Systemic absorption of nicotine following acute

International Journal of Hygiene and Environmental Health xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Hygiene and Environmental Health journal homepage: www.elsevier.com/locate/ijheh

Systemic absorption of nicotine following acute secondhand exposure to electronic cigarette aerosol in a realistic social setting ⁎

Paul Melstroma, , Connie Sosnoffb, Bartosz Koszowskic, Brian A. Kinga, Rebecca Bunnella, Grace Led, Lanqing Wangb, Meridith Hill Thannerc, Brandon Kenemera, Shanna Coxa, B. Rey DeCastrob, Tim McAfeea a

Office on Smoking and Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, GA, USA Division of Laboratory Services, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, USA c Battelle Public Health Center for Tobacco Research, Baltimore, MD, USA d Battelle Memorial Institute, Atlanta, GA, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Nicotine E-cigarette Secondhand Aerosol Cotinine

Evidence suggests exposure of nicotine-containing e-cigarette aerosol to nonusers leads to systemic absorption of nicotine. However, no studies have examined acute secondhand exposures that occur in public settings. Here, we measured the serum, saliva and urine of nonusers pre- and post-exposure to nicotine via e-cigarette aerosol. Secondarily, we recorded factors affecting the exposure. Six nonusers of nicotine-containing products were exposed to secondhand aerosol from ad libitum e-cigarette use by three e-cigarette users for 2 h during two separate sessions (disposables, tank-style). Pre-exposure (baseline) and post-exposure peak levels (Cmax) of cotinine were measured in nonusers’ serum, saliva, and urine over a 6-hour follow-up, plus a saliva sample the following morning. We also measured solution consumption, nicotine concentration, and pH, along with use behavior. Baseline cotinine levels were higher than typical for the US population (median serum session one = 0.089 ng/ml; session two = 0.052 ng/ml). Systemic absorption of nicotine occurred in nonusers with baselines indicative of no/low tobacco exposure, but not in nonusers with elevated baselines. Median changes in cotinine for disposable exposure were 0.007 ng/ml serum, 0.033 ng/ml saliva, and 0.316 ng/mg creatinine in urine. For tank-style exposure they were 0.041 ng/ml serum, 0.060 ng/ml saliva, and 0.948 ng/mg creatinine in urine. Finally, we measured substantial differences in solution nicotine concentrations, pH, use behavior and consumption. Our data show that although exposures may vary considerably, nonusers can systemically absorb nicotine following acute exposure to secondhand e-cigarette aerosol. This can particularly affect sensitive subpopulations, such as children and women of reproductive age.

1. Introduction Studies show a dramatic increase in experimentation and use of electronic cigarettes among US adults and youth (Arrazola et al., 2014; Giovenco et al., 2014; King et al., 2015). One area of particular concern to the public health community is nonusers’ exposure to the contents of e-cigarette aerosol, particularly sensitive subpopulations such as children, the developing fetus and pregnant women. In addition to nicotine, e-cigarettes’ aerosols can contain heavy metals, ultrafine particles, and cancer-causing agents like acrolein (Goniewicz et al., 2013a; McAuley et al., 2012; Pellegrino et al., 2012; Schober et al., 2013; Schripp et al.,

2013). The aerosol of e-cigarettes can also contain propylene glycol (PG) or vegetable glycerin (VG) and flavorings. The health effects of chronic inhalation of these substances are currently unknown. Moreover, fundamental questions remain about what compounds to measure, how best to measure them, and how best to describe the variable effects of e-cigarette solutions, uses, and devices when assessing health threats from e-cigarettes. Most e-cigarettes deliver aerosolized nicotine, which raises concerns for nonusers about the potential for acute poisonings, developmental and reproductive toxicity, and addiction (Chatham-Stephens et al., 2014; England et al., 2015; U.S. Department of Health and Human

⁎ Corresponding author at: Office on Smoking and Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, 4770 Buford Hwy, MS F-79, Atlanta, GA, USA. E-mail address: [email protected] (P. Melstrom).

https://doi.org/10.1016/j.ijheh.2018.05.003 Received 18 January 2018; Received in revised form 27 April 2018; Accepted 15 May 2018 1438-4639/ Published by Elsevier GmbH.

Please cite this article as: Melstrom, P., International Journal of Hygiene and Environmental Health (2018), https://doi.org/10.1016/j.ijheh.2018.05.003


P. Melstrom et al.

Services, Printed with corrections, January 2014). Studies have measured nicotine in air following e-cigarette aerosol generation (Czogala et al., 2013; Schripp et al., 2013), and Flouris and colleagues exposed never-smokers to machine-generated e-cigarette aerosol in an exposure chamber for a single hour and measured serum cotinine concentrations immediately after the exposure and an hour later (Flouris et al., 2013). Although the values were not published, cotinine concentrations in the never-smokers passively exposed to e-cigarette aerosol were significantly higher than in a control group. Similarly, Ballbe and colleagues measured urine and salivary cotinine in nonusers who lived with exclusive e-cigarette users (n = 5) and found significantly higher levels of cotinine in these nonusers compared to nonusers living in homes where no smoker or e-cigarette user was living (n = 24), both in urine and saliva (p-values 0.008 and 0.003, respectively) (Ballbe et al., 2014). However, these studies did not simulate realistic short-term exposures that occur in social or public settings, nor did they describe the factors affecting the exposures. To address this gap in the scientific literature, we examined acute secondhand nicotine exposure to nonusers by using three habitual e-cigarette users (active users) vaping ad libitum in a room co-occupied by six nonusers for two hours. The number of e-cigarette users for each exposure represented a plausible number of users in a physical space comparable to public spaces such as bars or restaurants, or to a private space such as a household room. We also accounted for other sources of nicotine exposure and the extent to which certain factors, such as solution and device characteristics, as well as how the e-cigarette users used their device (use behavior) might affect exposure.

