Evaluation of electronic cigarette liquids and vapour for the presence of selected inhalation toxins.
Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:
Keywords:
Nicotine & Tobacco Research
NTR-2014-374.R2 Original Investigation 18-Aug-2014 Farsalinos, Konstantinos; Konstantinos; Onassis Cardiac Surgery Center, Cardiology Kistler, Kurt; The Pennsylvania State University, Chemistry Gillman, Gene; Enthalpy Analytical, Voudris, Vassilis; Onassis Cardiac Surgey C enter, Cardiology Public health, Biochemistry, Health consequences, Prevention
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Title: Evaluation of electronic cigarette liquids and aerosol for the presence of selected
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inhalation toxins.
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Authors: Konstantinos E. Farsalinos, MD , Kurt A. Kistler, PhD , Gene Gillman, PhD , Vassilis 1
Voudris, PhD
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Department of Cardiology, Onassis Cardiac Surgery Center, Sygrou 356, Kallithea 17674,
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Greece.
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Department of Chemistry, The Pennsylvania State University Brandywine, 25 Yearsley Mill
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Road, Media, Pennsylvania 19063, USA.
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Enthalpy Analytical, Inc., 800 Capitola Drive, Suite 1, Durham, NC 27713.
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Abstract
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Introduction. The purpose of this study was to evaluate sweet-flavoured electronic cigarette
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(EC) liquids for the presence of diacet yl (DA) and acetyl propionyl (AP), which are chemicals
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approved for food use but are associated with respiratory disease when inhaled.
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Methods. In total, 159 samples were purchased from 36 manufacturers and retailers from 7
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countries. Additionally, three liquids were prepared by dissolving a concentrated flavour sample
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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
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and AP.
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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
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exposure levels were 56µg/day 56µg/da y (IQR: 26-278µg/day) for DA and 91µg/day (IQR: 20-432µg/da y)
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INTRODUCTION
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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
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form through heating a nicotine-containing solution resulting in the production of visible
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“vapour”. Besides nicotine delivery, they address the whole smoking ritual and psycho behavioural dependence through sensory sensor y stimulation and motor simulation (Farsalinos &
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Stimson, 2014).
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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
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flavourings has resulted in a large debate among public health professionals and regulators,
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suggesting that they can be attractive to youth. A recent survey of dedicated users (vapers)
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to specific flavours (Farsalinos et al., 2013b; Romagna et al., 2013; Bahl et al., 2012), indicating
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that further research is certainly needed in this area.
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Besides the lack of studies for the effects of flavouring substances when inhaled, there
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are some chemicals which, although approved for ingestion, have already established adverse
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health effects when inhaled. A characteristic example of this is diacetyl (DA, Figure 1). This
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substance, also known as 2,3-butanedione, is a member of a general class of organic compounds referred to as diketones, α-diketones or o r α-dicarbonyls. It is responsible for providing a
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characteristic buttery flavour, and is both naturally found in foods and used as a synthetic
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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
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(National Institute for Occupational Safety and Health, 2011; FEMA Nr 2370), it has been
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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
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Manuscripts submitted to Nicotine & Tobacco Research
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induced bronchiolitis obliterans in popcorn workers, because it adds the desired flavour while
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claims of “diacetyl-free” can be made by the manufacturer. Unfortunately, the risks associated
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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
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and AP, regulatory agencies have set specific Occupational Exposure Limits (OELs). For DA,
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the National Institute on Occupational Safety and Hazards (NIOSH) has proposed an upper limit 3
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of 5ppb (18�g/m ) for 8h Time-Weighted Average exposure (TWA) and 25ppb (88�g/m ) as
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Short-Term Exposure Limit (STEL) for 15 minutes, while the Scientific Committee on
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Occupational Exposure Limits (SCOEL) of the European Commission considered the NIOSH3
defined limits for DA unnecessarily strict and has set upper limits of 20ppb (70�g/m ) and
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100ppb (360�g/m ) respectively (European Commission, 2013; National Institute for
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Occupational Safety and Health, 2011). For AP, NIOSH has set a TWA limit of 9.3ppb 3
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3
(38�g/m ) and an STEL of 31ppb (127�g/m ) (National Institute for Occupational Safety and Health, 2011).
Manuscripts submitted to Nicotine & Tobacco Research
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Samples of EC liquids were selected from European and US manufacturers and retailers.
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The selection was based on information from local or international EC consumers’ forums, in
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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,
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milky, cream, chocolate, coffee, caramel, etc). A total of 159 samples were selected from 36
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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
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concentrated flavours (n=46), which are diluted by users in “base” liquids (mixtures of propylene
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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
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statements in the manufacturers’ websites that no DA was present in their liquids. All samples
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were bought anonymously from internet shops, without mentioning that the purpose of the
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purchase was to be analyzed for a scientific study. All bottles were received sealed, and were
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immediately sent to the laboratory for analysis.
