A Single-Center Evaluation of Environmental Emissions from ENDS and Combustible Cigarettes

Three Vuse ENDS products consisting of a battery, sensor, and microchips, as well as a cartridge containing propylene glycol, glycerol, nicotine, flavorings, and water were evaluated in this study. Two of the products were Vuse Solo Original and contained 14 and 29 mg of nicotine per cartridge, respectively. The third was Vuse Solo Menthol and contained 29 mg of nicotine per cartridge. Two commercially available cigarette products (Marlboro Gold Pack Box, Newport Box) were also evaluated. These products were chosen because each is the leading cigarette brand style in its respective market segment (i.e., non-menthol and menthol). Finally, two ENDS brands (blu™, NJOY^®) were chosen to represent cig-a-like products as they accounted for approximately 60% of the market at the time of study conduct (32). Any commercially available blu™ and NJOY^® product and flavor were allowed for evaluation during the study.

This was a single-center, open-label, parallel-group study designed to measure the differences in levels of SHV constituents, including volatile organic compounds (VOCs) (Table 1), after ad libitum subject vaping of ENDS in an environmental test chamber. The compounds in Table 1 were chosen based on FDA's list of harmful and potentially harmful constituents (HPHCs), FDA's concern that some of these constituents could potentially cause health hazards, and published articles on secondhand smoke (33).

Results were compared both to after-subject smoking of CC, and to a non-smoking/non-vaping blank condition. Emission factors were also determined for each ENDS and CC product. The methodology employed in this study was largely based on previous studies that utilized chamber sampling to measure secondhand emissions from CC (28, 31). All subjects provided written informed consent, and the study was approved by MidLands Institutional Review Board (IRB) (Overland Park, KS, USA). Male and female ENDS users (for ENDS cohorts) and smokers (for cigarette smoking cohorts) age 21 years and older were recruited from Stockbridge, GA and the surrounding area, including Atlanta, GA. This study consisted of an enrollment visit, a product-distribution visit, and a total of 10 chamber visits held at the study site. The study took place between May 21, 2014 and December 12, 2014.

The enrollment target for this study was 72 subjects, assigned to 1 of 6 cohorts (12 subjects per cohort) based on their current usual brand (UB) preference: Marlboro Gold Pack Box [Cohort 1]; Newport Box [Cohort 2]; menthol ENDS consumers willing to switch to a different menthol ENDS brand during study (Vuse menthol ENDS (29 mg nicotine/cartridge) [Cohort 3]); current non-menthol ENDS consumers willing to switch to a different non-menthol ENDS brand during study (Vuse non-menthol ENDS (29 mg nicotine/cartridge) [Cohort 4]); current non-menthol ENDS consumers willing to switch to a different nonmenthol ENDS brand during study (Vuse non-menthol ENDS (14 mg nicotine/cartridge) [Cohort 5]); or market sample ENDS consumers (blu™ or NJOY^® [Cohort 6]). Investigational products were supplied to study subjects in Cohorts 3, 4, and 5. Subjects in Cohorts 1, 2, and 6 were responsible for providing their own UB cigarettes or ENDS. Subjects in the Vuse cohorts were instructed to completely substitute the Vuse products for their regular ENDS product and to use their assigned products ad libitum during the 2 weeks prior to their chamber visits. This was done to ensure that the subjects were fully acclimated to the test product and their product use behavior in the chamber test sessions would represent that of experienced users of the product.

All subjects within a cohort were scheduled for the same chamber visit. Separate randomization lists were generated for each chamber visit test session and contained subject numbers for all subjects within a cohort. The first four subjects within a cohort who were identified on the randomization list and who were present participated in a test session. This process was used to minimize subject allocation bias for the chamber visit test sessions and to ensure timely conduct of the chamber visit test sessions if a subject arrived late to the testing site or failed to return for a chamber visit.

