Sven-Eric Jordt, PhD1 Duke Yale Associate Professor University University Sairam Jabba, PhD1 Senior Research Associate Hanno C. Erythropel, PhD2 Associate Research Scientist

1Duke University School of Medicine Department of Anesthesiology Durham NC 27710

2Center for Green Chemistry & Green Engineering at Yale University New Haven, CT 06511

To: Dockets Management Staff (HFA-305) Food and Drug Administration 5630 Fishers Lane, Rm. 1061 Rockville, MD 20852.

RE: Docket No. FDA-2012-N-0143 - Harmful and Potentially Harmful Constituents in Tobacco Products - Formation of Flavorant–Solvent Adducts With Novel Toxicological Properties

October 4th, 2019 Dear Commissioner Sharpless: With this letter we would like to provide new scientific data in support of the proposed rulemaking by the Food and Drug Administration for Harmful and Potentially Harmful Constituents (HPHC) in Tobacco Products, where FDA has proposed to add 19 new chemical toxicants that are present in tobacco products. We appreciate and agree with FDA that these 19 chemicals should be regarded as HPHC’s. In our opinion, this proposed HPHC list does not include several hundreds of other chemicals and classes of chemicals (especially respiratory irritants) that are present in the tobacco products or delivered through use of these products. In this document, we specifically wanted to inform FDA about flavor-solvent adducts, a class of chemical compounds not declared previously in tobacco products and that have unexpected toxicological effects. These adducts that are generally called as flavor aldehyde- PG/VG acetals are rapidly formed in e-cigarette liquids after mixing of their constituents and under storage conditions between popular flavor-aldehydes like vanillin (vanilla), cinnamaldehyde (cinnamon), benzaldehyde (cherry), citral (citrus), anisaldehyde (anise/spice) and solvents propylene glycol and vegetable glycerine. Here, we present our most recently published research data on chemical, pharmacological and toxicological effects of these flavors-solvents adducts. [1] [2]

In summary, our research on formation of flavor-solvent adducts and their toxicological effects demonstrated that: 1. Flavor aldehydes rapidly undergo a chemical reaction with the common e-liquid solvents propylene glycol (PG) and glycerol (VG) at room temperature (storage condition) to form new chemical species called Flavor aldehyde-solvent Acetals [1]. 2. Flavor aldehyde-solvent acetals have potent, stronger and increased respiratory irritation potential than their parent aldehydes (e.g. vanillin PG acetal vs. vanillin) [1]. This is indicated by acetals potential to activate sensory irritant receptors, TRPA1 and TRPV1, that are expressed in sensory nerve endings innervating the lungs and airways. 3. Flavor aldehyde-solvent acetals were identified in commercial e-liquids in significant amounts, including in highly popular JUUL e-liquids [1,2]. 4. Flavor aldehyde-solvent acetals reach the aerosol with high transfer efficiency, including in Juul vapor [1]. 5. Detected acetals are sufficiently stable in aqueous environment (airway lining fluids) to exert a sensory and respiratory irritant effect [1]. Limitations: 1. These acetals potential for enhanced respiratory irritation was measured based on their ability to activate heterologously expressed sensory receptors (human TRPA1 and TRPV1) and not on any in-vivo rodent or human inhalation toxicity data.

Implications and Recommendations: Our data clearly demonstrate that many popular flavoring chemicals can form novel chemical compounds that can have potent and efficacious effects in the respiratory system of mammals, effects that are mediated largely by the sensory irritant receptors like TRPA1 and TRPV1 ion channels in sensory nerve endings, leading to increase in TRPA1 or TRPV1 sensation of irritation. Based on these findings we are concerned that new respiratory irritant chemicals formed in e-cigarette liquids may increase respiratory irritation in vapers in addition to existing flavor chemicals that act as respiratory irritants. Especially in initiating and youth e-cigarette users who are mostly using popular sweet and candy flavors, where the addition of flavor will likely increase e-cigarette use, resulting in absorbance of higher amounts of potential HPHCs and increase in deposition of irritant flavor chemicals and adducts in the respiratory airways that can elicit an immune or inflammatory response or cause/exacerbate health issues. Based on these findings, we recommend that, 1. FDA should require tobacco companies to list all the ingredients, including flavor chemicals, chemical byproducts formed either during storage or heating of the e-cigarette liquid or during generation of aerosol. 2. FDA should strictly regulate and not allow addition of flavor chemicals that are potential respiratory or sensory irritant or that elicit an immune or inflammatory response or cause/exacerbate health issues. 3. FDA should have a rule that directs tobacco companies to conduct inhalation toxicity studies for every chemical, especially flavor chemicals added to - and formed in e-cigarette liquids. 4. FDA mandate tobacco companies to determine NOAEL (No Observed Adverse Effect Levels) for inhalation, oral and dermal routes of various e-cigarette flavors and byproducts formed. The final flavor chemical concentrations in the e-cigarette liquid should be adjusted accordingly. 5. More importantly, FDA should include flavor aldehyde-solvent acetals that have stronger respiratory irritation potential in the HPHC’s list as respiratory irritants. 6. As solvent chemicals, PG and VG, are required to form these higher respiratory irritant acetals, it provides an additional rationale to include PG and VG in the HPHC’s list.

References:

[1] Erythropel HC, Jabba SV, de Winter TM, et al. (2019) Formation of flavorant-propylene glycol adducts with novel toxicological properties in chemically unstable e-cigarette liquids. Nicotine & Tobacco Research 21(9):1248-1258. DOI 10.1093/ntr/nty192. [2] Erythropel HC, Davis LM, De Winter TM, et al. (2019) Flavorant-Solvent Reaction Products and Menthol in JUUL E-Cigarettes and Aerosol. American Journal of Preventive Medicine 57(3):425-427. DOI 10.1016/j.amepre.2019.04.004. PMID: 31358341; PMCID: PMC6702051. Nicotine & Tobacco Research, 2019, 1248–1258 doi:10.1093/ntr/nty192 Original investigation Received March 28, 2018; Editorial Decision September 10, 2018; Accepted September 13, 2018

Original investigation Downloaded from https://academic.oup.com/ntr/article-abstract/21/9/1248/5134068/ by National Science and Technology Library -Root user on 05 October 2019 Formation of flavorant–propylene Glycol Adducts With Novel Toxicological Properties in Chemically Unstable E-Cigarette Liquids Hanno C. Erythropel PhD1,2,‡, , Sairam V. Jabba PhD2,3,‡, Tamara M. DeWinter PhD2,4, Melissa Mendizabal1, Paul T. Anastas PhD1,4–6, Sven E. Jordt PhD2,3, Julie B. Zimmerman PhD1,2,4

1Department of Chemical and Environmental Engineering, Yale University, New Haven, CT; 2Yale Tobacco Center of Regulatory Science, Department of Psychiatry, Yale School of Medicine, New Haven, CT; 3Department of Anesthesiology, Duke University School of Medicine, Durham, NC; 4Yale School of Forestry and Environmental Studies, Yale University, New Haven, CT; 5Department of Chemistry, Yale University, New Haven, CT; 6Yale School of Public Health, Yale University, New Haven, CT

‡Co-first authors.

