[Background] The electronic cigarette (e-cigarette) is a battery-powered device that vaporizes liquid for inhalation by the user. In general, the e-cigarette is perceived as being healthier than the conventional cigarette because no combustion process (and subsequently no formation of hazardous substances related to this process) occurs. However, the absence of the combustion process does not directly imply that the e-cigarette is a healthy substitute compared to the tobacco cigarette. While some studies indicate that the e-cigarette may be up to 95% healthier than the conventional cigarette, an important amount of hazardous substances are still present in the e-liquid, albeit at lower concentrations. Exposure to e-liquid substances has been associated with molecular changes, carcinogenicity and diseases such as heart malfunctions, delayed cellular differentiation and impaired immunological responses in animal models. Whether these findings are also relevant to humans remains unclear.
[Aim] The genotoxic potential of flavouring substances present in e-cigarettes was studied using animal-free approaches. In addition, the applicability of the Ames test and the in vitro micronucleus test to evaluate the genotoxic potential of e-liquids mixtures was investigated.
[Methods] The study was performed with flavouring substances that have previously been identified in e-liquids. First, the genotoxic potential of these compounds was assessed using in silico prediction software Derek and Sarah Nexus. For substances that showed a structural alert for genotoxicity in one of the models, genotoxicity data (if available) were collected from the databases of the European Food Safety Authority and European CHemicals Agency. In a next step, compounds for which no data were found and that were commercially available were studied in the Ames test and the in vitro micronucleus test (i.e. b-phellandrene, 2,3-pentanedione). However, for the majority of the compounds, no standard could be obtained and consequently, the Ames test and the in vitro micronucleus test were performed on the e-liquid mixtures. In order to assess the applicability of the in vitro tests for the e-liquid mixtures, three 2,3-pentanedione-containing e-liquids (i.e. Nutella Shake, Hyprtonic Mercedes, Miller’s Chocolate) and an e-liquid containing no substances with structural alerts for genotoxicity (i.e. Dark Turtle) were first tested. In a next step, two e-liquids containing components with a structural alert for genotoxicity but without in vitro or in vivo data (i.e. Ice Ice Baby, Candy Shop) were studied in the Ames test and the in vitro micronucleus test.
[Results and discussion] Based on the predictions obtained with the in silico models and the availability of genotoxicity data, 28 compounds detected in e-liquids were selected for in vitro genotoxicity testing. However, only two of these compounds were commercially available. b-phellandrene showed a slightly genotoxic effect in the in vitro micronucleus test in the presence of S9 metabolic fraction but was clearly negative in the Ames test. In contrast, 2,3-pentanedione induced a clear dose-dependent genotoxic effect, both in the Ames test and the in vitro micronucleus test. The effect did not require metabolic activation. All three 2,3-pentadione-containing e-liquids showed a negative result in the Ames test. However, they all induced a positive effect in the in vitro micronucleus test that was clearly more pronounced than the effect induced by the propylene glycol/glycerol matrix. Dark Turtle, an e-liquid containing solely nicotine, showed no or little additional genotoxic effect when compared to the matrix. This e-liquid was also negative in the Ames test. The results indicate that e-liquids containing potentially genotoxic compounds can be picked up in the in vitro micronucleus test. Interestingly, out of the two e-liquids containing components with a structural alert for genotoxicity, one was clearly positive whereas the other was negative in the in vitro micronucleus test. Both were also negative in the Ames test.
[Conclusion] Known and unknown potentially genotoxic compounds can be present in e-liquids. Furthermore, genotoxic effects of e-liquid mixtures can be picked up with the in vitro micronucleus test. Consequently, this test could be useful for ranking e-liquids based on their genotoxic concern. However, further research should be conducted.