Toxicology Mechanisms and Methods

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Toxicology Mechanisms and Methods
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Reductions in biomarkers of exposure (BoE) to
harmful or potentially harmful constituents
(HPHCs) following partial or complete substitution
of cigarettes with electronic cigarettes in adult
smokers
Grant O’Connell, Donald W. Graff & Carl D. D’Ruiz
To cite this article: Grant O’Connell, Donald W. Graff & Carl D. D’Ruiz (2016): Reductions in
biomarkers of exposure (BoE) to harmful or potentially harmful constituents (HPHCs) following
partial or complete substitution of cigarettes with electronic cigarettes in adult smokers,
Toxicology Mechanisms and Methods, DOI: 10.1080/15376516.2016.1196282
To link to this article: http://dx.doi.org/10.1080/15376516.2016.1196282
© 2016 Fontem Ventures, B.V.
Published online: 12 Jul 2016.
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RESEARCH ARTICLE
Reductions in biomarkers of exposure (BoE) to harmful or potentially harmful
constituents (HPHCs) following partial or complete substitution of cigarettes with
electronic cigarettes in adult smokers
Grant O’Connella
, Donald W. Graffb
and Carl D. D’Ruizc
a
Fontem Ventures, B.V, Scientific and Regulatory Affairs, Amsterdam, The Netherlands; b
Celerion Inc, Lincoln, USA; c
Fontem Ventures,
B.V, Greensboro, NC, USA
ABSTRACT
Changes in fifteen urine, blood and exhaled breath BoEs of HPHCs representing classes of compounds
reported by FDA to be significant contributors to smoking-associated disease risks were measured in
105 clinical-confined subjects following randomization and a five-day forced-switch from usual brand
conventional combustible cigarettes to: (i) exclusive commercial e-cigarette use; (ii) dual-use of commer-
cial e-cigarettes and the subject’s usual cigarette brand; or (iii) discontinued use of all tobacco or nicotine
products. Levels of urinary biomarkers in subjects that completely substituted their usual cigarette with
e-cigarettes were significantly lower (29–95%) after 5 days. Percent reductions in eight of nine urinary
BoEs were indistinguishable to smokers who had quit smoking, except for nicotine equivalents, which
declined by 25–40%. Dual users who halved self-reported daily cigarette consumption with
e-cigarettes exhibited reductions (7–38%) in eight of nine urinary biomarkers, but had increase (1–20%)
in nicotine equivalents. Reductions were broadly proportional to the reduced numbers of cigarettes
smoked. Dual user urinary nicotine equivalents were slightly higher, but not statistically significant. After
5 days, blood nicotine biomarker levels were lower in the cessation (75–96%) and exclusive use groups
(11–83%); with dual users experiencing no significant reductions. All subjects experienced significant
decreases in exhaled CO. Decreases in the cessation and exclusive groups ranged from 88–89% and
27–32% in dual users. Exhaled NO increased in the cessation and exclusive groups (46–63% respectively),
whereas the dual users experienced minimal changes. Overall, smokers who completely or partially
substituted conventional cigarettes with e-cigarettes over five days, experienced reductions in HPHCs.
ARTICLE HISTORY
Received 25 April 2016
Revised 27 May 2016
Accepted 27 May 2016
Published online 11 July 2016
KEYWORDS
Biomarkers of exposure;
cessation; e-cigarettes;
exclusive and dual use;
harm reduction
Introduction
Electronic cigarettes (e-cigarettes) represent a rapidly-
emerging product category that holds promise as a conven-
tional tobacco cigarette alternative as they simulate some of
the familiar behavioral and sensorial aspects of the smoking
experience which conventional nicotine replacement therapy
products do not. E-cigarettes do not contain tobacco, do not
require combustion, or generate side-stream smoke. E-ciga-
rettes are battery-powered devices that deliver an aerosol
(popularly referred to as ‘‘vapor’’) to users from an e-liquid of
known chemical composition. E-liquids typically contain gly-
cerol and propylene glycol in varying proportions from which
the aerosol is generated and may contain nicotine and vari-
ous flavors. In contrast, tobacco smoke has been reported to
contain many thousands of chemicals including HPHCs associ-
ated with the combustion process, as identified by the FDA
(USFDA, 2012). The types and concentrations of potential toxi-
cants associated with e-cigarette aerosols is a topic of current
research reported in literature. However, the limited number
of speculated constituents are ten to thousand times lower
than in conventional tobacco cigarette smoke with many of
the toxicants in tobacco products simply not present in
e-cigarette aerosol at detectable levels when assessed follow-
ing machine-based aerosol generation (e.g. Goniewicz et al.,
2014; Tayyarah & Long, 2014) or are at levels equivalent
to the tolerances allowed in medical products. As a result,
e-cigarette aerosols elicit minimal biological responses in con-
ventional regulatory in vitro toxicology assays compared with
conventional tobacco cigarettes (e.g. Misra et al., 2014).
Nevertheless, there is relatively little information available on
actual adult smoker’s potential exposure to HPHCs resulting
from the use of e-cigarettes compared to conventional
tobacco cigarettes (Burstyn, 2014; Cahn & Siegel, 2011; Hajek
et al., 2014; Polosa et al., 2013; Tayyarah & Long, 2014).
Recent scientific surveys and studies examining the habits
and practices of e-cigarette users indicate that the dual use
of cigarettes and e-cigarettes is common practice and that
dual users report using e-cigarettes to reduce, replace or help
stop smoking conventional tobacco cigarettes as well as
reduce family member exposure to secondhand cigarette
smoke (i.e. the mixture of the smoke that comes from the
burning end of a cigarette and the smoke breathed out by
the smoker) (Farsalinos et al., 2014; Farsalinos & Polosa, 2014;
McRobbie et al., 2014; Pepper & Brewer, 2014). Exposures to
CONTACT Carl D. D’Ruiz Carldruiz@gmail.com Clinical Study Consultant, 22 Rosebay Lane, Greensboro, NC 27455-3407, USA
ß 2016 Fontem Ventures, B.V. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecom-
mons.org/Licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered,
transformed, or built upon in any way.
TOXICOLOGY MECHANISMS AND METHODS, 2016
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HPHCs that result from dual use may be anticipated to
depend on both the extent of daily uses of the respective
products, as well as any conscious or subconscious changes
in puffing topography consequent to any nicotine or sensory
deficits that may derive from the partial substitution of con-
ventional tobacco cigarettes by e-cigarettes. Previously, it had
been reported that reductions in daily conventional tobacco
cigarettes smoked resulted in reduced urinary excretion of
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) (Hecht
et al., 2004), but those reductions fell short of those antici-
pated from the reduced number of conventional tobacco cig-
arettes consumed. However, a more recent study of adult
smokers who switched to using only e-cigarettes and to dual
use of e-cigarettes and conventional tobacco cigarettes
showed significant reductions in exposure to carbon monox-
ide (CO) and the toxicant acrolein over a four-week period
(McRobbie et al., 2015).
The primary objective of this study was to compare
changes in selected urine, blood, and exhaled breath biomar-
kers of exposure to HPHCs among different user groups follow-
ing a five-day forced-switch from usual brand conventional
tobacco cigarettes to: (i) exclusive use of commercial
e-cigarettes; (ii) dual-use of commercial e-cigarettes and the
subject’s usual conventional tobacco cigarette brand; or (iii)
discontinued use of all tobacco or nicotine products. The bio-
markers of exposure to the selected HPHCs included a number
of cigarette smoke constituents representing major classes of
compounds believed to be the most significant contributors to
smoking-associated disease risks as reported by the FDA.
