Nicotine & Tobacco Research, 2019, 1239–1247

doi:10.1093/ntr/nty140
Original investigation
Received December 5, 2017; Editorial Decision June 29, 2018; Accepted July 23, 2018

Original investigation

Biomarkers of Tobacco Exposure Decrease

After Smokers Switch to an E-Cigarette or
Nicotine Gum

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Elaine K. Round PhD1, Peter Chen PhD1, Anthony K. Taylor MSPharm2,
Eckhardt Schmidt PhD1
Scientific & Regulatory Affairs, RAI Services Company, Winston-Salem, NC; 2AbbVie, North Chicago, IL
1

Corresponding Author: Elaine K. Round, PhD, Scientific & Regulatory Affairs, RAI Services Company, 401 North Main
Street, Winston-Salem, NC 27101, USA. E-mail: [email protected]

Abstract
Introduction: The aerosol composition of electronic cigarettes (ECs) suggests that exposure to toxi-
cants during use is greatly reduced compared to exposure from combustible cigarettes (CCs).
Methods: This randomized, parallel-group, clinical study enrolled smokers to switch to Vuse Solo
(VS) Digital Vapor Cigarettes (Original or Menthol) or Nicorette 4 mg nicotine gum (NG) in a con-
trolled setting. Subjects who smoked CCs ad libitum for 2  days during a baseline period were
then randomized to ad libitum use of either VS or NG for 5 days. Biomarkers of 23 toxicants were
measured in 24-hour urine samples and blood collected at baseline and following product switch.
Results: A total of 153 subjects completed the study. Total nicotine equivalents decreased in all
groups, but higher levels were observed in the VS groups compared to the NG groups, with
decreases of 38% and 60%–67%, respectively. All other biomarkers were significantly decreased
in subjects switched to VS, and the magnitude of biomarker decreases was similar to subjects
switched to NG. Decreases ranged from 30% to greater than 85% for constituents such as benzene
and acrylonitrile.
Conclusions: These results indicate that exposure to toxicants when using VS is significantly
reduced compared to CC smoking, and these reductions are similar to those observed with use
of NG. Although statistically significantly decreased, nicotine exposure is maintained closer to CC
smoking with VS use compared to NG use. This research suggests that use of VS exposes consum-
ers to fewer and lower levels of smoke toxicants than CCs while still providing nicotine to the
consumer.
Implications: This is the first study to report changes in nicotine delivery and biomarkers of tobacco
exposure following a short-term product switch from CCs to either an EC or NG in a controlled
environment. The study shows that nicotine exposure decreased in both groups but was main-
tained closer to CC smoking with the EC groups. Biomarkers of tobacco combustion decreased to
similar levels in both EC and gum groups.

Introduction nicotine-containing products, as proposed by Kozlowski et al.,3 is the

concept that nicotine-delivering products vary widely in the risks to
Cigarette smoking is a leading cause of preventable death in the
the individual consumer and population health. This continuum has
United States and has been associated with several types of dis-
been explicitly adopted by the Strategic Dialogue on Tobacco Harm
eases including cancer and heart disease.1,2 A continuum of risk of

© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco. 1239
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Reduction and embodied by the 2014 Surgeon General’s report.2,4 by Chesapeake Institutional Review Board (Columbia, Maryland) in
This framework clarifies that combustible cigarettes (CCs), on one November 2014 and was performed in accordance with the prin-
end of the continuum, are the major cause of tobacco-related disease ciples of the Declaration of Helsinki 2013. Registration on www.
and are associated with the highest health risk. On the other end of clinicaltrials.gov (Identifier number NCT02323438) occurred on
the continuum are medicinal nicotine and noncombustible tobacco December 18, 2014. Written informed consent was obtained from
products including electronic cigarettes (ECs) that may contribute each subject before study procedures were performed.
to reducing tobacco-related disease. Over the last several years, ECs
have been increasingly used by smokers as an alternative to CCs.5–9 Participants
The Royal College of Physicians has stated that EC products are Generally healthy males and females, 21–60 years of age, inclusive,
95% less risky than CCs; however, the long-term effects of use will who reported smoking at least 10 combustible, filtered, menthol or
remain under study for years to come.10 non-menthol cigarettes per day and reported smoking their first cig-
Several studies have assessed the aerosol emitted from various arette within 30 minutes of waking were included in the study. In
brands of first-generation cig-alike EC products. Although constitu- addition, potential participants had to be willing to switch from their
ents such as carbonyls and metals are detected in these products, the usual brand (UB) cigarettes to VS Original flavor, VS Menthol flavor,

