Duke Yale Sven-Eric Jordt, PhD University University ... · Duke Yale University University 2 To: Dockets Management Staff (HFA-305) Food and Drug Administration 5630 Fishers Lane,

Duke Yale University University

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]

Sven-Eric Jordt, PhD1 Associate Professor

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

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, 1248–1258doi:10.1093/ntr/nty192Original investigation

Received March 28, 2018; Editorial Decision September 10, 2018; Accepted September 13, 2018

© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. All rights reserved. For permissions, please e-mail: [email protected].

1248

Original investigation

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

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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.

Introduction

The addition of characterizing flavors to combustible cigarettes is prohibited by regulatory measures in several jurisdictions, including the United States, Canada, and the European Union; however, these restrictions have not been extended to “e-liquids” used in electronic cigarettes (e-cigarettes). Along with the global increase in e-cigarette use,1–3 the variety of flavored e-liquids has risen, with currently more than 7000 flavors available.4 Among the most popular e-liquids are sweet and fruity flavors, a main driver for the appeal of e-cigarettes to youth.5–7 Fragrant aldehydes, such as vanillin and ethylvanillin (vanilla flavor), benzaldehyde (cherry flavor), or cinnamaldehyde (cinnamon flavor), commonly serve as the characteristic flavorants.8 E-liquids are prepared by mixing the flavorants and nicotine with different ratios of the solvents propylene glycol (PG) and glycerol (VG for vegetable glycerin), which contain two and three alcohol moieties, respectively.9

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-ing in the formation of at least three stereoisomers; molecules with different bond orientation and therefore possibly different proper-ties, including toxicological properties.10 Acetalization reactions are commonly used in synthetic organic chemistry to “protect” reactive aldehyde moieties from reacting further. Consequently, chemical reactions (ie, acetal formation) between e-liquid constituents are possible, suggesting that e-liquids can be unstable environments post-preparation. Indeed, recent analytical studies have reported the presence of flavor aldehyde acetals in e-liquids, in the headspace above e-liquids, and in e-cigarette vapors (e-vapor).11–13 In the cases where manufacturers published ingredient lists of e-liquids (avail-able online), only aldehydes, but not acetals, were listed among the flavorants, suggesting that these acetals form after mixing. The published analytical studies did not report the concentrations of the detected flavor aldehyde acetals in the respective e-liquids, and it remains unclear how frequently and how rapidly these compounds form and whether they remain stable during heating and vaporiza-tion in e-cigarettes. Beyond flavor aldehyde acetals, the presence of formaldehyde hemiacetals in e-vapors was reported, generated from formaldehyde reacting with a single alcohol moiety on PG or VG.14 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 products, has raised concerns because of their potential respiratory and cardiovascular toxicological effects. Respiratory sensory ir-ritation is a first sign of potential toxicity of an inhaled chemical exposure, induced by chemical activation of chemosensory recep-tors in airway-innervating nerves. Respiratory sensory irritants are 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-posure limits. Recent studies identified the transient receptor poten-tial (TRP) ion channels, TRPA1 and TRPV1, as the receptors for

irritant aldehydes in airway-innervating nerves, activated by the major tobacco smoke aldehyde, acrolein, and flavor aldehydes such as cinnamaldehyde, benzaldehyde, and vanillin, and eliciting irrita-tion responses, pain, and cardiovascular reflexes increasing stress and inflammation.16–19 In vitro tests quantifying the capability of a chemical to activate TRP irritant receptors are currently considered as replacements for the animal studies determining RD50s. It remains to be examined whether aldehyde PG acetals activate the same irri-tant mechanisms as their parent aldehydes and exhibit toxicological effects of concern.

The aims of this study were to determine whether: (1) aldehyde flavorants and PG yield acetals in e-liquid environments, (2) acetals are carried over into e-vapor, (3) acetals remain stable under physio-logical conditions, and (4) acetals activate TRP irritant receptors. A workflow is proposed for a comprehensive risk assessment of e-liquids and resulting e-vapors encompassing all compounds a user could be exposed to, including reaction products.

Materials and Methods

Acetal Formation ExperimentsOne hundred micrograms of benzaldehyde (100 µg, >99%; Sigma-Aldrich, St. Louis, MO), trans-cinnamaldehyde (98+%; Alfa Aeasar, Haverhill, MA), citral (mixture of the structural isomers neral and geranial; 95%, Fisher Scientific, Waltham, MA) ethylvanillin (99%), and vanillin (99%; both Sigma-Aldrich) were added to plastic vials containing 5 g PG (USP-FCC grade, Waltham, MA), or mixtures of PG/VG (anhydrous; AmericanBio, Natick, MA) at 70/30, 50/50, or 30/70 ratios by weight, to yield an aldehyde concentration of 20 mg/g. The experiments in pure PG were carried out in duplicate (light and dark) storage conditions. Vials were vigorously shaken once per day, and 100 µL samples were drawn, diluted, and injected into a gas chromato-graph (GC). Using standard curves, samples were analyzed for their aldehyde and PG acetal content (vanillin PG acetal [97%], ethylvanillin PG acetal [98%, both Bedoukian Research, Danbury, CT], benzalde-hyde PG acetal [(>98%], trans-cinnamaldehyde PG acetal [both Sigma-Aldrich], citral PG acetals synthesized in-house, see Supplementary Material for details). Results are presented as mole fraction of formed acetal, defined as moles of acetal divided by the combined number of moles of acetal and corresponding aldehyde.

Acetal Content of Commercial E-liquidsTwo flavored e-liquids (cherry and vanilla) were purchased with PG/VG ratios of 100/0, 70/30, 50/50, 30/70, and 0/100, contain-ing 12 mg/mL nicotine (AmericaneLiquidStore, Wauwatosa, WI). Samples of all e-liquids were taken, the mass recorded, and diluted for GC injection. Using standard curves, aldehyde, aldehyde PG acetal, and nicotine (99%, Alfa Aesar) concentrations were determined.

