1. Le mag’
Edition de décembre 2015 HS N°4
Le magazine de l’Association Indépendante
Des Utilisateurs de Cigarette Électronique
2015
Les publications scientifiques en
lien avec la cigarette électronique
Scientific studies about electronic cigarettes
2. AIDUCE – le mag’
Ce nouveau hors-série du magazine de l'Aiduce
recense toutes les études et publications scientifiques
que nous avons pu trouver à ce jour sur la cigarette
électronique et ses éléments clés – dépendance à la
nicotine, propylène glycol etc.
Ce recueil est la propriété exclusive de l'Aiduce, elle
est mise à votre disposition afin d'informer toute
personne qui le souhaite.
Edition réalisée en décembre 2015.
3. AIDUCE – le mag’
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Flavour chemicals in electronic cigarette fluids
Vapors Produced by Electronic Cigarettes and E-Juices with Flavorings Induce Toxicity, Oxidative Stress, and
Inflammatory Response in Lung Epithelial Cells and in Mouse Lung
Ecigarette Vapor—Even when NicotineFree— Found to Damage Lung Cells
Cell studies on e-cigarettes: don’t waste your time reading (at least most of) them
Are Metals Emitted from Electronic Cigarettes a Reason for Health Concern? A Risk-Assessment Analysis of Currently
Available Literature
Use of electronic cigarettes (vapourisers) among adults in Great Britain
Associations between e-cigarette access and smoking and drinking behaviours in teenagers
Associations Between Ecigarette Type, Frequency of Use, and Quitting Smoking: Findings From a Longitudinal Online
Panel Survey in Great Britain
Biochemically verified smoking cessation and vaping beliefs among vape store
Clive Bates : E-cigarettes, vaping and public health
Dependence levels in users of electronic cigarettes, nicotine gums and tobacco cigarettes.
"Direct Dripping": A HighTemperature, HighFormaldehyde Emission Electronic Cigarette Use Method
E-Cigarette Liquid Nicotine Ingestion in a Child: Case Report and Discussion
E-Cigarettes and Smoking Cessation: Evidence from a Systematic Review and Meta-Analysis
E-cigarettes generate high levels of aldehydes only in ‘dry puff’ conditions
Electronic cigarette use and harm reversal: emerging evidence in the lung
Electronic Cigarettes and Cannabis: An Exploratory Study
Electronic cigarettes: patterns of use, health effects, use in smoking cessation and regulatory issues
Endothelial disruptive pro-inflammatory effects of nicotine and e-cigarette vapor exposures
Enquête sur l’usage de la cigarette électronique et du tabac en milieu scolaire
Experts’ consensus on use of electronic cigarettes: a Delphi survey from Switzerland
Explaining the effects of electronic cigarettes on craving for tobacco in recent quitters
Exposure to Electronic Cigarettes Impairs Pulmonary Anti-Bacterial and Anti-Viral Defenses in a Mouse Model
Food and Drug Administration Tobacco Regulation and Product Judgments
Hidden Formaldehyde in Ecigarette Aerosols
Is the use of electronic cigarettes while smoking associated with smoking cessation attempts, cessation and reduced
cigarette consumption? A survey with a 1-year follow-up
More on Hidden Formaldehyde in E-Cigarette Aerosols
Nicotine absorption from electronic cigarette use: comparison between experienced consumers (vapers) and naïve
users (smokers)
Nicotine and toxicant yield ratings of electronic cigarette brands in New Zealand
Nicotine Levels and Presence of Selected Tobacco-Derived Toxins in Tobacco Flavoured Electronic Cigarette Refill Liquids
Does the magnitude of reduction in cigarettes per day predict smoking cessation? A qualitative review
NIOSH submits letter to the editor concerning our diacetyl study
Quit and Smoking Reduction Rates in Vape Shop Consumers: A Prospective 12-Month Survey
La cigarette électronique permet-elle de sortir la société du tabac ?
Exhaled Electronic Cigarette Emissions: What’s Your Secondhand Exposure?
Publications de la SNRT 2015
The deception of measuring formaldehyde in ecigarette aerosol: the difference between laboratory measurements and
true exposure
The impact of flavor descriptors on nonsmoking teens’ and adult smokers’ interest in electronic cigarettes
The importance of proper information: Risk perception about ecigarettes is the strongest predictor of dual use
Toxicity Assessment of Refill Liquids for Electronic Cigarettes
Verified: formaldehyde levels found in the NEJM study were associated with dry puff conditions. An update
Effect of cigarette smoke and e-liquide vapour on caliary beat frequency of freshly isolated human nasal epithelial cells
E-cigarettes : implications for harm reversal
Prohibition of profit motive : competing visions of the endgame
Experts’ consensus on use of electronic cigarettes: a Delphi survey from Switzerland
An approach to ingredient screening and toxicological risk assessment of flavours in eliquids
Comparison of select analytes in aerosol from e-cigarettes with smoke from conventional cigarettes and with ambient
air
Ecigarettes generate high levels of aldehydes only in ‘dry puff’ conditions
Acute effects of electronic and tobacco cigarette smoking on complete blood count
Government survey shows number of children trying smoking continues to decline and very few children regularly using
electronic cigarettes
Liste des articles
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Successful Nicotine Intake in Medical Assisted Use of E-Cigarettes: A Pilot Study
Evaluation of toxicant and carcinogen metabolites in the urine of ecigarette users versus cigarette smokers.