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levels are generally many times lower than the levels observed in CC or NG while in clinic. Subjects with controlled, chronic health con-
smoke.11–15 This research suggests that exposure to the harmful and ditions were included at the discretion of the investigator; however,
potentially harmful constituents (HPHCs) found in cigarette smoke diabetic subjects were excluded. A total of 385 subjects were screened
would be greatly reduced when CC smokers switch to an EC product. for the study, and 153 subjects completed all study activities.
ECs were brought under regulatory authority of the US Food
and Drug Administration (FDA) on August 8, 2016, and will require
Test Products and Product Use
regulatory filing and marketing authorization to be legally sold in
VS Digital Vapor Cigarettes were introduced commercially by R.J.
the United States after the initial compliance period ends. In its
Reynolds Vapor Company in March 2013. The product is a first-
Premarket Tobacco Product Applications for Electronic Nicotine
generation cig-alike product composed of a battery, heating element,
Delivery Systems Draft Guidance, the FDA Center for Tobacco
microchip, sensor, and a cartridge containing e-liquid composed of
Products states that marketing authorization will be granted if an
propylene glycol, glycerin, nicotine, flavorings, and water. During
electronic nicotine delivery system product, which includes ECs, is
use, the heating element aerosolizes the liquid in the cartridge and
demonstrated to be appropriate for the protection of public health.
produces a puff of aerosol that contains aerosol-forming excipients
Among the many types of research suggested to support such an
(propylene glycol and glycerin) and nicotine. A microchip in the cart-
application, an assessment of biomarkers of tobacco exposure and
ridge tracks puffing activation time to prevent depletion of e-liquid.
harm in consumers of these products is recommended.16 In addition,
Power wattage is the most informative parameter of an EC with
experts in the field have proposed research agendas to better under-
respect to the heating of the e-liquid. The effective power to the VS
stand the health effects of EC products.17,18 Biomarkers of tobacco
cartridge is controlled to approximately 3 W during a puff. The two
exposure have been extensively researched and many biomarkers
brand styles used in this study include VS Original, a tobacco flavor,
exist that distinguish among smokers, smokeless-tobacco consum-
and VS Menthol. Both brand styles contain approximately 600 µL of
ers, and nontobacco consumers.19,20 Several researchers have made
a 4.8% nicotine e-liquid, or approximately 29 mg of nicotine.
recommendations to apply these biomarkers to tobacco product
Nicorette nicotine polacrilex gum (GlaxoSmithKline Consumer
regulation and disease prevention.21–23
Healthcare, LP, Philadelphia, PA) is commercially available in 2 and
There has been limited exploration to date of biomarkers of
4  mg strengths. The 4-mg NG was chosen for use in this study in
tobacco exposure associated with the use of ECs. Study types have
order to include smokers who typically have higher levels of nicotine
included cross-sectional studies with varying cohort types including
exposure. Instructions on the package state: “If you smoke your first
smokers, exclusive EC users of differing product generations and
cigarette within 30 minutes of waking up, use 4  mg nicotine gum.”
durations of use, dual users, and nicotine replacement therapy users;
The White Ice Mint flavor was provided for use in this study. Subjects
a short-term in-clinic switching study; and two longitudinal studies
received written instructions for use based on the Nicorette gum pack-
of smokers who switched to ECs in their natural environments. In
age label. Nicorette gum was the current market leader among oral
general, these studies have shown that biomarker levels are markedly
nicotine replacement therapies at the time this study was conducted.
lower when consumers use ECs compared to CCs.24–31
All subjects provided their own UB cigarettes for use during the
This study evaluated the changes in nicotine biomarkers, product
baseline period. UB cigarettes were collected by site staff at check-in
use, and biomarkers of 22 carcinogens and toxicants found in cigar-
on day −3 and dispensed to subjects one at a time upon request until
ette smoke after smokers switch to ad libitum use of Vuse Solo (VS)
11:00 pm on day −3 and from 07:00 am to 07:30 pm on day −2.
Digital Vapor Cigarettes (Original or Menthol) for 5 days. A group who
Following randomization, subjects used VS or NG ad libitum from
switched to ad libitum use of nicotine gum (NG) was included for quali-
07:00 am to 11:00 pm on days 1, 2, 3, and 4. On day 5, ad libitum
tative comparison to the maximum possible reductions in biomarkers
use occurred from 07:00 am to 07:30 pm. Product use ended ear-
of tobacco exposure over the study period while still allowing for nico-
lier on Days −2 and 5 to start a 12-hour nicotine abstinence period
tine use. This is the first study to report changes in nicotine delivery and
in preparation for nicotine pharmacokinetic assessments on Days
biomarkers of tobacco exposure following a short-term product switch
−1 and 6 (data to be reported in a separate publication). Days −1
from CCs to either ECs or NG in a controlled environment.
and 6 involved a short duration of product use starting no earlier
than 07:30 am, followed by nicotine abstinence and blood collection
Methods over a 6-hour period. After completion of the 6 hours, subjects on
This was a randomized, controlled, open-label, parallel group study Day −1 smoked ad libitum until 11:00 pm, and subjects on Day 6
conducted by DaVita Clinical Research at one site in Minneapolis, were discharged from the study following completion of final safety
Minnesota, between January and May 2015. The study was approved procedures.
Study Design The calculation of total nicotine equivalents includes uncon-
A full schematic of the study design can be found in Supplementary jugated nicotine, unconjugated cotinine, unconjugated
Figure 1. Potential subjects completed a prescreening telephone trans-3′-hydroxycotinine, nicotine-N-glucuronide, cotinine-
interview and one screening visit to assess eligibility within 30 days N-glucuronide, trans-3′-hydroxycotinine-O-glucuronide, coti-
of study enrollment on Day −3. On Day −3, eligible subjects were nine-N-oxide, nicotine-N-oxide, norcotinine, nornicotine, and
enrolled in the study and started a 9-day in-clinic residence. Baseline 4-hydroxy-4-(3-pyridyl)-butanoic acid.36
assessments during smoking of subjects’ UB cigarettes occurred for Results for each biomarker were reported by the lab as a concen-
the first 3 days (Day −3 through Day −1). On Day 1, smokers were tration. Total daily excretion yields for each biomarker were deter-
randomized to one of four cohorts. mined by multiplying the observed biomarker concentration by the
Smokers of non-menthol cigarettes were randomized to one of total urine volume for each 24-hour collection to obtain biomarker
two cohorts: mass/24 h. When observed concentrations were below the limit of
quantification, a value of ½ limit of quantification was imputed and
• Cohort 1: EC − VS Original, or used for determination of 24-hour totals. When multiple metabo-
• Cohort 2: NG lites were included in the calculation of total constituent equivalents