Vaping Carryover ExperimentsFor the vaping experiments, V2 blank cartridges were purchased on-line and filled with 0.5 g e-liquid and used with the corresponding

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79 mm, 4.2 V battery (both VMR Products LLC, Miami, FL). Cartridges were allowed to settle for at least 60 minutes before test-ing. E-vapors were produced and captured using a custom-built vap-ing machine described previously, using a series of cold-finger traps chilled with liquid nitrogen.20 The puffing topography was 4-second puffs, 73 ml puff volume, 30-second intervals, for 20 puffs total. The trapped e-vapor was subsequently taken up in methanol (JT Baker, Center Valley, PA) and injected into a GC. Compound e-vapor con-centration was calculated as total amount trapped in vapor divided by the mass change of the cartridge after 20 puffs. Carryover per-centage was calculated by dividing e-vapor concentration by neat e-liquid concentration.

Acetal Aqueous Stability TestingTo assess the stability of the PG acetals in physiological solution, ultraviolet–visible (UV–VIS) and proton nuclear magnetic resonance (1H-NMR) spectroscopy were used. The breakdown of vanillin PG acetal (starting concentration 100 µg/mL) to vanillin in Hanks bal-anced salts solutions buffer (Sigma-Aldrich), adjusted to pH 7.4, was monitored over time by UV–VIS spectroscopy at λ = 347 nm (Cary-3E; Varian, Palo Alto, CA). Breakdown of the other PG acetals in D2O (99.9 atom% D; Sigma-Aldrich) was monitored over time by 1H-NMR spectroscopy, comparing the appearing aldehyde sig-nal at approximately δ = 9.6 ppm to the disappearing acetal doub-let between δ = 5.6–5.9 ppm. 1H-NMR studies were carried out at starting concentrations of 150 µg/mL (benzaldehyde- and vanillin PG acetal) and 200 µg/mL (ethylvanillin PG acetal).

Gas ChromatographyA GC (GC-2010 Plus; Shimadzu, Kyoto, Japan) was used to quan-tify aldehydes, PG acetals, and nicotine. Citral and citral PG acetals were quantified as a combination of cis- and trans-isomers (see Supplementary Figure 5 for an example of a chromatogram). For the acetal formation reaction, 100 µL samples were diluted with 1 mL of methanol (high-performance liquid chromatography [HPLC] grade, JT Baker) containing 1 g/L 1,4-dioxane (99+%, Alfa Aeasar) as an in-ternal standard. For the carryover experiments, the complete content of the cold-finger trap was taken up in 1 mL of methanol (JT Baker) containing internal standard. See Supplementary Material for further GC method details. Calibration curves were prepared to calculate concentrations from peak area ratios of the compounds of interest to the internal standard.

Liquid ChromatographyA liquid chromatography–refractive index (LC–RI) (Shimadzu 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 deionized water (HPLC grade, JT Baker) and 1,4-dioxane was added as an internal standard. Calibration curves of PG and VG were prepared to calculate concentrations from peak area ratios of compounds to the internal standard.

1H-NMR SpectroscopyAll 1H-NMR spectroscopy was carried out on an Agilent DD2 400 MHz NMR spectrometer, using D2O, methanol (CD3OD), or chloroform (CDCl3; all at least 99.8% D, Cambridge Isotope Labs., Andover, MA).

Cell CultureHEK-293T cells (ATCC, Manassas, VA; Cat# CRL-11268, RRID: CVCL_1926) were cultured in Gibco Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 0.1 mg/mL streptomycin (all Fisher Scientific). Cells were plated on 100 mm tissue culture plastic dishes and grown over-night to 60%–70% confluency. The cells were transiently transfected with either human TRPA1 or TRPV1 plasmid DNA (18 µg plas-mid DNA/100 mm dish) for 16–24 hours using Fugene 6 transfec-tion reagent (Promega, Madison, WI) and Gibco Opti-MEM (Fisher Scientific) according to manufacturer’s protocols. Cells were then resuspended and plated onto poly-d-lysine-coated (Sigma-Aldrich) 96-well black-walled plates at 50 000 cells per well and allowed to grow for another 16–24 hours before the experiments. Cells were maintained as monolayers at 5% CO2 and 37°C.

Calcium Influx ImagingCalcium imaging was performed as described earlier.21 Briefly, the fluorescence calcium indicator FLIPR Calcium 6 dye (Molecular Devices, San Jose, CA) was applied to the cells, which were recon-stituted in Hanks balanced salts solutions buffered at pH 7.4 using 20 mM HEPES buffer (both Sigma-Aldrich). A FlexStation III benchtop scanning fluorometer chamber (Molecular Devices) at 37°C was used to excite cells at 485 nm and the Ca2+ emission sig-nal was recorded at 525 nm in intervals of 1.8 seconds. The change in fluorescence was measured and expressed as Fmax–F0, where Fmax is the maximum fluorescence and F0 is the basal fluorescence. The responses were normalized to the known agonists cinnamaldehyde (1 mM; for TRPA1) or capsaicin (3 µM; for TRPV1). A Boltzmann function (3- or 4-parameter logistic fit) was used to generate con-centration–response curves and determine the half maximal effective concentration (EC50)values (Prism 7 software; GraphPad, La Jolla, CA). 95% confidence intervals (CI) of the logEC50 values were cal-culated to display the variability of concentration–response curves. Efficacies (maximal responses) for each of the flavor aldehydes and their corresponding PG acetals were reported as a percentage of the maximum response elicited by saturating concentrations of the full agonists, cinnamaldehyde (1 mM) for TRPA1 and capsaicin (3 µM) for TRPV1 (Table 1).

