rip t Manuscripts submitted to Nicotine & Tobacco Research Nicotine & Tobacco Research Advance Access published September 1, 2014 Date Submitted by the Author: Complete List of Authors: 18-Aug-2014 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 Public health, Biochemistry, Health consequences, Prevention Ac ce pt e d Keywords: Original Investigation an u Manuscript Type: NTR-2014-374.R2 http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 Manuscript ID: Nicotine & Tobacco Research M Journal: sc Evaluation of electronic cigarette liquids and vapour for the presence of selected inhalation toxins. Page 1 of 27 Title: Evaluation of electronic cigarette liquids and aerosol for the presence of selected rip t inhalation toxins. Authors: Konstantinos E. Farsalinos, MD1, Kurt A. Kistler, PhD2, Gene Gillman, PhD3, Vassilis 1 sc Voudris, PhD1 Department of Cardiology, Onassis Cardiac Surgery Center, Sygrou 356, Kallithea 17674, 2 Department of Chemistry, The Pennsylvania State University Brandywine, 25 Yearsley Mill Road, Media, Pennsylvania 19063, USA. Enthalpy Analytical, Inc., 800 Capitola Drive, Suite 1, Durham, NC 27713. pt e Corresponding author d M 3 an u Greece. Konstantinos E Farsalinos, MD ce Email: kfarsalinos@gmail.com Tel: +306977454837 Ac Fax: +302109493373 http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research Abstract Introduction. The purpose of this study was to evaluate sweet-flavoured electronic cigarette rip approved for food use but are associated with respiratory disease when inhaled. t (EC) liquids for the presence of diacetyl (DA) and acetyl propionyl (AP), which are chemicals sc 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 an u 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. M 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) d for AP. They were slightly lower than the strict NIOSH-defined safety limits for occupational pt e 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. ce 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 Ac 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. http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 2 of 27 Page 3 of 27 INTRODUCTION rip t 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 sc 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- an u 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 M 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, d 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 pt e 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 ce “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’ Ac 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 ecigarette liquids and aerosol, although significantly lower than tobacco smoke, may be attributed http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research 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. t Besides the lack of studies for the effects of flavouring substances when inhaled, there rip 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 sc 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 an u 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 M (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 d in 1s (FEV1), in subjects exposed to it through inhalation. Additionally it has been implicated in pt e 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 ce DA presence in EC liquids was first mentioned in 2008 in EC consumers’ forums (http://www.ecigarette-forum.com/forum/health-safety-e-smoking/2666-inhaling-flavouring-chemicals.html). Ac 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- http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 4 of 27 Page 5 of 27 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 t with inhalation of AP may well be as high as from DA, based on inhalation studies performed on rip 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, sc the National Institute on Occupational Safety and Hazards (NIOSH) has proposed an upper limit of 5ppb (18µg/m3) for 8h Time-Weighted Average exposure (TWA) and 25ppb (88µg/m3) as an u Short-Term Exposure Limit (STEL) for 15 minutes, while the Scientific Committee on Occupational Exposure Limits (SCOEL) of the European Commission considered the NIOSHdefined limits for DA unnecessarily strict and has set upper limits of 20ppb (70µg/m3) and M 100ppb (360µg/m3) respectively (European Commission, 2013; National Institute for Occupational Safety and Health, 2011). For AP, NIOSH has set a TWA limit of 9.3ppb pt e Health, 2011). d (38µg/m3) and an STEL of 31ppb (127µg/m3) (National Institute for Occupational Safety and 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 ce 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 Ac data evaluating exposure from smoking tobacco cigarettes. METHODS Sample selection http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research 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 t order to get samples from major or popular sources. Since the chemicals examined were more rip 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 