Method: For this study nicotine and 20 flavored ECs liquids (Amaretto, Bubble Gum, Cappuccino, Cola, Double Mint, Ice Cool, Menthol-3, mint, Pineapple, Apple, Banana, Pomegranate, Blueberry, Cherry, Peppermint, Vanilla, Lime, Hazelnut, Menthol, Mint Gum), with a final concentration of 10 µg/ml nicotine were used (controls: propylene glycol and PBS). The fibroblasts were incubated with the different solutions for up to 96h. After 24h the BCA protein assay and LIVE/DEAD cytotoxicity assay were conducted. Cell viability was measured by using the LUNA automated cell counter. Over an incubation period of 96h the cell viability was measured by using the PrestoBlue® reagent and after 72h the migration assay was performed. Fluorescence staining was carried out in order to visualize cell growth and morphology. Data were statistically analyzed by t tests.
Result: The cell viability assay showed distinct differences between the examined liquids (p<0.05) and the controls. After an incubation period of 24h, gingival fibroblast vitality decreased in the group of menthol containing liquids. The growth in the “non menthol” group was significantly higher. Hence, the results show the highest cytotoxic potential in the menthol group. After an incubation period of 96h with the menthol-flavored liquids the fibroblasts were statistically significantly reduced (t test; p<0.001). The cell visualization tests confirmed these findings.
Conclusion: The special situation of an in vitro study permits only limited predictions for in vivo conditions. However, there is clear evidence that the added flavors, in particular menthol supplements, lead to significant cell damage; therefore these additives should be avoided.