Methods
Participants
The study protocol and the informed consent forms were
approved by Chesapeake IRB, Columbia, MD. The clinical trial
was registered on 6 February 2015 at: http://ClinicalTrials.gov
identifier: NCT02385227. Two hundred and fourteen potential
smokers were recruited from the Lincoln, NE (USA) area using
standard advertising methods (i.e., print and radio advertise-
ments) and from a database of subjects who had previously
participated in a clinical research study or who had expressed
interest in participating in a study. All potential subjects were
provided details regarding the study and written informed
consent was obtained prior to initiation of any study proce-
dures. One hundred and two subjects were excluded for
not satisfying the predefined inclusion/exclusion criteria;
15 subjects declined to participate prior to enrollment; and
two subjects were excluded because the study had reached
the randomization target of 105 eligible subjects. The
105 subjects meeting the eligibility criteria were enrolled into
the study and randomized into one of six study groups. Two
subjects withdrew consent from the study following random-
ization for personal reasons unrelated to study participation.
All subjects participating in the study were paid a fee for their
participation.
The main criteria for inclusion in the study were as follows:
healthy adult male and female smokers, 21–65 years of age
inclusive; a smoker for at least 12 months and currently
smoked an average of 10 or more conventional manufactured
tobacco cigarettes per day (any brand, flavor or style); consist-
ent use of their current usual brand style for 14 days prior to
check-in; positive urine cotinine at screening (! 500 ng/mL);
and exhaled carbon monoxide CO >12 ppm at screening.
Prior use of an e-cigarette was not an exclusion criterion, pro-
vided all other criteria were met; however, none of the sub-
jects reported previous use of e-cigarettes. Exclusion criteria
included: history or presence of clinically significant mental
or physical health conditions; females who were pregnant
or breastfeeding; high blood pressure; body mass index
<18 kg/m2
or >40 kg/m2
; acute illnesses (e.g., upper respira-
tory infection, viral infection) requiring treatment within
2 weeks prior to check-in; use of prescription smoking cessa-
tion treatments, anti-diabetic or insulin drugs or medications
known to interact with Cytochrome P450 2A6; positive urine
screen for alcohol or drugs of abuse; and self-reported
mouth-hold smokers (i.e., smokers who draw smoke from the
conventional tobacco cigarette into the mouth and throat but
do not inhale). Subjects who had used any tobacco- or nico-
tine-containing products other than manufactured tobacco
cigarettes or e-cigarettes within 28 days of in-clinic product
use were also excluded.
Products tested
Three commercially available closed system bluTM
e-cigarette
products (manufacturer, Fontem Ventures B.V., The
Netherlands) were evaluated during this study: rechargeable
tobacco flavor, rechargeable cherry flavor, and disposable
cherry flavor. The rechargeable e-cigarettes consist of a bat-
tery segment and a cartomizer segment comprising the heat-
ing unit and a liquid reservoir which can be separated from
the battery for recharging or replaced when the e-liquid is
depleted. The disposable e-cigarette was similar in form with
the exception that the battery and cartomizer segments are
included as a single, non-separable unit. Both units operated
at a voltage of 3.7 volts (nominal). The resistance of the heat-
ing element was $3 ohms for the disposable unit and about
3.5 ohms for the rechargeable unit. The maximum operating
temperature of each unit was dependent on the charge level
of the battery, the state of reservoir fluid fill and on the man-
ner of use and was not recorded in this study.
All e-cigarette products contained 24 mg/mL (2.4%) USP
grade nicotine, USP grade vegetable glycerol ($50% in cherry
flavor and $80% in tobacco flavor), USP grade propylene gly-
col ($45% in cherry flavor and $10% in tobacco flavor), dis-
tilled water, and flavorings. Each e-cigarette contained $1 mL
of e-liquid by volume.
Subjects were provided unopened packs of their reported
usual brand of conventional tobacco cigarettes for use during
the study.
Study design
This was a randomized, open-label, forced-switch parallel arm
study conducted at a single independent research center
(Celerion, Lincoln, NE) to assess biomarkers of exposure to
2 G. O’CONNELL ET AL.

HPHCs (Gregg et al., 2013; Hecht et al., 2010) following short-
term ad libitum use of e-cigarettes by established adult
smokers. This proof-of-concept study evaluated the hypoth-
esis that use of e-cigarettes, either exclusively or with dual
use of conventional tobacco cigarettes (with a 50% reduction
in self-reported conventional tobacco cigarettes per day
[CPD]), can significantly reduce exposure to many of the
HPHCs commonly associated with use of combustible tobacco
cigarettes. A cessation arm served as a maximum effect
control group comparator.
Following successful screening and study qualification,
subjects checked into the clinic on Day À2 and continued to
smoke their usual conventional tobacco cigarette brand
ad libitum through the evening of Day À1. Subjects were
confined in the research clinic for the entire duration of the
study. During enrollment, subjects were also trained on how
to use the e-cigarettes and were also informed of how to
notify the clinical staff of situations involving non-operating
e-cigarettes. A Fagerstr€om Test for Cigarette Dependance
(FTCD) (Fagerstr€om, 2012; Heatherton et al., 1991) was also
administered to all subjects upon enrollment. Baseline assess-
ments occurred from the morning of Day À1 through the
morning of Day 1 prior to the start of randomized product
use and post-baseline assessments on the morning of Day 1
through the morning of Day 6.
On the morning of Day 1, subjects were randomized into
one of six groups (N ¼ 15 each):
Exclusive E-Cigarette Use Groups
Group A1 – Tobacco flavor rechargeable bluTM
e-cigarette
Group A2 – Cherry flavor rechargeable bluTM
e-cigarette
Group A3 – Cherry flavor disposable bluTM
e-cigarette
Dual Use Groups
Group B1 – Tobacco flavor rechargeable bluTM
e-cigarette-
þ usual brand combustible tobacco cigarette
Group B2 – Cherry flavor rechargeable bluTM
e-cigarette-
þ usual brand combustible tobacco cigarette
Group B3 – Cherry flavor disposable bluTM
e-cigarette þ
usual brand combustible tobacco cigarette
Cessation Group
Group C – Complete tobacco and nicotine product
cessation
Product use
Use of the assigned products was documented daily and sub-
jects were monitored during clinical confinement to ensure
that no illicit nicotine or tobacco products were used.
Subjects randomized to the cessation group were housed in
an area of the clinic separate from the other groups. With
limited exceptions, all product use was ad libitum from 07:30
to 23:00 on Days 2 to 5. These exceptions included meals,
15 min prior to blood sampling, and 30 min prior to exhaled
CO and nitric oxide (NO) measurements.
Subjects randomized to receive the e-cigarette products
were allowed to carry them throughout the day in designated
sections of the clinic. New e-cigarettes were supplied to the
subjects each morning and throughout the day if the e-liquid
solution was fully consumed or the product failed to work
properly. All e-cigarettes were weighed before and after use.
Subjects randomized to the dual use group were required
to request a conventional tobacco cigarette product from the
clinic staff and smoke only in specified sections of the clinic
away from nonsmoking subjects. In order to standardize cigar-
ette consumption during the study, subjects in the dual use
groups were required to reduce their daily cigarette consump-
tion on Days 1–5 by $50% from that reported at screening.
Determination of sample size
The sample size estimation was based on total NNAL because
the group difference in percent change-from-baseline was
expected to be smaller than the other biomarkers due to a lon-
ger half-life for elimination (approximately 45 days, Hecht et al.,
1999). In a previous study, adult smokers who replaced conven-
tional tobacco cigarettes with a snus product or discontinued
use of all tobacco products completely for 5 days excreted
$60–70% less NNAL, while subjects who reduced conventional
tobacco cigarette use by half excreted $30% less total NNAL
over the same timeframe (Sarkar et al., 2010). Based on these
results, a sample size of 12 was estimated to detect a 70%
reduction from baseline in the groups that stopped smoking
and to be able to detect the differences between groups with
at least 80% power using two-sided testing. Up to 15 subjects
were assigned to each group to maximize the likelihood of a
minimum of 12 subjects completing the study in each group.
