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Evaluation of Electronic Cigarette Liquids and Aerosol for the Presence of Selected Inhalation Toxins

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Introduction: The purpose of this study was to evaluate sweet-flavored electronic cigarette (EC) liquids for the presence of diacetyl (DA) and acetyl propionyl (AP), which are chemicals approved for food use but are associated with respiratory disease when inhaled. Methods: In total, 159 samples were purchased from 36 manufacturers and retailers in 7 countries. Additionally, 3 liquids were prepared by dissolving a concentrated flavor sample of known DA and AP levels at 5%, 10%, and 20% concentration in a mixture of propylene glycol and glycerol. Aerosol produced by an EC was analyzed to determine the concentration of DA and AP. Results: DA and AP were found in 74.2% of the samples, with more samples containing DA. Similar concentrations were found in liquid and aerosol for both chemicals. The median daily exposure levels were 56 μg/day (IQR: 26-278 μg/day) for DA and 91 μg/day (IQR: 20-432 μg/day) for AP. They were slightly lower than the strict NIOSH-defined safety limits for occupational exposure and 100 and 10 times lower compared with smoking respectively; however, 47.3% of DA and 41.5% of AP-containing samples exposed consumers to levels higher than the safety limits. Conclusions: DA and AP were found in a large proportion of sweet-flavored EC liquids, with many of them exposing users to higher than safety levels. Their presence in EC liquids represents an avoidable risk. Proper measures should be taken by EC liquid manufacturers and flavoring suppliers to eliminate these hazards from the products without necessarily limiting the availability of sweet flavors.
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Accepted Manuscript
Evaluation of electronic cigarette liquids and vapour for the
presence of selected inhalation toxins.
Journal:
Nicotine & Tobacco Research
Manuscript ID:
NTR-2014-374.R2
Manuscript Type:
Original Investigation
Date Submitted by the Author:
18-Aug-2014
Complete List of Authors:
Farsalinos, Konstantinos; Onassis Cardiac Surgery Center, Cardiology
Kistler, Kurt; The Pennsylvania State University, Chemistry
Gillman, Gene; Enthalpy Analytical,
Voudris, Vassilis; Onassis Cardiac Surgey Center, Cardiology
Keywords:
Public health, Biochemistry, Health consequences, Prevention
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Title: Evaluation of electronic cigarette liquids and aerosol for the presence of selected
inhalation toxins.
Authors: Konstantinos E. Farsalinos, MD1, Kurt A. Kistler, PhD2, Gene Gillman, PhD3, Vassilis
Voudris, PhD1
1 Department of Cardiology, Onassis Cardiac Surgery Center, Sygrou 356, Kallithea 17674,
Greece.
2 Department of Chemistry, The Pennsylvania State University Brandywine, 25 Yearsley Mill
Road, Media, Pennsylvania 19063, USA.
3 Enthalpy Analytical, Inc., 800 Capitola Drive, Suite 1, Durham, NC 27713.
Corresponding author
Konstantinos E Farsalinos, MD
Email: kfarsalinos@gmail.com
Tel: +306977454837
Fax: +302109493373
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Abstract
Introduction. The purpose of this study was to evaluate sweet-flavoured electronic cigarette
(EC) liquids for the presence of diacetyl (DA) and acetyl propionyl (AP), which are chemicals
approved for food use but are associated with respiratory disease when inhaled.
Methods. In total, 159 samples were purchased from 36 manufacturers and retailers from 7
countries. Additionally, three liquids were prepared by dissolving a concentrated flavour sample
of known DA and AP levels at 5%, 10% and 20% concentration in a mixture of propylene glycol
and glycerol. Aerosol produced by an EC was analyzed to determine the concentration of DA
and AP.
Results. DA and AP were found in 74.2% of the samples, with more samples containing DA.
Similar concentrations were found in liquid and aerosol for both chemicals. The median daily
exposure levels were 56µg/day (IQR: 26-278µg/day) for DA and 91µg/day (IQR: 20-432µg/day)
for AP. They were slightly lower than the strict NIOSH-defined safety limits for occupational
exposure and 100 and 10 times lower compared to smoking respectively; however, 47.3% of DA
and 41.5% of AP-containing samples exposed consumers to levels higher than the safety limits.
Conclusions. DA and AP were found in a large proportion of sweet-flavoured EC liquids, with
many of them exposing users to higher than safety levels. Their presence in EC liquids represents
an avoidable risk. Proper measures should be taken by EC liquid manufacturers and flavouring
suppliers to eliminate these hazards from the products, without necessarily limiting the
availability of sweet flavours.
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INTRODUCTION
Electronic cigarettes (ECs) are novel nicotine-delivery products which have gained
popularity among smokers in recent years (Regan et al., 2013). They deliver nicotine in aerosol
form through heating a nicotine-containing solution resulting in the production of visible
“vapour”. Besides nicotine delivery, they address the whole smoking ritual and psycho-
behavioural dependence through sensory stimulation and motor simulation (Farsalinos &
Stimson, 2014).
Sensory stimulation is perceived from EC use both by the “throat hit” induced during
aerosol inhalation (Farsalinos et al., 2014a) as well as by the use of flavoured liquids. The use of
flavourings has resulted in a large debate among public health professionals and regulators,
suggesting that they can be attractive to youth. A recent survey of dedicated users (vapers)
concluded that flavours variability contributes to both perceived pleasure and the effort to reduce
cigarette consumption or quit smoking, and showed that dedicated vapers switch between
flavours quite frequently (Farsalinos et al., 2013a). Although the majority of flavourings are
“Generally Recognized As Safe” (GRAS) for food use, these substances have not been
adequately tested for safety when inhaled. In fact, the Flavors and Extracts Manufacturers’
Association (FEMA) has issued an official statement mentioning that flavour ingredients are
evaluated for exposure through ingestion only; thus, any results cannot be extrapolated to use
through inhalation (FEMA, 2014). Studies have shown that any cytotoxic properties of e-
cigarette liquids and aerosol, although significantly lower than tobacco smoke, may be attributed
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to specific flavours (Farsalinos et al., 2013b; Romagna et al., 2013; Bahl et al., 2012), indicating
that further research is certainly needed in this area.
Besides the lack of studies for the effects of flavouring substances when inhaled, there
are some chemicals which, although approved for ingestion, have already established adverse
health effects when inhaled. A characteristic example of this is diacetyl (DA, Figure 1). This
substance, also known as 2,3-butanedione, is a member of a general class of organic compounds
referred to as diketones, α-diketones or α-dicarbonyls. It is responsible for providing a
characteristic buttery flavour, and is both naturally found in foods and used as a synthetic
flavouring agent in food products such as butter, caramel, cocoa, coffee, dairy products and
alcoholic beverages (Mathews et al., 2010). Although it is approved and safe when ingested
(National Institute for Occupational Safety and Health, 2011; FEMA Nr 2370), it has been
associated with decline in respiratory function, manifested as reduced Forced Expiratory Volume
in 1s (FEV1), in subjects exposed to it through inhalation. Additionally it has been implicated in
the development of bronchiolitis obliterans, an irreversible respiratory disease also called
“popcorn lung disease” because it was initially observed in workers of popcorn factories
(Kanwal et al., 2006; CDC, 2002; Kreiss et al., 2002). To the best of our knowledge, the issue of
DA presence in EC liquids was first mentioned in 2008 in EC consumers’ forums (http://www.e-
cigarette-forum.com/forum/health-safety-e-smoking/2666-inhaling-flavouring-chemicals.html).