Comparative In Vitro Toxicity Profile of Electronic and Tobacco Cigarettes, Smokeless Tobacco and Nicotine
Replacement Therapy Products: E-Liquids, Extracts and Collected Aerosols
Tobacco-specific nitrosamines in Electronic Cigarettes : comparaison between liquid and aerosols levels
Stopping smoking by using other sources of nicotine
A clinical laboratory model for evaluating the acute effects of electronic “cigarettes”: nicotine delivery profile and
cardiovascular and subjective effects
Association of Electronic Cigarette Use With Initiation of Combustible Tobacco Product Smoking in Early Adolescence
E-cigarettes: an evidence update A report commissioned by Public Health England
Effects of Switching to Electronic Cigarettes with and without Concurrent Smoking on Exposure to Nicotine, Carbon
Monoxide, and Acrolein
Contexts of cigarette and e-cigarette use among dual users: a qualitative study
A rapid method for the chromatographic analysis of volatile organic compounds in exhaled breath of tobacco cigarette
and electronic cigarette smokers.
The real challenge is to make e-cigarettes accessible for poor smokers
IASLC Issues New Statement on Tobacco Control and Smoking Cessation
Adult Behavior in Male Mice Exposed to Ecigarette Nicotine Vapors during Late Prenatal and Early Postnatal Life
Regulation in the face of uncertainty: the evidence on electronic nicotine delivery systems (e-cigarettes)
A rapid method for the chromatographic analysis of volatile organic compounds in exhaled breath of tobacco cigarette
and electronic cigarette smokers
Nicotine retention and PK from e-cigarettes
Particulate Matter from Electronic Cigarettes and Conventional Cigarettes: a Systematic Review and Observational
Study
Une nouvelle étude indique que l'ecigarette émet des toxines dans l'environnement, mais les auteurs n'en trouvent pas
vraiment...
Short-term effects of a nicotine-free e-cigarette compared to a traditional cigarette in smokers and non-smokers
What to Advise to Respiratory Patients Intending to Use Electronic Cigarettes
E-cigarettes: promise or peril?
E-cigarettes: Public Health England’s evidence based confusion?
How does Electronic Cigarette Access affect Adolescent Smoking?
Patterns of Electronic Cigarette Use Among Adults in the United States
Electronic cigarettes induce DNA strand breaks and cell death independently of nicotine in cell lines
Characteristics of users, and usage of different types of electronic cigarettes: findings from an online survey
Highly Reactive Free Radicals in Electronic Cigarette Aerosols
Electronic Cigarette Trial and Use among Young Adults: Reasons for Trial and Cessation of Vaping
Electronic cigarette use in France in 2014
E-cigarettes as smoking cessation aids: a survey among practitioners in Italy
New study doesn’t show e-cigarettes gives you cancer
5. Flavour chemicals in electronic cigarette fluids
Peyton A Tierney,1
Clarissa D Karpinski,2
Jessica E Brown,1
Wentai Luo,2
James F Pankow1,2
1
Department of Civil and
Environmental Engineering,
Portland State University,
Portland, Oregon, USA
2
Department of Chemistry,
Portland State University,
Portland, Oregon, USA
Correspondence to
Dr James F Pankow,
Department of Civil and
Environmental Engineering,
Portland State University,
PO 751, Portland,
OR 97207-0751, USA;
pankowj@pdx.edu
Received 2 December 2014
Accepted 25 February 2015
To cite: Tierney PA,
Karpinski CD, Brown JE,
et al. Tob Control Published
Online First: [please include
Day Month Year]
doi:10.1136/tobaccocontrol-
2014-052175
ABSTRACT
Background Most e-cigarette liquids contain flavour
chemicals. Flavour chemicals certified as safe for
ingestion by the Flavor Extracts Manufacturers
Association may not be safe for use in e-cigarettes.
This study identified and measured flavour chemicals in
30 e-cigarette fluids.
Methods Two brands of single-use e-cigarettes were
selected and their fluids in multiple flavour types
analysed by gas chromatography/mass spectrometry. For
the same flavour types, and for selected confectionary
flavours (eg, bubble gum and cotton candy), also
analysed were convenience samples of e-cigarette fluids
in refill bottles from local ‘vape’ shops and online
retailers.
Results In many liquids, total flavour chemicals were
found to be in the ∼1–4% range (10–40 mg/mL);
labelled levels of nicotine were in the range of 0.6–
2.4% (6 to 24 mg/mL). A significant number of the
flavour chemicals were aldehydes, a compound class
recognised as ‘primary irritants’ of mucosal tissue of the
respiratory tract. Many of the products contained the
same flavour chemicals: vanillin and/or ethyl vanillin was
found in 17 of the liquids as one of the top three
flavour chemicals, and/or at ≥0.5 mg/mL.
Conclusions The concentrations of some flavour
chemicals in e-cigarette fluids are sufficiently high for
inhalation exposure by vaping to be of toxicological
concern. Regulatory limits should be contemplated for
levels of some of the more worrisome chemicals as well
as for total flavour chemical levels. Ingredient labeling
should also be required.
INTRODUCTION
Use of electronic cigarettes (aka e-cigarettes, elec-
tronic nicotine delivery systems and ENDS) is
expanding rapidly, with global sales estimated at US
$1.5 billion in 2012 and US$3.5 billion in 2013;
sales for 2014 were projected to be US$7 billion.1
Adoption of e-cigarettes has far out-paced our
understanding of their implications for health,
including the initial composition of the e-cigarette
fluids as well as presence of harmful by-products
formed during ‘vaping’.2
In April, US Food and
Drug Administration issued a report in which it
deemed that it has regulatory authority over
e-cigarettes.3
No specific regulations were yet pro-
posed, except that sales to those under 18 should
be prohibited; final action is slated for June 2015.
