Objectives We examined two waterpipe tobacco smoking components advertised to reduce harm to determine if they result in lower levels of biomarkers of acute exposure.
Methods We conducted a crossover study of 34 experienced waterpipe smokers smoking a research-grade waterpipe in three configurations ad libitum in a controlled chamber: control (quick-light charcoal), electric (electric heating) and bubble diffuser (quick-light charcoal and bubble diffuser). We collected data on smoking topography, environmental carbon monoxide (CO), subjective effects, heart rate, plasma nicotine and exhaled CO and benzene.
Results Smokers’ mean plasma nicotine, heart rate, and exhaled benzene and CO boost were all significantly lower for electric compared with control. However, smokers puffed more intensely and took significantly more and larger volume puffs for a larger total puffing volume (2.0 times larger, p<0.0001) when smoking electric; machine yields indicate this was likely due to lower mainstream nicotine. Smokers rated electric smoking experience less satisfying and less pleasant. For charcoal heating, the mean mass of CO emitted into the chamber was ~1 g when participants smoked for a mean of 32 minutes at a typical residential ventilation rate (2.3 hr−1).
Conclusion Waterpipe smokers engaged in compensation (i.e., increased and more intense puffing) to make up for decreased mainstream nicotine delivery from the same tobacco heated two ways. Waterpipe components can affect human puffing behaviours, exposures and subjective effects. Evidence reported here supports regulation of waterpipe components, smoking bans in multifamily housing and the use of human studies to evaluate modified or reduced risk claims.
- secondhand smoke
- global health
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The WHO recognises the urgent need for regulation of waterpipe tobacco under the WHO Framework Convention on Tobacco Control (FCTC).1 Hookah lounges that encourage and thrive on adolescent and young adult early social experimentation with waterpipe and other combustible tobacco products are centrally located near college campuses.2 3
The rapid rise in prevalence of use among young adults may be partially attributed to the widespread perception that waterpipe tobacco smoking is less harmful and less addictive than cigarette smoking or other tobacco products.1 4–9 The misconception that the bowl water filters out toxicants from the smoke is commonly held by waterpipe smokers and non-smokers alike.7 10 11 Indirect measurements made using machine smoking indicate some of the nicotine is retained by the water.12 Nevertheless, waterpipe tobacco smoke contains addictive levels of nicotine,13 and surveys show this is not well understood by US college students.14 15 In less than a month from initiation, infrequent (6 days/month), low consumption (7.5 waterpipes/month), sole use of waterpipe has been associated with signs of nicotine dependence in youth (12–18 years old).16 This form of tobacco may facilitate initiation of cigarette smoking.17–20 Among dual-users, waterpipe smoking is linked to progression to regular cigarette smoking21 and may impede cigarette smoking cessation.20 22
Most waterpipe tobacco contains high levels of a viscous humectant, glycerin,23 and therefore does not burn without a heat source. A burning lump of charcoal is typically placed on top of the foil-covered head that contains the tobacco. Charcoal heating in waterpipe smoking results in higher mainstream smoke yields of carbon monoxide (CO; >15 times higher) and benzene (>6 times higher), compared with smoking a cigarette.24–26 Biomarkers in blood, urine and exhaled breath confirm that increases in levels of these two compounds from waterpipe smoking greatly exceed those from cigarette smoking.27–31 Short-term, acute exposure to CO is associated with neurobehavioural and cognitive impairment,32 33 and numerous cases of acute CO poisoning from waterpipe smoking are reported.34–44 Non-smoking bystanders in cafés and residences are also at risk for CO exposure, as levels of sidestream CO emitted from machine smoking a waterpipe in a controlled environmental chamber exceed those for a cigarette by a factor of 30.45 Benzene is an International Agency for Research on Cancer Group 1 carcinogen46 and a suspected cardiovascular and reproductive toxicant.46–49 Nicotine, CO and benzene are all included in the WHO FCTC priority list of toxic contents and emissions in tobacco products50 51 and the US Food and Drug Administration’s (FDA) list of harmful and potentially harmful constituents.48
The US statutory definition of a tobacco product includes components that are reasonably expected to: (1) alter or affect the tobacco product’s performance, composition, constituents or characteristics and (2) be used with or for the human consumption of a tobacco product.52 Components may not be sold or distributed with health/modified risk claims unless the FDA issues an authorisation order after careful review of the evidence provided in a modified risk tobacco product application.52 53 This study examined two commercially available components because of specific health claims in their marketing rhetoric: a puck-like device that sits atop the foil-covered head and uses electrical resistance to heat waterpipe tobacco, and a plastic bubble diffuser that is press-fit onto the end of the waterpipe stem. During puffing, the bubble diffuser forces large bubbles that contain the mainstream smoke through a plastic mesh, creating smaller bubbles and increasing the surface area of water that the smoke contacts prior to being inhaled by the smoker. Manufacturers claim that these components are for ‘the healthy hookah smoker’, and their use results in increased filtration of the smoke sample (bubble diffuser),54 and smokeless, odourless, heating with no CO (electric heater).55