Subsequently, several companies released statements mentioning that DA was removed from
their EC liquid products (e.g. http://clearstream.flavourart.it/site/?p=366&lang=en). Another
chemical of concern is acetyl propionyl (AP), also called 2,3-pentanedione (Figure 1). This is
also an α-diketone and is chemically and structurally very similar to DA. It has become a popular
replacement for DA (Day et al., 2011; FEMA Nr 2841) since the negative press surrounding DA-
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induced bronchiolitis obliterans in popcorn workers, because it adds the desired flavour while
claims of “diacetyl-free” can be made by the manufacturer. Unfortunately, the risks associated
with inhalation of AP may well be as high as from DA, based on inhalation studies performed on
rats (Hubbs et al., 2012). Due to the potential hazards associated with inhalation exposure to DA
and AP, regulatory agencies have set specific Occupational Exposure Limits (OELs). For DA,
the National Institute on Occupational Safety and Hazards (NIOSH) has proposed an upper limit
of 5ppb (18g/m3) for 8h Time-Weighted Average exposure (TWA) and 25ppb (88g/m3) as
Short-Term Exposure Limit (STEL) for 15 minutes, while the Scientific Committee on
Occupational Exposure Limits (SCOEL) of the European Commission considered the NIOSH-
defined limits for DA unnecessarily strict and has set upper limits of 20ppb (70g/m3) and
100ppb (360g/m3) respectively (European Commission, 2013; National Institute for
Occupational Safety and Health, 2011). For AP, NIOSH has set a TWA limit of 9.3ppb
(38g/m3) and an STEL of 31ppb (127g/m3) (National Institute for Occupational Safety and
Health, 2011).
The purpose of this study was to examine the presence of DA and AP in a large sample of
EC liquids obtain from European and US manufacturers and retailers. Additionally, we sought to
measure the levels of these chemicals in aerosol produced from ECs, since this represents the
realistic use of ECs and the relevant exposure route of vapers, and compare this with literature
data evaluating exposure from smoking tobacco cigarettes.
METHODS
Sample selection
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Samples of EC liquids were selected from European and US manufacturers and retailers.
The selection was based on information from local or international EC consumers’ forums, in
order to get samples from major or popular sources. Since the chemicals examined were more
likely to be present in sweet flavourings, we chose samples with sweet flavours (butter, toffee,
milky, cream, chocolate, coffee, caramel, etc). A total of 159 samples were selected from 36
manufacturers and retailers from 6 European countries (France, Germany, Greece, Italy, Poland,
and UK, n=78) and from the US (n=81). Both refill liquids (“ready to use”, n=113) and
concentrated flavours (n=46), which are diluted by users in “base” liquids (mixtures of propylene
glycol, glycerol and nicotine), were obtained. Different number of samples per manufacturer was
obtained, depending on the availability of sweet flavourings. In several cases, there were clear
statements in the manufacturers’ websites that no DA was present in their liquids. All samples
were bought anonymously from internet shops, without mentioning that the purpose of the
purchase was to be analyzed for a scientific study. All bottles were received sealed, and were
immediately sent to the laboratory for analysis.
Methods of analysis
The samples were analyzed by High Performance Liquid Chromatography (HPLC). The
procedure followed was a modified version of the HPLC carbonyl compound analysis method
for mainstream cigarette smoke, by the Cooperation Centre for Scientific Research Relative to
Tobacco (CORESTA) (Cooperation Centre for Scientific Research Relative to Tobacco, 2013).
This method was previous validated by our laboratory for the analysis of carbonyls in EC liquids
and was expanded for the analysis of DA and AP. The performance of the method for diketones
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was evaluated for recovery from the sample matrix by addition of known amount of DA and AP
before derivatization. In all cases the recovery of both compounds was greater than 80%. To
prevent the formation of two carbonyl adducts, an aliquot of the sample for analysis was
combined with 1mL of a standard 2,4-dinitrophenylhydrazine (DNPH) trapping solution and
allowed to derivatize for 20 minutes, then quenched with 0.050mL of pyridine. This ensures that
only one of the two carbonyls is converted to its derivative. DA and AP standards were produced
by adding known amounts of DA and AP to the DNPH trapping solution. Standards were treated
in the same manner as samples, and were used to prepare a linear calibration curve which ranged
from 0.4-30µg/mL. All e-liquid samples were analyzed at an initial 22-fold dilution, while pure
flavour samples were analyzed at an initial 43-fold dilution. At these dilutions, the maximum
amount of propylene glycol and glycerol in the DNPH solution was less than 5% and had no
effect on derivatization. The efficient derivatization of DA and AP requires excess DNPH, and
all samples were evaluated for DNPH depletion by verifying that a large DNPH peak was
observed by HPLC. Any samples that were found to have depleted DNPH were prepared and
reanalyzed using a smaller sample aliquot (thus, DNPH trapping solution was used to dilute the
samples). An Agilent Model 1100, High Performance Liquid Chromatograph was equipped with
an Ultraviolet (UV) Detector operating at 365nm and a Waters Xterra MS C18, 3.0 x 250mm
column. Two solutions, A and B, were used as mobile phases in varying relative concentrations
over time. Mobile Phase A: 890mL water, 100mL of tetrahydrofuran and 10mL of isopropanol.
Mobile Phase B: 890mL acetonitrile, 100mL of tetrahydrofuran and 10mL of isopropanol.
Separation was accomplished with the following linear gradient: 0.00 minutes 65% A, 35% B;
11.00 minutes 40.0% A, 60% B; 18 minutes 0% A, 100% B. Flow rate was set to a constant
0.75mL/min.
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The materials used for the HPLC analysis were: deionized water – Millipore; phosphoric
acid (H3PO4), 85%, A.C.S Reagent, Sigma-Aldrich (P/N 438081); DNPH (50%), TCI America,
P/N D0845; acetonitrile (CAS #75-05-8), HPLC grade; tetrahydrofuran (CAS #109-99-9), HPLC
grade; isopropanol (CAS #67-63-0), distilled-in-glass; pyridine (CAS #110-86-1); diacetyl (97%)
Sigma-Aldrich (P/N B85307) (CAS #431-03-8); 2,3-pentanedione (97%) Sigma-Aldrich (P/N
241962) (CAS # 600-14-6).
Aerosol production and analysis
To evaluate the amount of DA and AP that is transferred from liquid to aerosol, three
liquids were prepared by diluting the sample of concentrated flavour with the highest level of
diacetyl to 5%, 10% and 20% in a mixture of 50% propylene glycol and 50% glycerol. These
dilutions were chosen because they represent the most common dilutions of concentrated
flavours used or recommended for EC use. The prepared liquids were analyzed by HPLC (with
the method described above), to determine the concentration of DA and AP. Aerosol was
produced by using a commonly used commercially-available EC device (eGo battery, Joyetech,
Shenzhen, China) with a bottom-coil clearomizer (EVOD, KangerTech, Shenzhen, China). The
device was fully charged before use and a new tank and atomizer was used for each sample.
Approximately 2mL of the prepared liquid was added to the tank. The device was weighed
before and after sample collection. A Cerulean SM 450 smoking machine was used to collect 50
puffs from all samples. The smoking machine was set to deliver a 55mL puff over 4 seconds
every 30 seconds (Farsalinos et al., 2013c) with a constant flow of 13.75mL per second. The EC
device was automatically triggered at the beginning of the puff for 4 seconds, by using a custom
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air-piston mechanism to push the activation button. The aerosol was passed through an impinger
containing 35mL of the DNPH trapping solution without the use of a filter pad. Once the aerosol
collection was complete, 5mL of this solution was quenched with 250 L of pyridine. The
samples were then analyzed by HPLC monitoring at 365 nm.
Interpreting NIOSH safety limits in the context of EC liquids
The TWA limits (8-hours exposure) defined by NIOSH (5ppb, i.e. 18µg/m3 for DA and
9.3ppb, i.e. 38µg/m3 for AP) were used as a guide to define potentially “acceptable” levels of DA
and AP in EC liquids. The average resting respiratory rate for an adult is 15 breaths per minute
while the tidal volume is 0.5L (Barrett and Ganong, 2012). Within 8 hours (480min), the total
volume of air inhaled is 3.6m3 ([0.5L x 15breaths/min x 480min] / 1000L/m3). Thus, the total
amount of DA that can be inhaled daily (according to NIOSH limits) is 65µg (18µg/m3 x 3.6m3),
while for AP it is 137µg (38µg/m3 x 3.6m3).
