A comparative toxicological evaluation of nicotine and its analog 6-methyl nicotine in E-cigarette aerosol utilizing a 3D in vitro human respiratory model

Under the U.S. Tobacco Regulatory Act of 2020, all novel nicotine-containing products require a Premarket Tobacco Product Application (PMTA) and FDA authorization before they can be marketed. However, lengthy PMTA review timelines have prompted some manufacturers to replace traditional nicotine with 6-methyl-nicotine (6-MN), a non-tobacco-derived analog that delivers comparable psychoactive effects while evading existing regulatory pathways. Despite its growing market presence as a purportedly “safer” alternative, the toxico-pharmacokinetic profile of 6-MN remains poorly characterized. This study assessed the toxicity after exposure to nicotine or 6-MN-containing e-liquid aerosols using a 3D EpiAirway tissue model. RT-qPCR analyses revealed differential effects on transcripts associated with DNA damage (53BP1, ATR), inflammation (NF-κB1), and cancer (MYCBP). Morphological evaluation of the airway tissues exposed to either aerosol showed an increase in epithelial thickness, a decrease in E-cadherin protein levels, increased goblet cell hypertrophy, evidenced by positive PAS staining and elevated mucus (MUC5AC protein) production, and a reduction in Occludin protein (part of the tight junction complex), which is suggestive of epithelial remodeling. Exposure to PG/VG aerosols alone significantly increased the release of MIP-1α, IFN-γ, and IL-4. Conversely, spearmint-flavored aerosols containing 6-MN or nicotine decreased several pro-inflammatory cytokines, significantly reducing TNF-α, Eotaxin, MCP-1, RANTES, and G-CSF levels, potentially via NF-κB and ERK1/2 pathways. Our findings reveal differential toxicological and chemical profiles for nicotine and 6-MN aerosols; however, flavorings may confer similar cytotoxicity, as measured by LDH and metabolic activity, in 6-MN formulations as they do in those with nicotine. Thus, 6-MN is not a “safer” nicotine alternative.