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.
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.
The mass spectrometric behavior of four pairs of stereoisomers was investigated by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The E- and Z-isomers of the pesticides chlorfenvinphos, dimethomorph, mevinphos and phosphamidon-each with one double bond-were chosen for this study. The MS response of the individual isomers was investigated by infusing the isomers individually into the MS or after the separation of isomer mixtures via high-performance liquid chromatography (HPLC). In the case of dimethomorph, the same MS response was found for the two isomers. In contrast to that, the individual isomers of chlorfenvinphos, mevinphos and phosphamidon showed different MS response both in the single ion monitoring (SIM) mode in single quadrupole MS and multiple reaction monitoring (MRM) mode in tandem MS. The MS response of the isomers partly depends on (1) the declustering potential of the precursor ion in the SIM mode, (2) the selected transition and (3) the collision energy in the MRM mode. Consequently, quantification by summation of the peak areas of the isomers is inaccurate due to over- or underestimating of one of the stereoisomers. Accurate quantitative results can only be achieved when the compound-specific MS parameters are separately determined for each isomer. This can be done by using pure isomers or by the determination of the MS parameters after HPLC separation and the measurement of the actual isomer ratio with an independent technique.
Three tobacco industry based laboratories determined selected mainstream components using their established in-house methods. Machine smoking was done according to the ISO smoking regime. The Test cigarettes smoked for this investigation were manufactured with different amounts of added glycerol, cocoa powder and sucrose. Variability between the three laboratories differed clearly for the analyzed smoke components. No overall effects due to the added ingredients on smoke components could be found. The high ‘tar’ products with the highest lodading of sucrose showed a slight increase in formaldehyde emissions among all three laboratories.
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