Biomarker analysis
The urine and blood biomarkers of exposure evaluated in this
study (Table 1) were chosen to represent major classes of
HPHCs that have previously been reported for conventional
tobacco cigarette smokers (Carmella et al., 2009; Gregg et al.,
2013; Hecht, 2002; Hecht et al., 2010; USDHHS, 2014). All urine
voided by each subject was collected in 24-h intervals from
07:30 on Day À1 through 07:30 on Day 1, and from 07:30 on
Day 5 through 07:30 on Day 6, and aliquots were prepared
from the 24-h collections. Blood samples were collected on
Days À1 and 5 in the evening following dinner to assess
exposure to CO and nicotine. Each biomarker was measured
using validated methods based on: FDA’s Guidance to
Industry for Bioanalytical Method Validation (2001); Good
Laboratory Practices per 21 CFR Part 58; and the EMEA
Guideline on Bioanalytical Method Validation (EMEA/CHMP/
EWP/192217/2009 Rev. 1 Corr.2).
Exhaled breath biomarkers
Exhaled CO and NO are measures of acute carbon monoxide
exposure and nitric oxide synthase activity, respectively
(Taylor et al., 2006). Smokers characteristically exhale higher
CO (Deveci et al., 2004) and lower NO (Kharitonov et al.,
TOXICOLOGY MECHANISMS AND METHODS 3

Table1.Urine,bloodandinhalationbiomarkersoftobaccosmokeexposure.
URINE:Biomarkersofexposureanalyzed
Nicotineequivalentsa
NNNNNAL1-OHP3-HPMAS-PMAMHBMAHMPMACEMA
Nicotineþ5majormetabolites
a
Nicotineequivalentsmeasured:includednicotineandfivemajornicotinemetabolites:nicotinegluc;cotinine;cotinine-gluc;trans-3’-hydroxycotinine;andtrans-3’-hydroxycotinine-gluc.
NNAL:4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol;1-HOP:1-hydroxypyrene;3-HPMA:3-hydroxypropylmercapturicacid;S-PMA:S-phenylmercapturicacid;MHBMA:Monohydroxy-3-butenylmercapturicacid;HMPMA:3-
hydroxy-1-ethylpropylmercapturicacid;CEMA:2-cyanoethylmercapturicacid.
HPHCs-associatedwithurinebiomarkers
NicotineTobaccoSpecificNitrosamines
(TSNA)
PAHVolatileOrganicCompounds(VOC’s)
NicotineNNNNNKPyreneAcroleinBenzene1,3-ButadieneCrotonaldehydeAcrylonitrile
PAH:PolycyclicAromaticHydrocarbons;NNN:N-Nitrosonornicotine;NNK:4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
ClinicalendpointsofurineHPHCs
NicotineExposureCancerCancerCancerCancerCancerCancerCancerCancer
AnalysismethodandLowerLimitofQuantification(LLOQ)
LC-MS-MS50–200ng/mLb
LC-MS-MS
0.2pg/mL
LC-MS-MS
5pg/mL
LC-MS-MS
10pg/mL
LC-MS-MS
20ng/mL
LC-MS-MS
25pg/mL
LC-MS-MS
0.1ng/mL
LC-MS-MS
20ng/mL
LC-MS-MS
0.275ng/mL
a
Nicotineequivalents:calculatedasthemolarsumofnicotineandfivemajornicotinemetabolitesexcretedinurineover24h(mgnicotineequivalents/24h).b
LLOQforNicotineequivalents:50ng/mLforNicotine,Nicotine
gluc,CotinineandTrans-3’-hydroxycotinine;200ng/mLforCotinine-glucandTrans-3’-hydroxycotinine-gluc.
BLOOD:Biomarkersofexposureanalyzed
BloodCarboxyHemoglobin(COHb)PlasmaNicotinePlasmaCotininePlasmaTrans-3’hydroxycotinine
Chemicalconstituentsassociatedwithbloodbiomarkers
CarbonOxideNicotine
Clinicalendpointsassociatedwithbloodbiomarkers
CarbonMonoxide(CO)exposureNicotineexposureNicotineexposureNicotineexposure
Spectrophotometric0.50%LC-MS-MS0.2ng/mLLC-MS-MS1.0ng/mLLC-MS-MS1.0ng/mL
INHALATION:Biomarkersofexposureanalyzed
ExhaledCarbonMonoxide(CO)ExhaledNitricOxide(NO)
BedfontMicroþSmokerlyzer:1ppm(0–500ppm)NioxMino:5ppb(5–300ppb)

1995) than nonsmokers. Exhaled CO and NO were measured
during the study in the afternoon on Days 1 and 5 using a
Bedfont Micro þ Smokerlyzer and Niox Mino, respectively.
Sampling was preceded by a 30-min (minimum) abstention
from study product use.
Data analyzes
Statistical analyzes were performed using SAS procedures in
SASVR
Version 9.3 (SAS Institute Inc., Cary, NC). A paired t-test
was used to make within-group comparisons between study
days and a linear mixed model was used to assess between-
group differences. Baseline values were included in the statis-
tical models for the between-group comparisons as a covari-
ate. Differences were considered statistically significant at an
alpha level of 5% and no adjustments were made for multiple
comparisons.
In addition, regression analyzes were performed for the
Day À1 to Day 5 % change in urine biomarker concentrations
against the Day À1 to Day 5 % change in CPD for the dual
use groups and to evaluate the relationship between urine
nicotine equivalents and the estimated amount of nicotine
delivered by the e-cigarette products (Day 5 exclusive and
dual use groups) and the number of cigarettes smoked
(Day 5 in dual use groups, Day À1 in all groups).
Results
Participant characteristics
A summary of the subjects’ demographics, tobacco product
use history, and FTCD scores for all study participants by
study product sequence and overall is presented in Table 2.
Urine and blood biomarker of exposure comparisons
Reducing consumption of conventional tobacco cigarettes
over 5 days according to the requirements of the study
tended to result in sizeable reductions in exposure to a num-
ber of HPHCs as measured by BoE (Tables 3 and 4, Figures 1
and 2). Smoking cessation lead to a 66–98% decrease in
excretion of the urine BoE evaluated in this study. The small-
est reduction was observed for NNAL, which has the longest
half-life of the individual biomarkers listed (Hecht et al., 1999).
Predictably, significant decreases were also observed in the
carboxyhemoglobin (COHb), nicotine, and the nicotine metab-
olites as the cessation subjects had no exposure to CO or
nicotine.
The changes from Day À1 observed in the exclusive use
groups were mostly comparable to those seen in the cessa-
tion group, with the notable exceptions of the nicotine and
nicotine metabolite urine and blood biomarkers as these sub-
jects continued to consume nicotine from the e-cigarettes.
Dual users who had substituted half of their self-reported
daily conventional tobacco cigarette consumption with
e-cigarettes exhibited reduced biomarkers levels that were
broadly proportional to the reduced numbers of cigarettes
smoked. Reductions in the urine BoE for these groups ranged
from $7% to 38% (Table 3).
Observed reductions in HPHCs
Measurable nicotine metabolites were present in the samples
from e-cigarette users, but levels of biomarkers for HPHCs
were significantly lower, and many were indistinguishable
from those of subjects who had quit smoking entirely.
Table 2. Summary of study demographics and FTCD scores by product use groups and overall.