WHO’s position is that misleading health claims on packaging and labelling of waterpipe components should be regulated to prohibit the use of such statements that infer waterpipe tobacco and charcoal are safe.1 Modified risk tobacco products in the US are now prohibited to make claims of ‘lower risk’, ‘less harmful’ or ‘contains a reduced level of a substance’ without an FDA order in effect.52 Protocols for testing waterpipe emissions are not yet established, and mainstream and sidestream toxicant emissions have been measured using a variety of waterpipes and machine smoking equipment.56 However, machine smoking emissions data are not valid measures of human exposure or risk.57 To address data gaps and inform regulation of waterpipe components, clinical research is needed to characterise human exposures resulting from use of so-called reduced harm components.
For this study, we applied a previously developed boost measurement paradigm for biomarkers of acute exposure58 combined with real-time analysis59 to estimate nicotine, benzene and CO exposures experienced by established waterpipe users smoking a research-grade waterpipe (RWP) in a standard configuration (control) and equipped with two different components (ie, bubble diffuser and electric heater) purported by the manufacturers to reduce the harm of waterpipe smoking. Human exposure was evaluated via continuous collection of puffing topography and heart rate, and discrete sampling of plasma nicotine and exhaled breath compounds in a crossover study of active waterpipe smokers engaging in laboratory smoking in a controlled chamber ventilated at typical residential levels. Environmental or secondhand exposures were estimated from continuous CO data collected in the participant’s breathing zone. Machine smoking according to a human-derived puffing regimen was used to inform compensation estimates.
Using Craigslist, print advertisements, flyers and referrals, we recruited a convenience sample (n=36) of healthy, established waterpipe users aged ≥18 years and screened for eligibility by telephone. Established user was defined as having smoked waterpipe tobacco ≥3 times in the previous 6 months and ≥1 time in the previous month. Exclusion criteria included pregnancy, evident intoxication, respiratory infection, mouth disease and any history of smoking-related disease. Participants refrained from drinking coffee and using any tobacco product 12 and 4 hours prior to their laboratory visits, respectively. Participants were remunerated $225 for completing all three laboratory smoking sessions.
For this repeated measures design, each participant undertook three laboratory visits to smoke the RWP with: (1) quick-light charcoal (control), (2) electric heating (electric) and (3) quick-light charcoal and stem fitted with bubble diffuser (bubble diffuser). Laboratory visits were scheduled according to Williams design randomisation scheme and separated by ≥1 week. Participants were instructed to smoke normally until satiated, with no minimum or maximum smoking time required. For the control or bubble diffuser, participants were limited to one quick-light charcoal. The RWP was prepared by laboratory staff, and participants were not allowed to manipulate the heat source during smoking.
During the first visit, smoking history and demographic information were collected, including the Minnesota Nicotine Withdrawal Scale, Brief Questionnaire of Smoking Urges, Direct Effect of Nicotine Scale and Direct Effects of Tobacco Scale. During each visit, participants completed these subjective effects questionnaires before and after smoking the RWP.
Before entering and immediately after exiting the chamber, blood and exhaled breath samples were collected. During smoking, participants were seated in a comfortable chair with a choice of videos for distraction. Participants’ puff topography and heart rate were measured and recorded during the smoking session.
Waterpipe configurations and smoking topography data collection
Details regarding the setup, precision and accuracy of the RWP are described elsewhere.60 Briefly, the RWP was fitted with a new hose and mouthpiece, the head was loaded with 10.0±0.2 g tobacco (Double Apple, Nakhla) and covered with foil (Reynolds 625, China, 23×23 cm sheet, perforated with 18 holes). For the control and bubble diffuser, a burning quick-light charcoal (40 mm diameter, Three Kings, Holland) was placed on top of the foil after being placed on an electric heater (Type RY0811, 28 mm diameter, Ren Headstream, China) for 2 min. For the electric, the same electric heater was heated for 2 min and then placed on top of the foil. As previously described,60 puffing flow rate data were continuously acquired (6 Hz) throughout the smoking session and later analysed to produce puffing topo-graphy. For the bubble diffuser, a Large Hookah Diffuser (Heba) was attached to the end of the stem in the bowl water.
