High-Nicotine Electronic Cigarette Products: Toxicity of JUUL Fluids and Aerosols Correlates Strongly with Nicotine and Some Flavor Chemical Concentrations

Abstract

Whereas JUUL electronic cigarettes (ECs) have captured the majority of the EC market, with a large fraction of their sales going to adolescents, little is known about their cytotoxicity and potential effects on health. The purpose of this study was to determine flavor chemical and nicotine concentrations in the eight currently marketed prefilled JUUL EC cartridges ("pods") and to evaluate the cytotoxicity of the different variants (e.g., "Cool Mint" and "Crème Brulee") using in vitro assays. Nicotine and flavor chemicals were analyzed using gas chromatography-mass spectrometry in pod fluid before and after vaping and in the corresponding aerosols. 59 flavor chemicals were identified in JUUL pod fluids, and 3 were >1 mg/mL. Duplicate pods were similar in flavor chemical composition and concentration. Nicotine concentrations (average 60.9 mg/mL) were significantly higher than those of any EC products we have previously analyzed. The transfer efficiency of individual flavor chemicals that were >1 mg/mL and nicotine from the pod fluid into aerosols was generally 35-80%. All pod fluids were cytotoxic at a 1:10 dilution (10%) in the MTT and neutral red uptake assays when tested with BEAS-2B lung epithelial cells. Most aerosols were cytotoxic in these assays at concentrations between 0.2 and 1.8%. The cytotoxicity of collected aerosol materials was highly correlated with nicotine and ethyl maltol concentrations and moderately to weakly correlated with total flavor chemical concentration and menthol concentration. Our study demonstrates that (1) some JUUL flavor pods have sufficiently high concentrations of flavor chemicals that may make them attractive to youth and (2) the concentrations of nicotine and some flavor chemicals (e.g., ethyl maltol) are high enough to be cytotoxic in acute in vitro assays, emphasizing the need to determine if JUUL products will lead to adverse health effects with chronic use.

Figure 1

Heat map of flavor chemicals in eight duplicate JUUL pod fluids. Chemicals are ordered on the y-axis according to their toxicity (Others, Harmful, Irritant) based on LC₅₀ data from rat oral exposures, and within each class, they are ranked from most to least toxic. The “Others” category on the y-axis represents chemicals that are corrosive, toxic, harmful, irritants as well as dangerous to the environment. JUUL products (x-axis) are ordered according to the total weight (mg/mL) of the flavor chemicals in each product with the highest concentration at the left. The total flavor chemical concentration (mg/mL) is indicated at the top of each column. The color gradient on the right shows the concentrations of the flavor chemicals in the heat map. Three chemicals (vanillin, ethyl maltol, and menthol) in the orange to red color gradient were ≥1 mg/mL in at least one product. JUUL pod code: Classic Tob. = “Classic Tobacco”; Virginia Tob. = “Virginia Tobacco”. The numbers 1 and 2 with the JUUL pod codes designate the first and second pod tested.

Figure 2

Nicotine and total flavor chemical concentrations in EC products. (a) Nicotine concentrations in 182 EC products. Red dots represent eight JUUL products; green dots represent 5 Vuse cartomizer fluids, and blue dots represent 169 refill fluids from 34 brands. The y-axis shows nicotine concentrations in each EC product listed on the x-axis. (b) The mean concentrations of nicotine in 169 EC refill fluids from 34 brands (blue bar), five Vuse cartomizers (green bar), and eight JUUL pods (red bar). The mean concentrations of nicotine were significantly different in each group. **** = p < 0.0001. (c) The mean concentrations of total flavor chemicals in 169 EC refill fluids from 34 brands (blue bar), five Vuse cartomizers (green bar), and eight pod JUUL pods (red bar).

Figure 3

Total flavor chemical and nicotine concentrations in JUUL pod fluids and aerosols. (a) The total flavor chemical concentrations in unvaped pod fluids, vaped pod fluids, and aerosols. (b) Concentrations of nicotine in unvaped pod fluids, vaped pod fluids, and aerosols. The total flavor chemical concentrations and nicotine concentrations were very similar in the unvaped and vaped pod fluids. Each bar is mean concentration of two independent experiments.

Figure 4

Concentrations of individual flavor chemicals in JUUL pod fluids and aerosols. (a) “Cool Mint”, (b) “Classic Menthol”, (c) “Crème Brulee”, (d) “Mango”, (e) “Cool Cucumber” (f) “Fruit Medley”, (g) “Classic Tobacco”, and (h) “Virginia Tobacco”. Most fluids contained 1–2 flavor chemicals >1 mg/mL, except the tobacco flavored products, which had very low concentrations of flavor chemicals. Flavor chemicals >1mg/mL transferred from unvaped pod fluids into the aerosols with 39 to 62% efficiency. Each bar is the mean concentration of two independent experiments.

Figure 5

Concentration-response curves for BEAS-2B cells treated with JUUL pod fluids and aerosols. (a-c) MTT assay, (d-f) NRU assay, and (g-i) LDH assay for all eight pod variants. The y-axis shows the response of cells in each assay as a percentage of the untreated control. Each point is the mean ± standard error of the mean for three independent experiments.

Figure 6

Relationship between cytoxicity of unvaped pod fluids and concentrations of nicotine and the flavor chemicals. Linear regression analysis for cytotoxicity (y-axis, expressed as a percentage of the untreated control) in the MTT and NRU assays versus the concentrations of: (a) total flavor chemicals and nicotine, (b) nicotine only, (c) total flavor chemicals only, (d) ethyl maltol, (e) menthol, and (f) vanillin. Blue dots and red triangles represent concentrations tested in the MTT and NRU assay, respectively. Cytotoxicity was strongly correlated with total concentration of chemicals (flavor chemicals and nicotine) and with nicotine concentration only and weakly to moderately correlated with the concentrations of total flavor chemicals, ethyl maltol, menthol and vanillin. All correlations were significant (p<0.05).

Figure 7

Relationship between cytoxicity of pod aerosols and the concentrations of nicotine and the flavor chemicals. Linear regression analysis for cytotoxicity in the MTT and NRU assays versus the concentrations of: (a) total flavor chemicals and nicotine, (b) nicotine only, (c) total flavor chemicals only, (d) ethyl maltol, (e) menthol, and (f) vanillin. Blue dots and red triangles represent the concentrations tested in the MTT and NRU assay. Cytotoxicity (percent of control) was strongly correlated with the total concentration of chemicals (flavor chemicals and nicotine), nicotine concentration only, and ethyl maltol concentration. The correlations between cytotoxicity and the concentrations of total flavor chemicals and menthol were moderate and weak, respectively. The correlation between cytotoxicity and vanillin concentration was not significant.