A Longitudinal Ambulatory Study to Assess Changes in Cigarette Consumption and Biomarkers of Exposure During a 6-week Switch to Very Low Nicotine Cigarettes
Phase: NA
Conditions: Tobacco Use
Sponsor: 22nd Century Group, Inc.

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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CA24914_FINALV1_09MAY2018 
 
 
 
 
 
 
A Longitudinal Ambulatory Study to Assess Changes in Cigarette Consumption Behavior 
and Biomarkers of Exposure during a 6-Week Switch to Very Low Nicotine Cigarettes 
 
 
 
 
Celerion Project No.: CA24914 
 
Final Protocol Version 1: 09 May 2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
GCP Statement 
This study is to be performed in full compliance with the protocol, Good Clinical Practice (GCP), and 
applicable regulatory requirements. All required study documentation will be archived as required by 
regulatory authorities. 
Confidentiality Statement 
This document is confidential. It contains proprietary information 22nd Century Group, Inc. Any viewing or 
disclosure of such information that is not authorized in writing by 22nd Century Group, Inc is strictly 
prohibited. Such information may be used solely for the purpose of reviewing or performing this study. 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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PROTOCOL HISTORY 
DATE/NAME 
DESCRIPTION 
19MAR2018/Ian Fearon 
Draft v0.1 Protocol for Internal Review 
20APR2018/Ian Fearon 
Draft v0.2 Protocol for Internal Review 
09MAY2018/Ian Fearon 
Final Protocol v1 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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CA24914_FINALV1_09MAY2018 
 
 
SIGNATURE PAGE 
 
Sponsor 
Representative: 
Dr. Juan Sanchez Tamburrino 
VP Research and Development 
22nd Century Group, Inc. 
8560 Main St 
Williamsville, NY 14221 
Tel.:  +1 336 749-1018 
E-mail:  jtamburrino@xxiicentury.com 
 
 
 
 
 
 
 
 
 
 
 
__________________________________ 
_________ 
Signature 
Date 
 
 
 
 
 
 
 
 
 
 
Principal 
Investigators: 
Dr. Philip Matthew 
621 Rose Street 
Lincoln, Nebraska 68502, USA 
Tel.: +1 402 476-2811  
Fax: +1 866 358-7993 
E-mail:  philip.matthew@celerion.com 
 
 
 
 
 
 
 
 
 
 
__________________________________ 
_________ 
 
Signature 
Date 
 
 
 
 
 
 
Dr. Melanie Fein 
Medical Director 
High Point Clinical Trials Center 
4160 Mendenhall Oaks Parkway, Suite 105 
High Point, NC 27265 
Tel:  +1 336 841-0700 ext 2530 
Mobile:  +1 336 455-3257 
Fax:  +1 336 841-0900 
E-mail:  mfein@HighPointCTC.com 
 
 
 
 
 
 
 
 
__________________________________ 
_________ 
 
Signature 
Date 
 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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CA24914_FINALV1_09MAY2018 
 
 
STUDY CONTACTS 
 
Sponsor Contact for  
Serious Adverse Events 
Dr. Juan Sanchez Tamburrino 
VP Research and Development 
22nd Century Group, Inc. 
8560 Main St 
Williamsville, NY 14221 
Tel.:  +1 215 285-7140 
E-mail:  jtamburrino@xxiicentury.com 
 
 
 
Sponsor Project Manager: 
Ed Carmines Ph.D. 
Principal 
Carmines Consulting LLC 
37220 N 102 St 
Scottsdale, AZ 85262 
Tel.:  +1 804 502-9275 
E-mail:  ed@carminesconsulting.com 
 
 
 
Clinical Laboratories 
Celerion 
621 Rose Street 
Lincoln, NE 68502 
Contact: Steve Hambek 
Tel.:  +1 402 476-2811 
Fax:  +1 402 939-0428 
E-mail:  steve.hambek@celerion.com 
 
 
 
 
Laboratory Corporation of America (Labcorp) 
1447 York Court 
Burlington, NC 27215 
Contact: Alicea Strzelczyk 
Tel.:  +1 512 225-1984 
Fax:  +1 512 225-1252 
E-mail:  pohlera@labcorp.com 
 
 
 
Bioanalytical Laboratory 
Celerion 
621 Peach Street  
Lincoln, NE 68502 
Contact:  Erica Nachi 
Tel.:  +1 402 437-6275 
Fax:  +1 402 939-0428 
E-mail:  erica.nachi@celerion.com 
 