Corresponding Author: Sven E. Jordt, PhD, Department of Anesthesiology, Duke School of Medicine, Durham, NC, USA. E-mail: [email protected]

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 nu- clear magnetic resonance spectroscopy, and their effects on irritant receptors determined by fluor- escent calcium imaging in HEK-293T cells. Results: Flavor aldehydes including benzaldehyde, cinnamaldehyde, citral, ethylvanillin, and van- illin 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

© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. 1248 For permissions, please e-mail: [email protected]. Nicotine & Tobacco Research, 2019, Vol. 21, No. 9 1249 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. Downloaded from https://academic.oup.com/ntr/article-abstract/21/9/1248/5134068/ by National Science and Technology Library -Root user on 05 October 2019

Introduction irritant aldehydes in airway-innervating nerves, activated by the major tobacco smoke aldehyde, acrolein, and flavor aldehydes such The addition of characterizing flavors to combustible cigarettes is as cinnamaldehyde, benzaldehyde, and vanillin, and eliciting irrita- prohibited by regulatory measures in several jurisdictions, including tion responses, pain, and cardiovascular reflexes increasing stress the United States, Canada, and the European Union; however, these and inflammation.16–19 In vitro tests quantifying the capability of a restrictions have not been extended to “e-liquids” used in electronic chemical to activate TRP irritant receptors are currently considered cigarettes (e-cigarettes). Along with the global increase in e-cigarette as replacements for the animal studies determining RD s. It remains use,1–3 the variety of flavored e-liquids has risen, with currently more 50 to be examined whether aldehyde PG acetals activate the same irri- than 7000 flavors available.4 Among the most popular e-liquids are tant mechanisms as their parent aldehydes and exhibit toxicological sweet and fruity flavors, a main driver for the appeal of e-cigarettes effects of concern. to youth.5–7 Fragrant aldehydes, such as vanillin and ethylvanillin The aims of this study were to determine whether: (1) aldehyde (vanilla flavor), benzaldehyde (cherry flavor), or cinnamaldehyde flavorants and PG yield acetals in e-liquid environments, (2) acetals (cinnamon flavor), commonly serve as the characteristic flavorants.8 are carried over into e-vapor, (3) acetals remain stable under physio- E-liquids are prepared by mixing the flavorants and nicotine with logical conditions, and (4) acetals activate TRP irritant receptors. different ratios of the solvents propylene glycol (PG) and glycerol A workflow is proposed for a comprehensive risk assessment of (VG for vegetable glycerin), which contain two and three alcohol e-liquids and resulting e-vapors encompassing all compounds a user moieties, respectively.9 could be exposed to, including reaction products. However, aldehydes and alcohols can undergo chemical reactions resulting in the formation of acetals (Figure 1). An acetalization re- action between an aldehyde (1) and PG (2) generally yields aldehyde PG acetals (3) with two chiral carbons (indicated with *), result- Materials and Methods ing in the formation of at least three stereoisomers; molecules with Acetal Formation Experiments different bond orientation and therefore possibly different proper- One hundred micrograms of benzaldehyde (100 µg, >99%; Sigma- ties, including toxicological properties.10 Acetalization reactions are Aldrich, St. Louis, MO), trans-cinnamaldehyde (98+%; Alfa Aeasar, commonly used in synthetic organic chemistry to “protect” reactive Haverhill, MA), citral (mixture of the structural isomers neral and aldehyde moieties from reacting further. Consequently, chemical geranial; 95%, Fisher Scientific, Waltham, MA) ethylvanillin (99%), reactions (ie, acetal formation) between e-liquid constituents are and vanillin (99%; both Sigma-Aldrich) were added to plastic vials possible, suggesting that e-liquids can be unstable environments containing 5 g PG (USP-FCC grade, Waltham, MA), or mixtures of PG/ post-preparation. Indeed, recent analytical studies have reported VG (anhydrous; AmericanBio, Natick, MA) at 70/30, 50/50, or 30/70 the presence of flavor aldehyde acetals in e-liquids, in the headspace ratios by weight, to yield an aldehyde concentration of 20 mg/g. The above e-liquids, and in e-cigarette vapors (e-vapor).11–13 In the cases experiments in pure PG were carried out in duplicate (light and dark) where manufacturers published ingredient lists of e-liquids (avail- storage conditions. Vials were vigorously shaken once per day, and able online), only aldehydes, but not acetals, were listed among 100 µL samples were drawn, diluted, and injected into a gas chromato- the flavorants, suggesting that these acetals form after mixing. The graph (GC). Using standard curves, samples were analyzed for their published analytical studies did not report the concentrations of the aldehyde and PG acetal content (vanillin PG acetal [97%], ethylvanillin detected flavor aldehyde acetals in the respective e-liquids, and it PG acetal [98%, both Bedoukian Research, Danbury, CT], benzalde- remains unclear how frequently and how rapidly these compounds hyde PG acetal [(>98%], trans-cinnamaldehyde PG acetal [both Sigma- form and whether they remain stable during heating and vaporiza- Aldrich], citral PG acetals synthesized in-house, see Supplementary tion in e-cigarettes. Beyond flavor aldehyde acetals, the presence of Material for details). Results are presented as mole fraction of formed formaldehyde hemiacetals in e-vapors was reported, generated from acetal, defined as moles of acetal divided by the combined number of formaldehyde reacting with a single alcohol moiety on PG or VG.14 moles of acetal and corresponding aldehyde. Formaldehyde and other small aldehydes have been shown to form from PG or VG during vaporization.15 The presence of aldehydes in e-liquids and e-vapor, either as flavorants or chemical reaction Acetal Content of Commercial E-liquids products, has raised concerns because of their potential respiratory Two flavored e-liquids (cherry and vanilla) were purchased with and cardiovascular toxicological effects. Respiratory sensory ir- PG/VG ratios of 100/0, 70/30, 50/50, 30/70, and 0/100, contain- ritation is a first sign of potential toxicity of an inhaled chemical ing 12 mg/mL nicotine (AmericaneLiquidStore, Wauwatosa, WI). exposure, induced by chemical activation of chemosensory recep- Samples of all e-liquids were taken, the mass recorded, and diluted for tors in airway-innervating nerves. Respiratory sensory irritants are GC injection. Using standard curves, aldehyde, aldehyde PG acetal, and nicotine (99%, Alfa Aesar) concentrations were determined. ranked according to their RD50, the toxicological parameter denot- ing the exposure concentration causing a 50% drop in respiratory rates in rodents, and used to determine occupational and other ex- Vaping Carryover Experiments posure limits. Recent studies identified the transient receptor poten- For the vaping experiments, V2 blank cartridges were purchased on- tial (TRP) ion channels, TRPA1 and TRPV1, as the receptors for line and filled with 0.5 g e-liquid and used with the corresponding 1250 Nicotine & Tobacco Research, 2019, Vol. 21, No. 9

79 mm, 4.2 V battery (both VMR Products LLC, Miami, FL). Cell Culture Cartridges were allowed to settle for at least 60 minutes before test- HEK-293T cells (ATCC, Manassas, VA; Cat# CRL-11268, RRID: ing. E-vapors were produced and captured using a custom-built vap- CVCL_1926) were cultured in Gibco Dulbecco’s modified Eagle’s ing machine described previously, using a series of cold-finger traps medium supplemented with 10% fetal bovine serum, 100 units/mL chilled with liquid nitrogen.20 The puffing topography was 4-second penicillin, and 0.1 mg/mL streptomycin (all Fisher Scientific). Cells puffs, 73 ml puff volume, 30-second intervals, for 20 puffs total. The were plated on 100 mm tissue culture plastic dishes and grown over- Downloaded from https://academic.oup.com/ntr/article-abstract/21/9/1248/5134068/ by National Science and Technology Library -Root user on 05 October 2019 trapped e-vapor was subsequently taken up in methanol (JT Baker, night to 60%–70% confluency. The cells were transiently transfected Center Valley, PA) and injected into a GC. Compound e-vapor con- with either human TRPA1 or TRPV1 plasmid DNA (18 µg plas- centration was calculated as total amount trapped in vapor divided mid DNA/100 mm dish) for 16–24 hours using Fugene 6 transfec- by the mass change of the cartridge after 20 puffs. Carryover per- tion reagent (Promega, Madison, WI) and Gibco Opti-MEM (Fisher centage was calculated by dividing e-vapor concentration by neat Scientific) according to manufacturer’s protocols. Cells were then e-liquid concentration. resuspended and plated onto poly-d-lysine-coated (Sigma-Aldrich) 96-well black-walled plates at 50 000 cells per well and allowed to Acetal Aqueous Stability Testing grow for another 16–24 hours before the experiments. Cells were

