Test substance depletion in in vitro genotoxicity testing

Assessment of potential genotoxicity is an essential part of the risk assessment for chemicals and is mandated by regulatory authorities worldwide. For this purpose, in vitro tests are used to assess different modes of action (MoA) for genotoxicity. However, interpretation of in vitro genotoxicity tests is usually based on calculated nominal concentrations which may not accurately reflect the exposure of the test system to the test substance. Based on the physicochemical properties of the test substance and assay setup, actual test substance concentrations may be significantly lower due to different pathways of substance depletion such as sorption to culture vessels, serum constituents or volatilization. If not accounted for, the extent of substance depletion can limit the assay sensitivity and the reliability of the results. The monoterpenes (R)-(+)-limonene (RLIM), β-myrcene (βMYR) and (±)-linalool (LIN) are expected to be prone to different depletion pathways due to their increased lipophilicity and volatility which has not been accounted for in previous genotoxicity studies. Therefore, this work aimed to quantify the extent of potential depletion pathways for these monoterpenes and to investigate the influence on the outcome of the Ames fluctuation test and the mouse lymphoma assay (MLA). Headspace gas chromatography-mass spectrometry (HS-GC-MS) measurements revealed quantitative losses of RLIM and βMYR in the first few minutes during conventional incubation for both assays while this was less significant for the more polar LIN. Measurements in modified incubation setups were used to identify volatilization as the main depletion pathway for the monoterpenes and to stabilize the exposure during incubation. These results were in accordance with the predicted distribution of the test substances from the biokinetic model of Kramer et al. (2010). Dichloromethane (DCM) and 1-bromopropane (1-BP) were included as suitable proof-of-concept positive controls to demonstrate the increased sensitivity of the modified incubation setups. Headspace-free (HS-free) incubation prevented false-negative results for DCM in the Ames fluctuation test while the sensitivity towards 1-BP mediated genotoxicity was five times higher in the HS-free incubation setup for the MLA. No signs for mutagenicity or genotoxicity were observed for the monoterpenes after improved exposure which underlines the safety of these chemicals.