Analysing the acute toxicity of e-cigarette liquids and their vapour on human lung epithelial (A549) cells in vitro

The use of alternative tobacco products such as vaping devices has significantly increased over the last 5-years, with the largest increase being amongst 18-25-year-olds. While the quantity of nicotine is tightly regulated, the composition of e-liquid flavourings is largely unregulated, and often absent from product labels. Herein, we compare the toxicity of carrier liquids propylene glycol (PG) & vegetable glycerine (VG) with five popular flavour concentrates: menthol, cherry, butterscotch, vanilla bourbon and tobacco on human alveolar type II cell-like A549 cells. The flavourings were tested in both liquid and vapour form and a vapour assay was developed to assess cytotoxicity of the flavourings. Our results conclude that menthol liquid was the most cytotoxic (LD₅₀ = <0.5 % over a <4 h exposure). Followed by cherry and vanilla bourbon which elicited a similar response at 4 % over 8 h exposure. Tobacco only reached 50 % toxicity at a concentration > 4 % over 24 h exposure. Butterscotch displayed similar toxicity profiles to PG and VG where cytotoxicity exceeded 20 % at 8 % concentration at all time points. The cytotoxicity of menthol was further evaluated as a vapour, with a significant reduction in viable cells and a 5-fold increase in the number of necrotic cells with only 11 % of cells remaining viable after 5 vaping episodes. Analysis revealed the presence of toxic chemicals and heavy metals in the fluids therefore further research is required to fully elucidate the long-term usage of flavourings with vaping devices and the impact this may have on human lung health.