Statistical analysis
Data were examined for distribution by Kolmogorov-Smirnov test. Continuous variables
were expressed as median (interquartile range [IQR]) while categorical variables were expressed
as number (%). For DA and AP levels, the medians were calculated from the samples which
contained the chemicals only (samples with non-detectable DA and AP were excluded). To
assess the difference in DA and AP levels between concentrated flavours and refill liquids,
Mann-Whitney U test was used. To assess the realistic exposure to DA and AP from
concentrated flavours, we multiplied the levels found in these samples with 0.2, assuming that
they are diluted to 20% in order to prepare a refill liquid. Chi-square test was used to assess the
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differences between European countries and US in the number of samples containing DA and
AP. Pearson’s correlation coefficient was used to assess the correlation between expected and
measured DA and AP levels in the aerosol analysis. To estimate the average daily exposure,
consumption of EC liquid was assumed to be 3mL/day, based on the results of a large survey of
vapers (Farsalinos et al., 2014b). To assess the difference in DA and AP daily exposure between
smoking and EC use, Mann-Whitney U test was also used. A two-tailed P value of <0.05 was
considered statistically significant. Commercially-available statistical software was used for the
analysis (SPSS v. 18, Chicago, IL, USA).
RESULTS
Analysis of liquid samples
In 41 (25.8%) samples DA and AP was not detected, while in 73 (45.9%) samples one of
the two chemicals was detected and in 45 (28.3%) samples both chemicals were detected. DA
was found in 110 (69.2%) samples, containing a median concentration of 29µg/mL (IQR: 10-
170µg/mL). Of those, 32 were concentrated flavours samples (69.6% of all concentrated flavours
samples) and 78 were refill samples (69.0% of all refill samples). Concentrated flavours
contained 3 times higher levels of DA compared to refill liquids (median: 68µg/mL vs. 20µg/mL,
P=0.001), with the highest levels being 32,115µg/mL in the former and 10,620µg/mL in the
latter. DA was detected in the samples of 33 manufacturers (91.6%) from all 7 countries (66.7%
of European and 71.6% of US samples, chi-square P=0.500). By converting the levels of DA
found in concentrated flavours to represent realistic exposure (see Statistical analysis section),
the median daily exposure level to DA from all DA-containing samples was calculated at
56µg/day (IQR: 26-278µg/day, Figure 2A). This is slightly lower than the NIOSH-defined safety
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limit (65µg/day). However, 52 samples (47.3% of the positive samples) would expose consumers
to levels higher than the NIOSH limits, with 26 of them (23.6%) having >5 times higher levels
than the safety limit. The sample with the highest level of DA would result in 490 times higher
daily intake compared to the NIOSH limit.
AP was found in 53 (33.3%) samples, containing a median concentration of 44µg/mL
(IQR: 7-172µg/mL). Of those, 10 were concentrated flavours samples (21.7% of all concentrated
flavours samples) and 43 were refill samples (38.1% of all refill samples). Concentrated flavours
contained 3 times higher levels of AP compared to refill liquids (median: 124µg/mL vs.
37µg/mL, P=0.114). The difference was not statistically significant, probably due to the low
number of concentrated flavours containing AP. The highest levels found were 3082µg/mL in
concentrated flavours and 1018µg/mL in refills. AP was detected in the samples of 24
manufacturers (66.7%) from 6 countries (23.1% of European and 43.2% of US samples, chi-
square P=0.007). By converting the levels of AP found in concentrated flavours to represent
realistic exposure (see Statistical analysis section), it was estimated that the median daily
exposure level to AP from all AP-containing samples was 91µg/day (IQR: 20-432µg/day, Figure
2B). This is lower than the NIOSH-defined safety limit (137µg/day). However, 22 samples
(41.5% of the positive samples) would expose consumers to levels higher than the NIOSH limits,
with 11 of them (20.8%) having >5 times higher levels than the safety limit. The sample with the
highest level of AP would result in 22 times higher daily intake compared to the NIOSH limit.
Analysis of aerosol
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One concentrated flavour sample was diluted to 5%, 10% and 20% into a mixture of 50%
propylene glycol and 50% glycerol, in order to prepare the 3 liquids used for the aerosol analysis.
The prepared liquids were analyzed by HPLC and were found to contain DA and AP at
respective levels of 1801µg/mL and 160µg/mL for the 5% sample, 3921µg/mL and 349µg/mL
for the 10% solution, and 7546µg/mL and 606µg/mL for the 20% solution. Based on the weight-
difference of the atomizer before and after the puffing session, we evaluated the volume of liquid
consumed in each puffing session by dividing the amount (mg) of liquid consumed with the
specific weight of the samples (which was determined to be 1.13). From that, the concentrations
of DA and AP per mL of liquid consumed were determined. Similar concentrations of DA and
AP were observed in the liquid and aerosol samples while a very strong correlation was observed
between the expected (based on the liquid consumption) and the observed (measured) DA and
AP concentrations (R2=0.997 and 0.995 respectively, Figure 3). These results indicate that both
DA and AP are readily delivered from the liquid to the aerosol.
Comparison with exposure from tobacco cigarettes
To compare DA and AP exposure from EC use and smoking, the study by Pierce et al.
(2014) was used. By using the ISO 3308 smoking regime, an average of 285µg of DA and 43µg
of AP (average values) was emitted in the smoke of a single cigarette. Considering a daily
consumption of 20 cigarettes, the median daily exposure would be 5870µg (4970-6195µg) for
DA and 894µg (713-965µg) for AP (we estimated the median values in order to be compared
with the data from our study, which were not normally distributed). As mentioned previously,
the median daily levels of DA and AP exposure from EC use were estimated to be 56µg and
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91µg respectively, which are 100 and 10 times lower compared to smoking (Mann-Whitney
P<0.001 for DA and P=0.020 for AP).
DISCUSSION
Main findings
This is the first study to analyze a large number of EC liquids with sweet flavours
obtained from a variety of manufacturers and retailers from Europe and the US for the presence
of DA and AP. The main findings were that these substances were present in the majority of the
samples tested, with a significant proportion containing both chemicals; they were detected even
in samples coming from manufacturers who clearly stated that they were not present in their
products. Additionally, it was determined that both DA and AP are readily delivered to the
aerosol that the vaper inhales, an expected finding considering the volatility of these compounds.
Although the median levels found were slightly lower than the strict NIOSH-defined safety
levels, a substantial proportion of the positive samples would expose consumers to levels higher
than the safety limits.
Flavourings in ECs
The issue of flavouring use in EC products is a matter of strong debate, mostly in terms
of being appealing to youth. A survey of more than 4000 dedicated users determined that the
reason for the availability of a large variety of flavours is the market demand by existing
consumers (vapers), and showed that sweet flavours were the most popular category used by this
population (Farsalinos et al., 2013a). Less attention has been given to the issue of safety when
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inhaling food-approved substances. While many food flavourings have never been tested for
inhalation safety, the focus here was on known inhalation toxins that are flavour compounds.