The use of flavourings in e-cigarette fluids has
become a central focus for those marketing
e-cigarettes4
and for those demanding regulatory
control, including 29 Attorneys General.5
Centers
for Disease Control and Prevention (CDC) reports
that the percentage of high school students who
acknowledged ever using an e-cigarette doubled
from 4.7% in 2011 to 10% in 2012.6
Supporters
of regulation note that cigarettes with ‘characteris-
ing flavours’ (other than with menthol) were
banned in 20097
due to evidence that they were
attracting youth to smoking. A recent report8
states
that an astonishing 7764 unique flavour names
were available online in January 2014, with 242
new flavours being added per month, and sales
occurring under 466 brands. For the 7764 flavour
names, only a small number relate to ‘tobacco’; the
vast majority are confectionary in nature, for
example, chocolate raspberry, cherry cheesecake,
cotton candy, vanilla, grape, apple, coffee, bubble
gum, etc. The NJOY brand had avoided explicitly
labelled confectionary flavour names, but due to
rapidly losing market share, it was recently
reported to have plans to offer products in ‘butter
crumble’ and ‘black and blue berry’.4
Some manufacturers of e-cigarette fluids have
cited that the ingredients, including the flavour che-
micals used, are all ‘food grade’, and/or ‘generally
recognised as safe’ (GRAS). However, GRAS certifi-
cation by the Flavor Extracts Manufacturers
Association (FEMA) pertains only to ingestion, not
inhalation. FEMA currently states9
The [FEMA] Expert Panel does not evaluate flavor
ingredients for use in tobacco products including
e-cigarettes or other products that are not human
food, or products that result in exposures other
than ingestion.
and
E-cigarette manufacturers should not represent or
suggest that the flavor ingredients used in their
products are safe because they have FEMA
GRAS™ status for use in food because such state-
ments are false and misleading.
While it is likely that virtually all flavour ingredi-
ents that are popular in confectionary and food
products have been included in multiple e-cigarette
products, very little has been published on the
levels of flavour chemicals in e-cigarette fluids.
Farsalinos et al10
analysed e-cigarette refill fluids
from seven countries for diacetyl (aka butanedione,
often described as giving a buttery flavour), and
acetyl propionyl (aka pentane-2,3-dione, often
described as giving a caramel or buttery flavour).
Both compounds were reported to be found in
74% of the samples tested, and the authors con-
cluded that 47% of the diacetyl-containing samples
and 42% of the acetyl propionyl-containing
samples could lead to exposures higher than
NIOSH safety limits. Bahl et al11
examined 41
e-cigarette refill fluids for cytotoxicity to human
pulmonary fibroblasts, human embryonic stem cells
and mouse neural stem cells, and concluded that
Tierney PA, et al. Tob Control 2015;0:1–6. doi:10.1136/tobaccocontrol-2014-052175 1
Brief report
TC Online First, published on April 15, 2015 as 10.1136/tobaccocontrol-2014-052175
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1
8. when present, the cytotoxicity was related to the flavour chemi-
cals, especially for cinnamon-flavoured refill fluids. A recent
opinion piece in JAMA12
states
Research is needed to characterize both the presence of toxic che-
micals in ENDS flavorings and the potential adverse respiratory
effects of exposure to e-liquids, especially flavorings.
Hutzler et al13
analysed 28 e-cigarette liquids from seven
manufacturers by gas chromatography/mass spectrometry (GC/
MS) and used comparisons with known compound-specific MS
patterns to tentatively (and qualitatively) identify the presence
of 141 flavour chemicals in one or more of the products.
Vanillin, ethyl maltol, ethyl vanillin and menthol were the four
most frequently found flavour chemicals, reported to be present
in 79%, 57%, 50% and 43% of the 28 samples, respectively.
However, since authentic standards were not used, actual con-
centrations could not be deduced. As follow-up to Bahl et al,11
Behar et al14 15
examined cytotoxicity and measured levels of
cinnamaldehyde, 4-methoxycinnamaldehyde and vanillin for 10
‘cinnamon’ flavoured refill fluids. For the three compounds, the
highest concentrations were ∼40, 3 and 8 mg/mL, respectively
(∼4%, 0.3% and 0.8% by weight or volume).
Product labels rarely provide ingredient information beyond
the level of nicotine, and the inclusion of propylene glycol and/
or glycerol. To provide additionally needed information, we
describe determinations of the levels of flavour chemicals in the
fluids of a convenience sample of disposable e-cigarettes and
refill bottles over a range of flavour types.
METHODS
We assumed that meaningful conclusions could be obtained by
analysing 30 products. The e-cigarette fluids examined were
selected from a vast and rapidly changing array of products.
BLU and NJOY, two brands of disposable-cartridge e-cigarettes,
were purchased in five flavours: tobacco, menthol, vanilla,
cherry and coffee. Also purchased in the same flavours (from
online retailers and local ‘vape’ shops in Portland, Oregon) were
refill bottles for tank systems. Refill bottles in five other confec-
tionary flavours (chocolate/cocoa, grape, apple, cotton candy
and bubble gum) were also purchased. After dilution with
methanol, the fluids were analysed by GC/MS. Using internal
standard-based calibration procedures similar to those described
elsewhere,16
analyses were performed using an Agilent (Santa
Clara, California, USA) 7693 autosampler, Agilent 7890A GC
and Agilent 5975C MS. The GC column type was Agilent
DB-5MS UI, of 30 m length, 0.25 mm id and 0.25 mm film
thickness. For each replicate sample, ∼50 mg of each fluid was
dissolved in 1 mL of methanol. One microlitre of the methanol
solution was then injected on the GC with a 25:1 split. The GC
temperature programme for all analyses was: 35°C hold for
5 min; 10°C/min to 300°C; then hold for 3.5 min at 300°C. No
analyses of aerosols generated from the fluids were carried out.
