Substance depletion of monoterpenes in the mouse lymphoma assay: Quantification, impact and mitigation

For test substances with unfavorable physicochemical properties, different pathways of substance depletion such as volatilization or sorption to polymers or serum constituents can decrease the bioavailable fraction during in vitro toxicity testing. If not accounted for, this can lead to underestimated toxicity or even false-negative results. Therefore a thorough understanding of the in vitro test system as well as potential pitfalls and analytical confirmation of substance concentrations are required for reliable results. Here, we investigated the genotoxicity of the monoterpenes (R)-(+)-limonene (RLIM) and β-myrcene (βMYR), the monoterpene alcohol (±)-linalool (LIN) and the known volatile mutagen 1-bromopopane (1-BP) in the mouse lymphoma assay (MLA) and quantified the exposure concentrations. Additionally, a headspace (HS)-free incubation setup is presented which allows for sufficient exposure of suspension cells with volatile test substances. RLim, βMYR and 1-BP, showed rapid and quantitative evaporation during incubation, potentially confounding the outcome of the genotoxicity test which could be minimized in the HS-free incubation setup. Furthermore, extensive binding of RLIM and βMYR to serum constituents was shown to decrease bioavailability during HS-free incubation. While the HS-free incubation setup increased the sensitivity of the MLA for volatile genotoxins, no signs for mutagenicity were observed for the monoterpenes, underscoring their safety.