Fig. 1. Electronic cigarettes used: first generation disposable e-cigarette (A) and second generation tank-style e-cigarette (B)*.

by estimating it to be a 50:50 ratio and averaging the specific density to 1.147 g/cm3. The volume could then be multiplied by the measured nicotine concentration to yield mass of nicotine consumed during the exposure. After each 2-hour exposure, the active users were discharged, and the nonusers monitored for an additional 6 h for collection of biological samples. Finally, the unused e-cigarette cartridges and solutions were collected and sent to the Centers for Disease Control and Prevention (CDC) for analysis of pH and nicotine concentrations, where the latter analysis was performed in a manner that aligned with the method described in Stanfill et al (Stanfill et al., 2009). Analysis of pH was performed as previously described with minor modifications.(U.S. federal register, 1999) Using the measured pH values, pKanicotine=8.02 (Rumble, 2017) and the Henderson-Hasselbalch equation pH = pKa + log10([Base]/[Acid]), the percentage of nicotine in the unionized, or free-base, form was calculated.

2. Methods 2.1. General procedure and setting Both sessions were conducted in a 6.10mX3.35 m X 2.57 m or 52.6m3 (20′x11′X8′5″ or 1858ft3) room kept at 26 °C (78.8 °F). The room was equipped with furnishings covered in fabric materials that allowed the aerosol to deposit on and recirculate from surfaces more commonly found in living and social quarters. The room was cleaned of background nicotine contamination before each exposure session by including wiping down hard surfaces and pre-washing fabric materials. Each participant wore a clean surgical top to prevent contamination from their clothes and to limit dermal absorption. The door and windows remained closed during the sessions. Room air ventilation measurements (∼5 air changes per hour), layout of the exposure room, and air nicotine concentrations for each nonuser were previously published in Melstrom, et al. (Melstrom et al., 2017). A 2-hour time length was chosen as a realistic duration for how long a member of the public who does not use nicotine-containing products might be exposed to e-cigarette aerosol in a social setting. All participants were allowed to perform normal activities during the sessions. In addition, because of the several different types of e-cigarette products now available, including first generation e-cigarettes (i.e., disposable, cig-a-like) and second generation tank systems (Fig. 1), we conducted two separate exposure sessions to account for e-cigarettes’ market diversity. During the first session, the active users used first generation e-cigarettes and tank-style second generation e-cigarettes during the second. Except for the type of e-cigarette used, both sessions were conducted identically. All participants were present for both sessions. On each study day, within 2 h prior to the 2-hour exposure, the following were obtained from the nonusers: blood, urine, and saliva samples; blood pressure; pulse; expired carbon monoxide and self-reported symptoms. Before and following each exposure, the masses of the e-cigarette products were measured to determine the amount of e-cigarette solution used during the exposure. The amount of nicotine consumed could then be calculated by converting the mass of solution consumed into volume by dividing the mass of solution by either the specific density of PG (1.032 g/cm3) or of VG (1.261 g/cm3) or, if the solution was a blend,

2.2. Participants Inclusion criteria for both nonusers and active users: male or nonpregnant, non-breastfeeding female; 21–55 years; healthy-by-history; able to commit to both sessions. Specific criteria for nonusers: neverusers of combustible tobacco products (never smoked more than 100 cigarettes in their lifetime), no use in the past year of non-combustible tobacco products (eg, smokeless tobacco) or nicotine replacement therapies. Nonusers agreed to abstain from exposure to secondhand tobacco smoke or e-cigarette aerosol for 6 days before each exposure. However, they must have had a history of tolerating exposure to secondhand tobacco smoke to establish that they were assuming no more risk by study participation than they had previously assumed without incident. At the screening visit and before each session, nonusers were given an exhaled carbon monoxide breath test and a spot urinalysis. They were required to have an expired carbon monoxide level < 6 ppm, an undetectable measured urine cotinine concentration (NicAlert, Nymox Pharmaceutical Corporation, Hasbrouck Heights, New Jersey), and for females, a negative pregnancy test (Sure-Vue, Fisher Scientific, Waltham, Massachusetts). Specific criteria for active users: having used an e-cigarette for more than 6 months, having used both a tank-style and a first generation e-cigarette, and currently using a tank-style at least 5 times per day with at least 18 mg/ml concentrated nicotine solution. Exclusion criteria included: having respiratory symptoms on study days; having chronic obstructive pulmonary disease; having had a stroke; having cancer within the past 5 years; having tuberculosis by history or chronic cough; having cardiac disease; having asthma or severe allergic rhinitis; having allergy or hypersensitivity to nicotine or a component of e-cigarette solution, including PG and VG; having hypertension (blood pressure > 150/95 after 5 min rest or by history); showing obvious intoxication or positive drug screen for cocaine, amphetamines, methamphetamine, or opiates (One Step Drug of Abuse 2