Qualitative analyses of the 30 e-cigarette fluids were first carried
out here using the NIST 14 MS library,17
and the results were com-
pared with data previously obtained for flavoured tobacco pro-
ducts.16
Quantitative analyses of the 30 fluids were then
undertaken, using authentic standards, for a specific list of com-
pounds, which formed the ‘target analyte list’. If reported here, the
presence of each target analyte was confirmed by matching GC
retention times and MS patterns with results obtained with the
authentic standards; the level was determined by comparison with
calibration standard runs. The target analyte list included the
70 compounds listed in Brown et al16
plus 20 others, namely aro-
madendrene, 1,4-cineol, trans-cinnamaldehyde, citronellal,
Table1Continued
Rankbytotal
flavourlevel
Flavourname
(numberforflavour)
Brand
Refillbottleor
disposablecartridge
Labellednicotine
(mg/mL)
Totalforflavour
chemicalsdetermined
(mg/mL)(mg/mL)
Individualflavour
chemicals
CASRegistry
numberClass
26‘TrueTobacco’
(4of7‘Tobacco’)
TasteE-Liquid
Refillbottle62.22.1Ethylmaltol4940-11-8Alcohol
0.1Vanillin121-33-5Aldehyde
NAUnknownminorconstituentsNAUnknown
27‘CherryCrush’
(3of3Cherry)
BLU
Disposablecartridge24‡1.20.6Benzylalcohol100-51-6Alcohol
0.3Piperonal120-57-0Aldehyde
0.3Vanillin121-33-5Aldehyde
28‘Torque56’
(5of7‘Tobacco’)
Halo
Refillbottle61.20.8Benzylalcohol100-51-6Alcohol
0.2β-Damascone23726-91-2Ketone
0.1Ethylbutyrate105-54-4Ester
29‘ClassicTobacco’
(6of7‘Tobacco’)
BLU
Disposablecartridge22†∼0.10.1Benzylalcohol100-51-6Alcohol
NAUnknownminorconstituentsNAUnknown
30‘Traditional’
(7of7‘Tobacco’)
NJOY
Disposablecartridge18lowNAUnknownminorconstituentsNAUnknown
LabelledLevelsforNicotineGiven.
*
Chiralityofanalytesnotdeterminedhere.MostwereprobablyLform.
†Basedonlabelledvaluefor‘mgnicotinepercartridge’andfluidvolumeestimatedhere.
‡Measuredhere.
CAS,ChemicalAbstractsService;NA,notapplicable;PG,propyleneglycol.
4 Tierney PA, et al. Tob Control 2015;0:1–6. doi:10.1136/tobaccocontrol-2014-052175
Brief report
group.bmj.comon April 16, 2015 - Published byhttp://tobaccocontrol.bmj.com/Downloaded from
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9. citronellyl propionate, coumarin, decanal, ethyl acetate, ethyl hex-
anoate, fenchol, limonene oxide, trans-linalyl propionate, maltol,
30
-methylacetophenone, neomenthol, 2-nonanone, pentyl propi-
onate, pulegone, γ-terpineol and 2,3,5,6-tetramethylpyrazine. The
vicinal diketone compounds diacetyl and 2,3-pentanedione were
not in the target analyte list.
RESULTS
Total flavour chemicals were greater than 1% by weight in 13 of
the liquids analysed (table 1). Concentration values in mg/mL are
nearly equivalent to values with units of mg/g; 10 mg/mL corre-
sponds to ∼1% by weight. Six of the 24 compounds in table 1
are aldehydes, a compound class recognised toxicologically to be
‘primary irritants’ of the mucosa of the respiratory tract.18
For
the ‘tobacco’ flavoured fluids, none of the flavour chemicals
reported are obtainable at the levels found by adding a tobacco
extract to the e-cigarette fluid; while extracts of tobacco may be
used in some ‘tobacco’ flavoured fluids, a majority of the
‘tobacco’ flavoured products were found to contain confection-
ary flavour chemicals. Figure 1 provides a bar plot for numbers
of fluids versus per cent by weight for the 30 e-cigarette liquids.
Thirteen of the liquids (43%) contained total determined flavour
chemical levels greater than 1% by weight. Seven of the liquids
(23%) contained levels greater than 2% by weight. Two of the
liquids (7%) contained levels greater than 3% by weight.
LIMITATIONS
The array of e-cigarette products is vast and growing daily. As
such, this study was unable to provide a comprehensive over-
view of the levels of flavour chemicals in such products cur-
rently on the market. Nevertheless, the results obtained are
likely to be similar to what a broad survey would have revealed,
and in any case strongly suggest that very high levels of some
flavour chemicals are undoubtedly present in a great number of
the thousands of products currently available.
DISCUSSION
Recommended 8 h occupational exposure limits by inhalation for
benzaldehyde and vanillin are ∼9 and 10 mg/m3
, respectively.19
Assuming respiration at 0.83 m3
/h (20 m3
/day), these values give
recommended work-place exposure limits of 60 and 67 mg/day,
respectively. For e-cigarette liquid consumption rates, ∼5 mL/day
is commonly self-reported in online ‘vaping’ forums. In our data,
the brand with rank 3 in total flavour chemicals contained benzal-
dehyde at 21 mg/mL; the rank 1 brand contained vanillin
(4-hydroxy-3-methoxybenzaldehyde) at 33 mg/mL; 5 mL/day
then suggests possible inhalation rates of ∼105 and ∼165 mg/day,
respectively, twice the recommended limits. Although the group of
fluids analysed here represents only a small sample of the available
products, the data suggest that a small number of flavour chemicals
are particularly popular among manufacturers: for example, vanil-
lin and ethyl vanillin, maltol and ethyl maltol, benzaldehyde and
benzyl alcohol, and ethyl butyrate and ethyl acetate. Regulatory
actions that should be considered include requiring ingredient
identification, limiting levels of some individual flavor chemicals,
and limiting total levels of flavor chemicals.
