Dosing Methods to Enable Cell-Based In Vitro Testing of Complex Substances: A Case Study with a PAH Mixture

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.