Statistical AnalysisStatistical 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-preted as significant, with p value less than .05 represented by *, p value of less than .01 by **, and p value less than .001 by ***.

Results

Acetal Formation and Presence in Laboratory-made and Commercial E-liquidsTo investigate the formation kinetics of flavor aldehyde-PG acetals, commonly used flavor aldehydes were mixed with pure PG and sampled over 14 days while stored at room temperature (Figure 1); aldehydes tested were benzaldehyde, cinnamaldehyde, citral (mixture of the two structural isomers neral and geranial), vanillin, and ethylvanillin, each at 20 mg/g, approximating the concentrations found in commercial e-liquids (Supplementary Figure 1). The PG acetal formation reactions were monitored in daylight and dark conditions with no significant

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Tab

le 1

. Ove

rvie

w o

f C

om

po

un

ds T

este

d: C

om

po

un

d a

nd

CA

S N

um

ber

, Bo

ilin

g P

oin

t (b

p),

Ob

serv

ed C

arry

over

fro

m E

-liq

uid

to

E-v

apo

r, C

alcu

late

d H

alf-

lives

Bas

ed o

n F

irst

-ord

er K

inet

ics

for

Pro

pyl

ene

Gly

col (

PG

) Ace

tals

in T

wo

Diff

eren

t Aq

ueo

us

Med

ia, a

nd

EC

50 (

Incl

ud

ing

95%

Co

nfi

den

ce In

terv

al [

CI]

) an

d E

ffica

cy C

om

par

ed t

o t

he

Kn

ow

n A

nta

go

nis

ts f

or

Hu

man

TR

PA1

and

TR

PV

1 Io

n C

han

nel

s (1

mM

Cin

nam

ald

ehyd

e an

d 3

µM

Cap

saic

in, R

esp

ecti

vely

). S

tati

stic

al A

nal

ysis

Ind

icat

ed f

or

Diff

eren

ces

Bet

wee

n P

aren

t Ald

ehyd

e an

d C

orr

esp

on

din

g A

ceta

l, fo

r E

C50

s an

d E

ffica

cies

, Res

pec

tive

ly P

aren

t Ald

ehyd

e an

d C

orr

esp

on

din

g A

ceta

l (n

s N

on

sig

nifi

can

t; *

, p <

.05;

**,

p <

.01;

***

, p <

.001

)

Com

poun

d (C

AS

no.)

bp (

°C)

Obs

erve

d ca

rryo

ver

rang

e to

e-v

apor

(%

)H

alf-

life

in D

2O

(HB

SS b

uffe

r) (

h)

Hum

an T

RPA

1H

uman

TR

PV1

EC

50 (

95%

CI)

Effi

cacy

(to

ci

nnam

alde

hyde

)E

C50

(95

% C

I)E

ffica

cy

(to

caps

aici

n)

Ben

zald

ehyd

e(1

00-5

2-7)

a

178

51%

–71%

c—

0.30

mM

***

(0.2

3 to

0.3

9)91

%n.

s.>

12 m

M36

%*

Ben

zald

ehyd

e PG

ace

tal

(256

8-25

-4)a

230–

232

47%

–80%

c44

h0.

88 m

M**

* (0

.70

to 1

.2)

93%

n.s.

2.4

mM

(1.

7 to

4.2

)47

%*

Eth

ylva

nilli

n(1

21-3

2-4)

a

295

62%

–71%

d—

0.17

mM

***

(0.1

3 to

0.2

2)74

%**

*>

5.6

mM

21%

***

Eth

ylva

nilli

n PG

ace

tal

(685

27-7

6-4)

b

346–

347

61%

–71%

d70

h0.

69 m

M**

* (0

.57

to 0

.81)

90%

***

3.5

mM

(3.

3 to

3.7

)40

%**

*

Van

illin

(121

-33-

5)a

285

63%

–71%

d—

0.18

mM

** (

0.12

to

0.28

)63

%**

*>

3 m

M9%

***

Van

illin

PG

ace

tal

(685

27-7

4-2)

b

353

59%

–65%

d34

–39

h (4

8 h)

0.91

mM

** (

0.76

to

1.1)

93%

***

1.1

mM

(0.

92 t

o 1.

3)56

%**

*

Nic

otin

e(5

4-11

-5)a

247

52%

–66%

c—

a Sou

rce:

Sig

ma-

Ald

rich

; b Sou

rce:

Bed

ouki

an R

esea

rch,

Dan

bury

, CT

; c e-l

iqui

d PG

/VG

ran

ge 1

00/0

to

5/95

; d e-l

iqui

d PG

/VG

ran

ge 1

00/0

to

40/6

0. H

BSS

= H

anks

bal

ance

d sa

lts

solu

tion

s; V

G =

gly

cero

l.


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ctober 2019

difference observed. Acetal mole fractions of the combined aldehyde and acetal content were measured by GC–flame ionization detector, and conversion rates and equilibrium concentrations were determined (Figure 1): cinnamaldehyde and citral conversions plateaued within the first day, at 92% and 88%, respectively. A small decline in PG acetal

mole fractions was observed subsequently. Benzaldehyde PG acetal formed more slowly, reaching equilibrium around day 5 and exceeding 95% mole fraction. Ethylvanillin and vanillin were converted to PG acetals more slowly, reaching equilibrium after approximately 7 days, at ca. 50% and 40% mole fractions, respectively.

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.


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The effect of varying PG/VG ratios, which are commonly offered in commercial e-liquids, on the formation of cinnamaldehyde PG acetal was also evaluated (Figure 1 inset). When the PG/VG ratio was decreased, lower equilibrium concentrations of cinnamaldehyde PG acetal were observed. These findings demonstrate that the rate and amount of PG acetal formed varies for different aldehydes and depends on the ratio of PG/VG present.