What this paper adds
▸ Flavour chemicals are present in almost all e-cigarette fluids
currently on the market in the USA and globally. Concerns
are rising among public health professionals that flavoured
e-cigarette products may make e-cigarette use attractive to
youth. Second, high doses of some flavour chemicals may be
safe when ingested, but quite unsafe when inhaled. Third,
toxic degradation products may be produced by reaction of
the flavour chemicals at the high temperatures present
during e-cigarette use (aka ‘vaping’).
▸ Flavoured e-cigarette products do not typically list the levels
of specific flavour chemicals present, and most do not
identify the major flavour chemicals present.
▸ The analyses of 30 products on the US market revealed that
13 were more than 1% by weight flavour chemicals.
Chemicals identified included aldehydes (eg, benzaldehyde
and vanillin) which could cause respiratory irritation.
Contributors JFP and PAT planned the study. JFP supervised the study. PAT
selected the e-cigarette fluids to be analysed. PAT, CDK, JEB and WL carried out the
analyses. WL managed the data QA/QC processes, and was assisted by PAT and
CDK. JFP and PAT drafted the manuscript. All authors reviewed the manuscript.
Funding This work was supported in part by Michael J Dowd, Regina M Dowd,
Patrick J Coughlin, the Penrose Foundation and the Cooley Family Fund for Critical
Research, of the Oregon Community Foundation.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.
Open Access This is an Open Access article distributed in accordance with the
Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which
permits others to distribute, remix, adapt, build upon this work non-commercially,
and license their derivative works on different terms, provided the original work is
properly cited and the use is non-commercial. See: http://creativecommons.org/
licenses/by-nc/4.0/
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(WEEL) Values. 2011. https://www.aiha.org/get-involved/AIHAGuidelineFoundation/
WEELs/Pages/default.aspx (accessed 24, Sep 2014).
6 Tierney PA, et al. Tob Control 2015;0:1–6. doi:10.1136/tobaccocontrol-2014-052175
Brief report
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11. fluids
Flavour chemicals in electronic cigarette
and James F Pankow
Peyton A Tierney, Clarissa D Karpinski, Jessica E Brown, Wentai Luo
published online April 15, 2015Tob Control
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15. 9/2/2015 PLOS ONE: Vapors Produced by Electronic Cigarettes and EJuices with Flavorings Induce Toxicity, Oxidative Stress, and Inflammatory Response …
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Mice were anesthetized by an intraperitoneal injection of pentobarbital sodium (100 mg/kg; Abbott Laboratories, Abbott Park, IL)
and then sacrificed by exsanguination in two different batches one immediately after 5 hrs ecig exposure on the 3 day and
another batch 24 hrs after last ecig exposure. The lungs were lavaged three times with 0.6 ml of saline via a cannula inserted into
the trachea. The aliquots were combined and centrifuged, and the bronchoalveolar lavage (BAL) fluid stored at 80°C for
cytokine/chemokine analysis and the cell pellet was resuspended in saline. The cells were stained with AO/PI stain and the total cell
number was counted using Cellometer 2000 (Nexcelom Bioscience, Lawrence MA). Cytospin slides (Thermo Shandon, Pittsburgh,
PA) were prepared using 50,000 cells per slide, and differential cell counts (~500 cells/slide) were performed on cytospinprepared
slides stained with DiffQuik (Siemens, DE).
Proinflammatory mediators analysis
Following 24 hrs humectant/eliquid treatment, conditioned media was collected and stored at 80°C for measuring pro
inflammatory mediators. IL8 and IL6 levels were measured by enzymelinked immunosorbent assay (ELISA) according to
manufacturer’s instructions (Life Technologies, Carlsbad, CA). Proinflammatory mediators in bronchoalveloar lavage fluid (BALF)
collected from room air and ecig aerosol exposed mice 24 hrs after the last Blu ecig exposure were measured using ELISA
according to the manufacturer’s instructions (MCP1 and IL6). Various cytokines/chemokines from BAL fluid were measured by the
Luminex Flexmap3D system (Austin, TX) using Milliplex mouse cytokine/chemokine magnetic bead panel for Luminex platform
according to manufacturer’s instructions (Billerica, MA).
Cotinine assay
Levels of cotinine in mouse plasma samples collected immediately after the 3 day Blu ecig aerosol exposure (5 hrs) was
measured by ELISA according to manufacturer’s instructions (Abnova, Taipei, TW).
Glutathione and glutathione disulfide measurements
Total and oxidized (disulfide) glutathione levels measured in mouse lung harvested immediately after the 5 hrs Blu ecig aerosol
exposure (3 day) as described previously [34]. In brief, the concentration of total glutathione in the supernatant of lung
homogenates was determined by comparison with the colorimetric rate of DTNB reduction by known standard concentrations of
reduced glutathione (GSH). For determining the concentration of oxidized glutathione/glutathione disulfide (GSSG), lung
homogenates were combined with 2% of 2vinylpyridine (VP) to derivatize (masking) endogenous GSH. Excess VP is neutralized
by triethanolamine so that in the subsequent reaction, glutathione reductase is able to recycle endogenous GSSG back into
underivatized GSH. GSSG levels are then indirectly measured by DTNB reduction by newly reduced GSH, which was produced
from endogenous GSSG in vitro. Results were expressed as the nmol of total glutathione and GSSG per mg protein as well as total
glutathione/GSSG ratio. All sample homogenates were prepared using RIPA buffer and underwent multiple freeze thaw cycles prior
to measuring glutathione levels.