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(eg, nicotine, acrylamide, and naphthalene), the individual metabo-
Smokers of menthol cigarettes were randomized to one of two
lites (mass/24 h) were converted to molar equivalents of the parent
cohorts:
compound and summed. The full list of biomarkers can be found in
• Cohort 3: EC − VS Menthol, or Tables 2 and 3.
• Cohort 4: NG
Statistical Analysis
Post–product switch assessments occurred for 6 days (Day 1 through
A sample size of 35 completed subjects per cohort was estimated to
Day 6). Upon completion of study procedures on Day 6, subjects
provide 80% power to detect a 25% reduction with a Bonferroni-
were discharged from the clinic. The Fagerström Test for Nicotine
adjusted p value of .05/36. The powering for the full clinical study
Dependence and a demographic questionnaire were administered to
used a Bonferroni-adjusted significance level that included 36
all potential subjects at the screening visit.32
comparisons, not all of which are presented here. Data from pre-
vious studies were used for the sample-size determination. Up to
Biological Sample Collection
41 subjects per cohort were enrolled to ensure 35 completed the
Whole blood samples were collected at approximately 07:00 pm on study. Demographic, Fagerström Test for Nicotine Dependence,
Days −2, 1, 3, and 5 for measurement of carboxyhemoglobin percent and product-use descriptive statistics were calculated for all rand-
saturation. Measurements were performed at LabCorp (Burlington, NC omized subjects. Biomarker data were summarized for all subjects
and Minneapolis, MN) using a carbon monoxide oximeter to spectro- who completed the study. Percent changes were calculated as the
photometrically measure carboxyhemoglobin and hemoglobin. percent difference between the mean biomarker values from baseline
Plasma was collected on Days −2, 1, 3, and 5 at approximately to post–product switch.
07:00 am (before product use began each day), 01:00 pm, and 07:00 A two-sided paired t test was used to determine the significance
pm for measurement of nicotine and cotinine. Additional plasma of differences between Day −2 and Day 5 urinary total nicotine
samples were collected on Days −1 and 6 for nicotine pharmaco- equivalents results within cohorts. A  two-sided test was employed
kinetic analysis just before and for 6 hours following the start of a here because neither an increase nor a decrease in nicotine exposure
single ad libitum use period (data to be reported elsewhere). Plasma could be predicted before conducting the study. A one-sided paired
samples were processed and aliquoted within 90 minutes of collec- t test was used to determine the significance of differences between
tion and stored at −70°C until shipment for analysis. Day −2 and Day 5 biomarker results within cohorts for all other
Urine samples were collected for 24-hour periods starting at urinary biomarkers and blood carboxyhemoglobin. A one-sided test
07:30 pm on Days −3 and 4 and ending at 07:30 pm on Days −2 was employed here because these biomarkers were all expected to
and 5. Urine was stored at 4°C until collection was complete. Total decrease based on chemical analysis of the aerosol produced by VS
24-hour volumes were recorded, and samples were aliquoted and during machine puffing. All calculations were performed using SAS
stored at −70°C until shipment for analysis. Version 9.1 or higher. All p values were adjusted using a Bonferroni
step-down method including 36 comparisons to maintain an overall
Urine Mutagenicity significance level of 0.05.
Aliquots of 24-hour urine samples were shipped on dry ice to
Covance Laboratories Limited (Harrogate, UK) for assessment of
urine mutagenicity using a modified Ames assay. Salmonella typh- Results
imurium strain YG1024 with S9 for metabolic activation was A total of 385 subjects were screened for the study, of which 162
used for assessment, and the assay and analysis were performed as were enrolled on Day −3, and 158 were randomized on Day 1. Of
described in Krautter et al., 2014.33 those randomized, 38, 39, 40, and 41 subjects were randomized to
Cohorts 1, 2, 3, and 4, respectively. Five subjects withdrew con-
Biomarker Analysis sent after randomization, resulting in 153 subjects who completed
Urinary biomarker analysis was performed by ABF GmbH (Munich, the study, with 37, 38, 38, and 40 subjects completing the study in
Germany), and plasma nicotine and cotinine analysis was performed Cohorts 1, 2, 3, and 4, respectively.
by Celerion, Inc. (Lincoln, NE). Methods are generally as described Table  1 summarizes the demographics and baseline charac-
in Theophilus et  al.34 and Round et  al.,35 with one exception. teristics of subjects by cohort. Subjects generally had similar
Table 1. Demographics and Baseline Characteristics (n [%]), or mean ± SD)

NM smoker—VS original NM smoker—nicotine gum M smoker—VS menthol M smoker—nicotine gum

(N = 38) (N = 39) (N = 40) (N = 41)

Age, mean ± SD 41.63 ± 11.22 40.18 ± 11.44 42.55 ± 10.87 41.46 ± 10.00

Gender, n (%)
 Female 11 (28.9) 14 (35.9) 15 (37.5) 11 (26.8)
 Male 27 (71.1) 25 (64.1) 25 (62.5) 30 (73.2)
Ethnicity, n (%)
  Hispanic or Latino 1 (2.6) 0 0 1 (2.4)
  Non-Hispanic or Latino 37 (97.4) 39 (100) 40 (100) 40 (97.6)
Race, n (%)
  American Indian or 0 0 4 (10.0) 1 (2.4)
Alaskan Native
 Asian 0 0 0 0

