An Analytical-to-In Vitro Approach: Deciphering the Bioactive Fraction of Complex Substances

Abstract

Natural and manmade disasters can be large-scale sources of environmental contamination, posing risks to human, animal, and environmental health. The circumstances surrounding each scenario are unique, as are the chemical compositions of substances dispersed variably in the environment, often yielding mixtures of unpredictable composition. Traditional risk assessment paradigms are formulated for analyzing individual chemicals, even though most often, people and the environment are exposed to complex substances and/or mixtures. Therefore, a critical challenge exists to inform and facilitate the risk assessment of multi-constituent substances and co-exposures. This is especially true when substances of unknown, variable, complex reaction byproduct, or biological (UVCB) origin are involved. Comprised of thousands of constituents, analytical characterization and toxicity testing of UVCBs for the purpose of exposure and risk assessment are challenging. Thus, we hypothesized that analytical techniques could be used to complement new approach methodologies (NAMs) to enable rapid characterization of the bioactive fraction of concern of complex substances and mixtures. The aims to accomplish this were four-fold: first, nontarget ion mobility spectrometry-mass spectrometry (IMS-MS) was used to characterize 195 geographically diverse complex substances. We determined the grouping efficiency of geographical and source rock groups and identified PAH compounds that drove groups by chemical similarity. Second, we analyzed the similarity in chemical and bioactivity profiles exhibited by two petroleum UVCB categories. This explored the “structural similarity” needed by regulatory agencies to apply category approaches in read-across of complex substances for registration and hazard evaluation. Third, we conducted a comparative analysis of three dosing methods to enable comprehensive exposure of complex substances and hydrophobic mixtures in high-throughput, small-volume in vitro assays: conventional solvent extraction with biocompatible DMSO, passive dosing via silicone micro-O-rings, and cell culture media accommodated fraction (MAF). We drew inspiration from passive dosing and water accommodated fraction (WAF) methods used in aquatic toxicity testing and detailed by Organization for Economic Co-operation and Development (OECD) Guideline 23. Results demonstrate the utility of nontarget analytical methods such as IMS-MS to rapidly characterize the chemical signatures of complex substances, as well as the capability to trace the origin of the substance. Further, we found that UVCBs belonging to the same category for hazard evaluation exhibit considerable chemical and bioactive variability. Finally, our comparative analysis of dosing methods found that MAF proved to be the most consistent, solvent-free dosing method for reliable toxicity testing of complex substances. Overall, these results supported the hypothesis that new approach methods (NAMs) coupled with analytical techniques offer an efficient approach to rapidly perform chemical characterization and toxicity testing to inform exposure and risk assessment for complex substances.