Comparing the cytotoxicity of electronic cigarette fluids, aerosols and solvents

Abstract

Background: As thousands of electronic cigarette (e-cigarette) refill fluids continue to be formulated and distributed, there is a growing need to understand the cytotoxicity of the flavouring chemicals and solvents used in these products to ensure they are safe. The purpose of this study was to compare the cytotoxicity of e-cigarette refill fluids/solvents and their corresponding aerosols using in vitro cultured cells.

Methods: E-cigarette refill fluids and do-it-yourself products were screened in liquid and aerosol form for cytotoxicity using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The sensitivity of human pulmonary fibroblasts, lung epithelial cells (A549) and human embryonic stem cells to liquids and aerosols was compared. Aerosols were produced using Johnson Creek's Vea cartomizer style e-cigarette.

Results: A hierarchy of potency was established for the aerosolised products. Our data show that (1) e-cigarette aerosols can produce cytotoxic effects in cultured cells, (2) four patterns of cytotoxicity were found when comparing refill fluids and their corresponding aerosols, (3) fluids accurately predicted aerosol cytotoxicity 74% of the time, (4) stem cells were often more sensitive to aerosols than differentiated cells and (5) 91% of the aerosols made from refill fluids containing only glycerin were cytotoxic, even when produced at a low voltage.

Conclusions: Our data show that various flavours/brands of e-cigarette refill fluids and their aerosols are cytotoxic and demonstrate the need for further evaluation of e-cigarette products to better understand their potential health effects.

Keywords: aerosol; cytotoxicity; electronic cigarettes; flavors; refill fluid; vegetable glycerin.

Figure 1

Four patterns of cytotoxicity for e-cigarette refill fluids (RF) and aerosols. (A and B) Both the RF and aerosol were non-cytotoxic. (C and d) Both the RF and the aerosol were cytotoxic. (e) The RF was cytotoxic but the aerosol was not. (F and G) The aerosol was cytotoxic but the RF was not. Red lines are RF previously screened with human pulmonary fibroblast (hPF). Blue lines are the same RF rescreened in this study to determine if storage effected cytotoxicity. Green lines are aerosols produced from each RF and used to treat hPF. For green lines, data are the means and SE of the mean of three independent experiments. Asterisks indicate the lowest concentrations that are significantly different from the untreated controls (lowest observed adverse effect level). *p<0.05, **p<0.01, ***p<0.001.

Figure 2

Most e-cigarette products containing glycerin produce cytotoxic aerosols. (A–L) Comparison of the 11 refill fluids (RF) and one do-ityourself purchased sample of vegetable glycerin. Red lines are RF previously screened with human pulmonary fibroblast (hPF). Blue lines are the same RF rescreened in this study to determine if storage effected cytotoxicity. Green lines are aerosols produced from each RF and used to treat hPF. For green lines, data are the means and SE of the mean of three independent experiments. Asterisks indicate the lowest concentrations that are significantly different and represent the lowest observed adverse effect level. *p<0.05, **p<0.01, ***p<0.001

Figure 3

Two adult respiratory cell types and a pluripotent cell compared for sensitivity to e-cigarette aerosols. H9 hESC (red lines), A549 cells (blue lines) and hPF (green lines) were treated with 10 e-cigarette aerosols (A–J). Data are plotted as the means and SE of the mean of three independent experiments. Asterisks indicate the lowest concentrations that are significantly different from the untreated control (lowest observed adverse effect level). *p<0.05, **p<0.01, ***p<0.001. hESC, human embryonic stem cell; hPF, human pulmonary fibroblast.

Figure 4

Relationship between solvent and aerosol cytotoxicity. The IC₅₀s (concentration in per cent) of the aerosols for the hPF are plotted for each product denoted by inventory number. Lower IC₅₀ values indicate higher cytotoxicity. Points plotted at 2.5 were not potent in the MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) assay, as an IC₅₀ value could not be generated. The ‘PG or VG only’ column denotes aerosols made from PG and VG purchased from Freedom Smoke USA. The ‘VG refill fluids’ column denotes aerosols made from refill fluids containing glycerin, and the ‘PG + VG refill fluids’ column denotes refill fluids that contain propylene glycol and glycerin. Each refill fluid was categorised based on the manufacturer’s labelling. hPF, human pulmonary fibroblast; IC₅₀, inhibitory concentration at 50%.