Toxicity of DA and AP
DA is a water soluble volatile α-diketone that is both a natural constituent of numerous
foods and an added ingredient used by the flavouring industry. In 1995, an estimated 96,000kg of
diacetyl were used in the food industry (Harber et al., 2006). It has been identified as a prominent
volatile organic compound in air samples from microwave popcorn plants and flavouring
manufacturing plants (Akpinar-Elci et al., 2004; Parmet & Von Essen, 2002). DA exposure
through inhalation has been associated with a decline in respiratory function (characterized by a
declined in FEV1) and the development of bronchiolitis obliterans, a rare irreversible obstructive
disease involving the respiratory bronchioles. Kreiss et al (2002) evaluated 117 workers in a
microwave popcorn production plant in Missouri and found that these workers had 2.6 times the
expected rate of respiratory symptoms such as chronic cough and shortness of breath and 3.3
times the expected rate of airway obstruction. Kanwal et al. (2006) examined workers in 6
popcorn plants and found that exposure to flavourings mixing for more than 12 months was
associated with higher prevalence of decline in respiratory function, while 3 cases of
bronchiolitis obliterans were documented by lung biopsy. Similar findings were observed by
Lockey et al. (2009). Three cases of clinical bronchiolitis obliterans were also diagnosed in a
diacetyl facility in the Netherlands (van Rooy et al., 2007). Finally, a cross-sectional analysis of
medical surveillance data from 16 companies confirmed the risk of lung disease among workers
at companies using diacetyl (Kim et al., 2010).
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AP is chemically and structurally almost identical to DA, has a similar buttery, creamy
flavour, and has been used as a DA substitute in many flavouring manufacturing facilities (Day
et al., 2011). Toxicological studies in animals have shown that it has adverse effects on
respiratory epithelium similar to DA and at similar levels (Hubbs et al., 2012; Morgan et al.,
2012).
Implications of the study findings
A wide range of DA and AP concentrations were found in the samples, indicating that in
some cases the chemicals were used deliberately as ingredients while in others they were
probably contaminants. Overall the estimated daily exposure from EC use was approximately
100 times lower for DA and 10 times lower for AP compared to tobacco cigarettes; therefore, it
is still plausible to classify ECs as tobacco harm reduction products (Polosa et al., 2013).
However, the major source of DA and AP in tobacco cigarette smoke is the combustion process
(Pierce et al., 2014); thus, it is an unavoidable risk. In EC liquids, these chemicals are introduced
during the production process, since there is no combustion. Production of DA and AP from
thermal decomposition is unlikely, and was not detected in this study. Since 25.8% of the
samples of similar flavours were DA and AP free, the findings indicate that vapers are exposed
to an avoidable risk. It is imperative that appropriate removal measures should be undertaken.
The major source of flavourings for EC liquid manufacturers is the food-flavouring industry,
with DA and AP being approved as ingredients. Establishment of an inhalation-specific
flavouring industry is recommended, with dedication to evaluate and choose appropriate
flavouring compounds for EC liquids, based on inhalation safety profiles. In any case, it is of
high priority for every manufacturer to properly examine the flavourings used in the production
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process. The results of the aerosol analysis, showing that DA and AP are readily delivered from
the liquid to the aerosol, indicate that analysis of the liquid is sufficient.
Limitations
Our selection was targeted to sweet-only flavours because it was expected that these are
more likely to contain DA and AP. Other classes of flavourings available in the market, such as
tobacco, mint/menthol, fruits, beverages and nuts, probably have lower prevalence of DA and
AP. However, we cannot exclude the possibility that there may be liquids from other flavour
types (besides sweets) which contain these compounds.
Fewer samples contained AP compared to DA. This was unexpected, since it has been
common practice for the flavouring industry to substitute DA with alternative chemicals due to
the criticism for the adverse effects of DA exposure to workers. It is unknown whether this is a
generalized finding in the EC liquid market or it is attributed to chance related to the selection of
the samples.
Although we tried to define the “acceptable” levels of DA and AP in EC liquids, there is
no clinical evidence indicating that the limit set by NIOSH is applicable to EC use. This limit is
set for occupational exposure, and no exposure limit has been set for continuous or recreational
exposure to e-cigarette aerosols. Therefore, this assessment should be approached with caution.
The cut-off level of risk calculated by NIOSH for the TWA limit is for 1 in 1000 chance of
suffering reduced lung function associated with lifelong diacetyl exposure. This is a very
conservative estimation; however, a significant proportion of the samples had >5 times higher
levels of DA and AP than NIOSH limits. Moreover, the finding that more than 25% of the
samples tested did not contain any of the two chemicals shows that it is feasible to prepare sweet
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flavourings with alternative chemicals; thus, there is no need to exclude them from the market,
since they have been found to be quite popular among dedicated users.
A recent study raised doubts about the association between DA and AP exposure and
development of bronchiolitis obliterans (Pierce et al., 2014); high levels of these chemicals were
found in tobacco smoke while smoking is not a risk factor for development the disease.
However, cigarette smoke contains many respiratory irritants, which probably act synergistically
and cause a different pattern of lung disease. The prevalence of chronic obstructive lung disease
in active smokers is estimated to be 15.4% (Raherison & Girodet, 2009), by far higher than the
prevalence of bronchiolitis obliterans in patients exposed to diacetyl. Moreover, it is quite
common that the condition is often misdiagnosed (Kreiss et al., 2002). Finally, post-mortem
examinations have shown that many smokers have histopathological features of respiratory
bronchiolitis (Niewoehner et al., 1974).
Conclusion
In conclusion, DA and AP were present in a large proportion of sweet-flavoured EC liquid
samples from both European and US manufacturers and retailers, and are readily delivered to the
aerosol inhaled by the users. The median level of exposure is lower compared to tobacco
cigarettes by 1-2 orders of magnitude, confirming their role as tobacco harm reduction products.
However, any risk from exposure to DA and AP by EC use is totally avoidable, by using
alternative compounds, and this was evident from the samples of similar flavour in which no DA
or AP was detected. Manufacturers and flavouring suppliers should take the necessary steps to
make sure that these chemicals are not present in EC liquid products, by regularly testing their
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products and changing formulations, without the need to limit the availability of sweet flavours
in the market.
Funding
This study was funded through an open internet crowd-funding campaign which was conducted
in the website www.indiegogo.com.
Declaration of interests
Some of the studies by KF and VV were performed using funds provided to the institution by e-
cigarette companies. KK and GG have no conflict of interest to report.
Acknowledgements
We would like to thank Dimitris Agrafiotis (a volunteer vaping advocate) for his assistance in
organising the crowd-funding campaign and in the selection of EC liquid samples.
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Figure legends
Figure 1. Chemical structures of diacetyl (DA) and acetyl propionyl (AP).
Figure 2. Box-plots of the estimated daily exposure to diacetyl (A) and acetyl propionyl (B)
from the liquid samples tested. The box represents the 25th and 75th percentiles, with the line
inside the box showing the median value. The error bars represent the 10th and 90th percentiles.
The dotted line represents the maximum acceptable levels of daily exposure estimated from the
NIOSH limit for occupational exposure.
Figure 3. Correlation between the expected (based on liquid consumption during aerosol
production) and the measured concentrations of diacetyl (DA) and acetyl propionyl (AP) in
aerosol. A strong correlation was observed, while the expected and measured values were almost
identical, verifying that DA and AP are readily delivered from the liquid to the aerosol and that
no additional DA and AP are produced during the evaporation process.
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Figure 2. Box-plots of the estimated daily exposure to diacetyl (A) and acetyl propionyl (B) from the liquid
samples tested. The box represents the 25th and 75th percentiles, with the line inside the box showing the
median value. The error bars represent the 10th and 90th percentiles. The dotted line represents the
maximum acceptable levels of daily exposure estimated from the NIOSH limit for occupational exposure.
230x96mm (300 x 300 DPI)
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y = 0.9526x + 182.64
R² = 0.9966
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(µg/mL)
Measured concentration
(µg/mL)
DA
y = 1.2435x - 25.483
R² = 0.9947
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(µg/mL)
Measured concentration
(µg/mL)
AP
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... As early as 2008 there were health concerns amongst vapers about the use of DA as a flavorant in e-liquids (Farsalinos et al., 2015;Vas et al., 2019). However, over the last decade a growing number of surveys have continued to identify the presence of DA, AC and AP in American, Canadian and European e-liquids (Farsalinos et al., 2015;Barhdadi et al., 2017;Moldoveanu et al., 2017;LeBouf et al., 2018;Vas et al., 2019;Czoli et al., 2019), (Supplementary Table S1), and aerosol emissions from commercial e-cigarettes (Allen et al., 2016;Margham et al., 2016;Sleiman et al., 2016;Klager et al., 2017;Moldoveanu et al., 2017;Melvin et al., 2020). ...