P. Melstrom et al.

serum or saliva. The sample was then basified with potassium hydroxide (50 μL, 0.2 N) and applied to a supported liquid extraction plate (Isolute SLE + 400 mg, Biotage, Charlotte, North Carolina). The analytes were eluted two times with 0.9 mL of 5% (v/v) isopropanol in methylene chloride, the organic extracts were dried, and the residue was reconstituted in 100 μl water. A 10 μl aliquot of the reconstituted extract was then injected onto a HPLC column (Hypersil Gold C18 Selectivity, 50 x 3 mm, 1.9 μm particle size, Thermo Scientific, Waltham, Massachusetts) in a liquid chromatograph (Nexera system, Shimadzu, Kyoto, Japan) coupled to a triple quadrupole mass spectrometer (API 6500, Sciex, Framingham, Massachusetts). Cotinine was monitored and identified by its retention time and precursor/product ion pairs using multiple reaction monitoring with atmospheric pressure chemical ionization. Analyte concentrations were derived from the area ratios of native to labeled compounds in the sample by comparison to a standard curve (11 concentrations ranging from 0.01 to 25 ng/mL) using 1/X weighted linear least-squares regression. The limit of detection for cotinine is 0.015 ng/ml. A water blank and two quality control samples were analyzed with each analytical run. All runs passed the quality control rules of the Division of Laboratory Science at CDC (Bernert et al., 2000, 1997). Urine samples were analyzed in the same way as serum and saliva samples, with the exception of the following: first, after spiking with internal standard solution, the urine sample was hydrolyzed (overnight at 60 °C) with 40 μl of β-glucuronidase solution (from Haliotis rufescens, 16.7 units/μL in ammonium acetate buffer, pH 5.1, 0.5 M) to free the bound analytes; second, chromatographic separation was performed using a UPLC column (Acquity UPLC BEH C18, 2.1 x 100 mm, 1.7 μm particle size, Waters, Milford, Massachusetts) with a 5 μl aliquot of the reconstituted sample extract; third, the urine standard curve was composed of 15 concentrations ranging from 0.025 to 200 ng/mL. The limit of detection (LOD) for cotinine in urine is 0.030 ng/mL.

Test, WHPM, Inc, Irwindale, California); being unable to speak or read English; females being pregnant or breastfeeding; and being unable to commit to both sessions. For the nonuser group, we also excluded those having a significant history of fainting, lightheadedness, hematoma, hemophilia or bleeding disorder, inaccessible veins, and discomfort with or significant bruising following blood draws, or taking any blood thinning medication. 2.3. Recruitment and enrollment The recruitment period began in February 2015. The first exposure session was conducted on March 19, and the second on March 26, 2015. The three active users were recruited by contacting potentially eligible individuals listed in a database owned by Battelle. Additional recruitment efforts for all participants were accomplished by advertisements in local newspapers. Eligibility was initially determined via telephone. Prospective participants underwent a brief clinical assessment (eg, heart rate, blood pressure, temperature) and urinalysis for tobacco use, illicit drug use (cocaine, amphetamines, methamphetamines, and opiates), and pregnancy for females. Prospective active users were asked to bring their tank-style e-cigarettes to ensure that they were tanks and to demonstrate tolerability of use. The first three and first six persons who met eligibility requirements for the active users and nonusers, respectively, were then enrolled. 2.4. E-cigarette products Each active user was given an iTaste (Innokin Technology Co. LTD, Shenzen, China PRC) variable voltage v3.0 tank (distributed by Mt Baker Vapor) and several selected blu (blu Ecigs, Imperial Tobacco Group, Bristol, UK) or Fling (White Cloud Electronic Cigarettes, Tarpon Springs, Florida) disposable e-cigarettes, based on the user’s preferred flavors. The devices were identical to those they would use during the exposure sessions and allowed each active user to become familiar with these products. The blu disposable e-cigarette brand was selected and the flavors offered for both study days were based on their position as most popular according to US market share, using existing retail scanner data at the time of the study. The Fling disposable e-cigarette was added to expand the flavor choices. Given the lack of retail scanner data for tank-style systems, the iTaste was selected as a common brand. No flavor ingredient that has known concerns based on a literature review was used (eg, known to contain diacetyl). The blu e-cigarettes were purchased at the same time from a local Baltimore tobacco retailer and the Fling e-cigarettes was purchased on-line from an e-cigarette retailer. All e-liquid for the second exposure was purchased from a local Baltimore “vape shop” at the same time as custom manufactured solutions. Flavors selected by the users were blu® classic tobacco and cherry crush, Fling® iced berry and custom manufactured solutions were java, swiss cherry and peach.

2.6. Use behavior during e-cigarette use Three cameras captured the use behavior of the active users. Following the sessions, the footage was reviewed and the number of puffs and duration of each puff was recorded. These were then analyzed to produce the total number of puffs and sum-puff durations (sum of the lengths of all individual puffs). 2.7. Statistical analysis Given the small sample size of this study, no statistical tests for differences were performed. 2.8. Research ethics considerations The study protocol was approved by the Institutional Review Boards of CDC and Battelle, Inc. Each participant also completed a written, signed informed consent form that was approved by both boards. Vital measurements and a review of self-reported subjective symptoms were collected solely to monitor for potential adverse health effects of the exposure.