Statistical analysis
Statistical analysis of significance was calculated using unpaired Student’s ttest. Probability of significance compared to control
was based on 2tail ttests and indicated in figure legends. The results are shown as the mean ± SD unless otherwise indicated. A
value of P < 0.05 is considered as statistically significant.
Results
ENDS/ecigarette OX/ROS generation
OX/ROS produced by ENDS/ecigs were detected by drawing the aerosols through a fluorescein derived dye (DCFH solution)
using an air flow pump (see Materials and Methods). The oxidized form of DCFH (DCF) emits green fluorescence following
excitation at 490 nm indicating OX/ROS or ROS activity. In both cell and cellfree systems, DCFH serves as a semiquantitative
indicator for presence of reactive OX/ROS and has been used previously to measure nanoparticle mediated oxidation in cell free
systems [35].
Detection for the presence of OX/ROS in Blu ecig vapor was performed using two different flavored Blu ecig cartomizers (Classic
Tobacco or Magnificent Menthol) (Fig. 1A). Each cartomizer varies in nicotine content (0 mg and 24 mg) and both were included to
assess if aerosols produced within the cartomizers give rise to major differences in DCF fluorescence intensity after they were
drawn through DCFH solution. Aerosols drawn through DCFH produced from the classic tobacco flavor cartomizer (16 mg of
nicotine) resulted in increased H O μM equivalents (equivalent to DCF fluorescence intensity units) as compared to airsham
group (Fig. 2A). The levels of H O μM equivalents from the menthol cartomizer aerosols were also significantly increased (Fig.
2A). Comparison of OX/ROS levels between both of the cartomizer aerosols showed that the one containing nicotine resulted in
significantly reduced levels of H O μM equivalents (Fig. 2A).
Fig 2. OX/ROS in ENDS vapor. Aerosols or airsham control drawn through DCFH OX/ROS indicator solution.
(A) Blu ecigarette cartomizers; Classic tobacco or Magnificent menthol flavor ecigs. Data are shown as mean ± SD (n =
3/group).* P < 0.05, *** P < 0.001 compared to airsham control (B) eGo refillable vaporizer. Humectants; propylene glycol
and glycerin. Commercial eliquid refills; Vape Dudes Classic tobacco flavor. Data are shown as mean ± SEM (air, n = 15;
propylene glycol, n = 23; glycerin, n = 21; Vape Dudes C. tobacco 0 mg nicotine, n = 7; Vape Dudes C. tobacco 24 mg
nicotine, n = 3; Heating element, n = 9). *** P < 0.001 compared to airsham control.
doi:10.1371/journal.pone.0116732.g002
Next, we exchanged the Blu ecigs with a different type of popular refillable ENDS to test for OX/ROS reactivity in ENDS aerosol.
The eGO Vision Spinner with a 2.2 ohm “wicked” heating element and clearomizer chamber capable of holding 4.5 ml of eliquid is
noticeably larger than the Blu ecig (Fig. 1A) and produces aerosols in a similar fashion. The eliquids, sold in a plethora of flavors
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are primarily comprised of humectants propylene glycol and glycerin [36]. Propylene glycol was filled into the clearomizer and
aerosols produced from the refillable ENDS elicited an increase in H O μM equivalents as compared to airsham group (Fig. 2B).
Similarly, aerosols produced exclusively from glycerin also reacted with DCFH leading to significantly increased H O μM
equivalent levels (Fig. 2B). Two of the commercially available eliquids (Vape Dudes and Classic tobacco flavor) were also tested
for OX/ROS reactivity using the refillable ENDS device. One of these samples contains 0 mg nicotine and the other contains 24 mg
nicotine in addition to undisclosed mixtures of propylene glycol, glycerin, and flavor additives. Both the nonnicotine and nicotine
containing commercially available eliquids produced aerosols that resulted in increased H O μM equivalents levels (Fig. 2B).
These results suggest OX/ROS are emanating from the ecigs/eliquids and are associated with the aerosols that are drawn
through the DCFH indicator, and nicotine was not likely a sole contributing factor in increased OX/ROS reactivity.
Source of OX/ROS generation from ENDS/ecigarettes
It was not clear if the OX/ROS we detected were exclusive to the aerosols of the ENDS or if they might emanate from another
source within the device. We determined that there are two possible sources of OX/ROS that are generated by the refillable ENDS
device. One of the sources of OX/ROS appears to be the heating element since there is an increase in OX/ROS when the heating
element is activated without eliquid filled into the clearomizer chamber (Fig. 2B). In this case, air is drawn through the device by
the pump as the ENDS device is activated, however, there was no visible sign of aerosol being produced. From this data, we
conclude it is possible to generate OX/ROS from ENDS independent of eliquid vaporization.
OX/ROS detection in ENDS aerosols overall yielded a rather broad range of measurements for DCF fluorescence including what
we defined as “high range” values. Attaining a high range value measurement required a 1:10 dilution in pristine DCFH solution to
extrapolate their final values. High range aerosolDCF fluorescence values, including the values for OX/ROS detected in ambient
air flow from activating the heating element without eliquids, were partitioned and compiled together (Table 2).