To assess the stability of the PG acetals in physiological solution, maintained as monolayers at 5% CO2 and 37°C. ultraviolet–visible (UV–VIS) and proton nuclear magnetic resonance (1H-NMR) spectroscopy were used. The breakdown of vanillin PG Calcium Influx Imaging acetal (starting concentration 100 µg/mL) to vanillin in Hanks bal- Calcium imaging was performed as described earlier.21 Briefly, the anced salts solutions buffer (Sigma-Aldrich), adjusted to pH 7.4, fluorescence calcium indicator FLIPR Calcium 6 dye (Molecular was monitored over time by UV–VIS spectroscopy at λ = 347 nm Devices, San Jose, CA) was applied to the cells, which were recon- (Cary-3E; Varian, Palo Alto, CA). Breakdown of the other PG acetals stituted in Hanks balanced salts solutions buffered at pH 7.4 using in D2O (99.9 atom% D; Sigma-Aldrich) was monitored over time 20 mM HEPES buffer (both Sigma-Aldrich). A FlexStation III by 1H-NMR spectroscopy, comparing the appearing aldehyde sig- benchtop scanning fluorometer chamber (Molecular Devices) at nal at approximately δ = 9.6 ppm to the disappearing acetal doub- 37°C was used to excite cells at 485 nm and the Ca2+ emission sig- let between δ = 5.6–5.9 ppm. 1H-NMR studies were carried out at nal was recorded at 525 nm in intervals of 1.8 seconds. The change starting concentrations of 150 µg/mL (benzaldehyde- and vanillin in fluorescence was measured and expressed as maxF –F0, where Fmax PG acetal) and 200 µg/mL (ethylvanillin PG acetal). is the maximum fluorescence and F0 is the basal fluorescence. The responses were normalized to the known agonists cinnamaldehyde Gas Chromatography (1 mM; for TRPA1) or capsaicin (3 µM; for TRPV1). A Boltzmann A GC (GC-2010 Plus; Shimadzu, Kyoto, Japan) was used to quan- function (3- or 4-parameter logistic fit) was used to generate con- tify aldehydes, PG acetals, and nicotine. Citral and citral PG acetals centration–response curves and determine the half maximal effective were quantified as a combination ofcis - and trans-isomers (see concentration (EC50)values (Prism 7 software; GraphPad, La Jolla,

Supplementary Figure 5 for an example of a chromatogram). For the CA). 95% confidence intervals (CI) of the logEC50 values were cal- acetal formation reaction, 100 µL samples were diluted with 1 mL of culated to display the variability of concentration–response curves. methanol (high-performance liquid chromatography [HPLC] grade, Efficacies (maximal responses) for each of the flavor aldehydes and JT Baker) containing 1 g/L 1,4-dioxane (99+%, Alfa Aeasar) as an in- their corresponding PG acetals were reported as a percentage of the ternal standard. For the carryover experiments, the complete content maximum response elicited by saturating concentrations of the full of the cold-finger trap was taken up in 1 mL of methanol (JT Baker) agonists, cinnamaldehyde (1 mM) for TRPA1 and capsaicin (3 µM) containing internal standard. See Supplementary Material for further for TRPV1 (Table 1). GC method details. Calibration curves were prepared to calculate concentrations from peak area ratios of the compounds of interest to Statistical Analysis the internal standard. Statistical analysis was performed using GraphPad Prism 7 software (La Jolla, CA), for one-way analysis of variance tests with Bonferroni post-tests, and Student’s t tests. A p value less than .05 was inter- Liquid Chromatography preted as significant, withp value less than .05 represented by *, A liquid chromatography–refractive index (LC–RI) (Shimadzu p value of less than .01 by **, and p value less than .001 by ***. LC-20 system, Shimadzu, Kyoto, Japan) was used to determine PG and VG contents of commercial e-liquids. Method details are pro- vided in the Supplementary Material. Samples were diluted with Results deionized water (HPLC grade, JT Baker) and 1,4-dioxane was Acetal Formation and Presence in Laboratory-made added as an internal standard. Calibration curves of PG and VG and Commercial E-liquids were prepared to calculate concentrations from peak area ratios of To investigate the formation kinetics of flavor aldehyde-PG acetals, compounds to the internal standard. commonly used flavor aldehydes were mixed with pure PG and sampled over 14 days while stored at room temperature (Figure 1); aldehydes 1H-NMR Spectroscopy tested were benzaldehyde, cinnamaldehyde, citral (mixture of the two All 1H-NMR spectroscopy was carried out on an Agilent DD2 structural isomers neral and geranial), vanillin, and ethylvanillin, each at 20 mg/g, approximating the concentrations found in commercial 400 MHz NMR spectrometer, using D2O, methanol (CD3OD), or e-liquids (Supplementary Figure 1). The PG acetal formation reactions chloroform (CDCl3; all at least 99.8% D, Cambridge Isotope Labs., Andover, MA). were monitored in daylight and dark conditions with no significant Nicotine & Tobacco Research, 2019, Vol. 21,No.9

Table 1. Overview of Compounds Tested: Compound and CAS Number, Boiling Point (bp), Observed Carryover from E-liquid to E-vapor, Calculated Half-lives Based on First-order Kinetics for Propylene Glycol (PG) Acetals in Two Different Aqueous Media, and EC50 (Including 95% Confidence Interval [CI]) and Efficacy Compared to the Known Antagonists for Human TRPA1 and TRPV1 Ion Channels (1 mM Cinnamaldehyde and 3 µM Capsaicin, Respectively). Statistical Analysis Indicated for Differences Between Parent Aldehyde and Corresponding Acetal, for

EC50s and Efficacies, Respectively Parent Aldehyde and Corresponding Acetal (ns Nonsignificant; *, p < .05; **, p < .01; ***, p < .001) Human TRPA1 Human TRPV1

Observed carryover Half-life in D2O Efficacy (to Efficacy

Compound (CAS no.) bp (°C) range to e-vapor (%) (HBSS buffer) (h) EC50 (95% CI) cinnamaldehyde) EC50 (95% CI) (to capsaicin)

Benzaldehyde 178 51%–71%c — 0.30 mM*** (0.23 to 0.39) 91%n.s. > 12 mM 36%* (100-52-7)a Benzaldehyde PG acetal 230–232 47%–80%c 44 h 0.88 mM*** (0.70 to 1.2) 93%n.s. 2.4 mM (1.7 to 4.2) 47%* (2568-25-4)a Ethylvanillin 295 62%–71%d — 0.17 mM*** (0.13 to 0.22) 74%*** > 5.6 mM 21%*** (121-32-4)a Ethylvanillin PG acetal 346–347 61%–71%d 70 h 0.69 mM*** (0.57 to 0.81) 90%*** 3.5 mM (3.3 to 3.7) 40%*** (68527-76-4)b Vanillin 285 63%–71%d — 0.18 mM** (0.12 to 0.28) 63%*** > 3 mM 9%*** (121-33-5)a Vanillin PG acetal 353 59%–65%d 34–39 h (48 h) 0.91 mM** (0.76 to 1.1) 93%*** 1.1 mM (0.92 to 1.3) 56%*** (68527-74-2)b Nicotine 247 52%–66%c — (54-11-5)a aSource: Sigma-Aldrich; bSource: Bedoukian Research, Danbury, CT; ce-liquid PG/VG range 100/0 to 5/95; de-liquid PG/VG range 100/0 to 40/60. HBSS = Hanks balanced salts solutions; VG = glycerol.