Secondhand vapor and secondhand smoke emissions were measured in a 3.04 m × 3.04 m × 3.04 m (approximately 28.3 m³) emission chamber equipped with twin banks of overhead light-emitting diode (LED) lights and four 6” box fans mounted on the ceiling which ran throughout the test session to ensure mixing of the air within the chamber. The chamber was manufactured from stainless steel with polished interior walls and was devoid of furnishings to minimize sink effects from surface adsorption of SHV and SHS constituents. A 3.04 m × 3.04 m wall panel, constructed of aluminum box cross members faced with polymer panels, covered the chamber opening. Panels were covered with an interior facing skin of polished stainless steel. A large glass viewing window mounted in the center of the panel allowed for viewing the chamber interior, and a hinged entrance door allowed ingress and egress to the chamber. A louvered vent, housing an activated charcoal filter, was mounted over the door. The vent was closed during chamber operation and was opened during chamber purges with High Efficiency-Particulate Air (HEPA)-filtered air. A schematic of the environmental chamber can be found in Additional Figure A1. Chamber conditions were set daily for static mode operation and were maintained at 25 ± 1 °C and relative humidity (RH) at 50 ± 2% during each chamber run (smoking/vaping and blank). The air exchange rate during a chamber test session was determined from the change in carbon dioxide (CO₂) concentration over time. A stainless-steel manifold in a corner of the chamber held sample collection devices and was attached via tubes to sampling pumps outside the chamber. Tubes were pre-calibrated and synchronized via digital timer to open or close simultaneously at the sampling start and end times.

The chamber visits for each cohort occurred over 2 weeks beginning with chamber visits 1 through 5, which occurred during the first week, and continuing with chamber visits 6 through 10, which occurred during the subsequent week. Chamber visits included five sessions in which four subjects smoked/vaped while in the environmental test chamber (smoking runs) and five sessions in which subjects did not smoke/vape while inside the environmental test chamber (blank runs). Each subject provided or received one UB cigarette, one Vuse investigational product cartridge and battery, or one UB ENDS (depending on the subject's cohort) for use during a test session. Butane lighters and ashtrays were provided to each subject in Cohorts 1 and 2 prior to entering the chamber for smoking runs. During a test session, the environmental test chamber was operated in static mode with the outside air and exhaust air dampers closed to allow for maximum collection of SHV and SHS. Subjects entered the test chamber (with their cigarette or ENDS for smoking runs), the test chamber entry was closed, and collection of real-time measurements of the test chamber atmosphere began. Subjects were continuously monitored for the 20 min they were in the chamber.

For smoking/vaping sessions, subjects were signaled 10 min after the start of the session and instructed to smoke/vape ad libitum. On days when smoking/vaping was not scheduled (blank runs), subjects remained in the environmental test chamber for 20 min without using product. At 19 min after the start of the session, subjects were signaled to indicate that it was almost time to exit the chamber; at 20 min, the staff monitoring the test opened the door and subjects quickly exited the test chamber (with the lighters and ashtrays [smoking runs only for Cohorts 1 and 2]). Subjects in Cohorts 3, 4, and 5 returned their used investigational product to the study staff upon exit. The test chamber entry was closed, and collection of TWA samples of the test chamber atmosphere began. Time-weighted average samples were collected over a 2-h period beginning 20 min after the start of the real-time data collection to determine concentrations of analytes in the chamber air, generated by subjects, and generated by products during the test runs and determination of concentration of analytes in chamber air and generated by subjects during the blank runs (the average difference being the amount of analyte generated by products).

