Toxicological assessment of e-liquid formulations using in vitro genotoxicity and cytotoxicity assays
In the electronic nicotine delivery systems (ENDS) Premarket Tobacco Application (PMTA; 2016) Draft Guidance, the U.S. Food and Drug Administration (FDA) recommends a full assessment of the toxicological profile associated with new tobacco products, using in vitro toxicology (e.g. genotoxicity and cytotoxicity) studies. As part of a toxicological hazard assessment, we tested flavor varieties of e-liquids used in MarkTen® e-vapor products (a total of 14 formulations) and two carrier formulations (propylene glycol, glycerin, with 0 % or 5 % nicotine) in a standard battery of in vitro cytotoxicity (neutral red uptake [NRU]) and genotoxicity (Ames and micronucleus [MN]) assays according to OECD guidelines, using the maximum doses suggested for mixtures. The e-liquid formulations were characterized for key ingredients (propylene glycol, glycerin, and nicotine). All the formulations were non-cytotoxic per NRU assay (viability >80 %). None of the e-liquids were mutagenic in the Ames assay, however some reduction in background lawn was observed with the carrier formulation at the high (5 %) nicotine content. In the MN assay, 3/14 MarkTen® flavor formulations induced a weak but statistically significant increase in micronuclei formation, resulting in positive or equivocal findings according to OECD 487. All three flavor formulations were further evaluated in an in vivo combined genotoxicity (MN and Comet; OECD 474/489) assay and found to be negative for genotoxic endpoints. Therefore, consistent with the International Conference on Harmonization (ICH) S2(R1) genotoxicity testing guideline (2012), the tested e-liquids were regarded as negative for genotoxicity under the conditions of the assays.