Table 2. DCF fluorescence values obtained for refillable ENDS aerosols or ambient air alone drawn through DCFH in cellfree ROS assay.
doi:10.1371/journal.pone.0116732.t002
To validate whether or not OX/ROS reactivity emanating from the refillable ENDS device occurs either by vaporizing e
liquids/humectants, or activating the heating element without eliquids/humectants, we hypothesized that the state of the heating
element (new versus multiuse) affects the capacity for OX/ROS to be generated by the refillable ENDS. We first cleaned and
refilled the removable clearomizer chambers with 2.0 ml of either propylene glycol, glycerin, or a commercial refill eliquid (Vape
Dudes Classic tobacco, 0 mg nicotine). A new set of new replacement 2.2 ohm heating elements was obtained from a local
merchant that sells ecigs and accessories and three of the heating elements that were used a number of times in previous
experiments (over 50 times use, exact number of uses unknown) retained. For each eliquid/humectant, two repeat trials were
conducted with a preused heating element, drawing aerosols into DCFH solution in exactly the same manner and timing as for our
previous DCFH experiments. Aerosols for each eliquid/humectant drawn through DCFH indicate the presence of OX/ROS
compared to airsham control which did not result in any appreciable level of OX/ROS reactivity (Table 3, Experiment 1). Next, in
order to determine if replacing the preused heating element with a new one is able to achieve “high range” range DCF
fluorescence, the same sample of eliquid/humectant in the clearomizer from Trial 1 and Trial 2 was retained. Each DCF
fluorescence value obtained after installing the new heating element required 1:10 dilutions in DCFH solution (Table 3, Experiment
1). These results suggest that the state of heating element after activation affects the generation of OX/ROS by the refillable ENDS.
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Table 3. State of the refillable ENDS heating element and its influence over successive use to generate OX/ROS in a cellfree ROS assay.
doi:10.1371/journal.pone.0116732.t003
We further confirmed that the state of the heating element affects OX/ROS generation by installing a new heating element and
activating it independently of eliquids (empty clearomizer) for three trials. As the state of the heating element transitions from new
to multiused between trials, it’s generation of OX/ROS approached airsham control level of DCF fluorescence (Table 3,
Experiment 2, New, 2 use, and 3 use). We then hypothesized that if the vaporization process of the eliquids is also a source
of OX/ROS generation, then adding eliquid for the 4 use of the used heating element will lead to a spike in DCF fluorescence
after the aerosols are drawn through DCFH solution. Rather than filling the clearomizer with eliquid, a single drop of efluid was
absorbed into the heating element wick. The 4 use heating element trial was completed after 5 minutes (half number of puffs)
rather than the usual 10 minutes for all other trials. The resultant DCF fluorescence value required a 1:10 dilution in DCFH solution
(Table 3, Experiment 2, 4 use). Overall, these results suggest that there are at least two possible sources of OX/ROS released
from ENDS, 1) from activation of the heating element, and 2) the process of vaporizing eliquids.
ENDS “dripping” technique and OX/ROS generation
The use of a refillable clearomizer chamber for ENDS is typical for securing eliquids while consumers inhale their aerosols. An
emerging trend abandons use of the clearomizer and replaces it for an inhalation tip that does not hold efluid. The “drip tip” allow
consumers to “drip” eliquid directly onto the heating element wick in the same manner as we applied eliquid to the heating
element for the 4 use (Table 3, Experiment 2). To determine whether or not the clearomizer filled with eliquid versus dripping the
eliquid onto the heating element wick leads to high range fluorescence values (requires 1:10 dilution in DCFH solution), a preused
functioning heating element was installed into the refillable ENDS. In the first two trials, aerosols produced by eliquid filled into the
clearomizer resulted in detection of OX/ROS and the DCF fluorescence values attained did not require 1:10 dilutions (Table 3,
Experiment 3). In contrast, aerosols produced in trials 3 and 4 were carried out by “dripping” small amounts of eliquid sufficient to
absorb into the wick without any liquid placed into the clearomizer. Aerosols produced in this manner resulted in high range DCF
fluorescence values which required 1:10 dilutions in DCFH solution to attain fluorometer readings (Table 3, Experiment 3). These
results suggest that the emerging trend of “dripping” eliquids to produce ENDS aerosols delivers a larger dose of OX/ROS to
consumers.
Reactivity of commercial efluids with DCFH
A variety of locally purchased commercially available eliquids differing in nicotine content and or flavor were reacted with the DCFH
solution directly. Water and the purified humectants propylene glycol and glycerin showed no appreciable indication of reactivity
with DCFH. All of the flavored eliquids exhibited various DCFH reactivity (Table 4). When eliquid DCF fluorescence values from
Table 4 were compared by nicotine content irrespective of brand or flavor, the nicotine containing eliquids exhibited significantly
less DCFH reactivity (Fig. 3A). Table 4 depicting eliquids that contained nontobacco flavor additives (dessert, fruit, and candy)
where on average significantly more reactive with DCFH than eliquids recreating tobacco flavors (Fig. 3B), suggesting more
oxidative reactivity and injurious response by flavored eliquids.
Fig 3. Eliquid reactivity with DCFH exhibits differences between nicotine content and flavor additives.
