Formation of flavorant-propylene Glycol Adducts With Novel Toxicological Properties in Chemically Unstable E-Cigarette Liquids

Abstract

Introduction: "Vaping" electronic cigarettes (e-cigarettes) is increasingly popular with youth, driven by the wide range of available flavors, often created using flavor aldehydes. The objective of this study was to examine whether flavor aldehydes remain stable in e-cigarette liquids or whether they undergo chemical reactions, forming novel chemical species that may cause harm to the user.

Methods: Gas chromatography was used to determine concentrations of flavor aldehydes and reaction products in e-liquids and vapor generated from a commercial e-cigarette. Stability of the detected reaction products in aqueous media was monitored by ultraviolet spectroscopy and nuclear magnetic resonance spectroscopy, and their effects on irritant receptors determined by fluorescent calcium imaging in HEK-293T cells.

Results: Flavor aldehydes including benzaldehyde, cinnamaldehyde, citral, ethylvanillin, and vanillin rapidly reacted with the e-liquid solvent propylene glycol (PG) after mixing, and upward of 40% of flavor aldehyde content was converted to flavor aldehyde PG acetals, which were also detected in commercial e-liquids. Vaping experiments showed carryover rates of 50%-80% of acetals to e-cigarette vapor. Acetals remained stable in physiological aqueous solution, with half-lives above 36 hours, suggesting they persist when inhaled by the user. Acetals activated aldehyde-sensitive TRPA1 irritant receptors and aldehyde-insensitive TRPV1 irritant receptors.

Conclusions: E-liquids are potentially reactive chemical systems in which new compounds can form after mixing of constituents and during storage, as demonstrated here for flavor aldehyde PG acetals, with unexpected toxicological effects. For regulatory purposes, a rigorous process is advised to monitor the potentially changing composition of e-liquids and e-vapors over time, to identify possible health hazards.

Implications: This study demonstrates that e-cigarette liquids can be chemically unstable, with reactions occurring between flavorant and solvent components immediately after mixing at room temperature. The resulting compounds have toxicological properties that differ from either the flavorants or solvent components. These findings suggest that the reporting of manufacturing ingredients of e-liquids is insufficient for a safety assessment. The establishment of an analytical workflow to detect newly formed compounds in e-liquids and their potential toxicological effects is imperative for regulatory risk analysis.

Figure 1.

Top: general aldehyde propylene glycol (PG) acetal formation reaction involving an aldehyde 1 and PG 2, to form the PG acetal 3. The reversible reaction yields compound 3 with two stereocenters indicated by *; middle: reaction kinetics of formation of aldehyde PG acetals from aldehydes dissolved in PG at a concentration of 20 µg/g of aldehyde, displayed as mole fraction of PG acetal against time. Citral PG acetals quantified as combined cis- and trans-isomers. Mean of n = 2 experiments in daylight and dark storage shown; error bars indicate the measured range; inset: formation of cinnamaldehyde PG acetal in mixtures of PG and glycerol; bottom: mole fractions of PG acetal to aldehyde in commercial cherry- and vanilla-flavored e-liquids as a function of PG content, for benzaldehyde, ethylvanillin, and vanillin.

Figure 2.

Carryover rates of benzaldehyde and benzaldehyde propylene glycol (PG) acetal from e-liquid to vapor generated by a first-generation e-cigarette, as a function of PG content of the neat e-liquid with the residual made up by glycerol (VG). Error bars represent the SD of at least n = 3 separate experiments (filled symbols), or the range when n = 2 (non-filled symbols). Statistical significance (*, p < .05) found only between benzaldehyde PG acetal 5% and 100%. Carryover results for vanillin, ethylvanillin, and nicotine are shown in Supplementary Figure 2.

Figure 3.

Effects of vanillin and vanillin propylene glycol (PG) acetal on human sensory irritant receptors, TRPA1 and TRPV1, measured by fluorescent calcium imaging in HEK-293T cells. Responses were normalized to maximal cinnamaldehyde (for TRPA1) or capsaicin (for TRPV1) responses. Each point represents mean values of 15–21 independent measures from 4 experiments, and error bars show standard error of the mean. Results for benzaldehyde, ethylvanillin, and their corresponding PG acetals are shown in Supplementary Figure 4.

Figure 4.

Proposed comprehensive testing scheme for e-liquid regulation: beyond e-liquid ingredients reported by the supplier, the final e-liquid and vapor generated from it should be characterized, all compounds therein identified, and quantified. Using this final list of compounds an e-cigarette user would be exposed to, a hazard and risk assessment needs to be carried out to approve or disapprove an e-liquid, based on known inhalation toxicity values. If such values do not yet exist, they need to be determined, including measurements as presented in this article: aqueous stability mimicking the environment in the airways, irritation potential, and various toxicity endpoints regarding inhalation toxicity.