sc 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 an u 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 M 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 pt e d immediately sent to the laboratory for analysis. Methods of analysis ce The samples were analyzed by High Performance Liquid Chromatography (HPLC). The procedure followed was a modified version of the HPLC carbonyl compound analysis method Ac 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 http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 6 of 27 Page 7 of 27 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 t prevent the formation of two carbonyl adducts, an aliquot of the sample for analysis was rip 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 sc 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 an u 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 M 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 d observed by HPLC. Any samples that were found to have depleted DNPH were prepared and pt e 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 ce 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. Ac 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. http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research 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, t P/N D0845; acetonitrile (CAS #75-05-8), HPLC grade; tetrahydrofuran (CAS #109-99-9), HPLC rip 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 an u sc 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 M 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 d dilutions were chosen because they represent the most common dilutions of concentrated pt e 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, ce 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. Ac 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 http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 8 of 27 Page 9 of 27 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 t collection was complete, 5mL of this solution was quenched with 250 µL of pyridine. The sc Interpreting NIOSH safety limits in the context of EC liquids rip samples were then analyzed by HPLC monitoring at 365 nm. The TWA limits (8-hours exposure) defined by NIOSH (5ppb, i.e. 18µg/m3 for DA and an u 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 M amount of DA that can be inhaled daily (according to NIOSH limits) is 65µg (18µg/m3 x 3.6m3), pt e Statistical analysis d while for AP it is 137µg (38µg/m3 x 3.6m3). Data were examined for distribution by Kolmogorov-Smirnov test. Continuous variables were expressed as median (interquartile range [IQR]) while categorical variables were expressed ce 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 Ac 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 http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research 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 t measured DA and AP levels in the aerosol analysis. To estimate the average daily exposure, rip 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 sc 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 an u analysis (SPSS v. 18, Chicago, IL, USA). RESULTS M 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 d was found in 110 (69.2%) samples, containing a median concentration of 29µg/mL (IQR: 10- pt e 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 ce 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% Ac 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 http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 10 of 27 Page 11 of 27 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 t than the safety limit. The sample with the highest level of DA would result in 490 times higher rip daily intake compared to the NIOSH limit. AP was found in 53 (33.3%) samples, containing a median concentration of 44µg/mL sc (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 an u 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 M 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, chisquare P=0.007). By converting the levels of AP found in concentrated flavours to represent d realistic exposure (see Statistical analysis section), it was estimated that the median daily pt e 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, ce with 11 of them (20.8%) having >5 times higher levels than the safety limit. The sample with the Ac highest level of AP would result in 22 times higher daily intake compared to the NIOSH limit. Analysis of aerosol http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research 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. t The prepared liquids were analyzed by HPLC and were found to contain DA and AP at rip 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- sc 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 an u 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 M AP concentrations (R2=0.997 and 0.995 respectively, Figure 3). These results indicate that both d DA and AP are readily delivered from the liquid to the aerosol. pt e 