2.5. Biological samples Eight samples of blood (for serum) and saliva were collected from each nonuser, once before the exposure and once immediately following it, as well as six hourly collections thereafter until discharge from the study. All urine was collected from the nonusers during the entire study day. Before the exposure, participants were asked to produce at least one void, but if more than one baseline urine was recorded, then a mean average was reported. Urine was collected ad lib during the exposure if needed and during the 6-hour follow-up period. Nonusers also collected their own saliva sample the morning following each exposure using a take home kit, and subsequently mailed the kit to researchers at CDC. Analysis of cotinine in serum and saliva was performed per an established CDC method, as follows. A 50 μl aliquot of internal standard solution containing d3-cotinine (10 ng/mL) was added to 200 μl of

3. Results 3.1. Demographics Characteristics of the six nonusers were as follows: four male and two female; ages 28–54; four white and two African-American. Three habitual e-cigarette users had a median length of e-cigarette use of 1 year and reported using e-cigarette liquid strength ≥18 mg/ml at a median of 50 puffs/hour. One active user currently used both first generation and tank systems. No active user reported using other tobacco products. 3


P. Melstrom et al.

Table 1 Pre-exposure baseline (BL), peak (CMax), and difference between values of cotinine (Δ) in serum, saliva, and urine, by nonuser for both exposure sessions. Session #1 – Disposable e-cigarettes

Nonuser # a

Salivaa (ng/ml)

Serum (ng/ml)

1 2 3 4 5 6 Mean Median SD

BL

Cmax

Δ

BL

Cmax

Δ

0.071 0.254 0.242 < LODc 0.015 0.107 0.117 0.089 0.107

0.096 0.243 0.215 0.049 0.040 0.096 0.123 0.096 0.085

0.025 −0.012 −0.027 0.034 0.025 −0.010 0.006 0.007 0.025

0.113 0.281 0.222 < LOD 0.023 0.091 0.124 0.102 0.107

0.137 0.308 0.332 0.069 0.047 0.129 0.170 0.133 0.121

0.024 0.027 0.110 0.054 0.024 0.039 0.046 0.033 0.033

BL 0.626 1.787 4.051 0.064 0.239 1.722 1.415 1.174 1.484

Cmax 1.057 2.043 3.817 0.441 0.369 2.187 1.652 1.550 1.311

Δ 0.431 0.256 −0.234 0.377 0.130 0.465 0.237 0.316 0.262

Session #2 – Tank-style e-cigarettes

Nonuser # Seruma (ng/ml)

1 2 3d 4 5 6 Mean Median SD

Urineb (ng/mg creatinine)

Salivaa (ng/ml)

Urineb (ng/mg creatinine)

BL

Cmax

Δ

BL

Cmax

Δ

0.052 0.894 2.944 < LODc 0.048 0.484 0.299 0.052 0.385

0.102 0.856 2.701 0.082 0.089 0.503 0.326 0.102 0.346

0.049 −0.037 −0.243 0.067 0.042 0.019 0.028 0.041 0.041

0.069 1.056 3.296 < LOD 0.058 0.554 0.350 0.069 0.452

0.133 1.026 3.844 0.089 0.112 0.614 0.395 0.133 0.415

0.064 −0.031 0.548 0.074 0.054 0.060 0.044 0.060 0.043

BL 0.568 5.613 49.363 0.074 0.563 0.930 1.550 0.568 2.292

Cmax 1.516 6.360 47.200 1.225 1.373 10.734 4.242 1.516 4.225

Δ 0.948 0.747 −2.163 1.151 0.810 9.804 2.692 0.948 3.979

Abbreviations: BL, pre-exposure baseline; Cmax, post-exposure peak; Δ, the difference between BL and Cmax; LOD, limit of detection; SD, standard deviation. a Limit of detection = 0.015 ng/ml for saliva and serum cotinine. b Limit of detection ≤ 0.030 ng/mg for urine cotinine. c When BLs were < LOD, then serum or saliva LOD of 0.015 ng/ml was used to calculate Δ. d Nonuser #3 in Session #2 was omitted from calculation of mean, median and standard deviation.

urine ranged from 0.074 to 49.36 ng/mg urine creatinine. Again, nonuser #3 in the second session was omitted from calculations. The median Cmax cotinine level in urine was 1.516 ng/mg urine creatinine (SD = 4.225).

3.2. Biomonitoring The nonusers’ levels of cotinine in serum, saliva, and urine are listed in Table 1. Systemic absorption (positive change from baseline to Cmax) was recorded in serum for three of six nonusers in session one (disposable e-cigarettes) and for four of six in session two (tank-style ecigarettes). In saliva, systemic absorption was recorded for all nonusers in both sessions except for one nonuser in the second session. For the first session, baseline serum cotinine values ranged from < lOD (< 0.015 ng/ml) to 0.254 ng/ml (SD = 0.107). In saliva, for the first session, baseline saliva cotinine values ranged from < lOD to 0.281 ng/ml (SD = 0.107). Specifically, our findings showed that nonusers with a baseline serum cotinine level of < 0.1 ng/ml showed systemic absorption of nicotine that could be attributed to inhalation of e-cigarette aerosol. In baseline serum cotinine levels above this, levels which most likely reflect recent exposure to nicotine, five of six levels decreased from baseline. The median Cmax for serum cotinine was 0.096 ng/ml (SD = 0.085 ng/ml) and for saliva cotinine was 0.133 ng/ ml (SD = 0.121 ng/ml). For the second session, baseline serum cotinine values ranged from < lOD to 2.94 ng/ml, while baseline saliva cotinine values ranged from < lOD to 3.30 ng/ml. Nonuser #3 from the second session clearly had prior exposure to nicotine, and was omitted from calculations of mean, median or standard deviation. The median Cmax for serum cotinine was 0.102 ng/ml (SD = 0.346 ng/ml) and for saliva cotinine was = 0.133 ng/ml (SD = 0.415 ng/ml) (Table 1). Systemic absorption was recorded in urine for all but one of the six nonusers in both the first and second sessions (Table 1). For the first session (disposable e-cigarettes), baseline cotinine values ranged from 0.064 to 4.05 ng/mg urine creatinine (SD = 1.484). The median Cmax cotinine level was 1.55 ng/mg urine creatinine (SD = 1.311). For the second session (tank-style e-cigarettes), baseline cotinine values in