Details on the controlled chamber, and methods used to measure exhaled breath CO and benzene, environmental CO levels, heart rate, subjective effects, mainstream smoke nicotine yields, and tobacco and heat source temperatures are described in the online supplementary material. RWP results are also compared with those obtained by other researchers with commercial waterpipes in the supplementary material.
Supplementary file 1
Data were analysed using a linear mixed effects model for the analysis of repeated measures crossover design to assess differences in outcomes between the types of configuration and to account for the correlated data within the same participant. The model includes two within-participant factors: configuration (control, bubble diffuser and electric) and period (1, 2 and 3), and one between-participant factor for order effects. The main effects of configuration and the interaction effects between configuration and period were estimated. None of the interactions were statistically significant, indicating no carryover effects. For paired comparisons, p values were adjusted for multiple testing using Tukey’s method. Because the study was predominantly exploratory, p values that are significant at the 0.10 significance level are marked. Data were analysed using SAS (V.9.4).
Data were collected in Columbus, Ohio, 3 November 2010–4 May 2011. Due to a technical error, two participants were excluded from topography analysis. Table 1 describes the study sample characteristics. Most participants first used waterpipe to smoke tobacco an average of 6 years prior to the study in a ‘café or restaurant' setting or ‘at a friend’s home’, but past 30-day use showed more smoking at home. Most participants described their waterpipe use as ‘at least once a month, but not weekly’. In the past 30 days, participants smoked waterpipe an average of 3 times for 71.5±44.4 min per session. No participants reported daily use, and only one participant responded ‘yes’ to the question ‘Do you consider yourself ‘hooked’ on waterpipe?’ Yet based on the Lebanon Waterpipe Dependence Scale,61 roughly one-third of the participants were considered dependent (a total score ≥10). More than half of the participants (56%) indicated they also smoked combustible cigarettes ‘every day’ or ‘some days’.
Objective study outcomes
Table 2 compares puffing behaviour, exposure measures and charcoal and tobacco consumption by configuration. Generally, there were no significant differences in objective outcomes when participants smoked the RWP using charcoal heating, with the exception being a small increase (14%–18%) in average puff flow rate for the bubble as compared with control.
There were significant differences in almost all the objective outcomes compared with the control when participants smoked the RWP with electric heating. On average, participants took 30% more puffs resulting in almost twice the total puffing volume when smoking electric compared with control. Participants’ puffs were 16% longer in duration and 38% larger in volume, with higher average and peak flow rates (18% and 12%, respectively) when smoking electric.
With electric heating, the mass of CO emitted into the chamber was over 1000 times lower compared with control. This was also reflected in the average boost in exhaled breath CO, which was only 3% of what was measured for participants using charcoal heating. Benzene was detected in the exhaled breath of participants after smoking with charcoal, but not with electric. Plasma nicotine and heart rate boost were both significantly lower for electric compared with control (38% and 32%, respectively). Recovery of nicotine spiked into plasma QC samples averaged 100.5%±10.3% (n=14), and the mean relative SD of participants’ duplicate plasma samples (n=3) was 5.3%±4.8% (mean±SD), indicating the analytical method performed well.
Figure 1 shows the amount of charcoal and tobacco consumed versus the time each participant spent smoking. Charcoal consumption correlates strongly with smoking time (Spearman r=0.905), indicating that the burn rate of the charcoal was hardly affected by the participants’ puffing behaviours. The relationship between tobacco consumption and smoking time is weaker (r=0.607), indicating puffing behaviour may play a more important role in how much tobacco is burned.
Temperature may have been the reason why mainstream nicotine yields were significantly lower for the electric configuration. We conducted machine smoking using a human-derived puffing regimen (see online supplementary table S–1) for the control and electric configurations while continuously monitoring the temperature of the tobacco and heating sources (see online supplementary figure S–1 and S–2). Charcoal tobacco temperatures exceed those of electric by as much as 17.5°C at the end of the 33 min smoking session. Curves for the heat sources are significantly different after the first minute, with the charcoal temperature being as much as 76°C higher than the electric device in the first 5 min.