To evaluate whether aldehyde PG acetals are present in com-mercial e-liquids, and whether their formation depends on PG con-tent, a range of commercial vanilla (vanillin and ethylvanillin) and cherry (benzaldehyde)-flavored e-liquids were examined (Figure 1 and Supplementary Figure 1). The vanilla e-liquids tested in this study were purchased with PG contents of 0%, 30%, 50%, 70%, and 100% labeled as such by the vendor, however, LC–IR revealed that the actual PG contents were 60%, 70%, 80%, 85%, and 100%,

respectively. For all three compounds shown in Figure 1, a trend to-ward increasing mole fraction of the respective acetals with increas-ing PG content of the e-liquid was observed, which means a higher proportion of flavor aldehyde was converted to the correspond-ing acetal. This effect was more pronounced for benzaldehyde PG acetal, especially at low PG content. These data demonstrate that the amounts of PG acetals present in commercial e-liquids are pro-portional to the ratio of PG/VG used to prepare the e-liquid. Further, they support the earlier finding that benzaldehyde reacts more readily with PG than vanillin or ethyl vanillin.

Compound Delivery to E-cigarette VaporDuring vaping, flavor aldehydes are aerosolized and deposited in the airways along with other constituents. To determine whether alde-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 StabilityFor 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 of vanillin PG acetal to vanillin was monitored by UV–VIS spectros-copy in physiological Hanks balanced salts solutions buffer at pH 7.4, resembling the pH of airway lining fluid of healthy humans.24 A series of UV–VIS measurements followed the hydrolysis of van-illin PG acetal to pure vanillin by the growth of an absorption peak at 347 nm (Supplementary Figure 3). This technique is used to de-termine vanillin concentrations in foods,25 yet it is pH-dependent,26

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.


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warranting the use of a buffered solution. Using first-order kinetics, the half-life of vanillin PG acetal under these conditions was deter-mined to be approximately 48 hours (Table 1). Due to the absence of characteristic UV–VIS absorption peaks for the other aldehyde PG acetals, their hydrolysis was monitored by 1H-NMR in unbuffered D2O. Vanillin PG acetal was used as control for comparison of meth-ods and buffer effects, with a series of a 1H-NMR spectra shown in Supplementary Figure 3 indicating the disappearance of vanillin PG acetal (doublet peak at δ = 5.6–5.8) because of its conversion to vanillin (singlet peak δ = 9.6) over time. Similar series of spectra were recorded for the PG acetals of benzaldehyde and ethylvanillin. After peak integration, the half-lives of these three compounds in D2O were calculated as 34–39 hours (vanillin PG acetal), 44 hours (benzaldehyde PG acetal), and 70 hours (ethylvanillin PG acetal) based on first-order kinetics (Table 1.) A similar attempt to determine the aqueous stability of cinnamaldehyde PG acetal failed because of the very low solubility of the compound in D2O. All measured half-lives are in the order of days, suggesting that the tested acetals are not instantly hydrolyzed once they reach the aqueous environment of the airway surfaces, warranting investigations of their potential biological effects.

Acetal Irritation PotentialTo compare the effects of flavor aldehydes and their corresponding PG acetals on TRP irritant receptors, HEK-293T cells were used for heterologous expression of human TRPA1 and TRPV1 ion channels and fluorometric measurements of TRP-mediated Ca2+ influx.

Indeed, ethyl vanillin PG acetal, vanillin PG acetal, and benzal-dehyde PG acetal robustly activated Ca2+ influx through TRPA1, with all three flavor aldehyde acetals as efficacious as the full TRPA1 agonist, cinnamaldehyde (>90% of maximum cinnamaldehyde response; Figures 3 and Supplementary Figure 4; Table 1). Further, all tested PG acetals activated TRPA1 with higher EC50 values compared to their corresponding aldehydes, and interestingly, high concentrations of vanillin PG acetal and ethylvanillin PG acetal (at 3 mM and 10 mM, respectively) were more efficacious at activat-ing TRPA1 than equivalent concentrations of their corresponding parent aldehydes (Table 1; Figure 3 and Supplementary Figure 4). The flavor aldehyde PG acetals also had surprising effects on TRPV1, the vanilloid receptor: While benzaldehyde, ethylvanillin, and vanillin weakly activated TRPV1 only at high concentrations (>3 mM) close to their solubility limits, their corresponding PG acetals robustly activated TRPV1 at lower concentrations and with higher efficacies (Table 1, Figure 3 and Supplementary Figure 4). This effect on TRPV1 was most conspicuous when comparing van-illin and vanillin PG acetal (Table 1, Figure 3). Cinnamaldehyde PG acetal robustly activated TRPA1 and significantly activated TRPV1 at a nominal concentration of 500 µM; however, a dose–response relation could not be established because of limited solubility of the compound.

Discussion

In this study, a reaction between e-liquid constituents of aldehydic flavorants and PG to form aldehyde PG acetals is described. These compounds form in situ and are shown to be present in commer-cial e-liquids. Carryover experiments reveal that quantities of 50%–80% of the initial acetal concentration in the neat e-liquid are transferred to the e-vapor generated by a first-generation e-cigarette. The relative stability of these acetals to hydrolysis at physiological pH is demonstrated, implying that they persist after inhalation by the e-cigarette user. Finally, this study shows that the tested acetals

activate respiratory irritant receptors with broader selectivities and higher potencies than their aldehyde precursors, suggesting that they may act as stronger airway irritants in e-cigarette users.