3.3. E-cigarette solution, use behavior and consumption The results of the analysis of e-cigarette solution are presented in Table 2. For the disposable e-cigarettes, the measured nicotine concentrations ranged from 12.0 to 20.5 mg/ml with a mean of 16.4 mg/ml (SD = 4.3). For the tank-style solution, the measured nicotine concentration ranged from 14.5 to 15.5 mg/ml, with a mean of 15.1 mg/ml (SD = 0.5). There was insufficient solution to measure pH in the poststudy analysis of the disposable e-cigarettes, but the pH of the tank ecigarette solutions ranged from 7.91 to 8.86. The corresponding calculated percentages of nicotine in the unionized form ranged from 45% to 88%. Results for number of puffs and sum-puff durations, as well as consumption data, are listed in Table 2. Use behavior and consumption data show large differences among the three active users. For example, in the first session using disposable e-cigarettes, active user #2 took 248 puffs over the 120-minute exposure period, while active users #1 and #2 took 132 puffs and 125 puffs, respectively. Similarly, the sum-puff durations differed between session one (28 min, 10 s) and session two (19 min, 43 s). The total mass of e-cigarette solution consumed across all users in the first session (disposable e-cigarettes) was 1.048 g, which corresponds to 14.3 mg nicotine. In contrast, the total mass of e-cigarette solution consumed during the second session (tank-style e-cigarettes) was 2.011 g (26.2 mg nicotine), approximately twice the amount of nicotine as consumed in the first. For this study, we also measured individual air nicotine 4


P. Melstrom et al.

Table 2 Product analysis, use behavior, and consumption data, by active e-cigarette user for both exposure sessions. Active user #

Brand

Flavor

PG, VG

Session #1 – Disposable e-cigarettes 1 blu Classic VG tobacco 2 blu Cherry PG, VG crush 3 Fling Iced berry PG, VG Mean (SD)

Labelled nicotine (mg/ ml)

Measured nicotine (mg/ml)

pHa

% unionizedb

Number of puffs

Sum-puff durationsc (mm:ss)

Mass solution consumed (g)

Mass nicotine consumed (mg)

18

12.0

NR

NR

132

12:16

0.404

3.8

18

16.7

NR

NR

248

09:40

0.309

4.5

18

20.5 16.4 (4.3)

NR

NR

125 168

06:14 09:23

0.335 0.349

6.0 4.8

505

28:10

1.048

14.3

Sum Session #2 – Tank-style e-cigarettes 1 iTaste Java PG 2 iTaste Swiss VG cherry 3 iTaste Peach VG Mean (SD)

18 18

15.5 15.2

7.91 8.36

45 69

98 219

05:45 06:44

0.760 0.734

11.4 8.9

18

14.5 15.1 (0.5)

8.86

88

116 144

07:14 06:34

0.517 0.670

5.9 8.7

433

19:43

2.011

26.2

Sum

Abbreviations: PG = propylene glycol; VG = vegetable glycerin; mm:ss = minutes: seconds; NR = not reported; SD = standard deviation. a Unable to determine pH of cartridges because of insufficient sample material. b Not possible to calculate where pH values not measured. c Sum of the durations of all individual puffs by active users.

persons who did not use nicotine-containing products and were not exposed to them. Yet, baseline values for our study sample were higher than would be expected for this population, particularly in the second session. Data from the 2007–2008 US National Health and Nutrition Examination Survey (NHANES) indicate that 63.3% of the nonsmoking U.S. population ≥20 years of age have serum cotinine values below 0.05 ng/ml (Centers for Disease and Prevention, 2010). In this study, only 33% of the nonusers (two/six) had levels below 0.05 ng/ml in each exposure. Pirkle and colleagues calculated a median serum cotinine of 0.034 ng/ml (95% confidence interval, 0.024–0.038) in nonsmokers ≥20 years of age using 2001–2002 NHANES data (Pirkle et al., 2006). Baseline median serum cotinine levels for this study were 0.089 ng/ml for session one and 0.052 ng/ml for session two. In fact, one nonuser’s serum cotinine level in the second session, 2.94 ng/ml, neared the baseline level of a smoker (> 3 ng/ml) and was omitted from analyses (Benowitz et al., 2009). It is likely that these nonusers had some exposure to secondhand sources of nicotine, despite their self-reported negative histories. These differences may limit the application of these results to nonusers who have little to no exposure, as well as highlight a challenge for similar future studies. Although a majority of the U.S. population has relatively low cotinine levels, measurement and statistical comparison is complicated, because cotinine levels are bound on the lower side by an LOD and can be easily influenced by secondhand exposures to conventional tobacco products. This creates a relatively small window of cotinine levels in which to test nonusers. Finally, it is uncertain how much the dermal pathway contributed to the systemic absorption of nicotine. Two recent studies demonstrate just how consequential the dermal pathway might be for absorption of nicotine under acute conditions (Beko et al., 2018, 2017). To limit dermal exposure to nicotine, this study dressed each nonuser in a clean long-sleeved surgical top, and the exposure room was cleaned before both exposure sessions. Although both Beko studies were conducted with fundamental design differences than this study (i.e. greater exposure concentrations and duration, longer collection times of urinary nicotine and metabolites), they demonstrated meaningful dermal uptake of nicotine directly from the air and from clothing contaminated with nicotine. We also measured the surface deposition of nicotine via wipe sampling, data in (Melstrom et al., 2017). The median wipe accumulation rates of nicotine were 2.1 ng/100cm2/hr for the first session and 4.0 ng/100cm2/hr for the second. These accumulation rates are

