Alexandra C. Cordova

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.