Gaining confidence in in vitro toxicity data with measured exposure concentrations

Cell-based in vitro bioassays are essential New Approach Methodologies (NAMs) that allow the bioactivity screening for thousands of chemicals in a time- and cost-efficient manner. One major obstacle for the widespread acceptance of in vitro bioassays is the undefined exposure in these tests. The present study aimed to complement in vitro testing of cytotoxicity as well as specific endpoints, such as the activation of oxidative stress response, with measured exposure concentrations as additional readout. Freely dissolved concentrations (C _free) of 24 test chemicals in four different cell-based in vitro bioassays were measured in 96-well plate format at the time point of dosing and after 24 h of exposure in the assay plates for all tested concentrations of the chemicals. Freely dissolved fractions (f _free) in the medium decreased with increasing hydrophobicity. Hydrophilic chemicals like atenolol had f _free of >90 %, while the most hydrophobic test chemical chrysene had an f _free of only 1.4 % at the beginning of the test and 0.7 % after 24 h of exposure to the cells. For 12 of the 24 chemicals C _free decreased >20 % during the bioassays. Depending on the chemical, the loss may be due to sorption to well plate plastic, cellular metabolism or abiotic degradation (e.g., hydrolysis). The simultaneous measurement of toxicity and exposure allows the identification of chemicals with unstable exposure concentrations and/or low bioavailability and provides a quantitative measure of bioactivity that can directly be applied for in vitro-to-in vivo extrapolation (IVIVE).