... As early as 2008 there were health concerns amongst vapers about the use of DA as a flavorant in e-liquids (Farsalinos et al., 2015;Vas et al., 2019). However, over the last decade a growing number of surveys have continued to identify the presence of DA, AC and AP in American, Canadian and European e-liquids (Farsalinos et al., 2015;Barhdadi et al., 2017;Moldoveanu et al., 2017;LeBouf et al., 2018;Vas et al., 2019;Czoli et al., 2019), (Supplementary Table S1), and aerosol emissions from commercial e-cigarettes (Allen et al., 2016;Margham et al., 2016;Sleiman et al., 2016;Klager et al., 2017;Moldoveanu et al., 2017;Melvin et al., 2020). ...
... However, despite the growing range of studies identifying these compounds in e-liquids or e-cigarette emissions, surprisingly no study has clearly evaluated emissions from e-cigarettes containing known e-liquid content at levels relevant to commercial e-liquids. The closest reported study was that of Farsalinos et al. (2015), who created three experimental e-liquids at very high DA and AP contents, and identified near-quantitative transfer to the aerosol even though the concentrations were significantly higher than measured in the great majority of commercial e-liquids. Moldoveanu et al. (2017) also measured both e-liquid and aerosol DA concentrations in their study. ...
Article
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Background: Concerns over the presence of the diketones 2,4 butanedione (DA) and 2,3 pentanedione (AP) in e-cigarettes arise from their potential to cause respiratory diseases. Their presence in e-liquids is a primary source, but they may potentially be generated by glycerol (VG) and propylene glycol (PG) when heated to produce aerosols. Factors leading to the presence of AP, DA and acetoin (AC) in e-cigarette aerosols were investigated. We quantified direct transfer from e-liquids, examined thermal degradation of major e-liquid constituents VG, PG and 1,3 propanediol (1,3 PD) and the potential for AC, AP and DA production from sugars and flavor additives when heated in e-cigarettes. Method: Transfers of AC, AP and DA from e-liquids to e-cigarette aerosols were quantified by comparing aerosol concentrations to e-liquid concentrations. Thermal generation from VG, PG or 1,3 PD e-liquids was investigated by measuring AC, AP and DA emissions as a function of temperature in an e-cigarette. Thermal generation of AC, AP and DA from sugars was examined by aerosolising e-liquids containing sucrose, fructose or glucose in an e-cigarette. Pyrolytic formation of AP and DA from a range of common flavors was assessed using flash pyrolysis techniques. Results: AC transfer efficiency was >90%, while AP and DA were transferred less efficiently (65%) indicating losses during aerosolisation. Quantifiable levels of DA were generated from VG and PG, and to a lesser extent 1,3 PD at coil temperatures >300°C. Above 350°C AP was generated from VG and 1,3 PD but not PG. AC was not generated from major constituents, although low levels were generated by thermal reduction of DA. Aerosols from e-liquids containing sucrose contained quantifiable (>6 ng/puff) levels of DA at all sucrose concentrations tested, with DA emissions increasing with increasing device power and concentration. 1% glucose, fructose or sucrose e-liquids gave comparable DA emissions. Furanose ring compounds also generate DA and AP when heated to 250°C. Conclusions: In addition to less than quantitative direct transfer from the e-liquid, DA and AP can be present in the e-cigarette aerosol due to thermal decomposition reactions of glycols, sugars and furanonse ring flavors under e-cigarette operating conditions.
... There is little consistency in puffing regimes being used for ENDS emission studies; studies have used 15 ml/s, 4 s puffs (1), 27 ml/s, 3 s puffs (49), 39 ml/s, 1.8 s puffs (2), 27.5 ml/s, 2 s puffs (3,4), 17.5 ml/s 2 s puffs (5-7), 10 ml/s, 4 s puffs (8), and in some articles the puffing protocol is unclear (9,10). It remains unclear how the puffing regimes used relate to the normal range of the device permitted by the manufacturer, or how the puffing regimes correlated with user behavior. ...
... To date, no standard emissions outcome measures have been agreed upon, while a wide variety of metrics have been reported. Emissions have frequently been reported as the total condensed aerosol, commonly referred to as the Total Particulate Matter (TPM) yield per puff [Y TPM , (mg/puff)] and the Harmful and Potentially Harmful Constituents (HPHC) Yield [Y HPHC , (mg/puff), i.e., the mass of selected HPHCs per number of puffs] (2,3,(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32). Some studies have reported emissions in terms of the HPHC mass ratio, f HPHC (mg/mg) (i.e., the mass of selected HPHCs per unit mass of TPM) (17,26,30,31,33). ...
Article
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Many Electronic Nicotine Delivery Systems (ENDS) employ integrated sensors to detect user puffing behavior and activate the heating coil to initiate aerosol generation. The minimum puff flow rate and duration at which the ENDS device begins to generate aerosol are important parameters in quantifying the viable operating envelope of the device and are essential to formulating a design of experiments for comprehensive emissions characterization. An accurate and unbiased method for quantifying the flow condition operating envelope of ENDS is needed to quantify product characteristics across research laboratories. This study reports an accurate, unbiased method for measuring the minimum and maximum aerosolization puff flow rate and duration of seven pod-style, four pen-style and two disposable ENDS. The minimum aerosolization flow rate ranged from 2.5 to 23 (mL/s) and the minimum aerosolization duration ranged from 0.5 to 1.0 (s) across the ENDS studied. The maximum aerosolization flow rate was defined to be when the onset of liquid aspiration was evident, at flow rates ranging from 50 to 88 (mL/s). Results are presented which provide preliminary estimates for the effective maximum aerosolization flow rate and duration envelope of each ENDS. The variation in operating envelope observed between ENDS products of differing design by various manufacturers has implications for development of standardized emissions testing protocols and data reporting required for regulatory approval of new products.
... Its presence in e-cigarette liquids; however, remains unregulated as the margin of exposure in some marketed e-cigarette liquids have been shown to far exceed that found in food . Further, acetoin is a known precursor to diacetyl formation in e-cigarette liquids (Vas et al., 2019), where diacetyl has been identified in a large number of sweet flavored liquids (Farsalinos et al., 2015). Importantly, occupational inhalation of diacetyl has previously demonstrated to cause serious human respiratory outcomes (bronchiolitis obliterans) (Kreiss et al., 2002). ...
