Using the Mouse Lymphoma TK Assay to Compare the Mutagenicity of Mainstream Smoke Condensate from Research Cigarettes
Presented at Society of Toxicology (SOT) Meeting
The mouse lymphoma TK assay -microtiter version- has been established and optimized to quantitatively determine the in vitro genotoxicity of mainstream smoke condensate (MSC) in order to complement our existing testing battery. The assay was validated with the positive control substances methyl methanesulfonate and benzo(a)pyrene and MSC from the reference cigarette LR4F with and without a metabolic activation consisting of the postmitochondrial (S9) fraction of the livers from rats treated with Aroclor 1254. Reproducible responses -with a coefficient of variation of less than 20%- were found for the positive control substances and for the LR4F-MSC. To test whether the assay is able to discriminate different cigarette types, MSC from five research cigarettes containing different tobacco blends was prepared and assayed. Positive responses were obtained for all cigarettes, with concentration-related increases in the mutant frequency. To compare the mutagenicity of the cigarettes, the dose-response curve for the mutant frequency was calculated by nonlinear regression analysis. From each dose-response curve, the effective concentration for a 3-fold increase over the spontaneous mutant frequency was calculated and compared. The comparison showed that without S9, the activity of MSC from a 100% burley blend cigarette was lower than that of the other blends; with S9 a similar trend was seen. This is different from the results seen in the s. Typhimurium reverse mutation assay in strains TA98 and ta100 with S9, where the burley cigarette was more active than the 100% bright blend cigarette. This supports the need for a genotoxicity testing battery of complementary assays for the comprehensive testing of cigarette smoke.