To pursue this study, several e-liquids with various PG/VG ratios were purchased online, however, their chemical analysis revealed that their contents were not accurately reflected by the compos-ition on the label. This was evidenced by the measured differences in the component concentrations reported by the vendor versus those measured in the purchased e-liquid products (eg, PG/VG ratios in vanilla e-liquids). Several recent studies reported incorrectly labeled nicotine contents of e-liquids, including substantial amounts of nico-tine detected in e-liquids labeled as “nicotine-free”, purchased in several different countries.27–29 In light of these reports, the deviat-ing PG/VG ratios measured in this study add to the concerns about inaccurate mixing, labeling and resulting unexpected toxicities of commercial e-liquids. Rapid regulatory action is needed to address the ongoing failure of some e-liquid manufacturers to provide labe-ling consistent with product contents to the public.29–31

Acetal Formation and Presence in Laboratory-made and Commercial E-liquidsThe reaction between aldehyde flavorants and PG to form an acetal occurs at conditions common during the mixing and storage of e-liquids by vendors and users alike. PG acetals formed from all of the tested aldehydic flavorants, including benzaldehyde, cinnamal-dehyde, citral (mixture of the structural isomers neral and geranial), vanillin, and ethylvanillin, when mixed with PG (Figure 1). As men-tioned in the Introduction section, aldehyde PG acetals contain two chiral carbons, and the two diastereomers can be distinguished in the NMR spectra (Supplementary Figure 3) and GC chromatograms (Supplementary Figure 5). It is interesting to note that the acetal for-mation kinetics differ for the tested aldehydes, which may be related to how quickly these molecules are solubilized by PG. Vanillin and ethylvanillin are both solid at room temperature, and while they par-tially dissolved in PG on initial mixing, dissolution was not com-plete until after 24 hours (observed visually). The slight decline in the concentrations of the PG acetals of cinnamaldehyde and citral at later times suggests that further reactions may occur in e-liquids: both compounds are α,β-unsaturated aldehydes, known to be rela-tively reactive.32

Further experiments investigated how the amount of acetal formed can vary as a function of the PG/VG ratio. When the PG/VG ratio is decreased, the equilibrium concentration of the correspond-ing PG acetal was reduced (Figure 1 inset). Besides PG, VG can also form acetals with aldehydes,8 yet because VG contains three alcohol moieties, a multitude of optical and structural isomers of VG acetals can form. At least four of such isomers are shown in a GC-MS chro-matogram for cinnamaldehyde VG acetal (Supplementary Figure 6) and vanillin VG acetal (Supplementary Figure 7). Since no standard for cinnamaldehyde VG acetal nor vanillin VG acetal was available, they were not quantified.

Although previous studies detected flavor aldehyde acetals in commercial e-liquids, these were not quantified.11,12 In this study, substantial amounts of flavor aldehyde PG acetals were detected in the analyzed commercial e-liquids, with 15%–30% of the aldehyde converted to its corresponding PG acetal, and increasing acetal concentrations at higher PG contents. It is noteworthy that the mole fractions of acetals in the commercial e-liquids did not reach the same equilibrium levels as in the experimental mixtures of only PG and the aldehyde in question (Figure 1). This can be explained by the higher complexity of the commercial e-liquids that contain additional ingredients, including water and other compounds that


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can compete with PG to react with the aldehydes, or influence the reaction equilibrium.5 However, the same order of mole fractions is found in both the experimental and commercial mixtures: ben-zaldehyde PG acetal > ethylvanillin PG acetal > vanillin PG acetal. This study also showed that the tested e-liquids contained lower amounts of flavor aldehydes as the ratio of PG/VG in the e-liquid decreased (Supplementary Figure 1). In this case, this likely means that the “flavor base” was mixed in pure PG, and the appropriate amount of VG was added to create the final product.

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. It is, therefore, possible that the acetals were added directly to the commercial e-liquids. However, acetals have never been listed in the e-liquid ingredient lists in the few cases where these were revealed, and are less economical than their corresponding aldehydes. They are prepared from the parent aldehyde, suggesting that the acetals would generally be more costly, without a requisite gain in aroma as acetals generally resemble the odor of the parent aldehyde, but are less pronounced.32 The odor is particularly important as the olfac-tory system (smell) is the dominating system with regards to a user’s flavor perception of e-vapor.

Acetal Carryover to Vapor in E-cigarettesThe fate of flavor aldehydes during heating and vaporization in e-cigarettes, and their resulting toxicities, has been the subject of intense controversy. All PG acetals discussed earlier exhibit higher boiling points than their corresponding aldehydes (Table 1), pos-sibly impacting vaporization. Heating may also affect the stability of acetals.

A substantial carryover of intact acetals from commercial e-liquids into the e-vapor was observed, higher than 50% in all cases, with no correlation between boiling points and carryo-ver ratios (Table 1; Figure 2 and Supplementary Figure 2). No major breakdown products of the acetals could be detected in the e-vapor. Aldehydes and nicotine, while having divergent boil-ing points, were carried over at similar rates. Among the studied compounds, benzaldehyde has the lowest boiling point (178°C), yet its carryover rate was in the same range as for most other compounds, including those with very high boiling points, such as vanillin PG acetal (bp: 353°C; Table 1). To the best of our know-ledge, only a few flavorant molecules with boiling points higher than 300°C are used in e-liquids,12 and boiling points of acetals would not be expected to be much higher than those of vanil-lin PG acetal. This suggests that the most important parameter for compound carryover from e-liquid to e-vapor is its solubil-ity in the solvents PG and VG rather than a compound’s boiling point. Therefore, most flavorants, including solvent adducts such as acetals, are likely to reach the e-vapor in appreciable amounts.