Article
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Numerous flavoring chemicals are added to e-cigarette liquids to create various flavors. Flavorings provide sensory experience to users and increase product appeal; however, concerns have been raised about their potential inhalation toxicity. Estimating potential health risk of inhaling these chemicals has been challenging since little is known about their actual concentrations in e-cigarette products. To date, a limited number of analytical methods exist to measure the concentrations of flavoring chemicals in e-cigarette products. We have developed an analytical method that accurately and precisely measures the concentrations of 20 flavoring chemicals of potential inhalation risk concerns: 2,3,5-trimethylpyrazine, acetoin, benzaldehyde, benzyl alcohol, butanoic acid, dl-limonene, ethyl maltol, ethyl salicylate, ethyl vanillin, eucalyptol, eugenol, furaneol, isovanillin, l-menthol, maltol, methyl salicylate, pulegone, trans-cinnamaldehyde, triacetin, and vanillin. Calibration and QC solutions were prepared in 50:50 propylene glycol (PG):vegetable glycerin (VG) and 5% H2O and flavoring concentrations ranging from 0.02 to 10.00 mg/ml. Samples of commercial e-cigarette liquids, calibration and QC solutions were combined with 30 µL of an internal standard mix (benzene-d6, pyridine-d5, chlorobenzene-d5, naphthalene-d8 and acenaphthene-d10; 1 mg/ml each) and were diluted 100-fold into methanol. Analysis was performed on an Agilent 7890B/7250 GC/Q-TOF using a DB-624UI column (30 m x 0.25 mmID x 1.4 μm film thickness), with a total runtime of 13.5 min. Calibration curves were fit using a weighted quadratic model and correlations of determination (r 2) values exceeded 0.990 for all chemicals. Bias and precision tests yielded values less than 20% and lower limits of quantitation (LLOQ) ranged from 0.02 to 0.63 mg/ml. Over 200 commercially available products, purchased or collected from adult e-cigarette users and spanning a range of flavor categories, were evaluated with this method. Concentrations of pulegone, a suspected carcinogen, varied from below limit of quantitation (BLOQ) to 0.32 mg/ml, while acetoin and vanillin, known precursors to more cytotoxic byproducts, ranged from BLOQ to 1.52 mg/ml and from BLOQ to 16.22 mg/ml, respectively. This method features a wide dynamic working range and allows for a rapid routine analysis of flavoring additives in commercial e-cigarette liquids.
Article
Objective Electronic cigarette (e-cigarette) use is growing significantly worldwide, especially among young people. This product has been associated with renormalizing smoking and hindering quit attempts in smokers. Moreover, among nonsmokers, it can lead to subsequent cigarette smoking and nicotine dependence. The present study aimed to assess the epidemiological profile of e-cigarette users worldwide. Study Design A systematic review was performed using 3 main electronic databases (Medline/PubMed, SCOPUS, EMBASE). Studies were independently assessed by 2 reviewers based on established eligibility criteria. The risk of bias was assessed using the MAStARI critical appraisal instrument. Results From 4,496 records, 43 were included. Among the 1,238,392 participants, 132,786 (10.72%) were e-cigarette users. The age range with the highest percentage of e-cigarette users was 18-24 years old, with 40,989 (30.86%) males, 34,875 (26.26%) females, and 33.6% being current cigarette smokers. The highest prevalence of users was 52.88% in Croatia, 49.62% in New Zealand. Other possible correlations were observed with e-cigarettes use, such as a high level of education. Conclusion Overall, e-cigarette users tended to be male young adults with a higher level of education. The highest prevalence of use was found in Croatia. This systematic review provides valuable information to improve the development of appropriate intervention strategies targeting e-cigarette users for more accurate anti-smoking actions.
E-cigarettes cause harm to adolescent users. The devices and constituents create multiple substances which are toxic on inhalation, including nicotine, metallic nanoparticles, particulate matter, and carbonyls. In addition, there is a robust relationship between youth vaping and use of combustible cigarettes as adults. This finding is based on longitudinal research and is found among youth who were at low risk for use of combustible cigarettes. Therefore, the most substantially confirmed health hazard of youth vaping is creating a new generation of smokers of combustible cigarettes and the documented health risks of such use. The physiological and psychological harms of nicotine dependence during adolescence also have been well documented. Additionally, population-based research has shown a consistent link between current vaping and respiratory issues during adolescence itself. Significant lung disease (EVALI) has occurred in adolescents and not all cases are linked to vitamin E acetate. Finally, extrapolating research on adults to adolescents raises the possibility that e-cigarette use is linked to pre-symptomatic cardiovascular dysfunction and may have a significant health impact during adulthood. The combination of this evidence, from pre-clinical to population-based longitudinal studies, conclusively demonstrates that e-cigarettes are not safe for youth.
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Various cardiopulmonary pathologies associated with electronic cigarette (EC) vaping have been reported. This study investigated the differential adverse effects of heating-associated by-products versus the intact components of EC aerosol to the lungs and heart of mice. We further dissected the roles of caspase recruitment domain-containing protein 9 (CARD9)-associated innate immune response and NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in EC exposure-induced cardiopulmonary injury. C57BL/6 wild type (WT), CARD9−/−, and NLRP3−/− mice were exposed to EC aerosol 3 h/day, 5 days/week for 6 month with or without heating the e-liquid with exposure to ambient air as the control. In WT mice, EC exposure with heating (EwH) significantly increased right ventricle (RV) free wall thickness at systole and diastole. However, EC exposure without heating (EwoH) caused a significant decrease in the wall thickness at systole. RV fractional shortening was also markedly reduced following EwH in WT and NLRP3−/− mice. Further, EwH activated NF-κB and p38 MAPK inflammatory signaling in the lungs, but not in the RV, in a CARD9- and NLRP3-dependent manner. Levels of circulatory inflammatory mediators were also elevated following EwH, indicating systemic inflammation. Moreover, EwoH activated TGF-β1/SMAD2/3/α-SMA fibrosis signaling in the lungs but not the RV of WT mice. In conclusion, EC aerosol exposure following EwH or EwoH induced differential cardiopulmonary remodeling and CARD9 innate immune and NLRP3 inflammasome contributed to the adverse effects.
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Explica por que no se puede asegurar que el cigarrillo electrónico sea seguro y su prohibición en Argentina desde 2011.
Article
Tobacco and cannabis use in pregnancy are associated with increased adverse perinatal and long-term offspring outcomes. Products for both have evolved with various forms available on the market, challenging accurate counseling of risks and quantification of tobacco and cannabis usage during the perinatal period. Health care providers are recommended to screen for any type of use, provide consistent messaging of harms of tobacco and cannabis use in pregnancy, and offer individualized interventions. The journey to cessation can be complicated by barriers and triggers, lack of social supports, and mental health challenges that should be addressed to prevent relapse and withdrawals.
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Electronic cigarettes (e-cigs) are used by millions of adolescents and adults worldwide. Commercial e-liquids typically contain flavorants, propylene glycol, and vegetable glycerin with or without nicotine. These chemical constituents are detected and evaluated by chemosensory systems to guide and modulate vaping behavior and product choices of e-cig users. The flavorants in e-liquids are marketing tools. They evoke sensory percepts of appealing flavors through activation of chemical sensory systems to promote the initiation and sustained use of e-cigs. The vast majority of flavorants in e-liquids are volatile odorants, and as such, the olfactory system plays a dominant role in perceiving these molecules that enter the nasal cavity either orthonasally or retronasally during vaping. In addition to flavorants, e-cig aerosol contains a variety of by-products generated through heating the e-liquids, including odorous irritants, toxicants, and heavy metals. These harmful substances can directly and adversely impact the main olfactory epithelium (MOE). In this article, we first discuss the olfactory contribution to e-cig flavor perception. We then provide information on MOE cell types and their major functions in olfaction and epithelial maintenance. Olfactory detection of flavorants, nicotine, and odorous irritants and toxicants are also discussed. Finally, we discuss the cumulated data on modification of the MOE by flavorant exposure and toxicological impacts of formaldehyde, acrolein, and heavy metals. Together, the information presented in this overview may provide insight into how e-cig exposure may modify the olfactory system and adversely impact human health through the alteration of the chemosensory factor driving e-cig use behavior and product selections. © 2021 American Physiological Society. Compr Physiol 11:2621-2644, 2021.
