Substance depletion of volatile monoterpenes – A confounding factor for toxicity testing in the Ames fluctuation test

In in vitro toxicology, reported test results are typically based on nominal concentrations, i.e., the calculated amounts of a substance added to a defined volume of the test system. Consequently, if a test system does not respond to a certain endpoint, the assay is interpreted as negative and the test substance is deemed to exert no toxicity at the tested nominal concentration. However, depending on the physicochemical properties of the test substance and assay setup, the actual exposure may differ widely from nominal concentrations due to different depletion processes. (R)-(+)-Limonene (RLIM), β-myrcene (βMYR) and linalool (LIN) are naturally occurring terpenes that are permitted as flavoring agents in the European Union without limitations based on their low toxicity. Nevertheless, their hydrophobicity and high volatility classifies them as difficult to test chemicals, which has not been considered in previous in vitro tests. To exclude possible false negative results, in the present study, we assessed the cytotoxic and mutagenic potential of the latter substances toward Salmonella Typhimurium in the Ames fluctuation test using different incubation setups to minimize possible substance losses due to sorption or volatilization. Actual substance concentrations during incubation were verified analytically at different time points via headspace gas chromatography-mass spectrometry (HS-GC-MS). Possible substance depletion due to sorption to well-plate material or volatilization was minimized using a polystyrene-free and headspace-free incubation setup, respectively. The results showed complete volatilization of the monoterpenes RLIM and βMYR in the conventional Ames fluctuation test, which may confound mutagenicity testing. The headspace-free incubation setup greatly improved substance exposure and showed cytotoxicity in low micromolar concentrations, but no signs of mutagenicity were observed.