Critical review of in vitro dosing methods for difficult-to-test substances and hydrocarbon UVCBs

Alternative approaches to traditional animal testing are being promoted to support regulatory chemical risk assessments for environment and human health. The Organisation for Economic Co-operation and Development has validated some in vitro test methods, but these are often suitable only for monoconstituent chemicals with a limited range of physicochemical properties. Most in vitro test methods are not suitable for poorly soluble, (semi)volatile, or multiconstituent chemical substances without significant methodological adaptations. In particular, substances of unknown or variable composition, complex reaction products, or biological materials (UVCBs), including hydrocarbon UVCBs and petroleum substances (PSs), can pose serious challenges for in vitro (eco)toxicity testing due to their complexity and variable chemical compositions. The choice of dosing method will depend on the purpose of the test as well as the physicochemical properties of the test substance. It remains difficult to establish and maintain stable exposures of PSs in in vitro test systems due to different factors: (a) the high surface area to volume ratios of multiwell plates that promote sorption; (b) the open test wells that allow (semi)volatile constituents to escape or contaminate neighboring plate wells; (c) the difficulty to analytically confirm exposure in small testing volumes; and (d) the presence of lipids and proteins in biological media, which bind PS constituents. This review maps the currently used dosing methods for hydrophobic and/or (semi)volatile chemicals and UVCBs in in vitro tests for environment and human health hazard assessments and outlines approaches and modifications to overcome various testing challenges associated with these test substances. Finally, research gaps are identified and recommendations are made for future development of in vitro assays for UVCBs.