Article
Our goal was to evaluate the effects of EC refill fluids and EC exhaled aerosol residue (ECEAR) on cultured human keratinocytes and MatTek EpiDerm™, a 3D air liquid interface human skin model. Quantification of flavor chemicals and nicotine in Dewberry Cream and Churrios refill fluids was done using GC–MS. The dominant flavor chemicals were maltol, ethyl maltol, vanillin, ethyl vanillin, benzyl alcohol, and furaneol. Cytotoxicity was determined with the MTT and LDH assays, and inflammatory markers were quantified with ELISAs. Churrios was cytotoxic to keratinocytes in the MTT assay, and both fluids induced ROS production in the medium (ROS-Glo™) and in cells (CellROX). Exposure of EpiDerm™ to relevant concentrations of Dewberry Cream and Churrios for 4 or 24 h caused secretion of inflammatory markers (IL-1α, IL-6, and MMP-9), without altering EpiDerm™ histology. Lab made fluids with propylene glycol (PG) or PG plus a flavor chemical did not produce cytotoxic effects, but increased secretion of IL-1α and MMP-9, which was attributed to PG. ECEAR derived from Dewberry Cream and Churrios did not produce cytotoxicity with Epiderm™, but Churrios ECEAR induced IL-1α secretion. These data support the conclusion that EC chemicals can cause oxidative damage and inflammation to human skin.
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Background: Electronic cigarette (EC) use has grown exponentially over the past few years. The purpose of this survey was to assess the characteristics and experiences of a large sample of EC users and examine the differences between those who partially and completely substituted smoking with EC use. Methods: A questionnaire was prepared, translated into 10 different languages and uploaded in an online survey tool. EC users were asked to participate irrespective of their current smoking status. Participants were divided according to their smoking status at the time of participation in two subgroups: former smokers and current smokers. Results: In total, 19,414 participants were included in the analysis, with 88 of them (0.5%) reported not being smokers at the time of EC use initiation. Complete substitution of smoking was reported by 81.0% of participants (former smokers) while current smokers had reduced smoking consumption from 20 to 4 cigarettes per day. They were using ECs for a median of 10 months. They initiated EC use with a median of 18 mg/mL nicotine-concentration liquids; 21.5% used higher than 20 mg/mL. Only 3.5% of participants were using 0-nicotine liquids at the time of the survey. Former smokers were highly dependent (Fagerström Test for Cigarette Dependence = 7) and were heavier smokers (21 cigarettes per day when smoking) compared to current smokers. The most important reasons for initiating EC use for both subgroups was to reduce the harm associated with smoking and to reduce exposure of family members to second-hand smoking. Most considered ECs as less harmful than tobacco cigarettes, while 11.0% considered them absolutely harmless. Side effects were reported by more than half of the participants (59.8%), with the most common being sore/dry mouth and throat; side effects were mild and in most cases were subsequently resolved (partially or completely). Participants experienced significant benefits in physical status and improvements in pre-existing disease conditions (including respiratory disease such as asthma and chronic obstructive lung disease). Being former smoker was independently associated with positive effects in health and improvements in disease conditions. Conclusions: The results of this worldwide survey of dedicated users indicate that ECs are mostly used to avoid the harm associated with smoking. They can be effective even in highly-dependent smokers and are used as long-term substitutes for smoking. High levels of nicotine are used at initiation; subsequently, users try to reduce nicotine consumption, with only a small minority using non-nicotine liquids. Side effects are minor and health benefits are substantial, especially for those who completely substitute smoking with EC use. Further population and interventional studies are warranted.
Article
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A wide range of electronic cigarette (EC) devices, from small cigarette-like (first-generation) to new-generation high-capacity batteries with electronic circuits that provide high energy to a refillable atomizer, are available for smokers to substitute smoking. Nicotine delivery to the bloodstream is important in determining the addictiveness of ECs, but also their efficacy as smoking substitutes. In this study, plasma nicotine levels were measured in experienced users using a first- vs. new-generation EC device for 1 hour with an 18 mg/ml nicotine-containing liquid. Plasma nicotine levels were higher by 35-72% when using the new- compared to the first-generation device. Compared to smoking one tobacco cigarette, the EC devices and liquid used in this study delivered one-third to one-fourth the amount of nicotine after 5 minutes of use. New-generation EC devices were more efficient in nicotine delivery, but still delivered nicotine much slower compared to tobacco cigarettes. The use of 18 mg/ml nicotine-concentration liquid probably compromises ECs' effectiveness as smoking substitutes; this study supports the need for higher levels of nicotine-containing liquids (approximately 50 mg/ml) in order to deliver nicotine more effectively and approach the nicotine-delivery profile of tobacco cigarettes.
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Background: A major characteristic of the electronic cigarette (EC) market is the availability of a large number of different flavours. This has been criticised by the public health authorities, some of whom believe that diverse flavours will attract young users and that ECs are a gateway to smoking. At the same time, several reports in the news media mention that the main purpose of flavour marketing is to attract youngsters. The importance of flavourings and their patterns of use by EC consumers have not been adequately evaluated, therefore, the purpose of this survey was to examine and understand the impact of flavourings in the EC experience of dedicated users. Methods: A questionnaire was prepared and uploaded in an online survey tool. EC users were asked to participate irrespective of their current smoking status. Participants were divided according to their smoking status at the time of participation in two subgroups: former smokers and current smokers. Results: In total, 4,618 participants were included in the analysis, with 4,515 reporting current smoking status. The vast majority (91.1%) were former smokers, while current smokers had reduced smoking consumption from 20 to 4 cigarettes per day. Both subgroups had a median smoking history of 22 years and had been using ECs for 12 months. On average they were using three different types of liquid flavours on a regular basis, with former smokers switching between flavours more frequently compared to current smokers; 69.2% of the former subgroup reported doing so on a daily basis or within the day. Fruit flavours were more popular at the time of participation, while tobacco flavours were more popular at initiation of EC use. On a scale from 1 (not at all important) to 5 (extremely important) participants answered that variability of flavours was "very important" (score = 4) in their effort to reduce or quit smoking. The majority reported that restricting variability will make ECs less enjoyable and more boring, while 48.5% mentioned that it would increase craving for cigarettes and 39.7% said that it would have been less likely for them to reduce or quit smoking. The number of flavours used was independently associated with smoking cessation. Conclusions: The results of this survey of dedicated users indicate that flavours are marketed in order to satisfy vapers' demand. They appear to contribute to both perceived pleasure and the effort to reduce cigarette consumption or quit smoking. Due to the fact that adoption of ECs by youngsters is currently minimal, it seems that implementing regulatory restrictions to flavours could cause harm to current vapers while no public health benefits would be observed in youngsters. Therefore, flavours variability should be maintained; any potential future risk for youngsters being attracted to ECs can be sufficiently minimized by strictly prohibiting EC sales in this population group.
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Background: Electronic cigarettes (ECs) have been marketed as an alternative-to-smoking habit. Besides chemical studies of the content of EC liquids or vapour, little research has been conducted on their in vitro effects. Smoking is an important risk factor for cardiovascular disease and cigarette smoke (CS) has well-established cytotoxic effects on myocardial cells. The purpose of this study was to evaluate the cytotoxic potential of the vapour of 20 EC liquid samples and a "base" liquid sample (50% glycerol and 50% propylene glycol, with no nicotine or flavourings) on cultured myocardial cells. Included were 4 samples produced by using cured tobacco leaves in order to extract the tobacco flavour. Methods: Cytotoxicity was tested according to the ISO 10993-5 standard. By activating an EC device at 3.7 volts (6.2 watts-all samples, including the "base" liquid) and at 4.5 volts (9.2 watts-four randomly selected samples), 200 mg of liquid evaporated and was extracted in 20 mL of culture medium. Cigarette smoke (CS) extract from three tobacco cigarettes was produced according to ISO 3308 method (2 s puffs of 35 mL volume, one puff every 60 s). The extracts, undiluted (100%) and in four dilutions (50%, 25%, 12.5%, and 6.25%), were applied to myocardial cells (H9c2); percent-viability was measured after 24 h incubation. According to ISO 10993-5, viability of <70% was considered cytotoxic. Results: CS extract was cytotoxic at extract concentrations >6.25% (viability: 76.9 ± 2.0% at 6.25%, 38.2 ± 0.5% at 12.5%, 3.1 ± 0.2% at 25%, 5.2 ± 0.8% at 50%, and 3.9 ± 0.2% at 100% extract concentration). Three EC extracts (produced by tobacco leaves) were cytotoxic at 100% and 50% extract concentrations (viability range: 2.2%-39.1% and 7.4%-66.9% respectively) and one ("Cinnamon-Cookies" flavour) was cytotoxic at 100% concentration only (viability: 64.8 ± 2.5%). Inhibitory concentration 50 was >3 times lower in CS extract compared to the worst-performing EC vapour extract. For EC extracts produced by high-voltage and energy, viability was reduced but no sample was cytotoxic according to ISO 10993-5 definition. Vapour produced by the "base" liquid was not cytotoxic at any extract concentration. Cell survival was not associated with nicotine concentration of EC liquids. Conclusions: This study indicates that some EC samples have cytotoxic properties on cultured cardiomyoblasts, associated with the production process and materials used in flavourings. However, all EC vapour extracts were significantly less cytotoxic compared to CS extract.
