AbstractThe aims were to develop a method to simulate extraction of toxic components into human saliva, to identify these components through bioassay‐driven analyses using the Microtox® test, and to quantify the toxic compounds. Chewing tobacco extractions into artificial saliva simulated oral chewing conditions. The Microtox bioassay‐directed subfractionation scheme identified acutely toxic components in chewing tobacco extracts. GC‐MS identified and quantified the toxic components. The subfractionation (pH 7 extraction, pH 2 extraction, then pH 10 extraction using organic solvents) scheme was shown to be comparable to biological digestive sequences and to simulate nicotine bioavailability. The low toxicity of artificial saliva allowed development of a sensitive bioassay. Nicotine was the major acutely toxic agent detected. Classification by Draize ocular irritative categories showed that leached nicotine concentrations were mildly to moderately irritating. Thus, the Microtox test can be used to detect irritative compounds and subfractions, and can be potentially applied to saliva biological monitoring in
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