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 ce 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 Ac 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 http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 12 of 27 Page 13 of 27 91µg respectively, which are 100 and 10 times lower compared to smoking (Mann-Whitney t P<0.001 for DA and P=0.020 for AP). rip DISCUSSION Main findings sc 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 an u 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 M 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 pt e than the safety limits. d levels, a substantial proportion of the positive samples would expose consumers to levels higher ce 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 Ac 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 http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research inhaling food-approved substances. While many food flavourings have never been tested for t inhalation safety, the focus here was on known inhalation toxins that are flavour compounds. rip Toxicity of DA and AP DA is a water soluble volatile α-diketone that is both a natural constituent of numerous sc 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 an u 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 M 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 d expected rate of respiratory symptoms such as chronic cough and shortness of breath and 3.3 pt e 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 ce 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 Ac 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). http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 14 of 27 Page 15 of 27 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 t et al., 2011). Toxicological studies in animals have shown that it has adverse effects on rip respiratory epithelium similar to DA and at similar levels (Hubbs et al., 2012; Morgan et al., sc 2012). Implications of the study findings an u 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 M 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 d (Pierce et al., 2014); thus, it is an unavoidable risk. In EC liquids, these chemicals are introduced pt e 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 ce 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, Ac 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 http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research process. The results of the aerosol analysis, showing that DA and AP are readily delivered from t the liquid to the aerosol, indicate that analysis of the liquid is sufficient. rip Limitations Our selection was targeted to sweet-only flavours because it was expected that these are sc 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 an u 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 M 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 d the samples. pt e 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 ce 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 Ac 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 http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 16 of 27 Page 17 of 27 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. t A recent study raised doubts about the association between DA and AP exposure and rip 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. sc 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 an u 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 M examinations have shown that many smokers have histopathological features of respiratory bronchiolitis (Niewoehner et al., 1974). d Conclusion pt e 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 ce 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 Ac 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 http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research products and changing formulations, without the need to limit the availability of sweet flavours t in the market. rip Funding This study was funded through an open internet crowd-funding campaign which was conducted sc in the website www.indiegogo.com. Declaration of interests an u Some of the studies by KF and VV were performed using funds provided to the institution by ecigarette companies. KK and GG have no conflict of interest to report. Acknowledgements M We would like to thank Dimitris Agrafiotis (a volunteer vaping advocate) for his assistance in pt e References d organising the crowd-funding campaign and in the selection of EC liquid samples. Akpinar-Elci, M., Travis, W. D., Lynch, D. A. & Kreiss, K. (2004). Bronchiolitis obliterans ce syndrome in popcorn production plant workers. European Respiratory Journal, 24, 298–302. doi:10.1183/09031936.04.00013903. Ac Bahl, V., Lin, S., Xu, N., Davis, B., Wang, Y. H. & Talbot, P. (2012) Comparison of electronic cigarette refill fluid cytotoxicity using embryonic and adult models. Reproductive Toxicology, 34, 529-537. doi: 10.1016/j.reprotox.2012.08.001. http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 18 of 27 Page 19 of 27 Barrett, K. E. & Ganong, W. F. (2012). Ganong's review of medical physiology. London, UK: McGraw-Hill Medical. t From the Centers for Disease Control and Prevention. (2002). Fixed obstructive lung disease rip in workers at a microwave popcorn factory--Missouri, 2000-2002. JAMA, 287, 2939-2940. sc doi:10.1001/jama.287.22.2939-JWR0612-2-1. Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA). (2013). an u CORESTA Recommended Method No. 75: Determination of Selected Carbonyls in Mainstream Cigarette Smoke by HPLC. Retrieved from: http://www.coresta.org/Recommended_Methods/CRM_75.pdf (accessed on March 19, M 2014). Day, G., LeBouf, R., Grote, A., Pendergrass, S., Cummings, K. J., Kreiss, K. & Kullman, G. (2011). Identification and measurement of diacetyl substitutes in dry bakery mix production. d Journal of Occupational and Environmental Hygiene, 8, 93-103. doi: pt e 10.1080/15459624.2011.547148. European Commission. (2013). Recommendation from the Scientific Committee on ce Occupational Exposure Limits for diacetyl. Retrieved from: http://www.ser.nl/documents/82310.pdf (accessed on May 27, 2014). Ac Farsalinos, K. E. & Stimson, G. V. (2014). Is there any legal and scientific basis for classifying electronic cigarettes as medications? International Journal of Drug Policy (published online). doi: 10.1016/j.drugpo.2014.03.003. http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research Farsalinos, K. E., Romagna, G., Tsiapras, D., Kyrzopoulos, S., Spyrou, A. & Voudris, V. (2013a). Impact of flavour variability on electronic cigarette use experience: an internet t survey. International Journal of Environmental Research and Public Health, 10, 7272-7282. rip doi: 10.3390/ijerph10127272. Farsalinos, K. E., Romagna, G., Allifranchini, E., Ripamonti, E., Bocchietto, E., Todeschi, sc S., ... Voudris, V. (2013b). Comparison of the cytotoxic potential of cigarette smoke and electronic cigarette vapour extract on cultured myocardial cells. International Journal of an u Environmental Research and Public Health, 10, 5146-5162. doi: 10.3390/ijerph10105146. Farsalinos, K. E., Romagna, G., Tsiapras, D., Kyrzopoulos, S. & Voudris, V. (2013c) Evaluation of electronic cigarette use (vaping) topography and estimation of liquid M consumption: implications for research protocol standards definition and for public health authorities' regulation. International Journal of Environmental Research and Public Health, d 10, 2500-2514. doi: 10.3390/ijerph10062500. pt e Farsalinos, K. E., Spyrou, A., Tsimopoulou, K., Stefopoulos, C., Romagna, G. & Voudris, V. (2014a). Nicotine absorption from electronic cigarette use: comparison between first and ce new-generation devices. Scientific Reports, 4, 4133. doi: 10.1038/srep04133. Farsalinos, K. E., Romagna, G., Tsiapras, D., Kyrzopoulos, S. & Voudris, V. (2014b). Ac Characteristics, perceived side effects and benefits of electronic cigarette use: a worldwide survey of more than 19,000 consumers. International Journal of Environmental Research and Public Health, 11, 4356-4373. doi: 10.3390/ijerph110404356. Flavors and Extracts Manufacturers’ Association (FEMA) (2014). Safety assessment and regulatory authority to use flavors: focus on e-cigarettes. Retrieved from: http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 20 of 27 Page 21 of 27 http://www.femaflavor.org/safety-assessment-and-regulatory-authority-use-flavors-focus-ecigarettes (accessed on May 31, 2014). rip Reviews, 25, 261–272. doi: 10.2165/00139709-200625040-00006 t Harber, P., Saechao, K. & Boomus, C. (2006). Diacetyl-induced lung disease. Toxicology sc Hubbs, A. F., Cumpston, A. M., Goldsmith, W. T., Battelli, L. A., Kashon, M. L., Jackson, M. C., ... Sriram, K. (2012). Respiratory and olfactory cytotoxicity of inhaled 2,3- an u pentanedione in Sprague-Dawley rats. American Journal of Pathology, 181, 829-844. doi: 10.1016/j.ajpath.2012.05.021. Kanwal, R., Kullman, G., Piacitelli, C., Boylstein, R., Sahakian, N., Martin, S., ... Kreiss, K. M (2006). Evaluation of flavorings-related lung disease risk at six microwave popcorn plants. Journal of Occupational and Environmental Medicine, 48, 149-157. doi: d 10.1097/01.jom.0000194152.48728.fb pt e Kim, T. J., Materna, B. L., Prudhomme, J. C., Fedan, K. B., Enright, P. L., Sahakian, N. M., ... Kreiss, K. (2010). Industry-wide medical surveillance of California flavoring manufacturing workers: cross-sectional results. American Journal of Industrial Medicine, 53, ce 857–865. doi: 10.1002/ajim.20858. Kreiss, K., Gomaa, A., Kullman, G., Fedan, K., Simoes, E. J. & Enright, P.L. (2002). Clinical Ac bronchiolitis obliterans in workers at a microwave-popcorn plant. New England Journal of Medicine, 347, 330-338. doi: 10.1056/NEJMoa020300. Lockey, J. E., Hilbert, T. J., Levin, L. P., Ryan, P. H., White, K. L., Borton, E. K., ... LeMasters, G. K. (2009). Airway obstruction related to diacetyl exposure at microwave http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research popcorn production facilities. European Respiratory Journal, 