Carryover of benzaldehyde PG acetal increased significantly with higher PG content of the e-liquid, with similar trends observed for the other PG acetals (Figure 2 and Supplementary Figure 2). Previous studies reported similar observations for nicotine carryover at vary-ing PG/VG ratios.33 It should be noted that the first-generation devices used in this study are not typically recommended for use with e-liquids with high VG contents because of their limited power, and this is likely to impact carryover rates, especially at low PG/VG ratios. Newer generation devices are much more powerful and are likely more effective in delivering flavorants as well as the described acetals to the e-vapor, as has already been described for nicotine.34

Acetal StabilityThe formation of acetals from an aldehyde and PG is reversible, with hydrolysis favored at acidic pH in aqueous solutions35 (Figure 1). The airway lining fluid in which e-vapor is deposited is slightly alka-line,17,24 therefore, acetal hydrolysis is expected to occur at slower rates. Indeed, the half-lives of the acetals measured in aqueous solu-tion exceeded 36 hours, sufficient for substantial amounts of acetals to be maintained in physiological fluids, potentially exerting direct effects on the airway lining fluids and underlying structures.

Recruitment of Additional Respiratory Irritant Receptors by Flavor Aldehyde PG AcetalsExposures to aldehydes cause respiratory irritation through acti-vation of irritant receptors in sensory nerve endings in the airway lining. TRPA1 is the major aldehyde-activated receptor, respond-ing during exposures to acrolein in tobacco smoke, formaldehyde, and flavor aldehydes such as cinnamaldehyde and benzalde-hyde.16,17,36 The vanilloid receptor, TRPV1, may also contribute to flavorant irritant effects as it is activated by vanillin-related compounds such as capsaicin, however, TRPV1 responds poorly to free aldehydes.37 The present study strongly suggests that flavor aldehyde PG acetals are delivered to the airways of e-cigarette users, however, except benzaldehyde PG acetal,38 it is unknown whether these compounds also activate irritant receptors. Our in vitro studies clearly demonstrate that the acetals activate human TRPA1 irritant receptors with increased efficacies at the high-est concentrations (Figure 3 and Supplementary Figure 4). Also, higher concentrations of vanillin and ethyl vanillin seem to be desensitizing TRPA1 and attenuating the ion-channel activation, whereas their corresponding PG acetals do not demonstrate ion-channel desensitization or attenuation of response. Although van-illin, ethylvanillin, and benzaldehyde had only negligible effects on TRPV1, their corresponding PG acetals robustly activated this receptor (Figure 3 and Supplementary Figure 4; Table 1). These observations suggest that the tested flavorant aldehyde PG acetals may produce stronger sensory irritation effects than the free alde-hydes alone, by recruiting additional sensory irritant receptors such as TRPV1. It remains to be investigated whether these effects occur in vivo, for example, by examining the respiratory irrita-tion response in rodents to e-vapors containing PG acetals, and by measuring inflammatory parameters.39,40

The aldehydes and acetals used in this study are listed as GRAS (generally regarded as safe) by the US Food and Drug Administration and the World Health Organization, as are sev-eral other PG acetals,41–43 but not furfural PG acetal.43 Yet, GRAS status is based purely on ingestion safety when used as food addi-tives, and cutaneous safety when used as scents. Currently, no data exist that would support inhalational safety of flavor aldehyde PG acetals. Since TRPA1 and TRPV1 contribute to chronic pulmonary conditions such as asthma, the formation of more strongly irritating compounds in e-liquids is of concern.39,44–47 Indeed, recent epidemio-logical and toxicological studies detected links between asthma frequency and e-cigarette use in adolescents and reported that vapor-ized e-liquids containing the same flavor aldehydes as tested in this study induce inflammation in human respiratory epithelia.48 The cur-rent study warrants further investigation into possible toxic effects of inhaling flavorant PG acetals by means of an e-cigarette, and one of the outcomes may be that GRAS status is not sufficient to deem certain flavor additives as safe for inhalation by an e-cigarette.


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ImplicationsThis study demonstrates the potential chemical instability of e-liq-uids: acetals can be formed between flavor aldehydes and the sol-vents PG and VG post-formulation, such as during mixing, shipping, and storage of e-liquids. This reaction starts almost immediately and continues over days, leading to significant acetal generation even though water is not removed actively from the reaction. Given the frequency at which aldehydes are used in e-liquids (eg, 11 different aldehydes found in 28 e-liquids,12 and a total of 126 aldehydes on the FEMA GRAS list8), and that at least two PG- and four VG-acetal stereoisomers (and structural isomers of the latter) are formed dur-ing the acetalization reaction, a large number of distinct acetal com-pounds could potentially be present in e-liquids, each possibly with different reaction kinetics, aqueous stability, and most importantly, potentially different physiological and toxicological effects. It should be noted that in this study, the potential to activate irritant receptors was not separately tested for optical isomers (enantiomers or dias-tereomers), but it is well known that these can have very different physiological effects.10,49

To comprehensively assess the risk of e-liquids, it is important to consider the complete life cycle of the e-liquid constituents from mixing through shipping and storage, heating, and oxidation in

e-cigarette devices, stability in biological environments (eg, mouth, airways, and lungs) to toxicity potential for a variety of endpoints (Figure 4). Without this comprehensive framework, important classes of compounds, such as the PG acetals studied here, could be unintentionally neglected if they are not directly added and disclosed by e-liquid manufacturers. To fully assess the risk potential of e-liq-uid use for regulatory purposes, it is imperative that the compounds that a user will actually be exposed to are reported and evaluated, and not only the initial ingredients combined during manufactur-ing.50 When selecting compounds to screen for potential toxicity concerns, the chemical instability of the given e-liquid, which will depend on the reactivity of the individual constituents, needs to be taken into account. These can undergo a multitude of reactions given the complex mixture environment of e-liquids, making the predic-tion of equilibrium concentrations of reaction products, including PG or VG acetals, extremely challenging. In addition, a comprehen-sive risk assessment should consider the potential reaction products that can result from heating and oxidation in the e-cigarette, requir-ing characterization and quantification of the e-vapor constituents for toxicity concerns.50 Finally, the biological stability of compounds present in the e-vapor should be considered, including interactions with pulmonary surfactants, as reactions may occur that change

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.


