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Introduction: Electronic cigarette (e-cigarette) aerosol is understood to provide reduced exposure to harmful toxicants compared with tobacco cigarette smoke, as it delivers nicotine and flavors without the use of tobacco. Published studies have shown that e-cigarette aerosol is chemically simple compared with tobacco smoke and corresponding reductions in toxicity in vitro have been demonstrated. However, comprehensive analytical and in vitro assessments of many widely available and currently marketed products, including pod-based systems, are limited. Materials and Methods: Here we report comparative data for aerosol emissions and in vitro toxicity, using the neutral red uptake, the bacterial reverse mutation, and in vitro micronucleus assays, for a pod system e-cigarette compared with 3R4F reference cigarette smoke. Results and Discussion: Many of the harmful and potentially harmful constituents found in cigarette smoke were not detected in e-cigarette aerosol. Using established in vitro biological tests, e-cigarette aerosol did not display any mutagenic or genotoxic activity under the conditions of test. By contrast, 3R4F cigarette smoke displayed mutagenic and genotoxic activity. E-cigarette aerosol was also found to be *300-fold less cytotoxic than cigarette smoke in the neutral red uptake assay. Conclusion: Data presented here show clear differences between a tobacco cigarette reference product and a commercially available nontobacco containing e-cigarette product in terms of emissions and in vitro toxicity profile. Our results demonstrate that high-quality e-cigarettes and e-liquids may offer the potential for substantially reduced exposure to cigarette toxicants in adult smokers who use such products as alternatives to cigarettes.

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In vitro (geno)toxicity assessment of electronic vapour products (EVPs), relative to conventional cigarette, currently uses assays, including the micronucleus and Ames tests. Whilst informative on induction of a finite endpoint and relative risk posed by test articles, such assays could benefit from mechanistic supplementation. The ToxTracker and Aneugen Clastogen Evaluation analysis can indicate the activation of reporters associated with (geno)toxicity, including DNA damage, oxidative stress, the p53-related stress response and protein damage. Here, we tested for the different effects of a selection of neat e-liquids, EVP aerosols and Kentucky reference 1R6F cigarette smoke samples in the ToxTracker assay. The assay was initially validated to assess whether a mixture of e-liquid base components, propylene glycol (PG) and vegetable glycerine (VG) had interfering effects within the system. This was achieved by spiking three positive controls into the system with neat PG/VG or phosphate-buffered saline bubbled (bPBS) PG/VG aerosol (nicotine and flavour free). PG/VG did not greatly affect responses induced by the compounds. Next, when compared to cigarette smoke samples, neat e-liquids and bPBS aerosols (tobacco flavour; 1.6% freebase nicotine, 1.6% nicotine salt or 0% nicotine) exhibited reduced and less complex responses. Tested up to a 10% concentration, EVP aerosol bPBS did not induce any ToxTracker reporters. Neat e-liquids, tested up to 1%, induced oxidative stress reporters, thought to be due to their effects on osmolarity in vitro. E-liquid nicotine content did not affect responses induced. Additionally, spiking nicotine alone only induced an oxidative stress response at a supraphysiological level. In conclusion, the ToxTracker assay is a quick, informative screen for genotoxic potential and mechanisms of a variety of (compositionally complex) samples, derived from cigarettes and EVPs. This assay has the potential for future application in the assessment battery for next-generation (smoking alternative) products, including EVPs.

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