In vitro toxicological assessment using NCI-H292 cells of the relationship between cytotoxic effects and chemical composition in flavored e-liquids.

The range of e-cigarette flavors has driven consumer interest while raising concerns about potential health risks. However, the toxicological impact and chemical composition of flavored e-liquids remain poorly characterized. This study investigates cytotoxicity differences between tobacco- and non-tobacco-flavored e-liquids and identifies key compounds contributing to toxicity through chemical correlation analysis. We assessed the cytotoxicity of 100 e-liquids (50 tobacco-flavored, 50 non-tobacco-flavored) using IC₅₀ values in NCI-H292 cells and selected representative samples for genotoxicity evaluation. Three component categories-principal constituents, organic acids, and cooling agents-were analyzed, alongside correlations between specific compounds and cytotoxicity. Our findings revealed that non-tobacco-flavored e-liquids exhibited significantly higher cytotoxicity than tobacco-flavored variants, with considerable variation across different flavors and nicotine concentrations. Among non-tobacco flavors, tropical fruit and melon varieties induced the greatest cytotoxicity. Genotoxicity analysis showed that while tobacco-flavored e-liquids (0.2 × to 1 × IC₅₀) did not cause significant DNA damage, certain non-tobacco flavors (0.2 × to 1 × IC₅₀) triggered pronounced DNA damage. Correlation analysis identified N,2,3-trimethyl-2-isopropylbutamide (WS-23), benzoic acid, and nicotine as negatively correlated with IC₅₀ values, with WS-23 displaying the strongest association. Further in vitro validation confirmed that WS-23 (0-2.4 mg/mL) induces DNA damage and elevates reactive oxygen species (ROS) levels in a dose-dependent manner. These findings highlight the need for more rigorous investigation and regulatory oversight of flavored e-liquids to mitigate potential health risks.