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Human cell-based test methods can be used to evaluate potential hazards of mixtures and products of petroleum refining ("unknown or variable composition, complex reaction products, or biological materials" substances, UVCBs). Analyses of bioactivity and detailed chemical characterization of petroleum UVCBs were used separately for grouping these substances; a combination of the approaches has not been undertaken. Therefore, we used a case example of representative high production volume categories of petroleum UVCBs, 25 lower olefin substances from low benzene naphtha and resin oils categories, to determine whether existing manufacturing-based category grouping can be supported. We collected two types of data: nontarget ion mobility spectrometry-mass spectrometry of both neat substances and their organic extracts and in vitro bioactivity of the organic extracts in five human cell types: umbilical vein endothelial cells and induced pluripotent stem cell-derived hepatocytes, endothelial cells, neurons, and cardiomyocytes. We found that while similarity in composition and bioactivity can be observed for some substances, existing categories are largely heterogeneous. Strong relationships between composition and bioactivity were observed, and individual constituents that determine these associations were identified. Overall, this study showed a promising approach that combines chemical composition and bioactivity data to better characterize the variability within manufacturing categories of petroleum UVCBs.

Cell-based testing of multi-constituent substances and mixtures for their potential adverse health effects is difficult due to their complex composition and physical-chemical characteristics. Various extraction methods are typically used to enable studies in vitro; however, a limited number of solvents are biocompatible with in vitro studies and the extracts may not fully represent the original test article's composition. While the methods for dosing with "difficult-to-test" substances in aquatic toxicity studies are well defined and widely used, they are largely unsuited for small-volume (100 microliters or less) in vitro studies with mammalian cells. Therefore, we aimed to evaluate suitability of various scaled-down dosing methods for high-throughput in vitro testing by using a mixture of polycyclic aromatic hydrocarbons (PAH). Specifically, we compared passive dosing via silicone micro-O-rings, cell culture media-accommodated fraction, and traditional solvent (dimethyl sulfoxide) extraction procedures. Gas chromatography-tandem mass spectrometry (GC-MS/MS) was used to evaluate kinetics of PAH absorption to micro-O-rings, as well as recovery of PAH and the extent of protein binding in cell culture media with and without cells for each dosing method. Bioavailability of the mixture from different dosing methods was also evaluated by characterizing in vitro cytotoxicity of the PAH mixture using EA.hy926 and HepG2 human cell lines. Of the tested dosing methods, media accommodated fraction (MAF) was determined to be the most appropriate method for cell-based studies of PAH-containing complex substances and mixtures. This conclusion is based on the observation that the highest fraction of the starting materials can be delivered using media accommodated fraction approach into cell culture media and thus enable concentration-response in vitro testing.

Although humans are continuously exposed to complex chemical mixtures in the environment, it has been extremely challenging to investigate the resulting cumulative risks and impacts. Recent studies proposed the use of “new approach methods,” in particular in vitro assays, for hazard and dose−response evaluation of mixtures. We previously found, using five human cell-based assays, that concentration addition (CA), the usual default approach to calculate cumulative risk, is mostly accurate to within an order of magnitude. Here, we extend these findings to further investigate how cell-based data can be used to quantify inter-individual variability in CA. Utilizing data from testing 42 Superfund priority chemicals separately and in 8 defined mixtures in a human cell-based population-wide in vitro model, we applied CA to predict effective concentrations for cytotoxicity for each individual, for “typical” (median) and “sensitive” (first percentile) members of the population, and for the median-to-sensitive individual ratio (defined as the toxicodynamic variability factor, TDVF). We quantified the accuracy of CA with the Loewe Additivity Index (LAI). We found that LAI varies more between different mixtures than between different individuals, and that predictions of the population median are generally more accurate than predictions for the “sensitive” individual or the TDVF. Moreover, LAI values were generally <1, indicating that the mixtures were more potent than predicted by CA. Together with our previous studies, we posit that new approach methods data from human cell-based in vitro assays, including multiple phenotypes in diverse cell types and studies in a population-wide model, can fill critical data gaps in cumulative risk assessment, but more sophisticated models of in vitro mixture additivity and bioavailability may be needed. In the meantime, because simple CA models may underestimate potency by an order of magnitude or more, either whole-mixture testing in vitro or, alternatively, more stringent benchmarks of cumulative risk indices (e.g., lower hazard index) may be needed to ensure public health protection.

Human cell-based population-wide in vitro models have been proposed as a strategy to derive chemical-specific estimates of inter-individual variability; however, the utility of this approach has not yet been tested for cumulative exposures in mixtures. This study aimed to test defined mixtures and their individual components and determine whether adverse effects of the mixtures were likely to be more variable in a population than those of the individual chemicals. The in vitro model comprised 146 human lymphoblastoid cell lines from four diverse subpopulations of European and African descent. Cells were exposed, in concentration−response, to 42 chemicals from diverse classes of environmental pollutants; in addition, eight defined mixtures were prepared from these chemicals using several exposure- or hazard-based scenarios. Points of departure for cytotoxicity were derived using Bayesian concentration−response modeling and population variability was quantified in the form of a toxicodynamic variability factor (TDVF). We found that 28 chemicals and all mixtures exhibited concentration−response cytotoxicity, enabling calculation of the TDVF. The median TDVF across test substances, for both individual chemicals or defined mixtures, ranged from a default assumption (101/2) of toxicodynamic variability in human population to >10. The data also provide a proof of principle for single-variant genome-wide association mapping for toxicity of the chemicals and mixtures, although replication would be necessary due to statistical power limitations with the current sample size. This study demonstrates the feasibility of using a set of human lymphoblastoid cell lines as an in vitro model to quantify the extent of inter-individual variability in hazardous properties of both individual chemicals and mixtures. The data show that population variability of the mixtures is unlikely to exceed that of the most variable component, and that similarity in genome-wide associations among components may be used to accrue additional evidence for grouping of constituents in a mixture for cumulative assessments.