Exclusive E-Cigarette use groups Dual use groups
Trait/Test
Tobacco
rechargeable
N ¼ 15
Cherry
rechargeable
N ¼ 15
Cherry
disposable
N ¼ 15
Tobacco
rechargeable
N ¼ 15
Cherry
rechargeable
N ¼ 15
Cherry
disposable
N ¼ 15
Nicotine
cessation
N ¼ 15
Overall
N ¼ 105
Gender
Female 6 (40%) 3 (20%) 9 (60%) 6 (40%) 3 (20%) 7 (47%) 3 (20%) 37 (35%)
Male 9 (60%) 12 (80%) 6 (40%) 9 (60%) 12 (80%) 8 (53%) 12 (80%) 68 (65%)
Race
American Indian/Alaska Native 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (7%) 0 (0%) 1 (1%)
Black or African American 2 (13%) 6 (40%) 1 (7%) 2 (13%) 4 (27%) 1 (7%) 1 (7%) 17 (16%)
Black or African American,
American Indian/Alaska
0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (7%) 1 (1%)
White 13 (87%) 9 (60%) 14 (93%) 13 (87%) 11 (73%) 13 (87%) 13 (87%) 86 (82%)
Age (years)
Mean 37.1 40.1 33.9 36.6 36.8 39.3 41.1 37.8
SD 11.4 10.6 11.8 10.8 11.6 10.6 11.2 11.1
BMI (kg/m2
)
Mean 28.2 26.2 28.7 28.9 27.2 27.8 27.8 27.8
SD 5.5 6.5 5.7 5.5 4.7 5.1 4.9 5.4
Cigarettes per Day
Mean 18.4 17.3 15.4 18.7 20.5 21.1 20.4 18.8
SD 7.1 6.2 3.3 6.6 7.3 5.8 7.5 6.5
Years Smoked
Mean 19.2 20.3 15.0 19.3 14.6 21.7 21.3 18.8
SD 12.9 10.5 10.9 10.1 11.6 8.7 10.6 10.8
Usual brand cigarette flavor
Menthol 6 (40%) 7 (47%) 8 (53%) 3 (20%) 7 (47%) 5 (33%) 3 (20%) 39 (37%)
Non-Menthol 9 (60%) 8 (53%) 7 (47%) 12 (80%) 8 (53%) 10 (67%) 12 (80%) 66 (63%)
FTCD Score
Mean 5.3 5.1 5.3 5.5 5.7 5.2 5.6 5.4
SD 1.5 2.0 1.5 2.0 1.1 1.7 2.0 1.7

Levels of polycyclic aromatic hydrocarbons (PAHs) such as
pyrene (as measured by the BoE: 1-OHP) were reduced by
70% in users that had ceased smoking or using any nicotine
product and by 62–69% in the exclusive use group. Similarly,
levels of Tobacco Specific Nitrosamines (TSNAs) such as NNAL
and NNN (as measured by the BoE: NNAL and NNN) were
reduced by 66% to 98%, respectively in the cessation group
and by 62–64% and 87–93%, respectively in the exclusive use
group. Moreover, levels of Volatile Organic Compounds
(VOCs) such as acrolein, benzene, 1-3-butadiene, crotonalde-
hyde and acyrlonitrile (as measured by the BoE: 3-HPMA,
S-PMA, MHBMA, HMPMA and CEMA) were reduced by 87%,
94%, 84%, 88% and 86%, respectively, in the cessation group
and by 82–83%, 93–94%, 86–90%, 82–85% and 85–87%,
respectively, in the exclusive use group.
As might be expected, the excretion and concentration of
all BoE evaluated in this study were significantly higher in the
dual use groups at Day 5 compared with the cessation group.
Levels of PAHs such as pyrene (as measured by the BoE:
1-OHP) were reduced by 25–35% in dual users versus 70% in
the cessation group. Levels of TSNAs such as NNN and NNN
(as measured by the BoE: NNAL and NNN) were reduced by
19–22% and 30–37%, respectively, in the dual use group ver-
sus 66% and 98%, respectively, in the cessation group. Levels
Table 3. Urine biomarker concentration summary and statistical comparisons.
Biomarker
Classic tobacco
rechargeable
N ¼ 15
Cherry
rechargeable
N ¼ 13
TCherry
disposable
N ¼ 13
Classic tobacco
rechargeable
N ¼ 14
Cherry
rechargeable
N ¼ 15
Cherry
disposable
N ¼ 13
Nicotine
cessation
N ¼ 13
NNAL (ng/24 h)
Day À1 427.6 ± 218.8 383.7 ± 178.8 299.1 ± 165.0 430.8 ± 217.1 422.0 ± 257.5 343.3 ± 123.3 481.6 ± 377.5
Day 5 174.3 ± 144.6 149.2 ± 80.3 111.1 ± 68.9 328.6 ± 178.9 321.1 ± 177.3 269.2 ± 96.3 175.1 ± 140.8
p value Day À1 vs Day 5 0.0001 0.0001 0.0001 0.0063 0.0042 0.0028 0.0004
p value Day 5 vs Cessation 0.1940 0.2456 0.2593 0.0001 0.0001 0.0001 NA
3-HPMA (lg/24 h)
Day À1 1521.7 ± 820.0 1903.0 ± 1132.7 1353.7 ± 598.8 1644.1 ± 501.3 1474.6 ± 519.9 1489.5 ± 567.1 2004.1 ± 1137.8
Day 5 214.4 ± 94.3 263.1 ± 64.7 246.7 ± 101.5 1046.2 ± 360.6 1070.7 ± 342.2 1155.4 ± 368.5 228.8 ± 84.2
p value Day À1 vs Day 5 0.0001 0.0001 0.0001 0.0001 0.0009 0.0062 .0001
HMPMA (lg/24 h)
Day À1 523.8 ± 225.3 657.2 ± 328.9 533.4 ± 208.3 590.7 ± 178.7 597.5 ± 198.0 504.5 ± 167.1 797.7 ± 429.4
Day 5 71.3 ± 33.1 83.2 ± 32.3 78.0 ± 20.7 391.8 ± 151.2 394.6 ± 119.3 386.8 ± 94.1 78.1 ± 18.6
p-value Day À1 vs Day 5 0.0001 0.0001 0.0001 0.0001 0.0001 0.0094 .0001
p-value Day 5 vs Cessation 0.4785 0.5206 0.4211 0.0001 0.0001 0.0001 NA
CEMA (lg/24 h)
Day À1 219.7 ± 98.5 266.1 ± 140.9 201.0 ± 72.8 256.0 ± 97.9 246.2 ± 109.8 223.5 ± 61.6 289.7 ± 132.2
Day 5 33.4 ± 21.8 41.3 ± 30.4 25.9 ± 11.2 172.8 ± 72.1 168.3 ± 50.9 173.0 ± 63.7 41.0 ± 19.7
1-OHP (ng/24 h)
Day À1 317.4 ± 138.7 302.9 ± 171.5 260.9 ± 166.8 363.6 ± 174.1 294.5 ± 145.5 304.1 ± 122.7 364.0 ± 200.7
Day 5 93.7 ± 52.9 85.9 ± 32.2 90.6 ± 38.4 235.1 ± 121.1 206.3 ± 90.9 224.1 ± 89.5 108.2 ± 55.0
p value Day À1 vs Day 5 .0001 0.0002 0.0007 0.0006 0.0004 0.0007 .0001
NNN (ng/24 h)
Day À1 18.6 ± 12.1 13.7 ± 11.5 13.9 ± 12.5 14.3 ± 8.6 12.3 ± 8.1 11.3 ± 5.4 16.2 ± 12.1
Day 5 1.2 ± 2.4 0.7 ± 0.9 1.2 ± 3.3 8.9 ± 7.7 7.6 ± 5.4 7.1 ± 3.1 0.2 ± 0.1
p value Day À1 vs Day 5 .0001 0.0011 0.0045 0.0001 0.0019 0.0032 0.0005
MHBMA (lg/24 h)
Day À1 4.9 ± 3.5 5.9 ± 3.8 4.6 ± 3.2 5.0 ± 2.9 3.4 ± 2.5 4.5 ± 2.8 5.6 ± 3.6
Day 5 0.3 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 3.5 ± 2.1 2.8 ± 1.8 4.3 ± 2.3 0.4 ± 0.1
S-PMA (lg/24 h)
Day À1 6.3 ± 3.7 8.1 ± 4.9 6.3 ± 3.9 7.0 ± 4.4 4.9 ± 3.5 6.9 ± 5.5 7.6 ± 4.3
Day 5 0.3 ± 0.1 0.3 ± 0.3 0.4 ± 0.2 4.9 ± 3.3 3.6 ± 2.3 6.0 ± 4.6 0.3 ± 0.2
p value Day À1 vs Day 5 .0001 .0001 0.0001 0.0031 0.0055 0.0072 0.0001
Nicotine equivalentsa
(mg/24 h)
Day À1 17.0 ± 6.6 17.6 ± 8.7 14.5 ± 4.4 16.6 ± 5.5 16.0 ± 6.2 15.7 ± 3.9 20.0 ± 8.9
Day 5 10.7 ± 9.1 12.7 ± 9.7 10.5 ± 9.6 18.4 ± 7.2 15.9 ± 5.5 15.8 ± 5.0 0.5 ± 0.2
Values are presented as mean ± SD.