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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 combinedcis - 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. difference observed. Acetal mole fractions of the combined aldehyde mole fractions was observed subsequently. Benzaldehyde PG acetal and acetal content were measured by GC–flame ionization detector, formed more slowly, reaching equilibrium around day 5 and exceeding and conversion rates and equilibrium concentrations were determined 95% mole fraction. Ethylvanillin and vanillin were converted to PG (Figure 1): cinnamaldehyde and citral conversions plateaued within the acetals more slowly, reaching equilibrium after approximately 7 days, first day, at 92% and 88%, respectively. A small decline in PG acetal at ca. 50% and 40% mole fractions, respectively. Nicotine & Tobacco Research, 2019, Vol. 21, No. 9 1253

The effect of varying PG/VG ratios, which are commonly offered respectively. For all three compounds shown in Figure 1, a trend to- in commercial e-liquids, on the formation of cinnamaldehyde PG ward increasing mole fraction of the respective acetals with increas- acetal was also evaluated (Figure 1 inset). When the PG/VG ratio ing PG content of the e-liquid was observed, which means a higher was decreased, lower equilibrium concentrations of cinnamaldehyde proportion of flavor aldehyde was converted to the correspond- PG acetal were observed. These findings demonstrate that the rate ing acetal. This effect was more pronounced for benzaldehyde PG and amount of PG acetal formed varies for different aldehydes and acetal, especially at low PG content. These data demonstrate that Downloaded from https://academic.oup.com/ntr/article-abstract/21/9/1248/5134068/ by National Science and Technology Library -Root user on 05 October 2019 depends on the ratio of PG/VG present. the amounts of PG acetals present in commercial e-liquids are pro- To evaluate whether aldehyde PG acetals are present in com- portional to the ratio of PG/VG used to prepare the e-liquid. Further, mercial e-liquids, and whether their formation depends on PG con- they support the earlier finding that benzaldehyde reacts more tent, a range of commercial vanilla (vanillin and ethylvanillin) and readily with PG than vanillin or ethyl vanillin. cherry (benzaldehyde)-flavored e-liquids were examined Figure ( 1 and Supplementary Figure 1). The vanilla e-liquids tested in this Compound Delivery to E-cigarette Vapor study were purchased with PG contents of 0%, 30%, 50%, 70%, During vaping, flavor aldehydes are aerosolized and deposited in the and 100% labeled as such by the vendor, however, LC–IR revealed airways along with other constituents. To determine whether alde- that the actual PG contents were 60%, 70%, 80%, 85%, and 100%, hyde PG acetals are also carried over into e-vapor, the concentrations of benzaldehyde, vanillin, ethylvanillin, their respective PG acetals, and nicotine were measured in cold-trapped e-vapor (Figure 2 and Supplementary Figure 2). Carryover rates for aldehydes and their corresponding acetals were very similar, amounting to approxi- mately 50%–80% of their concentration in neat e-liquid (Table 1). Carryover rates did not vary statistically significantly with change in PG content, except for benzaldehyde at 5% and 100% PG con- tent (Figure 2). Nicotine e-vapor carryover was also determined at rates close to 60% (Supplementary Figure 2) that is similar to rates reported in previous studies,22,23 thereby validating the used method- ology for quantifying carryover. Taken together, these results suggest that similar to flavor aldehydes, a significant proportion of the alde- hyde PG acetal will reach the airways during vaping.

Acetal Aqueous Stability For aldehyde PG acetals to exert biological effects, acetals need to persist on deposition in the respiratory system. To determine whether aldehyde PG acetals undergo hydrolysis in physiological solution, UV–VIS and 1H-NMR spectroscopy were used. First, the hydrolysis Figure 2. Carryover rates of benzaldehyde and benzaldehyde propylene glycol of vanillin PG acetal to vanillin was monitored by UV–VIS spectros- (PG) acetal from e-liquid to vapor generated by a first-generation e-cigarette, copy in physiological Hanks balanced salts solutions buffer at pH as a function of PG content of the neat e-liquid with the residual made 7.4, resembling the pH of airway lining fluid of healthy humans.24 up by glycerol (VG). Error bars represent the SD of at least n = 3 separate experiments (filled symbols), or the range whenn = 2 (non-filled symbols). A series of UV–VIS measurements followed the hydrolysis of van- Statistical significance (*, p < .05) found only between benzaldehyde PG illin PG acetal to pure vanillin by the growth of an absorption peak acetal 5% and 100%. Carryover results for vanillin, ethylvanillin, and nicotine at 347 nm (Supplementary Figure 3). This technique is used to de- are shown in Supplementary Figure 2. termine vanillin concentrations in foods,25 yet it is pH-dependent,26

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. 1254 Nicotine & Tobacco Research, 2019, Vol. 21, No. 9 warranting the use of a buffered solution. Using first-order kinetics, activate respiratory irritant receptors with broader selectivities and the half-life of vanillin PG acetal under these conditions was deter- higher potencies than their aldehyde precursors, suggesting that they mined to be approximately 48 hours (Table 1). Due to the absence may act as stronger airway irritants in e-cigarette users. of characteristic UV–VIS absorption peaks for the other aldehyde PG To pursue this study, several e-liquids with various PG/VG ratios acetals, their hydrolysis was monitored by 1H-NMR in unbuffered were purchased online, however, their chemical analysis revealed D O. Vanillin PG acetal was used as control for comparison of meth- that their contents were not accurately reflected by the compos-