The analysis of SHV and SHS constituents was performed by Materials Analytical Services, LLC (MAS) (Suwanee, GA, USA). The assays were validated according to applicable Good Laboratory Practices requirements and in compliance with Standard Operating Procedures (SOPs) in effect in the laboratory of MAS. The SOPs were based on the principles and requirements described in United States Food and Drug Administration Title 21 Code of Federal Regulations (CFR) Part 58, as well as the Center for Drug Evaluation and Research's (CDER) Guidance for Industry – Bioanalytical Method Validation and the European Medicines Agency's Guideline on Bio-analytical Method Validation that were in place at the time the validation took place (34, 35). These methods included a combination of real-time sample collection and TWA samples collected and analyzed by the appropriate analytical techniques. Along with the SHV/SHS constituents listed in Table 1, temperature (T), RH and CO₂ concentrations were measured for each run. A typical figure of the real-time measurements can be found in Additional Figure A2. Real-time measurement of total volatile organic compounds (TVOC), CO, CO₂, temperature, and RH was performed during all sessions using a WolfSense IQ-610 environmental probe (Graywolf^® Sensing Solutions, Shelton, CT, USA), which used an electrochemical sensor for detection of CO, a dual-wavelength non-dispersive infrared sensor for CO₂, and a photoionization detector with a krypton 10.6 eV ultraviolet (UV) emission source sensor for TVOC. The instrument sampled air through a port in the wall of the chamber and was started at the beginning of each test and allowed to operate and collect data during the entire test run. Sampling rate was one sample per 10 s for 140 min. Duplicate samples were obtained from each test run to measure benzene, toluene, styrene, ethylbenzene, o-, m-, p- xylene, pyridine, pyrrole, o-, m-, p- cresol, phenol, glycerol, and propylene glycol were obtained in accordance to US EPA method TO-17 (36) by air sampling through TENAX TA tubes at a flow rate of 0.05 L/min for 120 min for CC and at rates of 0.05 L/min for 120 min and 0.2 L/min for 120 min for ENDS. For ENDS, TENAX tubes sampled at the appropriate flow rate were selected in order to stay within the calibration range for the analytes. The TENAX tubes were thermally desorbed, and the compounds of interest were separated by gas chromatography (GC) and then identified and quantified by mass spectrometry (MS) (Agilent 6890N GC; Agilent 5973N; Santa Clara, CA, USA).

Duplicate samples for formaldehyde and acetaldehyde were captured during each test run in accordance to NIOSH method 2016 (37) using silica-DNPH (2,4-dinitrophenylhydrazine) cartridges (Waters Sep-Pak® DNPH-Silica cartridges; WAT047205; Waters Corporation, Milford, MA, USA) at a flow rate of 1.5 L/min for 120 min. The extracts were stored in a freezer until analysis with HPLC (high-pressure liquid chromatography) (Model LC-10AT, Shimadzu Corporation, Kyoto, Japan) with a UV detector (Model SPD-10A, Shimadzu) at 365 nm.

Duplicate ammonia samples were collected during each test run according to NIOSH method 6016 (38) by air sampling through sulfuric acid-treated silica gel tubes (SKC 226-10-03; SKC, Inc., Eighty Four, PA, USA) at a flow rate of 0.5 L/min for 120 min. Each tube was placed into a small centrifuge tube, and 10 mL of deionized water was added. The tubes were then agitated and allowed to extract for 45 min at room temperature. Sample tubes from the chamber runs were stored for 1 week at room temperature, until analysis by ion chromatography (Dionex DX-1100, Thermo Fisher Scientific, Waltham, MA, USA).

Duplicate samples of hydroquinone were collected and analyzed from each test run according to OSHA method PV2094 (39). Samples were recovered from the front and back portions of the OVS XAD-7 sorbent media tubes (SKC, Inc.), extracted with H₃PO₄-acidified methanol for 30 min at room temperature. The extracts from both sections were then analyzed by GC and flame ionization detection (Agilent 5890 + Dual Detector SVOA System, Agilent).

Nicotine samples were collected in duplicate during each test run on XAD-4 sorbent tubes according to CORESTA Recommended Method No. 50 (40). The compound was desorbed using ethyl acetate and triethylamine for 60 min at room temperature. The extracts from both sections were then analyzed by GC using a nitrogen phosphorus detector (Agilent 6890N GC with 5973N Model #G1530N, Agilent).

Five replicate samples for gravimetric respirable suspended particles (RSP) were collected and analyzed during each test run according to NIOSH 0501 (41), with filter type modified from polyvinyl chloride (PVC) to polytetrafluoroethylene (PTFE) for improved solvent resistance. Samples were collected on pre-weighed 37 mm diameter 1.0 μm pore size PTFE filters housed in five inertial impactor cassettes (Cat. No. 225-2705, 225-352; SKC, Inc.). For sample collection, a known volume of air (360 L) was drawn through all impactors during each sampling session. The impactors separated RSP measuring 1.0–4.0 μm (impactor cut point 2.5 μm) and smaller from the total suspended particulate matter. After sample collection, the PTFE filters were stored in sealed vials in a freezer (below 0 °C) until analysis. Following each test session, RSP on PTFE filters were retrieved from the sample cassette and re-weighed three times within 15 min on an analytical microbalance capable of ≥ 1.0 μg accuracy (Cahn C-35, Thermo Fisher Scientific); the average mass of the RSP was then determined. Two of the five PTFE filters containing RSP were extracted using 3.00 mL methanol for 60 min at room temperature.