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  Black or African American 14 (36.8) 13 (33.3) 25 (62.5) 29 (70.7)
  Native Hawaiian or Other 0 0 0 0
Pacific Islander
 White 21 (55.3) 25 (64.1) 11 (27.5) 6 (14.6)
 Multiple 2 (5.3) 0 0 5 (12.2)
 Other 1 (2.6) 1 (2.6) 0 0
Highest level of school completed
  Grade school 0 0 0 0
  High school (grades 9–11) 3 (7.9) 1 (2.6) 4 (10.0) 5 (12.2)
  High school graduate or 16 (42.1) 15 (38.5) 11 (27.5) 13 (31.7)
GED
  Technical school 5 (13.2) 3 (7.7) 6 (15.0) 3 (7.3)
  Some college 8 (21.1) 15 (38.5) 14 (35.0) 13 (31.7)
  College graduate 6 (15.8) 5 (12.8) 4 (10.0) 6 (14.6)
  Graduate school 0 0 0 0
  Decline to answer 0 0 1 (2.5) 1 (2.4)
  FTND, mean ± SD 6.0 (1.5) 6.3 (1.4) 6.0 (1.5) 6.2 (1.4)

FTND = Fagerström Test for Nicotine Dependence; GED = General Education Diploma; M = Menthol; NM = Non-menthol; SD = standard deviation; VS = Vuse
Solo.

Table 2. Biomarkers of Nicotine Exposure, Mean ± SD at Baseline and Day 5 and Percent Change

Baseline to Day 5
Nicotine biomarker Baseline (mean ± SD) Day 1 (mean ± SD) Day 3 (mean ± SD) Day 5 (mean ± SD) percent change

Urinary nicotine equivalents (mg/24 h)

  NM smoker—VS originala 20.9 ± 7.6 — — 12.9 ± 9.8 −38.3
  NM smoker—gumb 19.5 ± 5.7 — — 7.9 ± 6.1 −59.7
  M smoker—VS mentholc 21.5 ± 6.9 — — 13.4 ± 8.8 −37.8
  M smoker—gumd 21.7 ± 7.8 — — 7.2 ± 4.3 −66.7
Plasma cotinine at 07:00 pm (ng/mL)
  NM smoker—VS originala 269 ± 108 159 ± 83 160 ± 122 183 ± 153 −32.0
  NM smoker—gumb 264 ± 94 162 ± 75 113 ± 78 117 ± 95 −55.7
  M smoker—VS mentholc 311 ± 114 201 ± 117 186 ± 123 211 ± 148 −32.2
  M smoker—gumd 317 ± 111 184 ± 71 122 ± 74 110 ± 77 −65.3
Plasma nicotine at 07:00 pm (ng/mL)
  NM smoker—VS originala 19.2 ± 9.0 6.5 ± 5.9 9.6 ± 8.9 11.5 ± 10.4 −40.1
  NM smoker—gumb 19.0 ± 8.5 6.4 ± 4.3 5.2 ± 5.2 6.0 ± 5.4 −68.4
  M smoker—VS mentholc 20.3 ± 8.4 7.6 ± 4.2 10.5 ± 8.2 13.0 ± 9.8 −36.0
  M smoker—gumd 21.9 ± 8.3 5.6 ± 4.3 4.4 ± 3.8 5.3 ± 4.2 −75.8

Statistical significance for change in urinary nicotine equivalents was determined using a two-sided paired t test and adjusted using a Bonferroni step-down method.
All changes from baseline to Day 5 were statistically significant (p < .05). Statistical significance for trend in plasma cotinine and nicotine was determined using
mixed models with repeated measures for analysis of day effect. p values were adjusted using a step-down Bonferroni method. All trends were statistically signifi-
cant (p < .05). CPD = cigarettes per day; M = Menthol; NM = Non-menthol; SD = standard deviation; VS = Vuse Solo.
a
n = 37.
b
n = 38.
c
n = 38.
d
n = 40.
Table 3. Biomarkers of Tobacco Smoke Exposure in 24-hour Urine and Whole Blood (Mean ± SD) and Percent Change of the Mean Biomarker Amounts From Baseline to Day 5

Non-menthol smoker—VS original (n = 37) Non-menthol smoker—gum (n = 38) Menthol smoker—VS menthol (n = 38) Menthol smoker—gum (n = 40)

Baseline Day 5 Baseline Day 5 Baseline Day 5 Baseline Day 5

Biomarker Toxicant (M ± SD) (M ± SD) % change (M ± SD) (M ± SD) % change (M ± SD) (M ± SD) % change (M ± SD) (M ± SD) % change