concentrations for the nonusers. The data are published in (Melstrom et al., 2017). For the first exposure session, baseline median concentrations were 0.004 ng/l before the session, and 0.697 ng/l during the exposure. For the second exposure session, baseline median concentrations were 0.010 ng/l before the session, and 1.833 ng/l during the exposure. We measured individual air nicotine concentrations, because we wanted to ensure that there were no significant differences in air nicotine exposure concentrations among the non-users that might potentially be needed to explain significantly different biomonitoring levels. However, the air nicotine levels among the non-users were all within 1 ng/l of each other. Therefore, our air nicotine levels did not add useful information that helped to characterize individual exposures. 4. Discussion Our findings suggest that nonusers of e-cigarettes can experience systemic absorption of nicotine from acute exposure to secondhand ecigarette aerosol. However, the exposure may vary considerably depending upon solution, device, baseline cotinine levels, and use behavior of e-cigarette users. We recorded large variability among the measured nicotine concentrations and pH of e-cigarette solutions used, and there were also large differences in e-cigarette use behavior and solution consumption among the active users. 4.1. Biomonitoring Systemic absorption of nicotine can occur as a direct result of exposure to secondhand e-cigarette aerosol. However, this was dependent on baseline levels. In persons who had been recently exposed to nicotine before participating in the study, no additional increase was recorded. Further, our measured levels of systemic absorption of nicotine that were attributable to acute exposure to secondhand e-cigarette aerosol were less than would be expected from acute exposure to secondhand smoke from conventional cigarettes (Jones et al., 2013). Therefore, our results suggest that exposure to nicotine-containing ecigarette aerosol would likely lead to systemic absorption of nicotine for nonusers, but the absorption would be less than that from secondhand smoke from conventional cigarettes. The goal of this study was to examine nicotine exposure among 5


P. Melstrom et al.

secondhand aerosol that could increase air concentrations.

comparable to accumulation rates of nicotine from tobacco cigarettes (Matt et al., 2004). Therefore, it is likely that some of the systemic absorption recorded was via the dermal exposure route, despite our attempts to control for it.

6. Conclusion Nonusers of e-cigarettes can experience systemic absorption of nicotine from acute exposure to secondhand e-cigarette aerosol. Although less than levels produced by conventional cigarettes, the exposure is greater than in clean indoor air – the standard. Also, the exposure may vary considerably depending upon solution, device, baseline nicotine levels and the use behavior of e-cigarette users. In addition, aerosols produced from the use of e-cigarettes may contain other harmful constituents that were not measured in this study. These findings could have important implications for public health policy, planning, and practice. Since allowing e-cigarette use in public settings can result in systemic absorption of nicotine in nonusers, extending clean indoor air policies to include e-cigarettes would be expected to reduce nicotine exposure and absorption among nonusers, which is particularly important for subpopulations who are sensitive to nicotine’s effects, including children and women of reproductive age.

4.2. Solution, use behavior and consumption Although we used just a few e-cigarette products, the variability of the results indicate the potential for exposures to vary considerably. The mean nicotine concentrations measured for the custom mix e-cigarette solution used in the tanks were, on average, 16% lower than the labelled concentration; however, they showed relative consistency. In contrast, the measured nicotine concentrations in the first generation ecigarette solutions used in this experiment varied by as much as 33% from the labelled concentrations, a phenomenon previously reported (Cameron et al., 2014; Goniewicz et al., 2015, 2013a; Goniewicz et al., 2013b; Lisko et al., 2015). Measurements of pH were only available for the custom manufactured solutions used in the iTaste® device, as there were insufficient volumes in the pre-filled disposables. The marked differences in pH among the three solutions measured suggest that they were not buffered. Available evidence indicates that differences in nicotine concentrations can produce marked differences in the pH of various products (Lisko et al., 2015). However, certain flavors and other additives might also influence pH. The measured pH values would directly affect the calculated percentages of unionized nicotine and, in fact, the solutions used in the second session varied widely from 45% to 88% unionized nicotine. This large difference could have a correspondingly large effect on the amount of nicotine absorbed systemically, as nicotine in the unionized form is absorbed more effectively than in the ionized form (Armitage and Turner, 1970). Our data on use behavior and consumption point to wide variability in usage and, potentially, in the products’ ability to deliver nicotine. This latter point was recently explored in (Ruther et al., 2018), who demonstrated differences in blood nicotine concentrations among the use of tobacco cigarettes, tank model e-cigarettes and disposable e-cigarettes. Comparison of the use behavior among active users and assessment of the correlation with consumption of nicotine solution is limited in this study, because the inhalation volume was not collected in order to preserve the realism of the exposure, i.e. not hindering the active users ad lib use. However, it is important to note that the total of the active users’ sum-puff durations was almost 50% greater in session one (28 min, 10 s) than in session two (19 min, 43 s) (Table 1), despite the fact that e-cigarette solution consumption in the first session (14.3 mg) was about half that for the second (26.2 mg). This could be because the active users were current habitual users of tanks as opposed to first generation e-cigarettes and, therefore, knew how to use them more efficiently. More likely, the tanks were able to deliver nicotine more efficiently, given their increased size and battery power.