 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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SYNOPSIS 
Study Objectives 
Primary objectives: 
1. To characterize cigarette consumption behavior (cigarettes 
per day and smoking topography) before, during and after a 
switch from usual brand (UB) to very low nicotine (VLN) 
cigarettes for 6 weeks. 
Secondary objectives:  
1. To evaluate changes in tobacco-related biomarkers of 
exposure (BoE) before, during and after a 6-week switch 
from UB to VLN cigarettes.  
2. To evaluate nicotine pharmacokinetics (PK) before, during 
and after a 6-week switch from UB to VLN cigarettes. 
3. To evaluate changes in subjective effects before, during and 
after a 6-week switch from UB cigarettes to VLN cigarettes 
Study Design 
This is an open-label, non-randomized, forced-switching study to be 
conducted at multiple study sites. Seventy (70) self-affirmed 
exclusive filtered king size non-mentholated cigarette smokers and 
70 self-affirmed exclusive filtered king size mentholated cigarette 
smokers will be enrolled and begin the study at Week -1. 
All potential subjects will provide informed consent and successfully 
complete the Screening procedures prior to participation in the 
study. Subjects will also engage in a brief product trial with the VLN 
cigarettes. Subjects who react negatively (i.e., unwilling to use 
and/or cannot tolerate the product [experience adverse events 
(AEs) that will prevent them from continuing to use the product as 
judged by the Investigator]) to the VLN cigarettes during the product 
trial period will not continue in the study.  
At the start of Week -1, all subjects will be asked to smoke their UB 
cigarettes as per their usual daily consumption for the following 
week. Subjects will receive an electronic diary (e-diary) to record 
daily cigarette use (cigarettes per day [CPD]). Training in completion 
of the e-diary will be provided at the visit at the start of Week -1.  
Subjects will return at the end of Week -1, at the time indicated by 
the clinical research unit (CRU), for collection of blood and 24-hour 
urine samples for baseline BoE assessments. Subjective 
questionnaires for dependence, withdrawal symptoms, urges to 
smoke, and perceived health risk will also be completed at 
scheduled times. A subset of 18 non-menthol and 18 menthol 
smoker subjects will complete an assessment of puffing topography 
with their UB cigarettes during this visit. A further subset of 12 of the 
non-menthol and 12 of the menthol smoker subjects who complete 
the topography assessment will also complete a nicotine PK 
assessment at the end of this visit. Subjects who undergo 
topography and PK assessments will be assigned to switch to 
smoking VLN cigarettes.  
On Day 1 of Week 1, subjects will be randomly selected to either 
remain smoking their non-menthol (20 subjects) or menthol (20 
subjects) UB cigarettes, or to switch to smoking non-menthol (50 
subjects) or menthol (50 subjects) VLN cigarettes as per their UB 
cigarette flavor. Subjects will return at the end of Weeks 2 and 6, at 
the time indicated by the CRU, for collection of blood and 24-hour 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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urine 
samples 
for 
BoE 
assessments. 
Subjective 
effects 
questionnaires will also be completed at scheduled times. Subjects 
will continue recording their CPD in their e-diaries. A subset of 18 
non-menthol and 18 menthol smoker subjects will complete an 
assessment of puffing topography with the VLN cigarettes at these 
visits, and a further subset of 12 non-menthol and 12 menthol 
smoker subjects will also complete an assessment of nicotine PK at 
the end of these visits. Subjects undergoing topography and PK 
assessments will have been assigned to switch to smoking VLN 
cigarettes. 
Additionally, all subjects will visit the clinic at the end of Week 4 to 
receive further supplies of cigarettes (if assigned to the VLN groups) 
and to complete subjective effects questionnaires.  
Subjects randomized to the VLN groups will be provided with a 
supply of VLN cigarettes at each visit, which will be 150 % of their 
usual daily consumption as reported during Week -1. If a subject 
runs out of cigarettes between clinic visits, they may visit the clinic 
to receive further cigarettes. All subjects will be asked to smoke their 
cigarettes ad libitum, recording their actual daily consumption in 
their e-diaries. Non-compliant nicotine product consumption should 
also be recorded. Used cigarette butts will also be collected during 
ambulatory periods to verify product use and/or assess compliance. 
During Week -1 (all subjects) and all subsequent weeks (subjects 
randomized to continue smoking UB cigarettes) subjects will be 
asked not to change their UB cigarette brand or flavor.  
The CRU will attempt to contact all subjects who participated in the 
study (including subjects who terminate the study early) using their 
standard procedures approximately 7 days after the last contact to 
determine if any AE has occurred since the last study visit.  
 
Study Population 
Seventy 
(70) 
self-affirmed, 
exclusive 
smokers 
each 
of 
non-mentholated and mentholated king-size cigarettes will be 
enrolled into the study.  