Analysis of solanesol was performed according to CORESTA Recommended Method No. 52 (42) by HPLC (Model LC-10AT, Shimadzu). Ultraviolet particulate matter (UVPM) and fluorescent particulate matter (FPM) were analyzed according to CORESTA Recommended Method No. 51 (43) by HPLC with UV (Model SPD-10A, Shimadzu) and fluorescence detection (Model FL3000, Thermo Fisher Scientific).

Air exchange rate was determined from the decay of CO₂ measured in real-time in the chamber. Simple regression analysis was used to calculate decay rate for each session. Calculations of SHS analyte concentrations were based on TWA samples and background-corrected real-time data collected over specified testing periods. Background correction of real-time data was carried out by subtracting from measured data the average of the real-time data collected during a pre-defined period after the start of the chamber session (10 min), to eliminate background concentrations in the chamber atmosphere before product use. Values below the lower limit of quantification (LLOQ) were imputed as LLOQ for calculation. Emission factors were then determined for each analyte and reported for those analytes that were statistically significantly above the blank. Two-sample 2-tailed t-tests were performed on derived endpoints to determine if there were differences between TWA concentrations for product and reference, and TWA concentrations for smoking/vaping and blank runs. Comparisons for all measured SHV and SHS constituents (primary and secondary objectives) were made at a 0.05 level of significance.

Emission factors were calculated for each analyte for each investigational product with respect to per-product use and with respect to “per-mg e-liquid” used for Vuse (Cohorts 3, 4, and 5). For TWA analytes, blank-corrected (BC subscript in equations) concentrations were used for the emission factor calculations. Emission factors were calculated as:

With respect to per-product (for all cohorts): (TWABCμgm3)×(28.3 m3)×(PeakTWA)×1#products×mg1000μg×mg#products \left( {TW{A_{BC}}{{\mu g} \over {{m^3}}}} \right) \times \left( {28.3\,{m^3}} \right) \times \left( {{{Peak} \over {TWA}}} \right) \times {1 \over {\#\, products}} \times {{mg} \over {1000\,\mu g}} \times {{mg} \over {\#\, products}}

To adjust for decay of analytes in the chamber due to air infiltration resulting from the sampling process and low levels of air infiltration (the chamber could not be completely sealed), the emission rates were corrected based upon the ratio of peak concentration to TWA concentration of CO₂ in the chamber.

For real-time analytes, the emission factors were calculated directly from the background-corrected and blank-corrected peak concentration (PEAK_BGBC). For TVOC, the calculation was:

With respect to per-product (for all Cohorts): PEAKBGBCμgm3×(28.3 m3)×1#products×mg1000μg=mg#products PEA{K_{BGBC}}{{\mu g} \over {{m^3}}} \times \left( {28.3\,{m^3}} \right) \times {1 \over {\#\, products}} \times {{mg} \over {1000\mu g}} = {{mg} \over {\#\, products}}

For CO, the calculation was:

With respect to per-product (for all cohorts): PEAKBGBCmgm3×(28.3 m3)×1# products=mg# products PEA{K_{BGBC}}{{mg} \over {{m^3}}} \times \left( {28.3\,{m^3}} \right) \times {1 \over {\# \,products}} = {{mg} \over {\# \,products}}

A total of 71 subjects (12 in each of the Marlboro Gold Pack, Newport Box, Vuse non-menthol (14 mg nicotine/cartridge), Vuse non-menthol (29 mg nicotine/cartridge), and Vuse menthol (29 mg nicotine/cartridge) groups, and 11 in the market-sample ENDS group) were enrolled in the study. The mean (± standard deviation (SD)) age of subjects was 34 ± 9 years. Subjects were mostly Black or African American (64.8%) and White (32.4%); and 97.2% of subjects were not Hispanic or Latino. Gender distribution was similar for males (49.3%) and females (50.7%). Among the 24 subjects enrolled in the Marlboro Gold Pack and Newport Box groups, the average (± SD) self-reported number of cigarettes smoked per day was 15.1 ± 6.2; and two subjects were also dual users of ENDS. Subjects enrolled in the ENDS groups (n = 47) used an average (± SD) of 2.68 ± 2.2 ENDS per week; and 27 reported ongoing daily or weekly cigarette smoking of 3.9 ± 4.6 cigarettes per day.

