganoPlate® 3-lane 40 as compared to multi-well cultures are presence of media flow, albeit intermittent, and increased throughput compared to other microfluidic models. However, OrganoPlate® 3-lane 40 model appeared to offer only limited (e.g., MRP-mediated transport) advantages in terms of either gene expression or functional transport when compared to the multi-well plate culture conditions. While OrganoPlate® 3-lane 40 can be used to study cellular uptake and direct toxic effects of small molecules, it may have limited utility for studies for drug transport studies. Overall, this study offers refined experimental protocols and comprehensive comparative data on the function of RPETCs in traditional multi-well culture and micro-fluidic OrganoPlate® 3-lane 40, information that will be invaluable for the prospective end-users of in vitro models of the human proximal tubule.