2 Downloaded from https://academic.oup.com/ntr/article-abstract/21/9/1248/5134068/ by National Science and Technology Library -Root user on 05 October 2019 ods and buffer effects, with a series of a 1H-NMR spectra shown ition on the label. This was evidenced by the measured differences in in Supplementary Figure 3 indicating the disappearance of vanillin the component concentrations reported by the vendor versus those PG acetal (doublet peak at δ = 5.6–5.8) because of its conversion measured in the purchased e-liquid products (eg, PG/VG ratios in to vanillin (singlet peak δ = 9.6) over time. Similar series of spectra vanilla e-liquids). Several recent studies reported incorrectly labeled were recorded for the PG acetals of benzaldehyde and ethylvanillin. nicotine contents of e-liquids, including substantial amounts of nico- After peak integration, the half-lives of these three compounds in tine detected in e-liquids labeled as “nicotine-free”, purchased in 27–29 D2O were calculated as 34–39 hours (vanillin PG acetal), 44 hours several different countries. In light of these reports, the deviat- (benzaldehyde PG acetal), and 70 hours (ethylvanillin PG acetal) ing PG/VG ratios measured in this study add to the concerns about based on first-order kinetics (Table 1.) A similar attempt to determine inaccurate mixing, labeling and resulting unexpected toxicities of the aqueous stability of cinnamaldehyde PG acetal failed because of commercial e-liquids. Rapid regulatory action is needed to address the very low solubility of the compound in D2O. All measured half- the ongoing failure of some e-liquid manufacturers to provide labe- lives are in the order of days, suggesting that the tested acetals are ling consistent with product contents to the public.29–31 not instantly hydrolyzed once they reach the aqueous environment of the airway surfaces, warranting investigations of their potential Acetal Formation and Presence in Laboratory-made biological effects. and Commercial E-liquids The reaction between aldehyde flavorants and PG to form an acetal Acetal Irritation Potential occurs at conditions common during the mixing and storage of To compare the effects of flavor aldehydes and their corresponding e-liquids by vendors and users alike. PG acetals formed from all of PG acetals on TRP irritant receptors, HEK-293T cells were used for the tested aldehydic flavorants, including benzaldehyde, cinnamal- heterologous expression of human TRPA1 and TRPV1 ion channels dehyde, citral (mixture of the structural isomers neral and geranial), and fluorometric measurements of TRP-mediated Ca2+ influx. vanillin, and ethylvanillin, when mixed with PG (Figure 1). As men- Indeed, ethyl vanillin PG acetal, vanillin PG acetal, and benzal- tioned in the Introduction section, aldehyde PG acetals contain two dehyde PG acetal robustly activated Ca2+ influx through TRPA1, chiral carbons, and the two diastereomers can be distinguished in with all three flavor aldehyde acetals as efficacious as the full TRPA1 the NMR spectra (Supplementary Figure 3) and GC chromatograms agonist, cinnamaldehyde (>90% of maximum cinnamaldehyde (Supplementary Figure 5). It is interesting to note that the acetal for- response; Figures 3 and Supplementary Figure 4; Table 1). Further, mation kinetics differ for the tested aldehydes, which may be related to how quickly these molecules are solubilized by PG. Vanillin and all tested PG acetals activated TRPA1 with higher EC50 values compared to their corresponding aldehydes, and interestingly, high ethylvanillin are both solid at room temperature, and while they par- concentrations of vanillin PG acetal and ethylvanillin PG acetal (at tially dissolved in PG on initial mixing, dissolution was not com- 3 mM and 10 mM, respectively) were more efficacious at activat- plete until after 24 hours (observed visually). The slight decline in ing TRPA1 than equivalent concentrations of their corresponding the concentrations of the PG acetals of cinnamaldehyde and citral parent aldehydes (Table 1; Figure 3 and Supplementary Figure 4). at later times suggests that further reactions may occur in e-liquids: The flavor aldehyde PG acetals also had surprising effects on both compounds are α,β-unsaturated aldehydes, known to be rela- TRPV1, the vanilloid receptor: While benzaldehyde, ethylvanillin, tively reactive.32 and vanillin weakly activated TRPV1 only at high concentrations Further experiments investigated how the amount of acetal (>3 mM) close to their solubility limits, their corresponding PG formed can vary as a function of the PG/VG ratio. When the PG/VG acetals robustly activated TRPV1 at lower concentrations and with ratio is decreased, the equilibrium concentration of the correspond- higher efficacies (Table 1, Figure 3 and Supplementary Figure 4). ing PG acetal was reduced (Figure 1 inset). Besides PG, VG can also This effect on TRPV1 was most conspicuous when comparing van- form acetals with aldehydes,8 yet because VG contains three alcohol illin and vanillin PG acetal (Table 1, Figure 3). Cinnamaldehyde PG moieties, a multitude of optical and structural isomers of VG acetals acetal robustly activated TRPA1 and significantly activated TRPV1 can form. At least four of such isomers are shown in a GC-MS chro- at a nominal concentration of 500 µM; however, a dose–response matogram for cinnamaldehyde VG acetal (Supplementary Figure 6) relation could not be established because of limited solubility of and vanillin VG acetal (Supplementary Figure 7). Since no standard the compound. for cinnamaldehyde VG acetal nor vanillin VG acetal was available, they were not quantified. Although previous studies detected flavor aldehyde acetals in Discussion commercial e-liquids, these were not quantified.11,12 In this study, In this study, a reaction between e-liquid constituents of aldehydic substantial amounts of flavor aldehyde PG acetals were detected in flavorants and PG to form aldehyde PG acetals is described. These the analyzed commercial e-liquids, with 15%–30% of the aldehyde compounds form in situ and are shown to be present in commer- converted to its corresponding PG acetal, and increasing acetal cial e-liquids. Carryover experiments reveal that quantities of concentrations at higher PG contents. It is noteworthy that the 50%–80% of the initial acetal concentration in the neat e-liquid are mole fractions of acetals in the commercial e-liquids did not reach transferred to the e-vapor generated by a first-generation e-cigarette. the same equilibrium levels as in the experimental mixtures of only The relative stability of these acetals to hydrolysis at physiological PG and the aldehyde in question (Figure 1). This can be explained pH is demonstrated, implying that they persist after inhalation by by the higher complexity of the commercial e-liquids that contain the e-cigarette user. Finally, this study shows that the tested acetals additional ingredients, including water and other compounds that Nicotine & Tobacco Research, 2019, Vol. 21, No. 9 1255 can compete with PG to react with the aldehydes, or influence the Acetal Stability reaction equilibrium.5 However, the same order of mole fractions The formation of acetals from an aldehyde and PG is reversible, with is found in both the experimental and commercial mixtures: ben- hydrolysis favored at acidic pH in aqueous solutions35 (Figure 1). zaldehyde PG acetal > ethylvanillin PG acetal > vanillin PG acetal. The airway lining fluid in which e-vapor is deposited is slightly alka- This study also showed that the tested e-liquids contained lower line,17,24 therefore, acetal hydrolysis is expected to occur at slower amounts of flavor aldehydes as the ratio of PG/VG in the e-liquid rates. Indeed, the half-lives of the acetals measured in aqueous solu- Downloaded from https://academic.oup.com/ntr/article-abstract/21/9/1248/5134068/ by National Science and Technology Library -Root user on 05 October 2019 decreased (Supplementary Figure 1). In this case, this likely means tion exceeded 36 hours, sufficient for substantial amounts of acetals that the “flavor base” was mixed in pure PG, and the appropriate to be maintained in physiological fluids, potentially exerting direct amount of VG was added to create the final product. effects on the airway lining fluids and underlying structures. Flavor aldehyde PG acetals, including the compounds detected in this study (Table 1), are produced commercially as flavorants and scents for food, perfumes, cosmetics, and body care products. Recruitment of Additional Respiratory Irritant It is, therefore, possible that the acetals were added directly to the Receptors by Flavor Aldehyde PG Acetals commercial e-liquids. However, acetals have never been listed in the Exposures to aldehydes cause respiratory irritation through acti- e-liquid ingredient lists in the few cases where these were revealed, vation of irritant receptors in sensory nerve endings in the airway and are less economical than their corresponding aldehydes. They lining. TRPA1 is the major aldehyde-activated receptor, respond- are prepared from the parent aldehyde, suggesting that the acetals ing during exposures to acrolein in tobacco smoke, formaldehyde, would generally be more costly, without a requisite gain in aroma as and flavor aldehydes such as cinnamaldehyde and benzalde- acetals generally resemble the odor of the parent aldehyde, but are hyde.16,17,36 The vanilloid receptor, TRPV1, may also contribute less pronounced.32 The odor is particularly important as the olfac- to flavorant irritant effects as it is activated by vanillin-related tory system (smell) is the dominating system with regards to a user’s compounds such as capsaicin, however, TRPV1 responds poorly flavor perception of e-vapor. to free aldehydes.37 The present study strongly suggests that flavor aldehyde PG acetals are delivered to the airways of e-cigarette Acetal Carryover to Vapor in E-cigarettes users, however, except benzaldehyde PG acetal,38 it is unknown The fate of flavor aldehydes during heating and vaporization in whether these compounds also activate irritant receptors. Our in e-cigarettes, and their resulting toxicities, has been the subject of vitro studies clearly demonstrate that the acetals activate human intense controversy. All PG acetals discussed earlier exhibit higher TRPA1 irritant receptors with increased efficacies at the high- boiling points than their corresponding aldehydes (Table 1), pos- est concentrations (Figure 3 and Supplementary Figure 4). Also, sibly impacting vaporization. Heating may also affect the stability higher concentrations of vanillin and ethyl vanillin seem to be of acetals. desensitizing TRPA1 and attenuating the ion-channel activation, A substantial carryover of intact acetals from commercial whereas their corresponding PG acetals do not demonstrate ion- e-liquids into the e-vapor was observed, higher than 50% in all channel desensitization or attenuation of response. Although van- cases, with no correlation between boiling points and carryo- illin, ethylvanillin, and benzaldehyde had only negligible effects ver ratios (Table 1; Figure 2 and Supplementary Figure 2). No on TRPV1, their corresponding PG acetals robustly activated this major breakdown products of the acetals could be detected in receptor (Figure 3 and Supplementary Figure 4; Table 1). These the e-vapor. Aldehydes and nicotine, while having divergent boil- observations suggest that the tested flavorant aldehyde PG acetals ing points, were carried over at similar rates. Among the studied may produce stronger sensory irritation effects than the free alde- compounds, benzaldehyde has the lowest boiling point (178°C), hydes alone, by recruiting additional sensory irritant receptors yet its carryover rate was in the same range as for most other such as TRPV1. It remains to be investigated whether these effects compounds, including those with very high boiling points, such as occur in vivo, for example, by examining the respiratory irrita- vanillin PG acetal (bp: 353°C; Table 1). To the best of our know- tion response in rodents to e-vapors containing PG acetals, and by ledge, only a few flavorant molecules with boiling points higher measuring inflammatory parameters.39,40 than 300°C are used in e-liquids,12 and boiling points of acetals The aldehydes and acetals used in this study are listed as would not be expected to be much higher than those of vanil- GRAS (generally regarded as safe) by the US Food and Drug lin PG acetal. This suggests that the most important parameter Administration and the World Health Organization, as are sev- for compound carryover from e-liquid to e-vapor is its solubil- eral other PG acetals,41–43 but not furfural PG acetal.43 Yet, GRAS ity in the solvents PG and VG rather than a compound’s boiling status is based purely on ingestion safety when used as food addi- point. Therefore, most flavorants, including solvent adducts such tives, and cutaneous safety when used as scents. Currently, no data as acetals, are likely to reach the e-vapor in appreciable amounts. exist that would support inhalational safety of flavor aldehyde PG Carryover of benzaldehyde PG acetal increased significantly with acetals. Since TRPA1 and TRPV1 contribute to chronic pulmonary higher PG content of the e-liquid, with similar trends observed for conditions such as asthma, the formation of more strongly irritating the other PG acetals (Figure 2 and Supplementary Figure 2). Previous compounds in e-liquids is of concern.39,44–47 Indeed, recent epidemio- studies reported similar observations for nicotine carryover at vary- logical and toxicological studies detected links between asthma ing PG/VG ratios.33 It should be noted that the first-generation frequency and e-cigarette use in adolescents and reported that vapor- devices used in this study are not typically recommended for use ized e-liquids containing the same flavor aldehydes as tested in this with e-liquids with high VG contents because of their limited power, study induce inflammation in human respiratory epithelia.48 The cur- and this is likely to impact carryover rates, especially at low PG/VG rent study warrants further investigation into possible toxic effects ratios. Newer generation devices are much more powerful and are of inhaling flavorant PG acetals by means of an e-cigarette, and one likely more effective in delivering flavorants as well as the described of the outcomes may be that GRAS status is not sufficient to deem acetals to the e-vapor, as has already been described for nicotine.34 certain flavor additives as safe for inhalation by an e-cigarette. 1256 Nicotine & Tobacco Research, 2019, Vol. 21, No. 9 Downloaded from https://academic.oup.com/ntr/article-abstract/21/9/1248/5134068/ by National Science and Technology Library -Root user on 05 October 2019

