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

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).

In vitro and in silico genetic toxicity screening of flavor compounds and other ingredients in tobacco products with emphasis on ENDS.

Electronic nicotine delivery systems (ENDS) are regulated tobacco products and often contain flavor compounds. Given the concern of increased use and the appeal of ENDS by young people, evaluating the potential of flavors to induce DNA damage is important for health hazard identification. In this study, alternative methods were used as prioritization tools to study the genotoxic mode of action (MoA) of 150 flavor compounds. In particular, clastogen-sensitive (γH2AX and p53) and aneugen-sensitive (p-H3 and polyploidy) biomarkers of DNA damage in human TK6 cells were aggregated through a supervised three-pronged ensemble machine learning prediction model to prioritize chemicals based on genotoxicity. In addition, in silico quantitative structure-activity relationship (QSAR) models were used to predict genotoxicity and carcinogenic potential. The in vitro assay identified 25 flavors as positive for genotoxicity: 15 clastogenic, eight aneugenic and two with a mixed MoA (clastogenic and aneugenic). Twenty-three of these 25 flavors predicted to induce DNA damage in vitro are documented in public literature to be in e-liquid or in the aerosols produced by ENDS products with youth-appealing flavors and names. QSAR models predicted 46 (31%) of 150 compounds having at least one positive call for mutagenicity, clastogenicity or rodent carcinogenicity, 49 (33%) compounds were predicted negative for all three endpoints, and remaining compounds had no prediction call. The parallel use of these predictive technologies to elucidate MoAs for potential genetic damage, hold utility as a screening strategy. This study is the first high-content and high-throughput genotoxicity screening study with an emphasis on flavors in ENDS products.

Quantitative differentiation of whole smoke solution-induced mutagenicity in the mouse lymphoma assay.

In vitro genotoxicity dose-response data have been investigated for their utility in modeling and assessing potential differences in mutagenic responses between machine-generated whole smoke solutions (WSSs) from combusted cigarette tobacco products. Our previous study observed that potency ranking by benchmark dose (BMD) analysis was a useful modeling approach for quantitative assessment of differences between the mutagenicity of several structurally diverse chemical constituents of cigarette smoke. To follow-up on these observations, we used the mouse lymphoma assay (MLA) to evaluate the mutagenicity of WSSs prepared from two commercial cigarettes smoked under two different smoking machine regimens. L5178Y cells were exposed to ≥5 concentrations of each WSS for 4 hr ± S9 activation. S9 reduced the cytotoxicity and enhanced the mutagenicity of the WSSs. The resulting S9-mediated mutagenicity dose-responses were compared between test articles using BMD analysis, the lowest dose exceeding the Global Evaluation Factor, the no observed or lowest observed genotoxic effect level, and the mutagenic potency. The BMD10 , BMD50 , BMD100 , and BMD200 , indicating a 10%, 50%, 100%, or 200% increase in the background mutant frequency, respectively, were calculated using the PROAST software package. Overall, the quantitative approaches resulted in a similar rank order of mutagenic potency for the MLA tested WSSs, with potency increasing with the level of tar. The BMD approach using covariate analysis produced the most informative comparisons. Differences in potency were associated with the number of cigarettes smoked, the cigarette product smoked, and the smoking machine protocol used to prepare the sample. Under the conditions of this study, these results suggest that our hypothesis of modeling MLA data using the BMD approach to quantitatively discriminate between the mutagenic potential of WSSs from combustible cigarettes might be an useful method. Environ. Mol. Mutagen. 59:103-113, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA.