Comparative potency analysis of whole smoke solutions in the bacterial reverse mutation test

Abstract

Short-term in vitro genotoxicity assays are useful tools to assess whether new and emerging tobacco products potentially have reduced toxicity. We previously demonstrated that potency ranking by benchmark dose (BMD) analysis quantitatively identifies differences among several known carcinogens and toxic chemicals representing different chemical classes found in cigarette smoke. In this study, six whole smoke solution (WSS) samples containing both the particulate and gas phases of tobacco smoke were generated from two commercial cigarette brands under different smoking-machine regimens. Sixty test cigarettes of each brand were machine-smoked according to the International Organization for Standardization (ISO) puffing protocol. In addition, either 60 or 20 test cigarettes of each brand were machine-smoked with the Canadian Intense (CI) puffing protocol. All six WSSs were evaluated in the bacterial reverse mutation (Ames) test using Salmonella typhimurium strains, in the presence or absence of S9 metabolic activation. The resulting S9-mediated mutagenic concentration-responses for the four WSSs from 60 cigarettes were then compared using BMD modelling analysis and the mutagenic potency expressed as number of revertants per μl of the WSS. The quantitative approaches resulted in a similar rank order of mutagenic potency for the Ames test in both TA98 and TA100. Under the conditions of this study, these results indicate that quantitative analysis of the Ames test data can discriminate between the mutagenic potencies of WSSs on the basis of smoking-machine regimen (ISO vs. CI), and cigarette product (differences in smoke chemistry).

Figure 1.

Comparison of mutagenicity of the WSSs prepared with different regimens in Salmonella strains TA98 (A, C) and TA100 (B, D) with S9 activation. The WSSs were generated by smoking either MR (A, B) or MS (C, D) using the different smoke regimens (open circle, CI; closed circle, ISO). The data points (from Tables 1 and 2) represent the means ± SD from three independent experiments.

Figure 2.

Comparison of mutagenicity of the WSSs from different cigarette brands in Salmonella strains TA98 (A, B, C) and TA100 (D, E, F) with S9 activation. The WSSs were generated by smoking either MR (closed triangle) or MS (open triangle) using different smoke regimens (CI or ISO). The data points (from Tables 1 and 2) represent the means ± SD from three independent experiments.

Figure 3.

Comparison of BMD values for the four 60-cigarette WSSs in Salmonella strains TA98, TA100, YG1041 and YG1042 with S9 activation. The BMD₁₀₀ estimates, producing a 100% increase over the background revertant level, were calculated using PROAST software. The bars represent the calculated 90% confidence interval of each BMD value (i.e. from BMDL to BMDU). The lower and upper limits were used to differentiate between responses based on non-overlapping confidence intervals. For cigarette MR vs. MS, see black and red bars for each group in the same strain; for ISO vs. CI regimen, see top and bottom panels; and for TA98 (including YG1041) vs. TA100 (including YG1042), see left- and right-side panels.