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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Duration of Study 
Conduct 
Screening events will occur within 28 days prior to Day 1 of Week -1. 
Subjects will participate in the study for a period of 7 weeks after the 
start of Week -1, with a follow-up call approximately 7 days after the 
last study visit.  
Study Products 
and 
Summarization 
The study products are: 
• 
Subject’s UB non-mentholated filtered king size cigarettes  
• 
Subject’s UB mentholated filtered king size cigarettes  
• 
Non-mentholated VLN cigarettes  
• 
Mentholated VLN cigarettes  
VLN cigarettes contain 0.4mg nicotine per gram of tobacco.  
Subjects will smoke their assigned products ad libitum throughout 
the study. Product use will be listed by subject and summarized by 
time point (study week). 
Consumption and 
Biomarker Data 
Parameters and 
Summarization 
During ambulatory periods between clinic visits, subjects will record 
daily cigarette use (CPD) in their e-diary, including both compliant 
and non-compliant (nicotine containing products other than their 
assigned product) use. Subjects will also collect used cigarette butts 
during these periods.  
Twenty-four (24)-hour urine collections will be performed during 
clinic visits at the end of Weeks -1, 2 and 6, for BoE (total NNAL, 
total NNN, 3-HPMA, S-PMA, 1-OHP and Tneq) analysis. Creatinine 
will also be measured in each 24-hour collection and may be used 
to adjust the concentration values of the BoE if necessary. Blood 
samples for carboxyhemoglobin (COHb) and cotinine measurement 
will also be taken during these visits.  
All data will be listed by subject and summarized by time point using 
descriptive statistics.  
Puffing topography 
Data Parameters 
and 
Summarization 
Puffing topography will be measured in a randomly-selected subset 
of 18 non-menthol and 18 menthol cigarette smokers during an ad 
libitum usage session at the clinic visits at the end of Week -1 (UB 
cigarette) and Weeks 2 and 6 (VLN cigarettes). These subjects will 
be/have been assigned to switch to smoking VLN cigarettes. Puffing 
topography will be assessed using the mobile smoking puff analyzer 
(SPA-M; Sodim) during the confinement periods. Topography 
assessment will only be performed at one of the two clinical sites 
(Lincoln).  
The following topography parameters will be assessed:  
• 
Puff duration 
• 
Puff volume 
• 
Peak puff flow rate 
• 
Average flow rate 
• 
Inter-puff interval 
All data will be listed by subject and summarized using descriptive 
statistics.  

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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Nicotine PK Data 
Parameters and 
Summarization 
Plasma nicotine levels will be assessed in a subset of 12 non-
menthol and 12 menthol cigarette smokers before, during and after 
a 5-minute ad libitum smoking session with UB (Week -1) and VLN 
(Weeks 2 and 6) cigarettes. These subjects will be/have been 
assigned to switch to smoking VLN cigarettes and will be randomly 
selected from the subjects selected to undergo topography 
assessments. Blood samples will be drawn at -5, 2, 5, 7, 10, 12, 15, 
20, 30, 45, 60, 90, 120, 150 and 180 minutes relative to the start of 
cigarette smoking a single cigarette. Nicotine PK assessment will be 
performed on the day following 24-hour confined urine collection 
and after an overnight abstinence from cigarette smoking.  
Plasma nicotine PK parameters, Cmax, Tmax and AUC will be 
computed from the individual plasma concentrations.   
Nicotine concentrations and PK parameters will be listed by subject 
and summarized by study product and sex using descriptive 
statistics. 
Subjective Effects 
Data Collection 
and 
Summarization 
Measurement of subjective effects will be made during the clinic 
visits at Weeks -1, 2, 4 and 6, with the single administration of the 
Fagerström Test for Cigarette Dependence (FTCD), the Brief 
Questionnaire of Smoking Urges (QSU-Brief), the Minnesota 
Nicotine Withdrawal Scale - Revised (MNWS-R) and a perceived 
health risk scale.  
All data will be listed by subject and summarized by time point using 
descriptive statistics. 
Safety 
Assessments and 
Summarization 
Baseline visit: 
At the start of Week -1, evaluations will include vital signs, exhaled 
breath CO measurement, urine drug and alcohol screen, urine 
cotinine test and a serum pregnancy test (females only), and body 
weight.  
On study: 
At the end of Weeks -1, 2, 4 and 6, evaluations will include vital 
signs, a urine drug and alcohol screen, and a urine pregnancy test 
(females only). End of study (or early discontinuation) evaluations 
will include a 12-lead ECG and vital signs.  
Throughout the study: 
AEs spontaneously reported by the subjects or observed by the 
Investigator or other study personnel will be monitored from after the 
product trial at Screening until the End of Study (or upon early 
discontinuation), and will be queried by study staff at the follow-up 
call. Any concomitant medications taken from screening through the 
end of study (or early discontinuation) will also be recorded. A 
symptom-driven physical examination may be performed at any 
time, at the discretion of the Investigator or designee. 
 
