The mutagenic assessment of an electronic-cigarette and reference cigarette smoke using the Ames assay in strains TA98 and TA100

Abstract

Salmonella typhimurium strains TA98 and TA100 were used to assess the mutagenic potential of the aerosol from a commercially available, rechargeable, closed system electronic-cigarette. Results obtained were compared to those for the mainstream smoke from a Kentucky reference (3R4F) cigarette. Two different test matrices were assessed. Aerosol generated from the e-cigarette was trapped on a Cambridge filter pad, eluted in DMSO and compared to cigarette smoke total particulate matter (TPM), which was generated in the same manner for mutagenicity assessment in the Salmonella assay. Fresh e-cigarette and cigarette smoke aerosols were generated on the Vitrocell VC 10 smoking robot and compared using a modified scaled-down 35mm air agar interface (AAI) methodology. E-cigarette aerosol collected matter (ACM) was found to be non-mutagenic in the 85mm plate incorporation Ames assay in strains TA98 and TA100 conducted in accordance with OECD 471, when tested up to 2400μg/plate. Freshly generated e-cigarette aerosol was also found to be negative in both strains after an AAI aerosol exposure, when tested up to a 1L/min dilution for up to 3h. Positive control responses were observed in both strains, using benzo[a]pyrene, 2-nitrofluorene, sodium azide and 2-aminoanthracene in TA98 and TA100 in the presence and absence of metabolic activation respectively. In contrast, cigarette smoke TPM and aerosol from 3R4F reference cigarettes were found to be mutagenic in both tester strains, under comparable test conditions to that of e-cigarette exposure. Limited information exists on the mutagenic activity of captured e-cigarette particulates and whole aerosol AAI approaches. With the lower toxicant burden of e-cigarette aerosols compared to cigarette smoke, it is clear that a more comprehensive Ames package of data should be generated when assessing e-cigarettes, consisting of the standard OECD-five, TA98, TA100, TA1535, TA1537 (or TA97) and E. coli (or TA102). In addition, TA104 which is more sensitive to the carbonyl based compounds found in e-cigarette aerosols under dry-wicking conditions may also prove a useful addition in a testing battery. Regulatory standard product testing approaches as used in this study will become important when determining whether e-cigarette aerosols are in fact less biologically active than cigarette smoke, as this study suggests. Future studies should be supported by in vitro dosimetry approaches to draw more accurate comparisons between cigarette smoke, e-cigarette aerosol exposure and human use.

Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage

“…Moreover, oxidative DNA lesions have been proposed to cause the vast majority of human mutations [79]. Our data is in apparent contradiction with the fact that ECs have been deemed non-mutagenic to Ames Salmonella tester strains TA98 and TA100 [80, 81]. However, none of the previous studies included the strains TA102 or TA104 which have the highest sensitivity for the detection of mutagenesis induced by oxidative or cross-linking agents [82].…”

“…TA104 has also been reported to be particularly sensitive to carbonyl based compounds [83], as those reported to be present in EC aerosol. Moreover, the Organization for Economic Co-operation and Development (OECD) recommends testing a battery of at least five tester strains to capture the full range of chemical interactions and mutagenic events acting via different modes of action[81]. Another variable to consider, is the fact that toxicant yield varies with EC brand, product model, e-liquid flavor, e-liquid solvent, and device power [19, 21, 23, 84, 85] so it is not possible to directly compare our study to others using different ECs or e-liquids.…”

Limited mutagenicity of electronic cigarettes in mouse or human cells in vitro

“…[64] have recently reported that e-cig aerosol is not mutagenic in the Ames assay in bacterial strains TA98 and TA100. The limited mutagenicity of e-cig vapor found in this in vitro study as well as in those reports [65, 66] may imply that e-cig is worthy of further consideration as a putatively modified-risk tobacco substitute. The view that e-cig may pose a reduced risk as compared to combustible cigarettes is consistent with the lower concentrations of most toxicants and carcinogens in e-cig vapor than cigarette smoke [8–13].…”

A Review of Pulmonary Toxicity of Electronic Cigarettes in the Context of Smoking: A Focus on Inflammation

“…Existing studies suggest that the toxicological responses are qualitatively similar to smoking, e.g., exposing cell lines and cultures to the aerosols induces a pro-inflammatory effect (185, 186), disruption to epithelia barriers (187), oxidative stress (188), cytotoxicity (189), neutrophil inflammatory response (190) and DNA damage (191, 192). However, the magnitude of effect is low compared to cigarette smoke and aerosols were not found to be mutagenic (193). Normal human bronchial epithelial (NHBE) cells exposed to e-cig aerosols, with or without nicotine, increase IL-6 and IL-8 cytokine levels (194).…”