a
COHb (% saturation) Carbon monoxide 5.8 ± 1.6 1.4 ± 0.6 −75.3 5.4 ± 1.4 1.3 ± 0.4 −75.0 6.0 ± 1.7 1.4 ± 0.4 −77.1 5.7 ± 1.8 1.4 ± 0.5 −76.1
SPMA (µg/24 h)b Benzene 3.7 ± 2.2 0.4 + 0.2 −89.7 4.4 ± 3.8 0.4 ± 0.3 −90.1 3.9 ± 2.0 0.4 ± 0.2 −89.0 4.4 ± 4.8 0.5 ± 0.2 −89.3
3-HPMA (µg/24 h)b Acrolein 2052.8 ± 1274.3 605.6 ± 291.2 −70.5 1828.9 ± 518.5 512.5 ± 192.0 −72.0 2065.1 ± 790.9 598.3 ± 238.3 −71.0 1983.7 ± 632.6 623.5 ± 274.9 −68.6
HMPMA (µg/24 h)b Crotonaldehyde 578.2 ± 327.2 129.9 ± 75.7 −77.5 537.8 ± 186.2 118.9 ± 37.6 −77.9 564.1 ± 202.0 128.4 ± 58.2 −77.2 549.1 ± 204.8 128.9 ± 56.5 −76.5
MHBMA (µg/24 h)b 1,3-butadiene 4.9 ± 3.2 2.2 ± 2.6 −55.5 5.2 ± 2.9 1.9 ± 2.0 −63.4 4.2 ± 2.5 1.9 ± 1.8 −56.0 4.2 ± 2.2 2.6 ± 2.5 −37.7
CEMA (µg/24 h)b Acrylonitrile 261.2 ± 187.1 36.8 ± 21.7 −85.9 226.9 ± 72.3 29.0 ± 13.6 −87.2 254.0 ± 94.6 36.5 ± 19.8 −85.6 246.1 ± 106.2 34.7 ± 16.6 −85.9
HEMA (µg/24 h)b Ethylene oxide 16.4 ± 9.2 6.2 ± 3.1 −62.3 20.0 ± 17.5 7.9 ± 6.1 −60.4 16.7 ± 11.8 7.7 ± 4.6 −53.9 17.5 ± 11.2 9.4 ± 4.7 −46.0
NNAL-T (ng/24 h)b NNK 603.1 ± 428.9 249.4 ± 165.3 −58.7 483.8 ± 313.6 176.7 ± 113.1 −63.5 532.3 ± 365.6 239.7 ± 155.4 −55.0 503.1 ± 317.4 201.4 ± 115.8 −60.0
NNN-T (ng/24 h)b NNN 21.4 ± 17.1 2.7 ± 2.4 −87.4 27.8 ± 18.8 3.2 ± 4.9 −88.6 32.5 ± 32.9 2.7 ± 1.2 91.8 24.5 ± 15.7 2.5 ± 1.2 −89.8
NAT-T (ng/24 h)b NAT 303.8 ± 290.1 3.9 ± 7.9 −98.7 295.8 ± 223.4 2.4 ± 1.0 −99.2 264.5 ± 191.3 5.6 ± 7.9 −97.9 286.1 ± 225.3 4.6 ± 9.0 −98.4
NAB-T (ng/24 h)b NAB 54.5 ± 47.1 5.8 ± 2.7 −89.5 51.7 ± 34.3 6.1 ± 2.6 −88.3 47.5 ± 30.7 6.4 ± 2.2 −86.5 46.2 ± 33.4 6.9 ± 5.1 −85.0
1-AN (ng/24 h)b 1-aminonaphthalene 109.3 ± 43.0 4.5 ± 2.6 −95.5 103.1 ± 34.8 4.3 ± 1.7 −95.8 106.2 ± 41.5 5.3 ± 2.6 −95.0 109.1 ± 42.3 7.4 ± 13.5 −94.2
2-AN (ng/24 h)b 2-aminonaphthalene 27.4 ± 13.9 2.6 ± 1.4 −90.4 27.7 ± 10.4 2.5 ± 1.4 −90.9 29.2 ± 12.6 2.4 ± 0.7 −91.9 29.5 ± 12.7 2.5 ± 1.4 −91.5
3-ABP (ng/24 h)b 3-aminobiphenyl 10.6 ± 5.0 2.8 ± 1.4 −74.0 9.6 ± 3.9 2.1 ± 1.3 −78.0 10.1 ± 5.0 2.2 ± 1.0 −78.6 10.4 ± 5.3 2.0 ± 0.9 −80.6
4-ABP (ng/24 h)b 4-aminobiphenyl 21.2 ± 8.8 7.8 ± 3.4 −63.5 22.6 ± 9.3 7.2 ± 3.8 −68.3 22.4 ± 8.1 6.1 ± 2.6 −73.0 23.0 ± 8.0 6.6 ± 2.3 −71.4
o-toluidine (ng/24 h)b o-toluidine 259.3 ± 292.8 109.9 ± 108.4 −57.6 203.4 ± 75.0 97.8 ± 75.1 −51.9 203.6 ± 66.1 90.2 ± 33.3 −55.7 203.0 ± 68.3 96.0 ± 44.7 −52.7
Naphthalene equivalents Naphthalene 34.6 ± 14.1 5.7 ± 3.5 −83.6 33.8 ± 12.7 5.4 ± 2.5 −83.9 36.2 ± 13.0 10.6 ± 14.4 −70.1 42.3 ± 29.0 11.8 ± 18.3 −72.0
(µg/24 h)b
3-OH-B[a]P (pg/24 h)b Benzo[a]pyrene 258.1 ± 325.2 93.6 ± 73.6 −63.8 621.3 ± 2443.2 133.4 ± 171.6 −78.5* 270.2 ± 359.2 81.1 ± 52.9 −70.0 189.6 ± 157.6 104.5 ± 110.0 −44.9
2-OH-fluorene (µg/24 h)b Fluorene 1.7 ± 1.0 1.2 ± 0.9 −30.4 1.7 ± 0.8 1.0 ± 0.4 −43.0 1.7 ± 0.8 1.1 ± 0.6 −35.7 1.7 ± 0.8 1.1 ± 0.6 −34.2
1-OH-pyrene (ng/24 h)b Pyrene 503.5 ± 260.4 183.9 ± 128.5 −63.5 679.4 ± 1026.1 336.1 ± 731.0 −50.5 568.8 ± 407.9 186.5 ± 180.6 −67.2 529.3 ± 390.7 199.9 ± 158.9 −62.2
Acrylamide equivalents Acrylamide 106.0 ± 30.9 53.1 ± 19.4 −50.0 98.3 ± 30.1 49.7 ± 15.9 −49.2 108.8 ± 36.3 49.7 ± 16.0 −54.3 113.3 ± 34.6 55.9 ± 16.8 −50.6
(µg/24 h)b
Thiocyanate (µmol/24 h)b Hydrogen cyanide 171.3 ± 103.3 103.8 ± 45.7 −39.4 143.8 ± 68.3 101.6 ± 46.8 −29.3 186.8 ± 114.2 119.5 ± 54.5 −36.0 152.9 ± 81.9 108.5 ± 48.0 −29.0
Urine mutagenicity General measure of 272.5 ± 183.5 32.4 ± 25.8 −88.1 226.0 ± 155.5 40.2 ± 46.0 −82.2 296.6 ± 215.7 29.9 ± 24.9 −90.0 344.7 ± 290.4 38.6 ± 27.5 −88.8
(Revertants/103/24 h)b mutagenic properties
of urine