a
Nicotine equivalents: calculated as the molar sum of nicotine and five major nicotine metabolites (nicotine gluc, cotinine, cotinine-gluc, trans-3’-hydroxycotinine;
trans-3’-hydroxycotinine-gluc) excreted in urine over 24 h and reported as nicotine equivalents (mg/24 h). Nicotine equivalents (mg/24 h) ¼ [nicotine (mg/162.23
(mg/mmol) þ nicotine-gluc (mg/338.36 (mg/mmol) þ cotinine (mg/176.22 (mg/mmol) þ cotinine-gluc (mg/352.34 (mg/mmol) þ trans-3’-hydroxycotinine (mg/192.22
(mg/mmol) þ trans-3’-hydroxycotinine-gluc (mg/368.34 (mg/mmol)] Â 162.23 (mg/mmol)].

of VOCs such as Acrolein, Benzene, 1-3-Butadiene,
Crotonaldehyde and Acyrlonitrile (as measured by the BoE: 3-
HPMA, S-PMA, MHBMA, HMPMA and CEMA) were reduced by
20–33%, 21–30%, 7–26%, 20–33%, 24–32%, respectively, in
the dual use group versus 87%, 94%, 84%, 88% and 86%,
respectively, in the cessation group.
Relationship between product use and urine biomarker
Excretion
Statistically significant, positive linear relationships were
observed between percent change in biomarker excretion
and the percent change in CPD smoked for all urine BoE
Table 4. Blood biomarker concentration summary and statistical comparisons.
Biomarker
Classic tobacco
rechargeable
N ¼ 15
Cherry
rechargeable
N ¼ 13
Cherry
disposable
N ¼ 13
Classic tobacco
rechargeable
N ¼ 14
Cherry
rechargeable
N ¼ 15
Cherry
disposable
N ¼ 13
Nicotine
cessation
N ¼ 13
Blood COHb (%)
Day À1 6.3 ± 2.0 6.3 ± 2.0 6.0 ± 1.5 5.2 ± 2.0 4.8 ± 1.4 5.6 ± 1.9 7.4 ± 2.3
Day 5 1.2 ± 0.6 1.2 ± 0.5 1.0 ± 0.6 4.0 ± 1.5 3.8 ± 0.9 4.3 ± 1.3 1.6 ± 0.4
Plasma Nicotine (ng/mL)
Day À1 13.0 ± 6.1 14.7 ± 5.2 13.3 ± 6.5 12.5 ± 5.9 8.8 ± 3.2 11.1 ± 4.1 16.0 ± 7.0
Day 5 6.9 ± 6.3 8.4 ± 5.9 6.6 ± 5.6 9.5 ± 6.2 8.1 ± 5.2 7.9 ± 4.4 0.1 ± 0.0
Plasma Cotinine (ng/mL)
Day À1 260.1 ± 128.1 299.9 ± 93.7 250.1 ± 92.4 247.6 ± 99.0 213.6 ± 62.8 218.4 ± 58.3 282.2 ± 135.9
Day 5 164.5 ± 167.4 202.1 ± 103.2 149.2 ± 116.1 261.5 ± 119.4 211.5 ± 70.2 212.9 ± 89.3 5.49 ± 6.7
Plasma Trans-3’ hydroxycotinine (ng/mL)
Day À1 164.5 ± 167.4 202.1 ± 103.2 149.2 ± 116.1 261.5 ± 119.4 211.5 ± 70.2 212.9 ± 89.3 5.49 ± 6.7
Day 5 70.4 ± 59.0 85.0 ± 55.7 69.4 ± 56.5 102.2 ± 46.8 107.8 ± 50.7 98.5 ± 29.3 3.8 ± 2.7
Values are presented as mean ± SD.
Figure 1. Urine biomarkers – Day 5 percent change from Day À1.

(p ¼ 0.0001–0.0018) except nicotine equivalents (p ¼ 0.9316)
(Tables 5 and 6). These results may indicate that smokers
who reduce their conventional tobacco cigarette consump-
tion may predictably expect to see reduced exposure to a
number of HPHCs while replacing conventional tobacco ciga-
rettes with nicotine-containing e-cigarettes.
Furthermore, a statistically significant, positive linear rela-
tionship (p 0.0001) was observed between nicotine equiva-
lents excretion and the number of conventional tobacco
cigarettes smoked on Day À1 when all groups were included,
but not on Day 5 (p ¼ 0.2814) when the dual use groups
were included (Table 7). Within the exclusive and dual use
groups, the Day 5 relationship between nicotine equivalents
excreted and the estimated nicotine from the e-cigarettes
were statistically significant (p 0.0001). This finding may be
due to the relatively consistent use and constant nicotine
content in the e-cigarettes and the number of usual conven-
tional tobacco cigarettes smoked, coupled with differences in
individual smoking behaviors.
Exhaled Breath
Physiological changes associated with smoking reduction
were noted in the exhaled CO and NO endpoints. All groups
experienced statistically significant decreases in exhaled CO
at Day 5 compared to Day À1, with decreases in the cessa-
tion and exclusive use groups ranging from $88% to $89%
and in the dual use groups by $26–32% (Table 8 and Figure
3). Further, there were no differences between the cessation
and exclusive use group’s measurements on Day 5 whereas
the dual use groups had significantly higher exhaled CO com-
pared to cessation; this may be expected as this group con-
tinued to smoke conventional tobacco cigarettes.
Nitric oxide can be detected in expired breath and has
been identified in prior studies as a noninvasive biomarker of
inflammation (Birrell et al., 2006; Zhou et al., 2012).
Conventional tobacco cigarette has been reported to decrease
exhaled NO, possibly by the inhibition of the enzyme NO syn-
thase (Kharitonov et al., 1995), but the mechanism remains
incompletely understood. Exhaled NO was observed to
increase from Day À1 to Day 5 in the cessation and exclusive
use groups ($46–63%) whereas the dual use groups experi-
enced minimal changes. On Day 5, exhaled NO in the cessa-
tion and exclusive use groups was similar, but tended to be
lower in the dual use groups, though not all comparisons
were statistically significant (Table 7 and Figure 3).
Discussion
Dual use of electronic cigarettes and conventional cigarettes
has been cited as a potential public health concern because
of a possibility that it may expose smokers to greater health
risks than those encountered by smoking conventional
tobacco cigarettes alone (Grana et al., 2014). A more recent
study, however, reported that the dual use of e-cigarettes
while continuing to smoke did not result in reduced exposure
to known carcinogens and toxicants (Shahab et al., 2015).
