Emerging nicotine analog 6-methyl nicotine increases reactive oxygen species in aerosols and cytotoxicity in human bronchial epithelial cells.

Nicotine-contained e-cigarettes (E-cigs) generate reactive oxygen species (ROS), volatile organic compounds, and heavy metals. Inhalation toxicology studies suggest that exposure to these toxicants may adversely impact human health. These findings led to the U.S. Food and Drug Administration's (FDA) regulation of nicotine-containing E-cigs under the Tobacco Regulation Act (TRA) of 2020. Manufacturers aiming to sell nicotine products in the U.S. must submit a Premarket Tobacco Product Application (PMTA) and obtain FDA approval before marketing their products. However, due to the lengthy PMTA process, some companies have exploited a loophole in the TRA (2020) by introducing nicotine analogs, such as 6-methyl nicotine (6-MN) into E-cig products. 6-MN is marketed as a 'safer' alternative to nicotine, offering comparable satisfaction despite not being derived from tobacco or nicotine. Nonetheless, its safety profiles are unknown. Therefore, this study tested the toxicity of 6-MN compared to traditional nicotine in vitro. We observed that thermal degradation of 6-MN in e-liquids significantly generated more ROS in the aerosols than nicotine. We investigated the dose-response cytotoxicity of 6-MN vs nicotine when exposed to HBEC3-KT human bronchial epithelial cells. 6-MN-contained e-liquids significantly increased cytotoxicity and intracellular ROS induction in a dose-specific manner compared to nicotine. Further, we observed that 6-MN (pure compound) transiently increased metabolic activity significantly at all doses tested compared to nicotine. Given the potential risks associated with 6-MN, it cannot be deemed 'safer' than nicotine. Therefore, further primary toxicological research is urgently needed to provide regulatory agencies with more robust data to implement regulations.