Statistical significance was determined within cohort using a one-sided paired t test and was adjusted using a Bonferroni step-down method. All changes from baseline to Day 5 were statistically significant (p < .05) except
as indicated by *. 1-AN = 1-aminonaphthalene; 2-AN = 2-aminonaphthalene; 3-ABP = 3-aminobiphenyl; 3-HPMA = 3-hydroxypropyl mercapturic acid; 3-OH-B[a]P = 3-OH-benzo[a]pyrene; 4-ABP = 4-aminobiphenyl;
CEMA = 2-cyanoethylmercapturic acid; COHb = carboxyhemoglobin; HEMA = 2-hydroxyethylmercapturic acid; HMPMA = 3-hydroxy-1-methylpropylmercapturic acid; M = mean; MHBMA = monohydroxybutyl
mercapturic acid; NAB = N’-nitrosoanabasine; NAB-T = free plus N-glucuronidated (total) N’-nitrosoanabasine; NAT = N’-nitrosoanatabine; NAT-T = free plus N-glucuronidated (total) N’-nitrosoanatabine; NNAL-T
= free plus N-glucuronidated (total) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNK = 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN = N’-nitrosonornicotine; NNN-T = free plus N-glucuronidated (total)
N’-nitrosonornicotine; SD = standard deviation; SPMA = S-phenylmercapturic acid; VS = Vuse Solo.
a
Measured in whole blood.
b
Measured in urine.

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Fagerström Test for Nicotine Dependence scores, with means of 78.5% decrease in 3-hydroxy-benzo[a]pyrene, the difference was
6.0–6.3 per cohort, and smoked similar numbers of cigarettes per not statistically significantly different in the non-menthol smoker
day at baseline. Consistent with the US smoking population, non- gum group.
menthol smokers in this study were predominantly non-Hispanic
whites. A higher percentage of African American smokers smoke Product Use
menthol cigarettes, and the menthol smokers in this study were Product use amounts for each full day of ad libitum use are
predominantly black/African American, consistent with these summarized in Table  4, including the number of cigarettes
demographics.38 smoked over a 24-hour period at baseline and the amounts
of e-liquid and gum used per day for 5  days after product
Nicotine Biomarkers switch. Cigarettes per day at baseline were similar across the
Mean total nicotine equivalents in 24-hour urine samples decreased four cohorts, ranging from  means of 14.0 to 14.5. Following
38% in both VS groups and decreased 60% and 67% in the gum randomization, subjects chose to use the products to which
groups of non-menthol and menthol smokers, respectively (Table 2). they were assigned, as evidenced by the product-use results for
Reductions in plasma nicotine and cotinine concentrations measured individual subjects. Among subjects randomized to VS who

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at 07:00 pm on Days −2 and 5 mirrored the reductions in total urin- completed the study, 75 of 77 subjects used at least 0.10 g of
ary nicotine equivalents in all groups. e-liquid per day on at least 3 of 5 days. Among subjects rand-
omized to NG who completed the study, all used at least one
Biomarkers of Exposure piece on at least 3 of 5 days.
Biomarkers of toxicants decreased 30%–99% for all groups, and The mean daily amounts of e-liquid used by the VS groups
generally decreased by similar amounts whether subjects were increased from Day 1 to Day 3 and then the amounts used on Days
switched to an e-cigarette or NG (Table 3). 3, 4, and 5 were relatively consistent. Ad libitum use was permit-
Carboxyhemoglobin, both a biomarker of disease risk and ted for a shorter period of time on Day 5 than on the other days
a measure of CC cessation, decreased approximately 75% in post–product switch due to the start of the 12-hour abstinence at
all groups. Changes in other vapor-phase biomarkers decreased 07:30 pm, and the lower average amount of use on that day likely
38%–90% in all groups. In addition, tobacco-specific nitrosamines reflects this. In contrast to e-liquid use, average daily use of gum
decreased 55%–99%, aromatic amines decreased 52%–96%, and was relatively constant throughout the study: non-menthol smokers
polycyclic aromatic hydrocarbons decreased 30%–84% in all used approximately 4.5–6 pieces per day, and menthol smokers used
groups. Biomarkers of two constituents with longer half-lives, acryl- approximately 4–5 pieces per day.
amide and hydrogen cyanide, decreased approximately 50% and
30%–40%, respectively, consistent with expected decreases based Adverse Events
on results of similar studies with a smoking-cessation cohort.30,33,38,39 Eighty-three adverse events were reported among randomized
All biomarker decreases were statistically significant (p < .05) subjects during this study; 35 in the VS groups and 48 in the
in all cohorts, with the exception of the decrease observed in gum groups, all of which were mild or moderate. Of these, 43
3-hydroxy-benzo[a]pyrene in the non-menthol smoker gum group. were determined by the investigator to be possibly or definitely
One subject in that cohort showed a baseline value of nearly six related to product use: 17 in the VS groups and 26 in the gum
standard deviations above the mean. In addition, another subject groups. The most common adverse events reported by the VS
showed an approximately 20-fold higher value at Day 5 compared groups were headache, nausea, and cough. The most common
to Day −2. These results created a large variation among subjects, adverse events reported by the gum groups were dyspepsia, hic-
which decreased the sensitivity of the t test. Therefore, despite a cups, and oropharyngeal pain.