This study enforced a reduction in daily cigarettes smoked on
a dual use group as an initial examination of the responsive-
ness of the measured smoke exposure biomarkers to moder-
ately-reduced smoking combined with unlimited ad libitum
usage of e-cigarettes. Under these conditions, the study
showed that dual users’ experienced significant reductions in
most of the biomarkers assessed ($20–35% reduction in urine
biomarkers) and that the magnitude of reduction in exposure
to biomarkers of exposure in the dual use subject was
broadly proportional to the reduction of conventional tobacco
Figure 2. Blood biomarkers – Day 5 percent change from Day À1.
Table 5. Regression analyzes of the Day À1 to Day 5%
change in the amount of urine biomarker amount excreted
against the % change in cigarettes per day (CPD).
Urine biomarker Slope R-square p Value
NNAL 0.4154 0.1518 0.0108
3-HPMA 0.6940 0.4105 0.0001
HMPMA 0.7878 0.4289 0.0001
CEMA 0.7096 0.4891 0.0001
1-OHP 0.6297 0.4227 0.0001
NNN 0.7766 0.3181 0.0001
MHBMA 0.7469 0.2874 0.0003
S-PMA 0.7259 0.4636 0.0001
Nicotine Equivalents À0.0296 0.0002 0.9316

cigarettes smoked. As such, the study findings are consistent
with an expectation of significantly reduced exposures to
harmful smoke constituents in smokers who completely
replace their conventional tobacco cigarettes with
e-cigarettes.
The study also showed that subjects who switched to dual
use also experienced significantly reduced exposure to HPHCs
after partially replacing conventional tobacco cigarettes with
an e-cigarette product. As expected, the excretion and con-
centration of all biomarkers evaluated in this study were sig-
nificantly higher in the dual use group at Day 5 compared to
the cessation group. However, reductions of 25–35% in the
levels of PAHs such as pyrene were observed in dual users
(versus 70% in the cessation group); levels of TSNAs such as
Table 6. Regression analyzes of nicotine equivalents excretion and Day À1 and Day 5 product use.
Relationship assessed Slope R-square p Value
Day À1 nicotine equivalents amount excreted against cigarettes smoked (all Groups) 0.7485 0.2137 0.0001
Day 5 nicotine equivalents amount excreted against cigarettes smoked (dual use Groups) 0.3598 0.0289 0.2814
Day 5 nicotine equivalents amount excreted against estimated nicotine from e-cigarettes (dual use Groups) 0.3970 0.4502 0.0001
Day 5 nicotine equivalents amount excreted against estimated nicotine from e-cigarettes (exclusive use Groups) 0.4794 0.8538 0.0001
Table 7. Summary of regression analyzes of nicotine equivalents excretion and Day À1 and Day 5 product use.
Relationship assessed Slope p Value R-square
Day À1 nicotine equivalents amount excreted against cigarettes smoked (All Cohorts) 0.7485 0.0001 0.2137
Day 5 nicotine equivalents amount excreted against cigarettes smoked (Dual Use Cohorts) 0.3598 0.2814 0.0289
Day 5 nicotine equivalents amount excreted against estimated nicotine from blu products (Dual use Cohorts) 0.3970 0.0001 0.4502
Day 5 nicotine equivalents amount excreted against estimated nicotine from blu products (Exclusive Use Cohorts) 0.4794 0.0001 0.8538
Table 8. Exhaled breath biomarker summary and statistical comparisons.
Biomarker
Classic tobacco
rechargeable
N ¼ 15
Cherry
rechargeable
N ¼ 13
Cherry
disposable
N ¼ 13
Classic tobacco
rechargeable
N ¼ 14
Cherry
rechargeable
N ¼ 15
Cherry
disposable
N ¼ 13
Nicotine
cessation
N ¼ 13
CO (ppm)
Day À1 27.2 ± 10.5 27.3 ± 6.9 26.9 ± 6.4 25.1 ± 7.3 25.4 ± 7.7 24.7 ± 5.5 29.3 ± 10.4
Day 5 2.9 ± 0.8 2.9 ± 0.8 2.7 ± 0.9 17.3 ± 5.7 16.1 ± 3.3 18.2 ± 5.7 2.8 ± 0.7
NO (ppb)
Day À1 14.8 ± 12.8 11.5 ± 4.8 10.0 ± 4.0 14.9 ± 11.1 10.6 ± 4.6 14.3 ± 13.5 11.3 ± 4.0
Day 5 23.3 ± 21.6 15.5 ± 9.0 14.3 ± 6.5 12.9 ± 6.3 10.7 ± 4.4 11.4 ± 6.0 16.8 ± 10.1
Values are presented as mean ± SD. A paired t-test was used to make the Day À1 vs Day 5 within group comparisons, a linear mixed model was used to make the
Day 5 comparisons to the Cessation group.
Figure 3. Exhaled breath biomarkers – Day 5 percent change from Day À1.

NNAL and NNN were reduced by 19–22% and 30–37%,
respectively, in the dual use group versus 66% and 98%,
respectively in the cessation group; and levels of VOCs such
as acrolein, benzene, 1-3-butadiene, crotonaldehyde and acyr-
lonitrile were also observed to be reduced by 20–33%,
21–30%, 7–26%, 20–33%, 24–32%, respectively, in the dual
use group versus 87%, 94%, 84%, 88% and 86%, respectively,
in the cessation group.
The results of this study also demonstrated that smokers
who completely substitute conventional tobacco cigarettes
with e-cigarettes over a short period of time (5-days) experi-
ence reductions in exposure to a number of HPHCs and toxi-
cants as measured by urine, blood and exhaled breath BoE.
Reductions in the HPHCs (PAHs, TSNAs VOCs and nicotine)
analyzed showed that measurable nicotine metabolites were
present in the samples from e-cigarette users, which was
expected as subjects continued to consume nicotine in the e-
cigarettes. However, levels of biomarkers for HPHCs were sig-
nificantly lower, and many were similar to those of subjects
who had quit smoking entirely. For example, levels of PAHs
such as pyrene were reduced by 70% in users that had quit
smoking or using nicotine products altogether and by
62–69% in the group that used e-cigarettes exclusively. Levels
of TSNAs such as NNAL and NNN were also reduced by
66–98%, respectively, in the cessation group and by 62–64%
and 87–93%, respectively, in the exclusive use group.
Moreover, levels of VOCs such as acrolein, benzene, 1-3-buta-
diene, crotonaldehyde and acyrlonitrile were also reduced by
87%, 94%, 84%, 88% and 86%, respectively, in the cessation
group and by 82–83%, 93–94%, 86–90%, 82–85% and
85–87%, respectively, in the exclusive use group.
Moreover, the study findings associated with exhaled
breath biomarkers in the cessation and exclusive use groups
were consistent with other research findings associated with
reductions in exhaled CO and increases in NO following
smoking cessation (Chambers et al., 1998; Hogman et al.,
2002; Jarvis et al., 1980; Malinovschi et al., 2006; Ripoll
et al., 2012; Robbins et al., 1997; West et al., 2005; Yates et al.,
2001); both of which may be indicative of immediate and
future physiological benefits to the smoker.
The results of this study also support the findings of other
investigations which have demonstrated that e-cigarette use
results in a different aerosol exposure, including a decrease in
certain biomarkers typical for combusted tobacco cigarette
consumption (Caponnetto et al., 2013; McRobbie et al., 2015;
Polosa et al., 2011). Furthermore, a recently-published study
on e-cigarette emissions from the products evaluated in this
study, found the e-cigarette aerosol contained levels of
HPHCs such as carbonyl compounds, tobacco-specific nitros-
amines, polycyclic aromatic hydrocarbons and other constitu-
ents that were on the order of 1500 times lower than those
found in the smoke of conventional tobacco cigarettes
(2 lg/puff vs. $3,000 lg/puff) (Tayyarah Long, 2014). This
study extends those findings with the observation that the e-
cigarette produced markedly lower levels of BoE when used
by smokers in lieu of their preferred conventional tobacco
cigarette brand style for a period of 5 days.