As shown in table 3, all three configurations provided relief from urges to smoke. Compared with control, the bubble diffuser provided greater relief from ‘urges to smoke a waterpipe’ and ‘craving a waterpipe/nicotine’. Participants rated higher increased drowsiness scores using charcoal heating. ‘Desire for sweets’ increased slightly after smoking control, while it decreased very little after smoking bubble diffuser or electric.
The ‘intention to smoke WP’ and ‘relief from withdrawal’ decreased across all three configurations. Participants reported increased subjective nicotine effects after smoking all three configurations (see table 3). More specifically, participants showed significantly higher effects in dizzy and lightheaded when smoking control compared with electric. Participants rated satisfying, pleasant and sleepy higher after smoking the two charcoal configurations compared with electric. Between control and bubble diffuser, calm, awake, reduce hunger, and sick were significantly higher for bubble diffuser than for control.
Comparison of toxicants in residential-like chamber and café smoking
Table 4 compares increases in toxicant levels in participants’ exhaled breath and the chamber air to those reported for waterpipe café smokers and workers in the USA, Iran, Turkey, Russia, Egypt and Canada. Average boost in our participants’ exhaled benzene exceeded indoor café smokers’ exhaled62 and café environmental air benzene levels63 by a factor of 5. Our participants’ average boost in exhaled CO exceeded that measured in waterpipe café employees after a ~10-hour shift64 and that measured in field staff after being in a waterpipe café for 2 hours.65
The study results have several policy and regulatory implications regarding infrequent waterpipe smokers, tobacco product standards and waterpipe tobacco smoking consumer education, marketing regulations and passive exposure protections.
Smoker compensation and signs of addiction
The heating source had a significant effect on smokers’ puffing behaviours, toxicant exposures and subjective experiences. Participants rated the electric configuration less satisfying and pleasant, yet they took significantly more and larger puffs for almost twice the total puffing volume compared with charcoal heating. Although exposures associated with the burning charcoal, e.g., CO and benzene, were significantly lower, inhaling twice as much smoke when using the electric heat source would result in higher exposures to toxicants associated with the burning tobacco, such as furans and carbonyls. Outcomes related to nicotine delivery and the burning charcoal, e.g., plasma nicotine, heart rate, and exhaled benzene and CO boost, were significantly lower when using electric heating compared with charcoal. Initial symptoms of acute and chronic CO exposure, such as dizziness and lightheadedness,33 were also significantly reduced when participants used electric heating. Ironically, the reduction of these symptoms may enable smokers to better tolerate more aggressive puffing to obtain the nicotine they desire and/or frustrate smokers who consider these sensations a pleasurable part of waterpipe smoking. Testing tobaccos and components using machine smoking would not have revealed increased toxicant exposure due to human compensation, and therefore, human studies are critical to informing tobacco product standards.
Machine smoking according to a standardised puffing regimen is a useful tool for regulation because yields can be used to compare tobaccos and components, but they cannot predict actual human exposures.66 Nicotine yields indicated that the more aggressive puffing behaviour when using the electric component was likely in response to lower mainstream nicotine delivery associated with electric heating. Average boost in heart rate with electric heating was half that seen with charcoal heating, which may also be in response to lower mainstream nicotine delivery. We hypothesise that participants were engaging in compensation, or changing their puffing behaviour to compensate for lower nicotine levels in mainstream smoke.67 68 Mainstream nicotine yield was significantly lower for the electric configuration as compared with control (0.32 vs 1.44 mg/session). Although nicotine yield was almost a factor of 5 lower when using the electric heat source, participant plasma nicotine levels were only a factor of 2 lower. Average compensation level was 68% when participants smoked the RWP in the electric configuration compared with the control.67 68 Like addicted cigarette smokers,67 the study participants appeared to upregulate their nicotine intake when inhaling waterpipe tobacco smoke with lower levels of nicotine. Cobb et al 69 first reported evidence of increased puffing when high-use frequency waterpipe smokers (≥20 waterpipes/month) smoked a nicotine-free herbal versus a nicotine-containing product in the laboratory. Ours is the first study to quantify compensation in low-use frequency waterpipe smokers (3.1±3.0 waterpipes/month) when smoking the same tobacco heated using two different components (quick-light charcoal and electric). This finding supports previous reports that even infrequent waterpipe use is associated with signs of nicotine dependence.16 It also indicates that waterpipe components can affect the ‘elasticity’ of the waterpipe by causing the smoker to actively obtain more than the yield value of nicotine.70 The lower nicotine yield can be explained by the fact that the maximum temperature achieved by the electric heat source was lower than charcoal (see online supplementary figure S–1). Similar to what was reported for heated cigarettes,71 a direct relationship between temperature of the heated tobacco and mainstream nicotine yields is likely for waterpipe tobacco, but more data are needed. Should this relationship prove robust, product standards regarding the acceptable range of power for electric waterpipe heating devices would be warranted.