AEs will be tabulated and summary statistics for vital signs and 
clinical laboratory tests may be computed and provided, as deemed 
clinically appropriate. 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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SUMMARY OF STUDY EVENTS  
 
 
Clinic Visits 
Out-of-CRU 
Between 
Clinic Visits 
 
 
STUDY EVENTS/ASSESSMENTS 
Screening1 
Start of 
Week -1  
End of 
Week -1 
(baseline
) 
End of 
Week 2 
End of 
Week 4 
End of 
Week 6 
Weeks -1 to 
6 
End of 
study2 
Follow-up 
Call3 
Informed Consent 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
Smoking/Tobacco/Nicotine Product 
Use History 
X 
 
 
 
 
 
 
 
 
Usual Brand Cigarette and Flavor 
Documentation4 
X 
X 
X 
X5 
X6 
X7 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
Review of Inclusion / Exclusion 
Criteria 
X 
 
 
 
 
 
 
 
 
Review of Concomitant 
Medications 
X 
Physical Examination8 
X 
 
 
 
 
 
 
X9 
X10 
Height, Body Weight, BMI 
X 
X11 
 
 
 
 
 
X12 
 
Vital Signs 
X 
X 
X 
X 
X 
X 
 
X 
 
12-Lead ECG 
X 
 
 
 
 
 
 
X 
 
                                               
1 Up to 28 days prior to first study visit (start of Week -2).  
2 At end of Week 6 clinic visit or upon early discontinuation.  
3 Approximately 7 days after their last clinic visit, including subjects who discontinue from the study early.  
4 Including taking a color photocopy of the front and back of the subject’s UB cigarette pack along with a ruler.  
5 For subjects randomized to continue smoking UB cigarettes only.  
6 For subjects randomized to continue smoking UB cigarettes only. 
7 For subjects randomized to continue smoking UB cigarettes only. 
8 Standard physical examination including oral cavity and oropharynx. A symptom driven examination can be performed at other time, at Investigator or designee’s discretion. 
9 Symptom driven and if deemed necessary.  
10 Symptom driven and if deemed necessary based on the follow-up call. Subjects will visit the clinic for this to take place. 
11 Body weight only at Start of Week -1 
12 Body weight only at end of study 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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Clinic Visits 
Out-of-CRU 
Between 
Clinic Visits 
 
 
STUDY EVENTS/ASSESSMENTS 
Screening1 
Start of 
Week -1  
End of 
Week -1 
(baseline
) 
End of 
Week 2 
End of 
Week 4 
End of 
Week 6 
Weeks -1 to 
6 
End of 
study2 
Follow-up 
Call3 
Clinical Chemistry13, Hematology, 
Urinalysis 
X 
 
 
 
 
 
 
X 
 
Urine Drug and Alcohol Screen  
X 
X 
X 
X 
X 
X 
 
X 
 
HIV, HBsAg, and HCV Serology 
X 
 
 
 
 
 
 
 
 
Serum Pregnancy Test (females) 
X 
 
 
 
 
 
 
 
 
Serum FSH Test14 
X 
 
 
 
 
 
 
 
 
Urine Cotinine Screen 
X 
X 
 
 
 
 
 
 
 
Exhaled CO Measurement 
X 
X 
 
 
 
 
 
 
 
Test Product (VLN cigarette) Trial 
X 
 
 
 
 
 
 
 
 
Provide Smoking Cessation 
Information15 
X 
 
 
 
 
 
 
X 
X 
Review of AEs 
X16 
X 
X 
X 
X 
X 
 
X 
X 
Urine Pregnancy Test (females) 
 
X 
X 
 
 
 
 
 
 
Review Restrictions on Smoking 
and Nicotine Product Use 
X 
X 
X 
X 
X 
X 
 
 
 
Reminder Contacts to return to the 
CRU17 
 
X18 
 
 
 
 
X19 
 
 
Test Product Dispensing 
 
 
X 
X 
X 
 
 
 
 
                                               