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Manuscripts submitted to Nicotine & Tobacco Research
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was evaluated for recovery from the sample matrix by addition of known amount of DA and AP
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before derivatization. In all cases the recovery of both compounds was greater than 80%. To
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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
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allowed to derivatize for 20 minutes, then quenched with 0.050mL of pyridine. This ensures that
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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
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in the same manner as samples, and were used to prepare a linear calibration curve which ranged
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from 0.4-30µg/mL. All e-liquid samples were anal yzed at an initial 22-fold dilution, while pure flavour samples were analyzed at an initial 43-fold dilution. At these dilutions, the maximum
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amount of propylene glycol and glycerol in the DNPH solution was less than 5% and had no
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effect on derivatization. The efficient derivatization of DA and AP requires excess DNPH, and
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all samples were evaluated for DNPH depletion by verifying that a large DNPH peak was
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observed by HPLC. Any samples that were found to have depleted DNPH were prepared and
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The materials used for the HPLC analysis were: deionized water – Millipore; phosphoric
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acid (H3PO4), 85%, A.C.S Reagent, Sigma-Aldrich (P/N 438081); DNPH (50%), TCI America,
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P/N D0845; acetonitrile (CAS #75-05-8), HPLC grade; tetrah ydrofuran (CAS #109-99-9), HPLC grade; isopropanol (CAS #67-63-0), distilled-in-glass; pyridine (CAS #110 -86-1); diacetyl (97%)
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Sigma-Aldrich (P/N B85307) (CAS #431-03-8); 2,3-pentanedione (97%) S igma-Aldrich (P/N
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241962) (CAS # 600-14-6).
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Aerosol production and analysis To evaluate the amount of DA and AP that is transferred trans ferred from liquid to aerosol, three
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liquids were prepared by diluting the sample of concentrated flavour with the highest level of
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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
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air-piston mechanism to push the activation button. The aerosol was passed through an impinger
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containing 35mL of the DNPH trapping solution without the use of a filter pad. Once the aerosol
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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.
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Interpreting NIOSH safety limits in the context of EC liquids 3
The TWA limits (8-hours exposure) defined b y NIOSH (5ppb, i.e. 18µg/m for DA and
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9.3ppb, i.e. 38µg/m for AP) were used as a guide to define potentially “acceptable” levels of DA
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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 0.5 L (Barrett and Ganong, 2012). Within 8 hours (480min), the total
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3
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volume of air inhaled is 3.6m ([0.5L x 15breaths/min x 480min] / 1000L/m ). Thus, the total
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3
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amount of DA that can be inhaled daily (according to NIOSH limits) is 65µg 6 5µg (18µg/m x 3.6m ), 3
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while for AP it is 137µg (38µg/m x 3.6m ).
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differences between European countries and US in the number of samples containing DA and
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AP. Pearson’s correlation coefficient was used to assess the correlation between expected and
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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
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vapers (Farsalinos et al., 2014b). To assess the difference in DA and AP daily exposure between
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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
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analysis (SPSS v. 18, Chicago, IL, USA).
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RESULTS
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Analysis of liquid samples
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In 41 (25.8%) samples DA and AP was not detected, while in 73 (45.9%) samples one of
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the two chemicals was detected and in 45 (28.3%) samples both chemicals were detected. DA
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limit (65µg/day). However, 52 samples (47.3% of the positive samples) would expose consumers
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to levels higher than the NIOSH limits, with 26 of them (23.6%) having >5 times higher levels
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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.
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AP was found in 53 (33.3%) samples, containing a median concentration of 44µg/mL
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(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 flavo urs
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contained 3 times higher levels of AP compared to refill liquids (median: 124µg/mL vs.
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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
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concentrated flavours and 1018µg/mL in refills. AP was detected in the samples of 24
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manufacturers (66.7%) from 6 countries (23.1% of European and 43.2% of US samples, chi-
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square P=0.007). By converting the levels of AP found in concentrated flavours to represent
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realistic exposure (see Statistical analysis section), it was estimated that the median daily
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One concentrated flavour sample was diluted to 5%, 10% and 20% into a mixture of 50%
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propylene glycol and 50% glycerol, in order to prepare the 3 liquids used for the aerosol analysis.
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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
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for the 10% solution, and 7546µg/mL and 606µg/mL for the 20% solution. Based on the weight-
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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
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specific weight of the samples (which was determined to be 1.13). From that, the concentrations
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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
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between the expected (based on the liquid consumption) and the observed ( measured) DA and
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AP concentrations (R =0.997 and 0.995 respectively, Figure 3). These results indicate that both
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DA and AP are readily delivered from the liquid to the aerosol.
