Baseline effects of non-flavored e-liquids in the in vitro micronucleus assay

Abstract

Electronic nicotine delivery systems (ENDSs; e.g. e-cigarettes) are being developed as potentially reduced-risk alternatives to the continued use of conventional tobacco products. They typically comprise a device that heats an e-liquid to generate inhalable vapor. E-liquids and ENDS-derived vapor have been the focus of toxicological assessment; in particular, their DNA-damaging potential has been investigated with varying outcomes and conclusions. In vitro genetic toxicology assays have formed a part of these assessments. However, they are susceptible to producing misleading or false positive results, especially under extreme conditions. In the present study, we evaluated a series of six neat (non-vaporized) non-flavored e-liquids (NFEL-A to F) in a flow cytometry version of the in vitro micronucleus assay in order to characterize their baseline effects on Chinese hamster ovary cells under hazard identification conditions. The NFELs induced cytotoxicity universally despite differing in propylene glycol (PG), vegetable glycerin (VG), and nicotine content. In addition, significant genotoxic responses were also detected with the PG-predominant e-liquids NFEL-A, D, and F but not with NFEL-B, C, or E, which contained higher proportions of VG. All six NFELs induced extreme cell culture conditions (i.e. increases in pH and osmolality) at the concentrations assessed. They also exhibited nonbiologically relevant effects on the mechanistic endpoints (i.e. cell cycle and phosphorylated histones H2AX and H3). In conclusion, although the PG component of the NFELs drove micronucleus formation in the assay, data on the complementary mechanistic endpoints suggest that this apparent DNA damage is potentially misleading and of negligible biological relevance as a risk for DNA integrity. In future assessments, any adverse changes (such as signatures of micronuclei induction, G 2 M arrest, and increases in gH2AX) relative to this reference data set might indicate a possible genotoxic hazard and would prompt further investigations for exploring the extent of risk.

State-of-the-art methods and devices for generation, exposure, and collection of aerosols from e-vapor products

“…The threshold for a negative test outcome was a change in the GFP signal less than 2-fold above the background of the solvent control, while a positive result was a value greater than a 2-fold change above background. 120 The Toxtracker ® assay performed successfully and was able to discriminate among the different test solutions. A range of responses were obtained.…”

“…Other factors, including high test concentrations, liver microsome activation systems, or lysosomal nuclease release, potentially confound the performance and predictivity of in vitro assays that detect genetic damage. 113 Therefore, research on EVPs is turning to new in vitro assays being developed to complement the traditional battery of in vitro assays and that use physiologically relevant biological models of the respiratory tract and cardiovascular system. To test the progress toward toxicity reduction, endpoints are selected that are mechanistically linked to smoking-related chronic diseases.…”