Ventilation: residential versus indoor café smoking
This study’s average environmental CO concentration, when adjusted from a 1-hour time-weighted average (TWA) to a 10-hour or 8-hour workday (9.39 mg/m3and 11.7 mg/m3, respectively), does not exceed the US National Institute for Occupational Safety and Health (NIOSH) and the US Occupational Safety and Health Administration TWAs (40 mg/m3and 55 mg/m3, respectively).72 73 However, chamber air levels were very close to the NIOSH ceiling level (229 mg/m3), and therefore this ventilation level is not recommended for future work. The environmental CO levels measured here are cause for concern because the ventilation rate of the chamber (2.3 hour−1) was almost four times higher than median residential air exchange rates reported for three major US metropolitan areas,74 and the Passive House Standard75 for energy efficient construction (0.71 and 0.60 hr-1, respectively). Therefore, smoking a single waterpipe with charcoal heating at home with the windows closed may generate CO and benzene levels that significantly exceed permissible occupational exposure thresholds worldwide72 73 76–81 and, ironically, may also exceed those encountered in indoor hookah cafés where numerous people are smoking waterpipes.63 64 82–84
Benzene is generated primarily by the burning charcoal,25 and thus was not detected in participants’ breath when they smoked the electric configuration. This was corroborated by the lower average increase in heart rate and that they reported feeling less sleepy, drowsy and dizzy when smoking the electric configuration, all symptoms of acute exposure to high levels of benzene (700–3000 ppm).85 In our study, in a room that was ventilated at a rate comparable to most residences, smokers’ exhaled breath benzene levels for charcoal smoking were significantly higher than those reported by Samarghandi et al for waterpipe café smokers.62
Charcoal heating also resulted in a considerable mass of CO in the chamber, ranging from 0.34 g to 1.7 g, during the smoking session, whereas electric generated very little CO. Waterpipe smoking with charcoal generates levels of CO that are 25–30 times greater than those from a cigarette.26 45 Roughly 17% of the average mass of charcoal consumed during the smoking session (see table 2) was measured as environmental CO. Yet charcoal manufacturers are not required to provide warnings about smoking indoors or near residences, nor educational material regarding the importance of a smoke-free home as part of their consumer packaging.
CO emissions from waterpipe smoking are also concerning because CO is slightly lighter than air and can diffuse evenly throughout rooms in a house or units in multifamily housing, including into bedrooms where vulnerable occupants, such as children and older adults, are sleeping. A majority of our participants first smoked waterpipe in a café or restaurant setting, but their recent smoking shows a shift towards smoking more in the home. Smoking bans in public spaces are now prevalent throughout the world, yet passive exposure in multifamily homes is not similarly protected. One recent exception to this worldwide gap in legislation is that the US Department of Housing and Urban Development now prohibits smoking, including waterpipes, inside and within 25 feet of any public housing.86 Whether the US ban can be successfully implemented remains to be seen. In the case of waterpipe, to squarely address the mistaken perception that this form of tobacco use is less harmful, concurrent public education through the use of media campaigns and mandated warning labels and educational package inserts may be critical to the success of smoke-free housing.
Manufacturers’ marketing claims
Because participants engaged in compensation when smoking with the electric heat source, the anticipated higher active and passive particulate matter and tobacco smoke-related toxicants (besides nicotine) associated with that compensation, although not measured in this study, may offset any reduced risk that may be associated with the lower CO and benzene exposures. Puffing behaviour and exposures when using the bubble diffuser were not substantially different from the control, and thus the manufacturer’s health claim of greater filtration is unsubstantiated. However, the bubble diffuser did provide roughly twice as much relief from urges to smoke and craving a waterpipe, and reduced desiring sweets compared with the control, suggesting this component may make waterpipe tobacco smoking more appealing. More work is needed to understand the public health effects of using these components.
