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In the Electronic Nicotine Delivery Systems (ENDS) Premarket Tobacco Application (PMTA; 2016) Draft Guidance, the FDA recommends a full assessment of the toxicological profile associated with new tobacco products, using in vitro toxicology (e.g., genotoxicity and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) to a standard battery of in vitro cytotoxicity (Neutral Red Uptake [NRU]) and genotoxicity (Ames and micronucleus [MN]) assays according to OECD guidelines and 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 Ames assay, however some reduction in background lawn was observed with 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 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.