Table 4. Daily Consumption of Cigarettes, E-Liquid (g), and Nicotine Gum (Pieces)

NM smoker—VS original NM smoker—nicotine gum M smoker—VS menthol M smoker—nicotine gum

(mean gram e-liquid ± SD) (mean pieces ± SD) (mean gram e-liquid ± SD) (mean pieces ± SD)
N = 38a N = 39b N = 40c N = 41d

Baseline CPD 14.0 ± 4.0 14.4 ± 3.7 14.5 ± 4.6 14.3 ± 2.7

(mean ± SD)
Post–product switch
  Day 1 0.26 ± 0.23 5.6 ± 2.2 0.28 ± 0.21 4.8 ± 1.9
  Day 2 0.36 ± 0.30 4.7 ± 2.3 0.38 ± 0.27 4.5 ± 2.3
  Day 3 0.42 ± 0.30 5.0 ± 3.0 0.42 ± 0.30 4.6 ± 2.7
  Day 4 0.43 ± 0.32 4.6 ± 3.0 0.44 ± 0.32 4.4 ± 2.6
  Day 5 0.40 ± 0.30 4.4 ± 2.5 0.42 ± 0.29 3.9 ± 2.4

Daily nicotine intake for VS users may be calculated by multiplying the daily 4.8% nicotine e-liquid mass values above by 0.048. CPD  =  cigarettes per day;
M = Menthol; NM = non-menthol; SD = standard deviation; VS = Vuse Solo.
a
Includes a partial day of use for one subject on Day 5 due to withdrawal of consent.
b
Final n = 38 due to withdrawal of consent for one subject on Day 4.
c
Final n = 38 due to withdrawal of consent for one subject on Day 3 and one subject on Day 4.
d
Includes a partial day of use for one subject on Day 5 due to withdrawal of consent.
Discussion use were similar for those biomarkers with short elimination half-
lives. Differences were observed between the studies that assessed
This study was designed to evaluate changes in nicotine uptake and
short-term versus longer-term switching in results for NNAL, which
exposure to toxicants of tobacco combustion, and to understand
were expected given the long elimination half-life of that biomarker,
product-use behavior after a short-term switch from CCs to either
as discussed earlier.
VS ECs (non-menthol or menthol) or NG. Both products were gen-
Product-use patterns reported here showed similarity between
erally well tolerated by subjects.
the VS Original and Menthol groups. Both groups used an average
Nicotine uptake was higher during baseline than in either of the
of approximately 0.26–0.28 g of e-liquid on Day 1 (Table 4). Day
VS groups or NG groups. Although statistically significant decreases
2 showed similar increases in use for both groups, with an average
in total nicotine equivalents occurred in all groups, subjects in the
e-liquid use of approximately 0.36–0.38 g. Increase in e-liquid use
VS groups reduced their nicotine uptake by a lesser extent than sub-
was similar again on Day 3 at an average of approximately 0.42–
jects in the gum groups (a qualitative comparison of approximately
0.44  g. Average use remained similar for both groups and similar
40% versus approximately 60%, respectively). Plasma nicotine and
to Day 3 use on Days 4 and 5. Although product use was slightly
cotinine concentrations observed at 07:00 pm on Day −2 and Day
5 showed similar results (Table  2). Although substantial nicotine less for both groups on Day 5, participants were permitted to use