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Smokers of any age can reap substantial health benefits by quitting. In fact, no other single public health effort is likely to achieve a benefit comparable to large-scale smoking cessation. Surveys document that most smokers would like to quit, and many have made repeated efforts to do so. However, conventional smoking cessation approaches require nicotine addicted smokers to abstain from tobacco and nicotine entirely. Many smokers are unable -- or at least unwilling -- to achieve this goal, and so they continue smoking in the face of impending adverse health consequences. In effect, the status quo in smoking cessation presents smokers with just two unpleasant alternatives: quit or suffer the harmful effects of continuing smoking. But, there is a third choice for smokers: tobacco harm reduction. It involves the use of alternative sources of nicotine, including modern smokeless tobacco products like snus and the electronic cigarette (E-cig), or even pharmaceutical nicotine products, as a replacement for smoking. E-cigs might be the most promising product for tobacco harm reduction to date, because, besides delivering nicotine vapour without the combustion products that are responsible for nearly all of smoking's damaging effect, they also replace some of the rituals associated with smoking behaviour. Thus it is likely that smokers who switch to E-cigs will achieve large health gains. The focus of this article is on the health effects of using an E-cig, with consideration given to the acceptability, safety and effectiveness of this product as a long-term substitute for smoking.
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Background: Although millions of people are using electronic cigarettes (ECs) and research on this topic has intensified in recent years, the pattern of EC use has not been systematically studied. Additionally, no comparative measure of exposure and nicotine delivery between EC and tobacco cigarette or nicotine replacement therapy (NRTs) has been established. This is important, especially in the context of the proposal for a new Tobacco Product Directive issued by the European Commission. Methods: A second generation EC device, consisting of a higher capacity battery and tank atomiser design compared to smaller cigarette-like batteries and cartomizers, and a 9 mg/mL nicotine-concentration liquid were used in this study. Eighty subjects were recruited; 45 experienced EC users and 35 smokers. EC users were video-recorded when using the device (ECIG group), while smokers were recorded when smoking (SM-S group) and when using the EC (SM-E group) in a randomized cross-over design. Puff, inhalation and exhalation duration were measured. Additionally, the amount of EC liquid consumed by experienced EC users was measured at 5 min (similar to the time needed to smoke one tobacco cigarette) and at 20 min (similar to the time needed for a nicotine inhaler to deliver 4 mg nicotine). Results: Puff duration was significantly higher in ECIG (4.2 ± 0.7 s) compared to SM-S (2.1 ± 0.4 s) and SM-E (2.3 ± 0.5 s), while inhalation time was lower (1.3 ± 0.4, 2.1 ± 0.4 and 2.1 ± 0.4 respectively). No difference was observed in exhalation duration. EC users took 13 puffs and consumed 62 ± 16 mg liquid in 5 min; they took 43 puffs and consumed 219 ± 56 mg liquid in 20 min. Nicotine delivery was estimated at 0.46 ± 0.12 mg after 5 min and 1.63 ± 0.41 mg after 20 min of use. Therefore, 20.8 mg/mL and 23.8 mg/mL nicotine-containing liquids would deliver 1 mg of nicotine in 5 min and 4 mg nicotine in 20 min, respectively. Since the ISO method significantly underestimates nicotine delivery by tobacco cigarettes, it seems that liquids with even higher than 24 mg/mL nicotine concentration would be comparable to one tobacco cigarette. Conclusions: EC use topography is significantly different compared to smoking. Four-second puffs with 20-30 s interpuff interval should be used when assessing EC effects in laboratory experiments, provided that the equipment used does not get overheated. Based on the characteristics of the device used in this study, a 20 mg/mL nicotine concentration liquid would be needed in order to deliver nicotine at amounts similar to the maximum allowable content of one tobacco cigarette (as measured by the ISO 3308 method). The results of this study do not support the statement of the European Commission Tobacco Product Directive that liquids with nicotine concentration of 4 mg/mL are comparable to NRTs in the amount of nicotine delivered to the user.
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The rapid growth in the use of electronic cigarettes has been accompanied by substantial discussions by governments, international organisations, consumers and public health experts about how they might be regulated. In the European Union they are currently regulated under consumer legislation but new legislation will regulate them under the Tobacco Products Directive. However, several countries have sought to regulate them under medicines regulations. These claims have been successfully challenged in 6 court cases in European states. Under European legislation a product may be deemed to be a medicine by function if it is used in or administered to human beings either with a view to restoring, correcting or modifying physiological functions by exerting a pharmacological, immunological or metabolic action, or to making a medical diagnosis. It is a medicine by presentation if it is presented (eg by a manufacturer or distributor) as having properties for treating or preventing disease in human beings. We assess the legal and scientific basis for the claim that electronic cigarettes should be regulated as medicines. We conclude that they are neither medicine by function nor necessarily by presentation The main reason for their existence is as a harm reduction product in which the liking for and/or dependence on nicotine is maintained, and adoption of use is as a substitute for smoking and not as a smoking cessation product. In reality, they are used as consumer products providing pleasure to the user. They are not used to treat nicotine addiction or other disease, but to enable continued use of nicotine. Their use is adjusted individually by each consumer according to his or her perceived pleasure and satisfaction. Gaps in current regulation regarding safety and quality can be met by tailored regulations.
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Context: Electronic cigarettes (ECs) are used as alternatives to smoking; however, data on their cytotoxic potential are scarce. Objective: To evaluate the cytotoxic potential of 21 EC liquids compared to the effects of cigarette smoke (CS). Methods: Cytotoxicity was evaluated according to UNI EN ISO 10993-5 standard. By activating an EC device, 200 mg of liquid was evaporated and was extracted in 20 ml of culture medium. CS extract from one cigarette was also produced. The extracts, undiluted (100%) and in five dilutions (50%, 25%, 12.5%, 6.25% and 3.125%), were applied to cultured murine fibroblasts (3T3), and viability was measured after 24-hour incubation by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Viability of less than 70% was considered cytotoxic. Results: CS extract showed cytotoxic effects at extract concentrations above 12.5% (viability: 89.1 ± 3.5% at 3.125%, 77.8 ± 1.8% at 6.25%, 72.8 ± 9.7% at 12.5%, 5.9 ± 0.9% at 25%, 9.4 ± 5.3% at 50% and 5.7 ± 0.7% at 100% extract concentration). Range of fibroblast viability for EC vapor extracts was 88.5-117.8% at 3.125%, 86.4-115.3% at 6.25%, 85.8-111.7% at 12.5%, 78.1-106.2% at 25%, 79.0-103.7% at 50% and 51.0-102.2% at 100% extract concentration. One vapor extract was cytotoxic at 100% extract concentration only (viability: 51.0 ± 2.6%). However, even for that liquid, viability was 795% higher relative to CS extract. Conclusions: This study indicates that EC vapor is significantly less cytotoxic compared tobacco CS. These results should be validated by clinical studies.