There are limitations to the data reported for this study. Because of the convenience sample, our results are not generalisable to the larger waterpipe tobacco smoking community. Although we believe the study provides convincing evidence of compensatory smoking in waterpipe tobacco smokers, this conclusion is not robust because mainstream charcoal emissions were not present when participants smoked the electric configuration. It is possible that charcoal emissions by themselves are reinforcing and established waterpipe smokers might puff more intensely because they are missing this stimulus when using electric heating. However, we would expect this behaviour to decay quickly because, unlike increases in nicotine delivery, there would be no reward for this more intense puffing. To further study compensation, one could test participants smoking the RWP with the same tobacco being electrically heated at two different temperatures, or in the control configuration using two different plastic hoses, one with and one without air ventilation holes.
Although the RWP is a predictable and precise tool that is well accepted by established waterpipe tobacco smokers,60 87 the data presented cannot be generalised for the variety of commercial waterpipes sold worldwide. However, where available, our study outcomes agree with those reported by other researchers using commercial waterpipes. A laboratory study may not yield puffing behaviour representative of behaviour in more relaxed, social settings. Nevertheless, the reliability of this tool, combined with the crossover study design, allows us to make valid comparisons of exposures when smokers use different waterpipe components.
Considering misconceptions about waterpipe being non-addictive, and increasing prevalence of use, youth and young adults are a vulnerable population that is susceptible to the promise of reduced harm. Results from this study suggest that waterpipe smokers exhibit compensatory behaviour associated with nicotine addiction by inhaling much larger volumes of smoke per smoking session when using a component that results in lower levels of mainstream nicotine. In addition, smoking waterpipe at home may result in smokers and non-smokers being exposed to harmful levels of benzene and CO that significantly exceed worldwide occupational exposure limits. Future work should include a wider panel of biomarkers representing toxicants generated by the tobacco to more thoroughly understand exposures associated with electric heating. Data reported here support regulating waterpipe components as stringently as tobacco products to enhance public health.
To combat the mistaken perception that waterpipe smoking is a less harmful form of tobacco use and increase consumer education, we recommend mandating indelible (non-removable) health warning labels on components that warn users of the product’s potential to initiate nicotine addiction (for the waterpipe itself), cause smokers to inhale more smoke (for heating sources) and produce poisonous levels of CO and benzene indoors (for charcoal). These data also strongly support banning manufacturers’ claims that any component results in a lower risk from waterpipe smoking until regulatory agencies can evaluate carefully designed human studies to determine the real toxicant exposures associated with components purported to reduce harm.
What this paper adds
It is widely accepted in the tobacco regulatory science community that waterpipe tobacco smoking delivers sufficient levels of nicotine to initiate and sustain addiction, and active and passive exposures to CO and benzene exceed those from smoking a cigarette. Our results have regulatory implications in that they:
Suggest that waterpipe smokers are addicted to nicotine and therefore will engage in more frequent and aggressive puffing to compensate for lower mainstream nicotine delivery. Thus, reduced harm or modified risk claims cannot be evaluated based on machine smoking yield data alone and require testing with human cohorts to better understand waterpipe tobacco smokers’ actual exposures when using these components.
Demonstrate the importance of waterpipe components to the active and passive toxicant exposures experienced by waterpipe smokers and passively exposed non-smokers. These data support the regulation and controlled testing of waterpipe components.
Suggest that waterpipe smoking at home exposes smokers and non-smokers to harmful levels of benzene, CO and other toxicants. These data support the inclusion of ‘waterpipe tobacco smoking’ as banned behaviour in and near multifamily public housing.
The authors would like to thank Dawn M Deojay, Robyn R Kroeger and Iza L Reyes for their assistance with processing participants and data collection.
Contributors MCB, SMG and PIC were responsible for the conception and design of the study. SSB was responsible for the chemical analysis, and HK and AMA were responsible for the statistical analysis. All contributed to the analysis and interpretation of the data and the preparation of the manuscript.
Funding Research was supported by grant number R01CA133149; the preparation of the manuscript was supported by P50CA180523 from the Office of the Director, National Institutes of Health (OD), National Cancer Institute and the FDA Center for Tobacco Products.
Disclaimer The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Food and Drug Administration.
Competing interests None declared.
Patient consent Not required.
Ethics approval The study was approved by the Battelle Internal Review Board (FWA 0004696).
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement In accordance with P.L. 110-161, this article is publicly available on PubMed Central. Additional methodology and data are available in the supplementary file.
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