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.

Implications e-cigarette devices, stability in biological environments (eg, mouth, This study demonstrates the potential chemical instability of e-liq- airways, and lungs) to toxicity potential for a variety of endpoints uids: acetals can be formed between flavor aldehydes and the sol- (Figure 4). Without this comprehensive framework, important vents PG and VG post-formulation, such as during mixing, shipping, classes of compounds, such as the PG acetals studied here, could be and storage of e-liquids. This reaction starts almost immediately and unintentionally neglected if they are not directly added and disclosed continues over days, leading to significant acetal generation even by e-liquid manufacturers. To fully assess the risk potential of e-liq- though water is not removed actively from the reaction. Given the uid use for regulatory purposes, it is imperative that the compounds frequency at which aldehydes are used in e-liquids (eg, 11 different that a user will actually be exposed to are reported and evaluated, aldehydes found in 28 e-liquids,12 and a total of 126 aldehydes on and not only the initial ingredients combined during manufactur- the FEMA GRAS list8), and that at least two PG- and four VG-acetal ing.50 When selecting compounds to screen for potential toxicity stereoisomers (and structural isomers of the latter) are formed dur- concerns, the chemical instability of the given e-liquid, which will ing the acetalization reaction, a large number of distinct acetal com- depend on the reactivity of the individual constituents, needs to be pounds could potentially be present in e-liquids, each possibly with taken into account. These can undergo a multitude of reactions given different reaction kinetics, aqueous stability, and most importantly, the complex mixture environment of e-liquids, making the predic- potentially different physiological and toxicological effects. It should tion of equilibrium concentrations of reaction products, including be noted that in this study, the potential to activate irritant receptors PG or VG acetals, extremely challenging. In addition, a comprehen- was not separately tested for optical isomers (enantiomers or dias- sive risk assessment should consider the potential reaction products tereomers), but it is well known that these can have very different that can result from heating and oxidation in the e-cigarette, requir- physiological effects.10,49 ing characterization and quantification of the e-vapor constituents To comprehensively assess the risk of e-liquids, it is important for toxicity concerns.50 Finally, the biological stability of compounds to consider the complete life cycle of the e-liquid constituents from present in the e-vapor should be considered, including interactions mixing through shipping and storage, heating, and oxidation in with pulmonary surfactants, as reactions may occur that change Nicotine & Tobacco Research, 2019, Vol. 21, No. 9 1257 the composition of the e-vapor and could result in new chemical 11. Geiss O, Bianchi I, Barahona F, Barrero-Moreno J. Characterisation of products with unknown toxicities. This suggests that ingredient dis- mainstream and passive vapours emitted by selected electronic cigarettes. closure of e-liquids by suppliers may be insufficient to inform a com- Int J Hyg Environ Health. 2015;218(1):169–180. prehensive risk assessment of e-liquids and vaping more generally. 12. Hutzler C, Paschke M, Kruschinski S, Henkler F, Hahn J, Luch A. Chemical hazards present in liquids and vapors of electronic cigarettes. Arch Toxicol. 2014;88(7):1295–1308.

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Hanno C. Erythropel, Ph.D.1,2, Sairam V. Jabba, Ph.D.2,3, Tamara M. deWinter, Ph.D.2,4, Melissa Mendizabal1, Paul T. Anastas, Ph.D.1,4,5,6, Sven E. Jordt, Ph.D.2,3*, Julie B. Zimmerman, Ph.D.1,2,4*

1 Department of Chemical and Environmental Engineering, Yale University, New Haven, CT 2 Yale Tobacco Center of Regulatory Science (TCORS), Department of Psychiatry, Yale School of Medicine, New Haven, CT. 3 Department of Anesthesiology, Duke University School of Medicine, Durham, NC 4 School of Forestry and Environmental Studies, Yale University, New Haven, CT 5 Department of Chemistry, Yale University, New Haven, CT 6 School of Public Health, Yale University, New Haven, CT

Supplemental Material

Figure S1: Absolute concentrations of benzaldehyde and benzaldehyde PG acetal vs. PG content of tested cherry e-liquid (top row), and of vanillin, vanillin PG acetal, ethylvanillin, and ethylvanillin PG acetal vs. PG content of tested vanilla e- liquid.