Furthermore, this investigation also further confirms and
extends the findings of several prior, smaller studies that
have compared levels of BoE in users of e-cigarettes to those
of conventional tobacco cigarette smokers. Vansickel et al.
(2010), Farsalinos et al. (2014) and Walele et al. (2016)
reported moderate plasma nicotine values in users of first
generation e-cigarette devices similar to those used in this
study. Typical use of later-generation, tank-style e-cigarettes
or intensive use of the first-generation cigarette-like devices
has been reported to produce plasma nicotine values similar
to those from conventional tobacco cigarettes (D’Ruiz et al.,
2015; Yan D’Ruiz, 2015). Hecht et al. (2015) also have
recently reported combined findings from three independent
studies of smokers whose biomarkers levels were compared
to those of 28 self-reported users of a variety of commercial
cartridge- and tank-based e-cigarettes under uncontrolled ad
libitum conditions. These authors concluded, with respect to
the biomarkers analyzed, that the e-cigarettes had a more
favorable toxicity profile than conventional tobacco
cigarettes.
BoE to tobacco toxicants are well established and numer-
ous studies exist that have utilized biomarkers of exposure to
assess exposure to tobacco smoke constituents in humans
and have generated meaningful measures of tobacco toxicant
exposure. This has been useful for evaluating the individual
potential risks associated with different classes of tobacco
products (Mattes et al., 2014). Whether the reductions in
exposure to toxicants such as those observed in this study
may have the potential to reduce risks for chronic, smoking-
caused diseases for long-term e-cigarette users who have par-
tially or completely discontinued cigarette smoking warrants
further investigation. It has been previously shown that for
those Swedish smokers who completely switch from ciga-
rettes to a noncombustible form of tobacco (snus, which
delivers nicotine without smoke) there appears to be an
association with lower relative risks for major smoking-related
disease, including cancer (Round et al., 2015).
The study’s main limitation was that it was only a short-
term (5-day) trial looking at select BoE associated with a sin-
gle product type (i.e., closed system e-cigarette). Nevertheless,
the study was able to provide data to address a deficit in sci-
entific knowledge with regard to HPHC levels in different
types of e-cigarette users by showing that reducing conven-
tional cigarette smoking leads to reductions in HPHC expos-
ure in individuals who exclusively use electronic cigarettes or
are dual users (electronic cigarettes and conventional cigar-
ette smokers) under short-term use conditions. Longer-term
BoE or biomarker of effect studies may be informative for
assessing the long-term implications and physiological rele-
vance of reduced exposure to HPHCs in individuals who
exclusively use e-cigarettes or are dual users. Information
from longer-term e-cigarette product tolerability and adverse
event surveillance studies may also be informative.
Data associated with several other secondary objectives
were also collected during the course of this study but are
not reported in this paper. These include estimates of daily
nicotine delivery following exclusive e-cigarette and dual use
of e-cigarettes and the subject’s usual brand combustible cig-
arette over a 5-day period; subjective effects related to urge
to smoke and satisfaction; changes in selected physiological
endpoints such as blood pressure and pulse; and tolerability

and adverse events. Given the importance of our findings in
these areas, the results will be reported under separate publi-
cations. Furthermore, participant biofluids were collected and
frozen as part of this study in anticipation of further research
related to targeted and untargeted biomarkers of effect. The
results of our future investigations in this area will also be
published when available.
Conclusions
This study provides data to address a deficit in scientific
knowledge in regards to HPHC levels in different types of
electronic cigarette users. The study showed that reducing
conventional tobacco cigarette consumption over 5 days
resulted in significant reductions in exposure to known bio-
markers of HPHCs reported in tobacco smoke, with greater
reductions observed in subjects who ceased consumption of
all nicotine product use or switched to e-cigarette products
compared to subjects who switched to dual use. The magni-
tude of reduction in exposure to biomarkers in dual use
group was broadly proportional to reduction in conventional
tobacco cigarettes smoked. Physiological effects of reducing
cigarette consumption were also observed in the exhaled
breath endpoints. This study also illustrates that biomarkers
of exposure may play a role in assessing and comparing
exposure to HPHCs across different product categories and
exemplifies their potential for informing product regulatory
reviews and tobacco product regulation. Overall, this data
indicate the great potential that e-cigarettes may provide for
smokers seeking an alternative to tobacco products and sup-
ports the case for regulating e-cigarettes differently from
combustible tobacco-containing products.
Acknowledgements
We gratefully acknowledge the support and ideas from our current and
past colleagues from the A.W. Spears Research Center and the study
investigators at Celerion in Lincoln, Nebraska. We also thank the science
team at Imperial Brands plc for critical review of the manuscript.
Disclosure statement
CDD is consultant for Fontem Ventures U.S. Inc.; GOC is an employee of
Fontem Ventures, B.V.; DWG is an employee of Celerion, the contract
research organization (CRO) that conducted the study.
Funding
The work in this manuscript was supported by Fontem Ventures B.V., a
fully owned subsidiary of Imperial Brands plc, and the manufacturer of
the e-cigarette products used in this study.
References
Birrell MA, McCluskie K, Hardaker E, et al. (2006). Utility of exhaled nitric
oxide as a noninvasive biomarker of lung inflammation in a disease
model. Eur Respir J 28:1236–44.
Burstyn I. (2014). Peering through the mist: systematic review of what the
chemistry of contaminants in electronic cigarettes tells us about health
risks. BMC Public Health 14:18.
Cahn Z, Siegel M. (2011). Electronic cigarettes as a harm reduction strat-
egy for tobacco control: a step forward or a repeat of past mistakes.
J Public Health Policy 32:16–31.
Caponnetto P, Auditore R, Russo C, et al. (2013). Impact of an electronic
cigarette on smoking reduction and cessation in schizophrenic smok-
ers: a prospective 12-month pilot study. Int J Environ Res Public Health
10:446–61.
Carmella SG, Chen M, Han S, et al. (2009). Effects of smoking cessation on
eight urinary tobacco carcinogen and toxicant biomarkers. Chem Res
Toxicol 22:734–41.
Chambers DC, Tunnicliffe WS, Ayres JG. (1998). Acute inhalation of cigar-
ette smoke increases lower respiratory tract nitric oxide concentrations.
Thorax 53:677–9.
Deveci SE, Deveci F, Ac¸ik Y, Ozan AT. (2004). The measurement of exhaled
carbon monoxide in healthy smokers and non-smokers. Respir Med
98:551–6.
D’Ruiz CD, Graff DW, Yan XS. (2015). Nicotine delivery, tolerability and
reduction of smoking urge in smokers following short-term use of one
brand of electronic cigarettes. BMC Public Health 15:991.
Fagerstr€om K. (2012). Determinants of tobacco use and renaming the
FTND to the Fagerstrom test for cigarette dependence. Nicotine Tob
Res 14:75–8.
Farsalinos KE, Polosa R. (2014). Safety evaluation and risk assessment of
electronic cigarettes as tobacco cigarette substitutes: a systematic
review. Ther Adv Drug Saf 5:67–86.
Farsalinos KE, Romagna G, Tsiapras D, et al. (2014). Characteristics, per-
ceived side effects and benefits of electronic cigarette use: a world-
wide survey of more than 19,000 consumers. Int J Environ Res Public
Health 11:4356–73.
Farsalinos KE, Spyrou A, Tsimopoulou K, et al. (2014). Nicotine absorption
from electronic cigarette use: comparison between first and new-
generation devices. Sci Rep 4:4133.