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the composition of the e-vapor and could result in new chemical products with unknown toxicities. This suggests that ingredient dis-closure of e-liquids by suppliers may be insufficient to inform a com-prehensive risk assessment of e-liquids and vaping more generally.

Supplementary Material

Supplementary data are available at Nicotine and Tobacco Research online.

FundingThis work was supported by the Yale Tobacco Center of Regulatory Sciences (TCORS) of the National Institutes of Health (P50-DA-036151) and the Center for Tobacco Products (CTP). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the Food and Drug Administration.

Declaration of InterestsNone declared.

AcknowledgmentsThe authors would like to express their gratitude to Barry Green (PhD, Yale School of Medicine) and Kathryn Rosbrook from the John B. Pierce Laboratory for their valuable advice as well as for supplying the vanilla- and cherry-flavored e-liquids with varying PG/VG ratios. Dr Green and Ms Rosbrook did not receive financial compensation.

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http://apps.who.int/iris/handle/10665/77752

1

Formation of flavorant-propylene glycol adducts with novel toxicological properties

in chemically unstable e-cigarette liquids

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.

2

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).

3

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.

4

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.

5

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.

6

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.

7

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.

8

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.

9

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

© 2019 American Journal of Preventive Medicine. Publisreserved.

RESEARCH LETTER

From the 1DepaUniversity, NewScience (TCORSMedicine, New Hpore; 4School oNew Haven, Cosity School of MHealth, Yale Uni

Address corrChemical and EAvenue, New Ha

0749-3797/$3https://doi.or

hed by Elsevier Inc. All rights

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

The “JUUL” e-cigarette is the best-selling e-ciga-rette on the U.S. market.1,2 JUUL refill “pods”contain nicotine benzoate salt and flavorants

dissolved in a 30:70 ratio of propylene glycol (PG) andglycerol (VG for vegetable glycerin). Nicotine benzoateis perceived as more satisfactory and less harsh, enablingthe delivery of higher amounts of nicotine to users. Assuch, nicotine concentrations in JUUL e-liquids arehigher (5%; 3% since August 2018) than in non-JUUL e-liquids (typically 0.3%−2.4%). JUUL e-liquids are avail-able in several fruity flavors, known to be particularlyappealing to youth.3 Common e-cigarette (includingJUUL) flavorants include menthol and various aldehydes(e.g., vanillin); aldehydes are known to react with alco-hols (e.g., PG and VG) to form acetals (structural andoptical isomers).4 The inhalational safety of flavor alde-hyde PG/VG acetals is unknown; however, a recentstudy found that several acetals, including vanillin PGacetal, activate pro-inflammatory irritant receptors morestrongly than their parent compounds (e.g., vanillin).4,5

Despite the popularity of JUUL, little is known aboutthe composition of JUUL aerosol. The aim of this studywas to evaluate the carryover of vanillin and its reactionproducts with PG and VG, menthol, and nicotine benzo-ate from JUUL e-liquid to aerosol to understand poten-tial human exposures.

rtment of Chemical and Environmental Engineering, YaleHaven, Connecticut; 2Yale Tobacco Center of Regulatory), Department of Psychiatry, Yale University School ofaven, Connecticut; 3Yale-NUS College, Singapore, Singa-

f Forestry and Environmental Studies, Yale University,nnecticut; 5Department of Anesthesiology, Duke Univer-edicine, Durham, North Carolina; and 6School of Publicversity School of Medicine, New Haven, Connecticutespondence to: Julie B. Zimmerman, PhD, Department ofnvironmental Engineering, Yale University, 17 Hillhouseven CT 06511. E-mail: [email protected]/10.1016/j.amepre.2019.04.004

METHODSA JUUL device and pods of all eight flavors (Figure 1) were pur-chased online in 2018. For aerosol capture (both gas phase andmicrodroplets6), a custom-built vaping machine with liquidnitrogen−chilled traps was used as described previously.7 Thepuffing regime was 20 puffs of 2.8-second length, 79-mL vol-ume, and a 30-second cooldown between puffs. Neat e-liquidsand captured aerosol were diluted and analyzed by gas chroma-tography mass spectroscopy.4 Commercially available standardswere used for quantification except for vanillin VG acetals,which were synthesized in house. Aerosol concentrations and

percentage carryover were calculated per experiment by divid-ing the amount of compound trapped by the pod mass change,and as aerosol concentration over neat e-liquid concentration,respectively (Figure 1).

RESULTS

The reaction products vanillin PG acetal and vanillin VGacetals were detected in JUUL “Cr�eme Brul�ee” e-liquidand carried over to aerosol at 68§4% (mean§95% CI,all n=3) and 59§20%, or 0.8§0.04 mg/puff and 2.0§0.5mg/puff, respectively (Figure 1). Vanillin was carriedover at 79§17%, resulting in the delivery of 7.9§0.8 mg/puff. Menthol was found in four of the eight tested fla-vors, and the menthol aerosol concentration of “ClassicMenthol” and “Cool Mint” was 34§3 mg/puff and 38§12 mg/puff, respectively, which is comparable to men-tholated cigarettes (29−392 mg/puff8 for ten puffs/ciga-rette; 10%−20% carryover to cigarette smoke9). Nicotineand benzoic acid carryover were 98§6% and 82§5%(5%-pods, n=21), and 102§4% and 80§14% (3%-pods,n=5), respectively. However, a statistical significancebetween e-liquid and aerosol concentrations was foundonly for benzoic acid (Figure 1). Absolute nicotine aero-sol content (114§13 mg/puff, 5%-pods, 65§15 mg/puff,3%-pods) was comparable to previous reports analyzingJUUL or combustible cigarettes (50−180 mg/puff; tenpuffs/cigarette).10−12

Am J Prev Med 2019;57(3):425−427 425

http://crossmark.crossref.org/dialog/?doi=10.1016/j.amepre.2019.04.004&domain=pdf

mailto:[email protected]

https://doi.org/10.1016/j.amepre.2019.04.004

Figure 1. Comparison of the concentrations of six compounds in neat e-liquid (background bars, varying shading) and capturedaerosol (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 percompound were carried out using GraphPad Prism 8 software. A p-value of <0.05 was interpreted as significant; ***p<0.001.a5%-nicotine flavors: Cr�eme Brul�ee, 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.