1

Figure S2: Carryover rates of vanillin, ethylvanillin, their respective PG acetals, and nicotine from e-liquid to vapor generated by a first generation e-cigarette, as a function of PG content of the neat e-liquid. Error bars represent the standard deviation (SD) of at least n=3 separate experiments (filled symbols), or the range when n=2 (non-filled symbols).

2

Figure S3: top: UV/VIS spectra of vanillin PG acetal stored in HBSS buffer at pH=7.4 over time, and of an equimolar solution of pure vanillin at in the same solution (orange line); bottom: 1H-NMR spectra of vanillin PG acetal stored in

D2O over time. The disappearing doublet between δ=5.6-5.8 stems from the PG acetal diastereomers as shown, and the singlet at ca. δ=9.6 represents the aldehydic hydrogen as indicated.

3

Figure S4: Effects of benzaldehyde, ethylvanillin, and their corresponding PG acetals on human sensory irritant receptors, TRPA1 and TRPV1, measured by fluorescent calcium imaging in HEK293T cells. Responses were normalized to maximal cinnamaldehyde (TRPA1) or capsaicin (for TRPV1) responses. Each point represents mean values of 15-21 independent measures from 4 experiments, and error bars show SEM.

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Figure S5: Part of GC chromatograms of cinnamaldehyde (CAD; black), vanillin (red), and citral (blue; mixture of cis and trans structural isomers neral and geranial) after storage in propylene glycol (PG) for 4.2 days, showing the formation of two diastereomers of the corresponding PG acetals for each aldehyde.

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Figure S6: bottom: Part of GC chromatograms showing at least 2 stereoisomers of cinnamaldehyde propylene glycol acetal (blue; CAD PG acetal) and at least 4 stereo- and/or structural isomers of cinnamaldehyde glycerol acetal (purple; CAD VG acetal), respectively, after storage in PG and VG, respectively, for 3 days. Top insets show mass spectroscopy (MS) spectra of the specified peaks.

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Figure S7: top: Part of GC chromatograms showing at least 2 stereoisomers of vanillin propylene glycol acetal (Vanillin PG acetal) and at least 4 stereo- and/or structural isomers of vanillin glycerol acetal (Vanillin VG acetal), respectively, after storage in a mixture of PG and VG for 4 days. Below, mass spectroscopy (MS) spectra of the specified peaks are shown.

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Gas chromatography – Flame Ionization Detector (GC-FID) method:

The injection volume was 1µL and the injection was carried out with a split ratio of 300 at 250 ºC. To separate the injected sample an Agilent J&W DB-5 column (length 60 m, id 0.25 mm, 0.25µm film) was used under the following conditions: the oven was initially held at a temperature of 30 ºC for 7 min, then heated to 50 ºC at a rate of 10 ºC/min and held constant for 20 min, followed by a heating ramp to 310 ºC at a rate of 10 ºC/min and held constant for another 7 min. Helium (Airgas, Radnor, PA) was used as carrier gas and the detector was of the flame ionization type (FID) run at 325 ºC. Limit of quantification (LOQ) 50 µg/mL, limit of detection (LOD) 15 µg/mL , precision 1.38 %RSD (N=6), recovery 99.13% (N=9)

Liquid chromatography – Refractive Index (LC-RI) method:

For compound separation, an Aminex HPX-87H ion exclusion column (length 300 mm, id 7.8 mm, 9µm particle size, Bio-Rad, Hercules, CA) was used in isocratic mode, the mobile phase was a 5mM solution of sulphuric acid (95-98%) in HPLC grade water (both JT Baker, Center Valley, PA) at a flow rate of 0.6 mL/min, the injection loop used was 20 µL, the total run time was 30 min, and the system was coupled to a refractive index (RI) detector (Shimadzu RID-10A, Kyoto, Japan) set to 50 ºC. Limit of quantification (LOQ) 10 µg/mL, limit of detection (LOD) 3 µg/mL, precision 0.18 %RSD (N=6), recovery 99.84% (N=9).

Gas chromatography - mass spectroscopy (GC-MS):

A Perkin Elmer Clarus 580 with an Elite-5MS column (length 60 m, id 0.25 mm, 0.25µm film) coupled with a SQ 8 S MS detector was used to obtain compound separation and mass spectra shown in Figures S5 and S6. The above-described GC method was used, and the MS settings were EI+ mode with a 5min initial solvent delay.

Synthesis of citral PG acetals:

Citral (5 g, 32.8 mmol; mixture of cis and trans, 95 %, Fisher Scientific, Waltham, MA) and propylene glycol (4 g, 26.3 mmol; USP/FCC grade, Fisher Scientific) were mixed in a hermetically sealed vial and vigorously stirred at room temperature for 96 hours. The aqueous layer was removed in a separatory funnel, and the organic phase was injected at known concentrations (150, 300, 750, 1500, 3000 µg/mL) into the GC-FID. Since the injected mixture contained both citral PG acetals and pure citral, the respective citral PG acetals concentration was calculated by subtracting the amount of citral in the mixture (determined by a separate calibration curve) to obtain the values shown in Table S1 below (40, 89, 225, 452, 934 µg/mL). These values were used to construct the calibration curve of citral PG acetals.

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Calibration curves Calibration curves for were obtained by serial dilution of the compounds in question, using an appropriate solvent, which was methanol (JT Baker) containing 1 g/L 1,4-dioxane (Sigma-Aldrich) as internal standard for GC- FID, and water (HPLC grade, JT Baker) containing 1 g/L 1,4-dioxane as internal standard for LC-RI. The specific intervals of concentrations to construct the calibration curves for each compound are shown in Tables S1 and S2 below.

Table S1: Concentration intervals of compounds for calibration curve construction, analyzed by GC-FID. Concentrations (µg/mL) Benzaldehyde 50, 100, 250, 500, 1000, 2000 Cinnamaldehyde 50, 100, 250, 500, 1000, 2500, 5000 Citrala 50, 100, 200, 500, 1000, 2000 Ethylvanillin 100, 250, 500, 1000 Vanillin 50, 100, 250, 500, 1000 Benzaldehyde PG acetal 100, 250, 500, 1000, 2000 Cinnamaldehyde PG acetal 50, 100, 500, 1000 Citral PG acetalsb 40, 89, 225, 452, 934 Ethylvanillin PG acetal 50, 100, 250, 500, 1000, 2000 Vanillin PG acetal 50, 100, 250, 500, 1000, 2000 Nicotine 100, 250, 500, 1000, 2500 a combined peak areas of the structural isomers neral and geranial utilized. b combined peak areas of respective PG acetals of the structural isomers neral and geranial utilized.