Average chamber conditions (air changes per h (ACH), T, and RH) during smoking/vaping runs were consistent across all six cohorts and comparable to the values from all blank runs. Mean ACH values ranged from 0.06 to 0.10 for smoking/vaping runs and 0.07 to 0.10 for blank runs.

Average blank-corrected constituent concentrations by cohort are presented in Table 2. Uncorrected mean chamber concentrations are summarized in Additional Table A1. Statistical comparisons were made between uncorrected chamber concentrations of secondhand constituents from a product and its respective blank, and the results are presented in Additional Table A2. Notations were made in Table 2 where uncorrected secondhand constituent emissions from a product were statistically significantly different than from its respective blank. In general, mean uncorrected TWA concentrations of secondhand constituents from an ENDS were not statistically significantly different than from its respective blank. Exceptions were observed for nicotine emissions from each ENDS, for glycerol and propylene glycol emissions from both Vuse Menthol and Vuse non-menthol (29 mg nicotine/cartridge), and for ammonia emissions from Vuse non-menthol (29 mg nicotine/cartridge). For each of these exceptions, secondhand constituent concentration from the ENDS was statistically significantly higher than from its respective blank. Mean uncorrected TWA chamber concentrations of SHS constituents from the CC products were higher than from their respective blanks, and most differences were statistically significant (p < 0.05) (Additional Tables A1 and A2). The only exception to these findings was glycerol, for which the difference between smoking and blank runs was not statistically significant.

The results of the statistical comparison (p-values) of mean TWA_BC (TWA_BGBC for CO and TVOC) concentrations from the ENDS and their respective reference CC are summarized in Table 3. Comparisons in Table 3 are like-to-like; i.e., menthol products were compared to menthol products and non-menthol products were compared to non-menthol products. Because the market sample contained menthol and non-menthol products results were compared to both reference CCs.

For the majority of the SHV and SHS constituents evaluated in this study, mean TWA_BC concentrations from ENDS were statistically significantly lower than CC (p < 0.05), with many of the chamber concentrations after ENDS vaping close to 0 or negative. Two exceptions were glycerol and propylene glycol. Glycerol levels following the market-sample ENDS and Vuse non-menthol (14 mg nicotine/cartridge) vaping runs were higher than following CC smoking runs but were not statistically significant. When compared to CC, differences in glycerol levels for Vuse nonmenthol (29 mg nicotine/cartridge) and Vuse menthol were significant (p < 0.05) (Table 3). Mean TWA_BC for propylene glycol was nearly five times higher for Vuse menthol than for Newport Box CC, a comparison that was also statistically significant (p < 0.05).

Emission factors were calculated for all SHV and SHS constituents. Emission factors were only reported for constituents for which the uncorrected chamber concentration from the study product was statistically significantly greater than that of its respective blank. Table 4 summarizes the emission factor results for CC and ENDS (mg/cigarette or mg/10 min vaping).

A total of 20 cartridges each of Vuse Menthol, Vuse Original, and Vuse Original Silver were provided to 12 participants in each of Cohorts 3, 4, and 5, respectively. These cartridges were used for product use (vaping) runs in the chamber. The mass of e-liquid (g) used by participants in Cohorts 3, 4, and 5 is summarized in Table 5. The mass of e-liquid consumed during use of the market sample products was not measured. Table 6 lists the emission factor results for Vuse products (only) in units of μg/mg e-liquid consumed. Reporting as μg/mg e-liquid may be useful for modeling of emissions from the Vuse products over a typical day's usage. Among the three Vuse products, mean emission factors (μg/mg of e-liquid) were similar with the exception of propylene glycol, which was higher for Vuse menthol than for Vuse non-menthol (29 mg nicotine/cartridge) and Vuse non-menthol (14 mg nicotine/cartridge) (171, 22, and 22 μg/mg of e-liquid, respectively) (Table 6).