To date most toxicological testing of cigarettes has been performed on the particulate phase of cigarette smoke using standard genotoxic and cytotoxic methods, which include the AMES reverse mutation assay, neutral red uptake, mouse lymphoma and micronucleus assays. However, traditional test methods are based on a particulate test material and under submerged conditions and are not suitable for the testing of aerosols; including cigarette smoke. As a result there is a requirement for new methodologies which facilitate the testing of aerosols in vitro.

In this study we have modified the Ames reverse mutation assay, using a spread plate methodology, to allow exposure to a cigarette smoke aerosol at an air-agar interface (AAI). The methodology was evaluated using cigarette smoke generated from 3R4F reference cigarettes on a Vitrocell^® VC 10 Smoking Robot. Four strains of S. typhimurium and one strain of E. coli were tested individually on 6 independent occasions in the presence of S-9. A dose-related increase in revertant numbers was observed in strains TA98, TA100, YG1024 and YG1042 up to mean fold increases of 5.6, 1.7, 24.8 and 5.5-fold, respectively. E. coli strain WP2 uvrA pKM101 was unresponsive at all concentrations tested. To enable us to accurately quantify dose, we measured deposited particulate mass using Quartz Crystal Microbalance technology in situ of exposure.

In conclusion, we have modified the traditional Ames reverse mutation assay using an aerosol-based exposure system for the assessment of cigarette smoke toxicology. Furthermore, this method is not restricted to the testing of whole smoke and could be applied to the testing of other gases, mixtures or aerosols.