Conflict of interest statement The authors declare that there are no conflicts of interest. Disclaimer The findings and conclusions in this presentation are those of the author(s) and do not necessarily represent the views of the Centers for Disease Control and Prevention. Acknowledgements The authors would like to acknowledge the contributions of Neal Benowitz (University of California San Francisco), Sarah Edwards (Centers for Disease Control and Prevention), Wallace B. Pickworth and Jennifer L. Potts (Battelle Public Health Center for Tobacco Research. References Armitage, A.K., Turner, D.M., 1970. Absorption of nicotine in cigarette and cigar smoke through the oral mucosa. Nature 226, 1231–1232. Arrazola, R.A., Neff, L.J., Kennedy, S.M., Holder-Hayes, E., Jones, C.D., 2014. Tobacco use among middle and high school students–United States, 2013. MMWR Morb. Mortal. Wkly. Rep. 63, 1021–1026. Ballbe, M., Martinez-Sanchez, J.M., Sureda, X., Fu, M., Perez-Ortuno, R., Pascual, J.A., Salto, E., Fernandez, E., 2014. Cigarettes vs. e-cigarettes: passive exposure at home measured by means of airborne marker and biomarkers. Environ. Res. 135C, 76–80. Beko, G., Morrison, G., Weschler, C.J., Koch, H.M., Palmke, C., Salthammer, T., Schripp, T., Eftekhari, A., Toftum, J., Clausen, G., 2018. Dermal uptake of nicotine from air and clothing: experimental verification. Indoor Air 28, 247–257. Beko, G., Morrison, G., Weschler, C.J., Koch, H.M., Palmke, C., Salthammer, T., Schripp, T., Toftum, J., Clausen, G., 2017. Measurements of dermal uptake of nicotine directly from air and clothing. Indoor Air 27, 427–433. Benowitz, N.L., Bernert, J.T., Caraballo, R.S., Holiday, D.B., Wang, J., 2009. Optimal serum cotinine levels for distinguishing cigarette smokers and nonsmokers within different racial/ethnic groups in the United States between 1999 and 2004. Am. J. Epidemiol. 169, 236–248. Bernert Jr., J.T., McGuffey, J.E., Morrison, M.A., Pirkle, J.L., 2000. Comparison of serum and salivary cotinine measurements by a sensitive high-performance liquid chromatography-tandem mass spectrometry method as an indicator of exposure to tobacco smoke among smokers and nonsmokers. J. Anal. Toxicol. 24, 333–339. Bernert Jr., J.T., Turner, W.E., Pirkle, J.L., Sosnoff, C.S., Akins, J.R., Waldrep, M.K., Ann, Q., Covey, T.R., Whitfield, W.E., Gunter, E.W., Miller, B.B., Patterson Jr., D.G., Needham, L.L., Hannon, W.H., Sampson, E.J., 1997. Development and validation of sensitive method for determination of serum cotinine in smokers and nonsmokers by liquid chromatography/atmospheric pressure ionization tandem mass spectrometry. Clin. Chem. 43, 2281–2291. Cameron, J.M., Howell, D.N., White, J.R., Andrenyak, D.M., Layton, M.E., Roll, J.M., 2014. Variable and potentially fatal amounts of nicotine in e-cigarette nicotine solutions. Tob. Control 23, 77–78. Centers for Disease, C., Prevention, 2010. Vital signs: nonsmokers’ exposure to secondhand smoke –United States, 1999-2008. MMWR Morb. Mortal. Wkly. Rep. 59, 1141–1146. Chatham-Stephens, K., Law, R., Taylor, E., Melstrom, P., Bunnell, R., Wang, B., Apelberg,

5. Limitations This study is subject to limitations. First, there is great heterogeneity among e-cigarette devices and flavors, as well as in the behavior of users that make it challenging to select representative products and active users, as well as apply the results from the exposure detailed in our study to the existing landscape of available devices and flavors. Future studies should describe the devices used, as well as measure the pH and nicotine concentration of e-cigarette solutions. Second, the elevated average baseline values of our study’s nonusers were not representative of the majority of the U.S. population. Therefore, it is important for future studies targeting nonusers of nicotine-containing products to employ a high sensitivity analysis for nicotine or metabolites as part of the screening process. Third, some real-world settings where disposable e-cigarettes and tank-style devices are used could result in higher exposure levels, such as smaller enclosed spaces with less ventilation (eg, vehicles), or spaces with ongoing deposition of 6