(A) Commercially available eliquids with different nicotine content. No nicotine (0 mg), low nicotine (6–12 mg) and high
nicotine (16–24 mg). Data are shown as mean ± SD. *** P < 0.001. (B) Comparison of commercially available eliquids,
tobacco flavors (Tobacco, American tobacco, Classic tobacco 9x Tobacco, Marbo) versus nontobacco flavors (Very berry,
AMP, Mountain dew, Cinnamon roll, Grape vape, Cotton candy, Strawberry zing, Strawberry fields, Peaches n cream, Berry
intense, Pineapple express, Melon mania, and Coconut). Data are shown as mean ± SD of n = 3, *** P < 0.001. Yaxis equal
to DCF fluorescence Intensity Units (FIU).
doi:10.1371/journal.pone.0116732.g003
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Both total and oxidized forms of glutathione were assessed. Glutathione levels in mouse lung lysates following animal exposure to
sidestream Blu ecig aerosols were depleted. Air group levels of glutathione averaged 5.89 ± 3.40 nM/mg protein while the
average glutathione level for animals exposed to ecig aerosols was reduced to 1.69 ± 8.10 nM/mg protein (Fig. 8A). Oxidized
(glutathione disulfide) levels of glutathione (GSSG) for air group averaged 3.54 ± 2.25 nM/mg protein and were decreased to 0.68 ±
0.32 nM/mg protein in exposed animals (Fig. 8B).
Fig 8. Intracellular glutathione levels in mouse lung following acute ecigarette aerosol exposure.
Mice were exposed to ecig aerosol exposure (200 mg/m TPM) for 3 days and sacrificed immediately after the last exposure
(3 day after 5 hrs exposure). Levels of (A) Total glutathione. (B) glutathione disulfide GSSG. (C) Total glutathione to GSSG
ratio and (D) GSSG to total glutathione ratio were measured in lung homogenates. Data are shown as mean ± SD (n =
3/group).* P < 0.05 compared to air group mice (C57BL/6J).
doi:10.1371/journal.pone.0116732.g008
Ratios of total Glutathione to GSSG and vice versa were measured to determine if there was an effect from the sidestream ecig
aerosols on the balance between reduced and oxidized forms of glutathione within the lung. The ratio for total glutathione to GSSG
was not significantly different between Air group and ecig exposed animals (Fig. 8C). There was however, a small decrease in the
ratio for GSSG to total glutathione (Fig. 8D). These results suggests that total glutathione levels are reduced by ecig aerosols and
the redox balance between the reduced and oxidized forms of glutathione is affected by sidestream ecig aerosol inhalation as
well.
Discussion
ENDS/ecigs have become prominent fixture in the consumer landscape. Habitually inhaling their aerosols has been implicated by
manufacturers as a safer alternative to smoking conventional cigarettes and many electronic cigarette users have adopted similar
perspectives [40]. However, recent ecigs studies showing that there are substantial levels of nanoscale particles in addition to
detectable levels of metals with toxic materials (e.g., aluminum, copper, magnesium, zinc, lead, chromium, manganese, and nickel)
in ecig aerosols brings this view into question [10]. At the nanoscale size, particles may reach the alveolar epithelium and mediate
oxidative stress and inflammation [41,42].
It is not yet certain what the exact factors are associated with ENDS that might mediate oxidative stress. The eGO Vision ENDS
vaporizer with refillable chamber and exchangeable heating element were employed to detect reactive OX/ROS under a variable
set of parameters, such as the ratios of pure humectant mixtures, commercially available eliquids, changeable voltage settings,
and state of heating element. The ability to manipulate these parameters individually facilitates determination of the OX/ROS
source from the vaporizer.
Using the above parameters, our results indicate that there are a number of variables that affect OX/ROS production in ENDS/e
cigs that are not exclusive to eliquid aerosols. For example, it was observed that OX/ROS reactivity in aerosols produced from the
refillable ENDS device varied between relatively high or low levels. For instance, in some cases DCF fluorescence values from
refillable ENDS aerosols approached or overlapped airsham control values despite aerosols being sufficiently produced during
experimental trials that included vaporization of eliquids/humectants. Multiple batches of DCFH solution prepared for additional
experimental replicates may account for some of the variability seen for OX/ROS reactivity. The attainment of a number of
unusually high fluorescence DCF measurements prompted us to question if the state of the heating element also influenced
OX/ROS release from the device. We also noticed that each time a new heating element was installed into the eGo ENDS, a small
amount of aerosol could be produced without addition of any eliquid suggesting there may be volatile substances associated with
ENDS heating elements following manufacturing.
The trend called “dripping” is intended to allow the user to achieve stronger ‘hits’ and also gives the option to more easily switch
between flavors, brands, or nicotine content without frequently emptying and refilling the clearomizer chamber (communication with
Mr. Douglas Done University of Rochester Department of Public Health Sciences) [43,44]. The design of refillable ENDS is
suggested to incorporate “dripping” as an option for consumers [45]. The spike in OX/ROS release that resulted in high range DCF
fluorescence after dripping eliquid onto the 4 use heating element wick led us to hypothesize that “dripping” rather than filling the
clearomizer with eliquid, which completely submerges the heating element, is potentially more hazardous. Our results indicate that
the dripping method for ENDS usage is likely to generate a larger amount of OX/ROS.
As Goniewicz et al reports, heating eliquids with sufficient temperatures produces detectible levels of formaldehyde, acrolein, and
acetaldehyde carbonyls with the possibility that these compounds form due to the pyrolysis of glycerin [5,6]. However, in
comparison to conventional cigarette smoke, levels of these carbonyls were found to be between 9 and 807 times lower suggesting
that ENDS/ecigs may be a safer alternative to conventional cigarettes [5,6]. Carbonyl levels in ENDS/ecigs also appeared to
depend on the brand of the device while the black deposits that we see from the heating elements following our experiments are
consistent with what has been found associated with other devices [10,46]. Therefore, although toxic carbonyl byproducts
measured in ENDS aerosols may be orders of magnitude lower than conventional cigarettes as reported by Goniewicz et al. and
Kosmider et al., the potential for delivering oxidizing agents as measured here may be currently underappreciated. The OX/ROS
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