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91µg respectively, which are 100 and 10 times lower compared to smoking (Mann-Whitney
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P<0.001 for DA and P=0.020 for AP).
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DISCUSSION
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Main findings This is the first study to analyze a large number of EC liquids with sweet flavours
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obtained from a variety of manufacturers and retailers from Europe and the US for the presence
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of DA and AP. The main findings were that these substances were present in the majority of the
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samples tested, with a significant proportion containing both chemicals; they were detected even
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in samples coming from manufacturers who clearly stated that they were not present in their
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products. Additionally, it was determined that both DA and AP are readily delivered to the
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aerosol that the vaper inhales, an expected finding considering the volatility of these compounds.
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Although the median levels found were slightly lower than the strict NIOSH-defined safety
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inhaling food-approved substances. While many food flavourings have never been tested for
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inhalation safety, the focus here was on known inhalation toxins that are flavour compounds.
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Toxicity of DA and AP
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DA is a water soluble volatile α-diketone that is both a natural constituent of numerous
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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
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volatile organic compound in air samples from microwave popcorn plants and flavouring
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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
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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
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microwave popcorn production plant in Missouri and found that these workers had 2.6 times the
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expected rate of respiratory symptoms such as chronic cough and shortness of breath and 3.3
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AP is chemically and structurally s tructurally almost identical to DA, has a similar s imilar buttery, creamy
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flavour, and has been used as a DA substitute in many flavouring manufacturing facilities (Day
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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.,
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2012).
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Implications of the study findings
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A wide range of DA and AP concentrations were found in the samples, indicating that in
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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
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100 times lower for DA and 10 times lower for AP compared to tobacco tobac co cigarettes; therefore, it
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is still plausible to classify ECs as tobacco harm reduction products (Polosa et al., 2013).
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However, the major source of DA and AP in tobacco cigarette smoke is the combustion process
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(Pierce et al., 2014); thus, it is an unavoidable risk. In EC liquids, these chemicals are introduced
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process. The results of the aerosol analysis, showing that DA and AP are readily delivered from
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the liquid to the aerosol, indicate that analysis of the liquid is sufficient.
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Limitations
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Our selection was targeted to sweet-only flavours because it was expected that these are
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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
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AP. However, we cannot exclude the possibility that there may be liquids from other flavour
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types (besides sweets) which contain these compounds. Fewer samples contained AP compared to DA. This was unexpected, since it has been
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common practice for the flavouring industry to substitute DA with alternative chemicals due to
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the criticism for the adverse effects of DA exposure to workers. It is unknown whether this is a
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generalized finding in the EC liquid market or it is attributed to chance related to the selection of
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the samples.
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flavourings with alternative chemicals; thus, there is no need to exclude them from the market,
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since they have been found to be quite popular among dedicated users.
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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
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found in tobacco smoke while smoking is not a risk factor for development the disease.
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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
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in active smokers is estimated to be 15.4% (Raherison & Girodet, 2009), by far higher than the
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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
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examinations have shown that many smokers have histopathological features of respiratory
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bronchiolitis (Niewoehner et al., 1974).
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Conclusion
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products and changing formulations, without the need to limit the availability of sweet flavours
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in the market.
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Funding
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This study was funded through an open internet crowd-funding campaign which was conducted
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in the website www.indiegogo.com. Declaration of interests
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Some of the studies by KF and VV were performed using funds provided to the institution by e-
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cigarette companies. KK and GG have no conflict of interest to report.
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Acknowledgements
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We would like to thank Dimitris Agrafiotis (a volunteer vaping advocate) for his as sistance in
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organising the crowd-funding campaign and in the selection of EC liquid samples.
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Figure legends
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Figure 1. Chemical structures of diacetyl diacet yl (DA) and acetyl propionyl (AP).
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Figure 2. Box-plots of the estimated daily exposure to diacetyl (A) and acetyl propionyl (B)
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th
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th
from the liquid samples tested. The box represents the 25 and 75 percentiles, with the line th
th
inside the box showing the median value. The error bars represent the 10 and 90 percentiles.
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The dotted line represents the maximum acceptable levels of daily exposure estimated from the NIOSH limit for occupational exposure.
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Figure 3. Correlation between the expected (based on liquid consumption during aerosol
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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
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F o r P e e r R e v i e w
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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 l ine 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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DA
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AP
8000
800
n o i t a r t 6000 n e ) c L n m o / c g 4000 µ d ( e t c e p 2000 x E
y = 0.9526x + 182.64 R² = 0.9966
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
y = 1.2435x - 25.483 R² = 0.9947
n o i t 600 a r t n e ) c L n m o / 400 c g µ d ( e t c e 200 p x E
0
0 0
2000
4000
6000
Measured concentration (µg/mL)
8000
0
200
400
600
Measured concentration (µg/mL)
800