P. Melstrom et al. B., Schier, J.G., 2014. Notes from the field: calls to poison centers for exposures to electronic cigarettes - United States, september 2010-february 2014. MMWR Morb. Mortal. Wkly. Rep. 63, 292–293. Czogala, J., Goniewicz, M.L., Fidelus, B., Zielinska-Danch, W., Travers, M.J., Sobczak, A., 2013. Secondhand exposure to vapors from electronic cigarettes. Nicotine Tob. Res. 16 (6), 655–662. England, L.J., Bunnell, R.E., Pechacek, T.F., Tong, V.T., McAfee, T.A., 2015. Nicotine and the developing human: a neglected element in the electronic cigarette debate. Am. J. Prev. Med. 7 00035-00035. Flouris, A.D., Chorti, M.S., Poulianiti, K.P., Jamurtas, A.Z., Kostikas, K., Tzatzarakis, M.N., Wallace Hayes, A., Tsatsaki, A.M., Koutedakis, Y., 2013. Acute impact of active and passive electronic cigarette smoking on serum cotinine and lung function. Inhal. Toxicol. 25, 91–101. Giovenco, D.P., Hammond, D., Corey, C.G., Ambrose, B.K., Delnevo, C.D., 2014. E-cigarette Market trends in traditional U.S. Retail channels, 2012–2013. Nicotine Tob. Res. 17, 1279–1283. Goniewicz, M.L., Gupta, R., Lee, Y.H., Reinhardt, S., Kim, S., Kim, B., Kosmider, L., Sobczak, A., 2015. Nicotine levels in electronic cigarette refill solutions: a comparative analysis of products from the U.S., Korea, and Poland. Int. J. Drug Policy 26, 583–588. Goniewicz, M.L., Knysak, J., Gawron, M., Kosmider, L., Sobczak, A., Kurek, J., Prokopowicz, A., Jablonska-Czapla, M., Rosik-Dulewska, C., Havel, C., Jacob 3rd, P., Benowitz, N., 2013a. Levels of selected carcinogens and toxicants in vapour from electronic cigarettes. Tob. Control. 23 (2), 133–139. Goniewicz, M.L., Kuma, T., Gawron, M., Knysak, J., Kosmider, L., 2013b. Nicotine levels in electronic cigarettes. Nicotine Tob. Res. 15, 158–166. Jones, I.A., St Helen, G., Meyers, M.J., Dempsey, D.A., Havel, C., Jacob 3rd, P., Northcross, A., Hammond, S.K., Benowitz, N.L., 2013. Biomarkers of secondhand smoke exposure in automobiles. Tob. Control. 23 (1), 51–57. King, B.A., Patel, R., Nguyen, K.H., Dube, S.R., 2015. Trends in awareness and use of electronic cigarettes among US adults, 2010–2013. Nicotine Tob. Res. 17, 219–227. Lisko, J.G., Tran, H., Stanfill, S.B., Blount, B.C., Watson, C.H., 2015. Chemical composition and evaluation of nicotine, tobacco alkaloids, pH, and selected flavors in e-cigarette cartridges and refill solutions. Nicotine Tob. Res. 17, 1270–1278. Matt, G.E., Quintana, P.J.E., Hovell, M.F., Bernert, J.T., Song, S., Novianti, N., Juarez, T., Floro, J., Gehrman, C., Garcia, M., Larson, S., 2004. Households contaminated by environmental tobacco smoke: sources of infant exposures. Tob. Control 13, 29–37.

McAuley, T.R., Hopke, P.K., Zhao, J., Babaian, S., 2012. Comparison of the effects of ecigarette vapor and cigarette smoke on indoor air quality. Inhal. Toxicol. 24, 850–857. Melstrom, P., Koszowski, B., Thanner, M.H., Hoh, E., King, B., Bunnell, R., McAfee, T., 2017. Measuring PM2.5, ultrafine particles, nicotine air and wipe samples following the use of electronic cigarettes. Nicotine Tob. Res. 19, 1055–1061. Pellegrino, R.M., Tinghino, B., Mangiaracina, G., Marani, A., Vitali, M., Protano, C., Osborn, J.F., Cattaruzza, M.S., 2012. Electronic cigarettes: an evaluation of exposure to chemicals and fine particulate matter (PM). Ann. Ig. 24, 279–288. Pirkle, J.L., Bernert, J.T., Caudill, S.P., Sosnoff, C.S., Pechacek, T.F., 2006. Trends in the exposure of nonsmokers in the U.S. Population to secondhand smoke: 1988-2002. Environ. Health Perspect. 114, 853–858. Rumble, J.R., 2017. Handbook of Chemistry and Physics, 98 ed. CRC Press, Boca Raton, Florida. Ruther, T., Hagedorn, D., Schiela, K., Schettgen, T., Osiander-Fuchs, H., Schober, W., 2018. Nicotine delivery efficiency of first- and second-generation e-cigarettes and its impact on relief of craving during the acute phase of use. Int. J. Hyg. Environ. Health 221, 191–198. Schober, W., Szendrei, K., Matzen, W., Osiander-Fuchs, H., Heitmann, D., Schettgen, T., Jorres, R.A., Fromme, H., 2013. Use of electronic cigarettes (e-cigarettes) impairs indoor air quality and increases FeNO levels of e-cigarette consumers. Int. J. Hyg. Environ. Health 217, 628–637. Schripp, T., Markewitz, D., Uhde, E., Salthammer, T., 2013. Does e-cigarette consumption cause passive vaping? Indoor Air 23, 25–31. Stanfill, S.B., Jia, L.T., Ashley, D.J., Watson, C.H., 2009. Rapid and chemically selective nicotine quantification in smokeless tobacco products using GC-MS. J. Chromatogr. Sci. 47, 902–909. U.S. Department of Health and Human Services, 2014. C.F.D.C.a.P., National Center For Chronic Disease Prevention and Health Promotion, Office on Smoking and Health, Printed With Corrections, January 2014. Nicotine, The Health Consequences of Smoking: 50 Years of Progress. A Report of the Surgeon General. U.S. Department of Health and Human Services, Atlanta, GA, pp. 914. U.S. federal register, 1999. In: Services, D.o.H.a.H. (Ed.), Notice Regarding Requirement for Annual Submission of the Quantity of Nicotine Contained in Smokeless Tobacco Products Manufactured, Imported, or Packaged in the United States, pp. 14086–14096.

7