34, 63-71. doi: 10.1183/09031936.00050808. t Mathews, J. M., Watson, S. L., Snyder, R. W., Burgess, J. P. & Morgan, D.L. (2010). rip Reaction of the butter flavorant diacetyl (2-3 butanedione) with N-α-Acetylarginine: a model for epitope formation of pulmonary protein in the etiology of obliterative bronchiolitis. sc Journal of Agricultural and Food Chemistry, 58, 12761-12768. doi: 10.1021/jf103251w. an u Morgan, D.L., Jokinen, M.P., Price, H.C., Gwinn, W.M., Palmer, S.M. & Flake, G.P. (2012). Bronchial and bronchiolar fibrosis in rats exposed to 2,3-pentanedione vapors: implication for bronchiolitis obliterans in humans. Toxicologic Pathology, 40, 448-465. doi: M 10.1177/0192623311431946. National Institute for Occupational Safety and Health (NIOSH). (2011). Criteria for a recommended standard: Occupational Exposure to Diacetyl and 2,3-Pentanedione. Retrieved d from: http://www.cdc.gov/niosh/docket/archive/pdfs/NIOSH- pt e 245/DraftDiacetylCriteriaDocument081211.pdf (accessed on May 27, 2014). Niewoehner, D. E., Kleinerman, J. & Rice, D. B. (1974). Pathologic changes in the ce peripheral airways of young cigarette smokers. The New England Journal of Medicine, 291, 755–758. doi: 10.1056/NEJM197410102911503 Ac Parmet, A. J. & Von Essen, S. (2002). Rapidly progressive, fixed airway obstructive disease in popcorn workers: a new occupational pulmonary illness? Journal of Occupational and Environmental Medicine, 44, 216–218. Retrieved from: http://journals.lww.com/joem/Citation/2002/03000/Rapidly_Progressive,_Fixed_Airway_Ob structive.2.aspx (accessed on June 4, 2014). http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 22 of 27 Page 23 of 27 Pierce, J. S., Abelmann, A., Spicer, L. J., Adams, R.E. & Finley, B. L. (2014). Diacetyl and 2,3-pentanedione exposures associated with cigarette smoking: implications for risk t assessment of food and flavoring workers. Critical Reviews in Toxicology, 44, 420-35. doi: rip 10.3109/10408444.2014.882292. Polosa, R., Rodu, B., Caponnetto, P., Maglia, M. & Raciti C. (2013). A fresh look at tobacco sc harm reduction: the case for the electronic cigarette. Harm Reduction Journal, 10, 19. an u doi:10.1186/1477-7517-10-19. Raherison, C. & Girodet, P-O. (2009). Epidemiology of COPD. European Respiratory Review, 18, 213-221. doi:10.1183/09059180.00003609 M Regan, A. K., Promoff, G., Dube, S. R. & Arrazola, R. (2013). Electronic nicotine delivery systems: adult use and awareness of the 'e-cigarette' in the USA. Tobacco Control, 22, 19– d 23. doi: 10.1136/tobaccocontrol-2011-050044. pt e Romagna, G., Allifranchini, E., Bocchietto, E., Todeschi, S., Esposito, M. & Farsalinos, K. E. (2013). Cytotoxicity evaluation of electronic cigarette vapor extract on cultured mammalian fibroblasts (ClearStream-LIFE): comparison with tobacco cigarette smoke ce extract. Inhalation Toxicology, 25, 354-361. doi: 10.3109/08958378.2013.793439. van Rooy, F. G., Rooyackers, J. M., Prokop, M., Houba, R., Smit, L. A. & Heederik, D.J. Ac (2007). Bronchiolitis obliterans syndrome in chemical workers producing diacetyl for food flavorings. American Journal of Respiratory and Critical Care Medicine, 176, 498-504. doi: 10.1164/rccm.200611-1620OC. http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Manuscripts submitted to Nicotine & Tobacco Research Manuscripts submitted to Nicotine & Tobacco Research Figure legends rip t 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) sc 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. NIOSH limit for occupational exposure. an u The dotted line represents the maximum acceptable levels of daily exposure estimated from the M 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 d aerosol. A strong correlation was observed, while the expected and measured values were almost pt e identical, verifying that DA and AP are readily delivered from the liquid to the aerosol and that Ac ce no additional DA and AP are produced during the evaporation process. http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 24 of 27 sc an u Fo rP ee pt e d M rR cc e 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 rip t Manuscripts submitted to Nicotine & Tobacco Research Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 Page 25 of 27 ev iew http://mc.manuscriptcentral.com/ntr rip sc Ac ce pt e d M an u 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) http://mc.manuscriptcentral.com/ntr Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 26 of 27 t Manuscripts submitted to Nicotine & Tobacco Research AP 8000 800 2000 0 4000 6000 400 200 8000 0 0 d 2000 600 M 4000 0 y = 1.2435x - 25.483 R² = 0.9947 an u Expected concentration (µg/mL) y = 0.9526x + 182.64 R² = 0.9966 6000 sc DA pt e Measured concentration (µg/mL) cc e Expected concentration (µg/mL) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 rip t Manuscripts submitted to Nicotine & Tobacco Research Downloaded from http://ntr.oxfordjournals.org/ by guest on September 2, 2014 Page 27 of 27 http://mc.manuscriptcentral.com/ntr 200 400 600 Measured concentration (µg/mL) 800