Table S2: Concentration intervals of compounds for calibration curve construction, analyzed by LC-RI. Concentrations (µg/mL) Propylene glycol (PG) 10, 100, 1000, 10000 Glycerin (VG) 10, 100, 1000, 10000

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RESEARCH LETTER

Flavorant−Solvent Reaction Products and Menthol in JUUL E-Cigarettes and Aerosol Hanno C. Erythropel, PhD,1,2 Lucy M. Davis3, Tamara M. de Winter, PhD,2,4 Sven E. Jordt, PhD,2,5 Paul T. Anastas, PhD,4,6 Stephanie S. O’Malley, PhD,2 Suchitra Krishnan-Sarin, PhD,2 Julie B. Zimmerman, PhD1,2,4

INTRODUCTION percentage carryover were calculated per experiment by divid- ing the amount of compound trapped by the pod mass change, he “JUUL” e-cigarette is the best-selling e-ciga- and as aerosol concentration over neat e-liquid concentration, rette on the U.S. market.1,2 JUUL refill “pods” respectively (Figure 1). T contain nicotine benzoate salt and flavorants dissolved in a 30:70 ratio of propylene glycol (PG) and RESULTS glycerol (VG for vegetable glycerin). Nicotine benzoate is perceived as more satisfactory and less harsh, enabling The reaction products vanillin PG acetal and vanillin VG “   ” the delivery of higher amounts of nicotine to users. As acetals were detected in JUUL Creme Brulee e-liquid § § such, nicotine concentrations in JUUL e-liquids are and carried over to aerosol at 68 4% (mean 95% CI, n § § m § higher (5%; 3% since August 2018) than in non-JUUL e- all =3) and 59 20%, or 0.8 0.04 g/puff and 2.0 0.5 m liquids (typically 0.3%−2.4%). JUUL e-liquids are avail- g/puff, respectively (Figure 1). Vanillin was carried § § m able in several fruity flavors, known to be particularly over at 79 17%, resulting in the delivery of 7.9 0.8 g/ fl appealing to youth.3 Common e-cigarette (including puff. Menthol was found in four of the eight tested a- “ JUUL) flavorants include menthol and various aldehydes vors, and the menthol aerosol concentration of Classic ” “ ” § m § (e.g., vanillin); aldehydes are known to react with alco- Menthol and Cool Mint was 34 3 g/puff and 38 m hols (e.g., PG and VG) to form acetals (structural and 12 g/puff, respectively, which is comparable to men- − m 8 optical isomers).4 The inhalational safety of flavor alde- tholated cigarettes (29 392 g/puff for ten puffs/ciga- − 9 hyde PG/VG acetals is unknown; however, a recent rette; 10% 20% carryover to cigarette smoke ). Nicotine § § study found that several acetals, including vanillin PG and benzoic acid carryover were 98 6% and 82 5% n § § acetal, activate pro-inflammatory irritant receptors more (5%-pods, =21), and 102 4% and 80 14% (3%-pods, n fi strongly than their parent compounds (e.g., vanillin).4,5 =5), respectively. However, a statistical signi cance Despite the popularity of JUUL, little is known about between e-liquid and aerosol concentrations was found the composition of JUUL aerosol. The aim of this study only for benzoic acid (Figure 1). Absolute nicotine aero- § m § m was to evaluate the carryover of vanillin and its reaction sol content (114 13 g/puff, 5%-pods, 65 15 g/puff, products with PG and VG, menthol, and nicotine benzo- 3%-pods) was comparable to previous reports analyzing − m ate from JUUL e-liquid to aerosol to understand poten- JUUL or combustible cigarettes (50 180 g/puff; ten 10−12 tial human exposures. puffs/cigarette).

METHODS From the 1Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut; 2Yale Tobacco Center of Regulatory A JUUL device and pods of all eight flavors (Figure 1) were pur- Science (TCORS), Department of Psychiatry, Yale University School of 3 chased online in 2018. For aerosol capture (both gas phase and Medicine, New Haven, Connecticut; Yale-NUS College, Singapore, Singa- 6 pore; 4School of Forestry and Environmental Studies, Yale University, microdroplets ), a custom-built vaping machine with liquid 5 nitrogen−chilled traps was used as described previously.7 The New Haven, Connecticut; Department of Anesthesiology, Duke Univer- sity School of Medicine, Durham, North Carolina; and 6School of Public puffing regime was 20 puffs of 2.8-second length, 79-mL vol- Health, Yale University School of Medicine, New Haven, Connecticut ume, and a 30-second cooldown between puffs. Neat e-liquids Address correspondence to: Julie B. Zimmerman, PhD, Department of andcapturedaerosolweredilutedandanalyzedbygaschroma- Chemical and Environmental Engineering, Yale University, 17 Hillhouse 4 tography mass spectroscopy. Commercially available standards Avenue, New Haven CT 06511. E-mail: [email protected]. were used for quantification except for vanillin VG acetals, 0749-3797/$36.00 which were synthesized in house. Aerosol concentrations and https://doi.org/10.1016/j.amepre.2019.04.004

© 2019 American Journal of Preventive Medicine. Published by Elsevier Inc. All rights Am J Prev Med 2019;57(3):425−427 425 reserved. 426 Erythropel et al / Am J Prev Med 2019;57(3):425−427

Figure 1. Comparison of the concentrations of six compounds in neat e-liquid (background bars, varying shading) and captured aerosol (foreground bars, striped) for different JUUL flavors. Note: Error bars represent 95% CI. Two-way ANOVAs with Bonferroni post-test results to compare neat e-liquids versus aerosol concentrations per compound were carried out using GraphPad Prism 8 software. A p-value of <0.05 was interpreted as significant; ***p<0.001. a5%-nicotine flavors: Creme Brulee, Fruit Medley, Mango, Cool Cucumber, Cool Mint, Classic Menthol, Classic Tobacco, and Virginia Tobacco. b3%-nicotine flavors: Cool Mint, and Virginia Tobacco. PG, propylene glycol; VG, glycerol.

DISCUSSION (“Creme Brulee”). Although vanillin PG/VG acetal aero- sol carryover is slightly lower than nicotine, indicating This study is the first to report the presence of flavor possible acetal hydrolysis, appreciable amounts of acetals aldehyde VG acetals in e-liquids and aerosols and are present in the aerosol, which, if inhaled, may cause expands the authors’ prior finding of flavor aldehyde irritation and contribute to inflammatory responses.4,5 PG acetals in commercial e-liquids.4 JUUL e-liquids The average vanillin puff concentration was 101 mg/m3. contain higher levels of vanillin VG acetals compared In comparison, chronic inhalational exposure to vanillin with vanillin PG acetal because of the higher VG:PG in occupational environments is limited to 10 mg/m3,13 ratio. Flavor aldehyde−solvent acetal formation can be raising the question of what long-term effects regular expected in any e-liquid−containing flavor aldehydes, inhalation of vanillin at such doses and frequency (200 including JUUL, at room temperature (e.g., without puffs/pod) might have. Aerosol menthol levels from heating in the e-cigarette).4 Furthermore, the possibility “Fruit Medley” (5.3 ppm), which is not labeled as men- of other unintended chemical reactions between e-liquid tholated, and the other mentholated JUUL e-liquids constituents should be considered in future research. (“Cool Cucumber”: 7.5 ppm, “Classic Menthol”: 63 ppm, Compounds present in JUUL e-liquids are delivered and “Cool Mint”: 70 ppm) are sufficient to suppress efficiently to the aerosol, exposing users to similar quan- respiratory irritation responses to aldehydes and tobacco tities of nicotine as cigarettes, to menthol in four of eight smoke and increase nicotine intake.14,15 flavors, and to the PG and VG acetals of vanillin

www.ajpmonline.org Erythropel et al / Am J Prev Med 2019;57(3):425−427 427 Future e-cigarette regulatory policy should address (1) REFERENCES the formation of new compounds with potential toxico- 1. Huang J, Duan Z, Kwok J, et al. Vaping versus JUULing: how the logic properties within e-liquids, (2) JUUL menthol levels extraordinary growth and marketing of JUUL transformed the U.S. that may increase nicotine intake, and (3) flavorant expo- retail e-cigarette market. Tob Control. 2018;28(2):146‒151. https://doi. sure effects in e-cigarette users as also recently highlighted org/10.1136/tobaccocontrol-2018-054382. 16 2. Willett JG, Bennett M, Hair EC, et al. Recognition, use and percep- by the U.S. Food and Drug Administration. tions of JUUL among youth and young adults. Tob Control. 2019;28 (1):115‒116. https://doi.org/10.1136/tobaccocontrol-2018-054273. ACKNOWLEDGMENTS 3. HHS. E-cigarette use among youth and young adults: a report of the Surgeon General. 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September 2019