Critical review of in vitro dosing methods for hydrocarbon UVCBs

Summary

Alternative approaches to traditional animal testing are being promoted to support regulatory chemical assessments. These approaches include in vitro test methods and can be used in multiple contexts, from prioritization and screening to supporting chemical grouping and read-across. It is anticipated that in vitro tests will eventually replace whole organism in vivo testing. An important challenge of most in vitro testing methods is establishing, maintaining, and confirming defined exposure concentrations throughout typical test durations. This is particularly challenging for petroleum UVCBs (substances of Unknown, Variable composition, Complex reaction products, or Biological origin) that typically contain a large number and variety of hydrophobic and (semi)volatile hydrocarbon constituents (HC) that are prone to evaporative and sorptive losses. The ability to deliver and maintain stable petroleum substance (PS) exposures in in vitro test systems is challenged by several factors, including: the high surface area to volume ratios of most multi-well plates, which increases the likelihood of sorption to plate walls; the inability to seal some test vessels (e.g. volatile constituents can escape open test vessels and may contaminate neighboring plate wells); poor solubility of hydrophobic constituents in biological media; compatibility with small testing volumes; and presence of lipids and proteins in biological media which may differentially bind individual constituents. Here we present the findings from a critical review describing the challenges of dosing UVCBs into conventional in vitro test setups and identifies relevant assays, dosing methods, and potential adaptations that may improve in vitro testing of UVCBs and other difficult-to-test substances. Approaches best suited for regulatory testing of PS and areas of future research are also identified.