The Ames methodology is governed by clear international regulatory guidelines (e.g. OECD 471), which recommend the use of at least five bacterial strains in an in vitro test battery. Salmonella typhimurium strains TA1535; TA1537 or TA97 or TA97a; TA98 and TA100 between them detect frameshift and base-pair substitutions. Either strain TA102 or an Escherichia coli (E. coli) strain (WP2 uvrA or WP2 uvrA (pKM101)) are accepted as a fifth strain and between them detect certain hydrazines, oxidising mutagens and cross-linking agents.

In this study we have modified the Ames assay, using a spread plate methodology, to allow exposure to cigarette smoke at the air-agar interface, which facilitates the assessment of the complete cigarette smoke aerosol. In total, nine S. typhimurium strains and one E. coli strain were investigated using varying dilutions of cigarette smoke (12.0, 8.0, 4.0 and 1 L/min), generated from a VC 10 Smoking Robot. Of the assessed strains, five tested positive (TA98, TA100, YG1024, YG1042 and TA104), four were negative (TA102, WP2 uvrA pKM101, TA97 and TA1535) and one strain (TA1537) was deemed incompatible with this scaled-down methodology and was not assessed with cigarette smoke. A response was considered positive if greater than a two-fold increase over background spontaneous revertants was observed, combined with statistical differences (p<0.01). In the case of a negative response, smoke exposures were increased from 24 to 64 minutes to further assess mutagenic activity. Finally, to support in vitro exposure and to quantify dose, deposited particulate mass (µg/cm²) was assessed using Quartz Crystal Microbalance technology in situ of exposure.

In conclusion, we have assessed an OECD acceptable battery and additional Ames strains for their responsiveness to mainstream cigarette smoke. Based on these data, we propose that a selection of these strains could be appropriate to assess the genotoxicity of current and future aerosol-based tobacco products.