426 Erythropel et al / Am J Prev Med 2019;57(3):425−427

DISCUSSION

This study is the first to report the presence of flavoraldehyde VG acetals in e-liquids and aerosols andexpands the authors’ prior finding of flavor aldehydePG acetals in commercial e-liquids.4 JUUL e-liquidscontain higher levels of vanillin VG acetals comparedwith vanillin PG acetal because of the higher VG:PGratio. Flavor aldehyde−solvent acetal formation can beexpected in any e-liquid−containing flavor aldehydes,including JUUL, at room temperature (e.g., withoutheating in the e-cigarette).4 Furthermore, the possibilityof other unintended chemical reactions between e-liquidconstituents should be considered in future research.Compounds present in JUUL e-liquids are delivered

efficiently to the aerosol, exposing users to similar quan-tities of nicotine as cigarettes, to menthol in four of eightflavors, and to the PG and VG acetals of vanillin

(“Cr�eme Brul�ee”). Although vanillin PG/VG acetal aero-sol carryover is slightly lower than nicotine, indicatingpossible acetal hydrolysis, appreciable amounts of acetalsare present in the aerosol, which, if inhaled, may causeirritation and contribute to inflammatory responses.4,5

The average vanillin puff concentration was 101 mg/m3.In comparison, chronic inhalational exposure to vanillinin occupational environments is limited to 10 mg/m3,13

raising the question of what long-term effects regularinhalation of vanillin at such doses and frequency (200puffs/pod) might have. Aerosol menthol levels from“Fruit Medley” (5.3 ppm), which is not labeled as men-tholated, and the other mentholated JUUL e-liquids(“Cool Cucumber”: 7.5 ppm, “Classic Menthol”: 63 ppm,and “Cool Mint”: 70 ppm) are sufficient to suppressrespiratory irritation responses to aldehydes and tobaccosmoke and increase nicotine intake.14,15

www.ajpmonline.org

Erythropel et al / Am J Prev Med 2019;57(3):425−427 427

Future e-cigarette regulatory policy should address (1)the formation of new compounds with potential toxico-logic properties within e-liquids, (2) JUUL menthol levelsthat may increase nicotine intake, and (3) flavorant expo-sure effects in e-cigarette users as also recently highlightedby the U.S. Food and Drug Administration.16

ACKNOWLEDGMENTSThe authors wish to thank Sofya Zeylikman, BFA, Dante Archan-geli, BS, and Larry Wilen, PhD, at the Center for EngineeringInnovation & Design within the Yale School of Engineering &Applied Science for their advice and support to design and 3D-print custom-made connector pieces. Ms. Zeylikman, Mr. Arch-angeli, and Dr. Wilen did not receive compensation.

The funding organization had no role in the design and con-duct of the study; the collection, management, analysis, andinterpretation of the data; the preparation, review, or approval ofthe manuscript; nor in the decision to submit the manuscript forpublication. The content is solely the responsibility of the authorsand does not necessarily represent the views of National insti-tutes of Health (NIH) or the Food and Drug Administration (FDA).

LMD received funding from the Yale-NUS Summer ResearchProgramme. This work was supported by the National Instituteon Drug Abuse of NIH and the Center for Tobacco Products ofthe U.S. FDA under awards number P50DA036151 andU54DA036151 (Yale Center for the Study of Tobacco ProductsUse and Addiction: Flavors, Nicotine, and other Constituents;YCSTP).

Author contributions were as follows: HCE, SEJ, PTA, andJBZ conceptualized and designed the study; HCE and LMDacquired and analyzed the data; HCE interpreted the data;HCE and JBZ drafted the manuscript; HCE and JBZ revised themanuscript; HCE carried out statistical analysis; HCE, PTA andJBZ provided supervision; LMD, TMdW, SEJ, PTA, SSO, SK-S,and JBZ critically revised the manuscript for important intel-lectual content; TMdW supplied technical support; and SSO,SK-S, and JBZ obtained funding.

Dr. Jordt reports receiving personal fees from Hydra Bio-sciences LLC and Sanofi S.A. and nonfinancial support fromGlaxoSmithKline Pharmaceuticals outside the submitted work.Dr. Krishnan-Sarin reports donated study medications fromNovartis and Astra Zeneca for research project unrelated tothe current work. She was also a member of the FDA TobaccoProduct Scientific Advisory committee in 2012−2014 and2015−2016. Dr. O’Malley reports the following unrelated to thecurrent work: consultant/advisory board member for Alkermes,Amygdala, Indivior, Mitsubishi Tanabe, Opiant; a member of theAmerican Society of Clinical Psychopharmacology Alcohol Clini-cal Trials Initiative supported by Alkermes, Amygdala, ArborPharmaceuticals, Ethypharm, Lilly, Lundbeck, Otsuka, Pfizer,and Indivior; donated medications from Astra Zeneca, Novartis,and Pfizer; a grant from Lilly; and Data and Safety MonitoringBoard member for the National Institute on Drug Abuse(Emmes Corporation). No other financial disclosures werereported by the authors of this paper.

September 2019

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