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uptake occurred in both the VS and gum groups, these results indi- product for 3.5 fewer hours than on Days 1–4. These results suggest
cate that neither product, when used ad libitum, resulted in the same that acclimation to the new product occurred over the first 2 days
level of nicotine uptake as CCs under the conditions of this study. and that subjects reached their typical product use on Day 3 that
In general, large, significant reductions were seen in biomarkers continued through Day 5. Further research will be needed to confirm
of carcinogens and toxicants. These results indicate that exposure to whether use after a 3-day acclimation period reflects how an EC con-
many HPHCs present in cigarette smoke is greatly reduced by using sumer may use the product long term.
VS and that those reductions are similar to the reductions that occur Average daily gum use was slightly less than the average reported
when a smoker switches to NG. Several biomarkers measured in use by smokers during their first week of cessation: approximately
this study are known to have longer elimination half-lives, including five pieces per day here compared to 7.7 pieces reported by Shiffman
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), acrylamide et al.,40 but was greater than the reported average of 3.2 pieces per
equivalents, and thiocyanate, and the smaller reductions observed day after 24 weeks of cessation.
for those are as expected. This study was intended to determine the maximum biomarker
Biomarker reductions in all groups were similar to the reductions reductions possible when smokers fully switched to VS. Several lim-
observed in tobacco-abstinent groups of studies of a similar design, itations of this study exist. First, the study was conducted in-clinic
with two exceptions. Biomarkers of fluorene and 1,3-butadiene and did not examine subjects in their normal environments, which
decreased less than expected in all groups, with decreases of approxi- would allow for a more realistic picture of their choice of product use.
mately 35% and 55%, respectively, compared to 70% and 90%, Second, a continue-smoking cohort was not included, which would
respectively, in smokers randomized to tobacco abstinent groups in address whether subjects’ UB cigarettes per day might change in clinic.
other studies.30,33,38,39 Although less than expected, the reductions In addition, this study recruited smokers who were not postponing a
here were consistent across all groups, which suggests that the results decision to quit smoking; therefore, subjects may not have been highly
were not related to VS or NG use. An influence such as an unknown motivated to switch to a product other than CCs.
environmental exposure may have occurred equally for all groups, This was a short-term study that focused on acute reductions
preventing reductions to levels observed in other studies. in biomarkers with generally short elimination half-lives. The bio-
The results for 3-OH-B[a]P generally showed larger variability markers measured were generally representative of smoke constitu-
across cohorts than other biomarkers. This is especially so for the ents to demonstrate how a switch from smoking to VS would affect
non-menthol smoker gum cohort, which resulted in a nonstatisti- exposure. In fact, 20 of the 23 biomarkers measured here represent
cally significant decrease after product switch. This variability is compounds found on FDA’s established list of HPHCs found in
largely due to 52% of subjects across all cohorts whose urinary tobacco products and tobacco smoke.37 In addition, 11 of the bio-
B[a]P concentrations were measured as below the limit of quantifi- markers measured are representative of constituents included on
cation of 50 fg/mL for the method at baseline while smoking CCs. the list of 29 HPHCs that FDA recommends for analysis in elec-
Similarly, 79% of subjects across all cohorts showed 3-OH-B[a]P tronic nicotine delivery system aerosols per their Premarket Tobacco
levels below the limit of quantification after product switch; there- Product Applications for Electronic Nicotine Delivery System Draft
fore, the change in mass/24  h for subjects with values below the Guidance.16 Longer-term studies to assess biomarkers of potential
limit of quantification for both time points is dependent on the vol- harm and health effects will be important to understand the changes
ume of the urine collected. Increasing the sensitivity of the method in health risk associated when smokers switch to VS, and ECs in
may not be feasible because it is already validated to a very low general, for longer periods of time. Such studies might also include
level. Therefore, although B[a]P has been determined to be an EC-specific biomarkers, such as nickel and chromium, as they
HPHC by the FDA Center for Tobacco Products, and a metabolite become qualified to distinguish among tobacco-use groups.41
unique to that compound exists, it may not be an appropriate bio- In summary, this study examined nicotine exposure, product-use
marker of tobacco exposure due the very low levels detected in the patterns, and biomarkers of carcinogens and toxicants in smokers
urine of smokers. who exclusively switched to VS or NG for 5  days in a controlled
The results presented here are similar to results observed in other setting. Results indicate that exposure to toxicants from VS use are
studies that assessed biomarkers either after a 5-day switch, a 4- significantly reduced and appear to be reduced to the same degree as
to 12-week switch, or in cross-sectional studies in which subjects seen with NG use, and adds to the research that suggests use of ECs,
reported short or longer-term EC use.24–31 Although the study designs represented here by VS, exposes consumers to fewer and lower levels
differed, the biomarker differences between exclusive EC use and CC of smoke toxicants than CCs.
Supplementary Material 11. Goniewicz ML, Knysak J, Gawron M, et  al. Levels of selected carcino-
gens and toxicants in vapour from electronic cigarettes. Tob Control.
Supplementary data are available at Nicotine & Tobacco Research online.
2014;23(2):133–139.
12. Kosmider L, Sobczak A, Fik M, et al. Carbonyl compounds in electronic
cigarette vapors: effects of nicotine solvent and battery output voltage.
Funding Nicotine Tob Res. 2014;16(10):1319–1326.
This study was funded by R.J. Reynolds Vapor Company through R.J. 13. Ohta K, Uchiyama S, Inaba Y, Nakagome H, Kunugita N. Determination
Reynolds Tobacco Company. of carbonyl compounds generated from the electronic cigarette using cou-
pled silica cartridges impregnated with hydroquinone and 2,4-dinitrophe-
nylhydrazine. Bunseki Kagaku. 2011;60:791–797.
Declaration of Interests 14. Geiss O, Bianchi I, Barahona F, Barrero-Moreno J. Characterisation of
mainstream and passive vapours emitted by selected electronic cigarettes.
ER, PC, and ES are full-time employees of RAI Services Company. AT was
Int J Hyg Environ Health. 2015;218(1):169–180.
a full-time employee of RAI Services Company at the time this study was
15. Blair SL, Epstein SA, Nizkorodov SA, Staimer N. A real-time fast-flow
conducted. RAI Services Company is a wholly owned subsidiary of Reynolds
tube study of VOC and particulate emissions from electronic, potentially
American Inc., which is a wholly owned subsidiary of British American
reduced-harm, conventional, and reference cigarettes. Aerosol Sci Technol.

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Tobacco plc.
2015;49(9):816–827.
16. US Department of Health and Human Services. Premarket Tobacco
Acknowledgments Product Applications for Electronic Nicotine Delivery Systems,
Guidance for Industry, Draft Guidance. Rockville, MD: Food and Drug
The authors wish to thank Michael Borgerding for significant contributions Administration, Center for Tobacco Products; May 2016.
to the design of this study and Harry Alcorn for additional protocol sugges- 17. Abrams DB, Niaura R. The importance of science-informed policy and
tions. Angela Slater, Herman Krebs, Frank Terschan, Kristen Fitzpatrick, Amy what the data really tell us about e-cigarettes. Isr J Health Policy Res.
Denvir, Colleen Hebranson, and Jenny Ellefson provided significant contribu- 2015;4:22.
tions to the execution of this study. Megan Whelen provided significant editing 18. Walton KM, Abrams DB, Bailey WC, et al. NIH electronic cigarette work-
contributions to the manuscript. The authors also wish to thank Kimberly shop: developing a research agenda. Nicotine Tob Res. 2015;17(2):259–269.
Frost-Pineda, Paul Nelson, Christopher Cook, Ryan Potts, Dan Heck, and Jack 19. Gregg EO, Minet E, McEwan M. Urinary biomarkers of smokers’ ex-
Henningfield for careful review of this manuscript. posure to tobacco smoke constituents in tobacco products assessment: a
fit for purpose approach. Biomarkers. 2013;18(6):467–486.
20. Hecht SS. Tobacco carcinogens, their biomarkers and tobacco-induced
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