Goniewicz ML, Knysak J, Gawron M, et al. (2014). Levels of selected carci-
nogens and toxicants in vapour from electronic cigarettes. Tob Control
23:133–9.
Grana R, Benowitz N, Glantz SA. (2014). E-cigarettes: a scientific review.
Circulation 129:1972–86.
Gregg EO, Minet E, McEwan M. (2013). Urinary biomarkers of
smokers’ exposure to tobacco smoke constituents in tobacco
products assessment: a fit for purpose approach. Biomarkers
18:467–86.
Hajek P, Etter J-F, Benowitz N, et al. (2014). Electronic cigarettes: review of
use, content, safety, effects on smokers and potential for harm and
benefit. Addiction 109:1801–10.
Heatherton TF, Kozlowski LT, Frecker RC, Fagerstr€om KO. (1991). The
Fagerstr€om test for nicotine dependence: a revision of the Fagerstr€om
tolerance questionnaire. Br J Addict 86:1119–27.
Hecht SS, Carmella SG, Kotandeniya D, et al. (2015). Evaluation of toxicant
and carcinogen metabolites in the urine of E-cigarette users versus cig-
arette smokers. Nicotine Tob Res 17:704–9.
Hecht SS, Yuan J-M, Hatsukami D. (2010). Applying tobacco carcinogen
and toxicant biomarkers in product regulation and cancer prevention.
Chem Res Toxicol 23:1001–8.
Hecht SS, Carmella SG, Chen M, et al. (1999). Quantitation of urinary
metabolites of a tobacco-specific lung carcinogen after smoking cessa-
tion. Cancer Res 59:590–6.
Hecht SS, Murphy SE, Carmella SG, et al. (2004). Effects of reduced
cigarette smoking on the uptake of a tobacco-specific lung carcinogen.
J Natl Cancer Inst 96:107–15.
Hecht SS. (2002). Human urinary carcinogen metabolites: biomarkers for
investigating tobacco and cancer. Carcinogenesis 23:907–22.
Hogman M, Holmkvist T, Walinder R, et al. (2002). Increased nitric oxide
elimination from the airways after smoking cessation. Clin Sci (Lond)
103:15–19.
Jarvis JM, Rusell MAH, Saloojee Y. (1980). Expired air carbon monoxide: a
simple breath test of tobacco smoke intake. BMJ 281:484–5.
Kharitonov SA, Robbins RA, Yates D, et al. (1995). Acute and chronic
effects of cigarette smoking on exhaled nitric oxide. Am J Respir Crit
Care Med 152:609–12.

Malinovschi A, Janson C, Holmkvist T, et al. (2006). Effect of smoking on
exhaled nitric oxide and flow-independent nitric oxide exchange
parameters. Eur Respir J 28:339–45.
Mattes W, Yang X, Orr MS, et al. (2014). Biomarkers of tobacco smoke
exposure. Adv Clin Chem 67:1–45.
McRobbie H, Phillips A, Goniewicz ML, et al. (2015). Effects of switching to
electronic cigarettes with and without concurrent smoking on expos-
ure to nicotine, carbon monoxide, and acrolein. Cancer Prev Res (Phila)
8:873–8.
McRobbie H, Bullen C, Hartmann-Boyce J, Hajek P. (2014). Electronic ciga-
rettes for smoking cessation and reduction. Cochrane Database Syst
Rev 12:CD010216.
Misra M, Leverette RD, Cooper BT, et al. (2014). E Comparative in vitro
toxicity profile of electronic and tobacco cigarettes, smokeless tobacco
and nicotine replacement therapy products: e-liquids, extracts and col-
lected aerosols. Int J Environ Res Public Health 11:11325–47.
Pepper JK, Brewer NT. (2014). Electronic nicotine delivery system (elec-
tronic cigarette) awareness, use, reactions and beliefs: a systematic
review. Tob Control 23:375–84.
Polosa R, Rodu B, Caponnetto P, et al. (2013). A fresh look at tobacco
harm reduction: the case for the electronic cigarette. Harm Reduct J
10:19. doi:10.1186/1477-7517-10-19.
Polosa R, Caponnetto P, Morjaria JB, et al. (2011). Effect of an electronic
nicotine delivery device (e-cigarette) on smoking reduction and cessa-
tion: a prospective 6-month pilot study. BMC Public Health 11:786.
Ripoll J, Girauta H, Ramos M, et al. (2012). Clinical trial on the efficacy of
exhaled carbon monoxide measurement in smoking cessation in pri-
mary health care. BMC Public Health 12:322.
Robbins RA, Millatmal T, Lassi K, et al. (1997). Smoking cessation is associ-
ated with an increase in exhaled nitric oxide. Chest 112:313–18.
Round EK, Campbell LR, Stiles MF, et al. (2015). Changes in biomarkers of
exposure and subjective effects when smokers switch to dual use of
cigarettes and either snus or a dissolvable tobacco product: a sum-
mary of three clinical studies. Beitr Tabakforsch Int 26:242–60.
Sarkar M, Liu J, Koval T, et al. (2010). Evaluation of biomarkers of exposure
in adult cigarette smokers using Marlboro Snus. Nicotine Tob Res
12:105–16.
Shahab L, Goniewicz M, Alwis U, et al. (2015). Exposure to selected toxi-
cants and carcinogens as a function of smoking status and long-term
use of nicotine replacement therapy or electronic cigarettes. Abstract
presented at the 21st annual meeting of the Society for Research on
Nicotine and Tobacco, Philadelphia, PA.
Taylor DR, Pijnenburg MW, Smith AD. (2006). Exhaled nitric oxide
measurements: clinical application and interpretation. Thorax
61:817–27.
Tayyarah R, Long GA. (2014). Comparison of select analytes in aerosol
from e-cigarettes with smoke from conventional cigarettes and with
ambient air. Reg Toxicol Pharmacol 70:704–10.
USFDA (2012). U.S. Food and Drug Administration, Center for Tobacco
Products (CTP), Harmful and Potentially Harmful Constituents in
Tobacco Products and Tobacco Smoke; Established List, April 2012.
Available from: http://www.fda.gov/TobaccoProducts/Guidance
ComplianceRegulatoryInformation/ucm297786.htm [last accessed 17
Apr 2015].
USDHHS (2014). US Department of Health and Human Services. The
Health Consequences of Smoking–50 Years of Progress: A Report of
the Surgeon General. Atlanta, GA: US Department of Health and
Human Services, Centers for Disease Control and Prevention, National
Center for Chronic Disease Prevention and Health Promotion, Office
on Smoking and Health.
Vansickel AR, Cobb CO, Weaver MF, Eissenberg TE. (2010). A clinical
laboratory model for evaluating the acute effects of electronic ‘‘ciga-
rettes’’: nicotine delivery profile and cardiovascular and subjective
effects. Cancer Epidem Biomar 19:1945–53.
Walele T, Sharma G, Savioz R, et al. (2016). A randomised, crossover study
on an electronic vapour product, a nicotine inhalator and a conven-
tional cigarette. Part B: safety and subjective effects. Reg Toxicol
Pharmacol 74:187–92.
West R, Hajek P, Stead L, Stapleton J. (2005). Outcome criteria in smoking
cessation trials: proposal for a common standard. Addiction
100:299–303.
Yan XS, D’Ruiz CD. (2015). Effects of using electronic cigarettes on nico-
tine delivery and cardiovascular function in comparison with regular
cigarettes. Regul Toxicol Pharmacol 71:24–34.
Yates DH, Breen H, Thomas PS. (2001). Passive smoke inhalation decreases
exhaled nitric oxide in normal subjects. Am J Respir Crit Care Med
164:1043–6.
Zhou M, Liu Y, Duan Y. (2012). Breath biomarkers in diagnosis of pulmon-
ary diseases. Clin Chim Acta 413:1770–80.

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