13 Clinical chemistry tests will be performed after at least an 8 hour fast; however, in case of dropouts or rechecks, subjects may not have fasted for 8 hours prior to the clinical 
chemistry sample being taken. 
14 Postmenopausal females only 
15 Subjects will be referred to the Quit Assist® website 
16 AE recording to begin immediately after the product trial at Screening.  
17 Phone calls or texts or instant messages (as per site preference) reminding subjects of clinic visits. 
18 The day prior to clinic visit at start of Week -1 subjects will be contacted. 
19The day prior to clinic visits at end of Weeks -1, 2, 4 and 6 subjects will be contacted. 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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CA24914_FINALV1_09MAY2018 
 
 
 
 
Clinic Visits 
Out-of-CRU 
Between 
Clinic Visits 
 
 
STUDY EVENTS/ASSESSMENTS 
Screening1 
Start of 
Week -1  
End of 
Week -1 
(baseline
) 
End of 
Week 2 
End of 
Week 4 
End of 
Week 6 
Weeks -1 to 
6 
End of 
study2 
Follow-up 
Call3 
Dispense Canisters for Used 
Cigarette Butt Collection 
 
X 
X 
X 
X 
 
 
 
 
Used Cigarette Butt Collection 
 
 
 
 
 
 
X 
 
 
Used Cigarette Butt Canister 
Return 
 
 
X 
X 
X 
X 
 
 
 
24-hour Urine Collection for BoE 
analysis 
 
 
X 
X 
 
X 
 
 
 
Plasma Sample for Cotinine 
Analysis 
 
 
X 
X 
 
X 
 
 
 
Blood Sample for COHb Analysis 
 
 
X 
X 
 
X 
 
 
 
Puffing Topography20 
 
 
X 
X 
 
X 
 
 
 
Blood Samples for Nicotine PK 
Analysis21 
 
 
X 
X 
 
X 
 
 
 
Training on e-diary use 
 
X 
 
 
 
 
 
 
 
Daily e-diary use to record 
cigarette consumption 
 
X 
X 
X 
X 
X 
X 
 
 
Fagerström Test for Cigarette 
Dependence (FTCD) 
 
 
X 
X 
X 
X 
 
 
 
Brief Questionnaire of Smoking 
Urges (QSU-brief) 
 
 
X 
X 
X 
X 
 
 
 
Minnesota Nicotine Withdrawal 
Scale - Revised (MNWS-R) 
 
 
X 
X 
X 
X 
 
 
 
                                               
20 In a randomly-selected subset of 18 non-menthol and 18 menthol cigarette smokers at a single site (Lincoln). These subjects will be/have been assigned to switch to smoking VLN 
cigarettes. To be performed during the clinic confinement period.  
21 In a randomly-selected subset of 12 non-menthol and 12 menthol cigarette smokers at a single site (Lincoln). These subjects will be/have been assigned to switch to smoking VLN 
cigarettes and and will be randomly selected from the subjects selected to undergo topography assessments. To be performed on the day following 24-hour confined urine collection 
and after an overnight abstinence from cigarette smoking/nicotine product use. 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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Clinic Visits 
Out-of-CRU 
Between 
Clinic Visits 
 
 
STUDY EVENTS/ASSESSMENTS 
Screening1 
Start of 
Week -1  
End of 
Week -1 
(baseline
) 
End of 
Week 2 
End of 
Week 4 
End of 
Week 6 
Weeks -1 to 
6 
End of 
study2 
Follow-up 
Call3 
Perceived health risk scale 
 
 
X 
X 
X 
X 
 
 
 
Confinement to CRU for 24 hours 
 
 
X 
X 
 
X 
 
 
 
Ambulatory Visits to CRU 
X 
X 
 
 
X 
 
X 
 
 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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ABBREVIATIONS 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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CA24914_FINALV1_09MAY2018 
 
 
1-OHP 
1-hydroxypyrene 
3-HPMA 
3-hydroxypropylmercapturic acid 
AE 
Adverse event 
ANPRM 
Advanced Notice of Proposed Rule Making  
AUC 
Area under the concentration curve 
BMI 
Body mass index 
BoE 
Biomarker of exposure 
bpm 
Beats per minute 
C 
Degree Celsius 
CDMS 
Clinical Data Management System  
CFR 
Code of Federal Regulations 
CPD 
Cigarettes per day 
Cmax 
Maximum plasma concentration 
CO 
Carbon monoxide 
COHb 
Carboxyhemoglobin 
CRF 
Case report form 
CRU 
Clinical research unit 
CV 
Coefficient of variability 
CYP 
Cytochrome P450 
e-diary 
Electronic diary 
ECG 
Electrocardiogram 
eCO 
Exhaled carbon monoxide 
eCRF 
Electronic case report form 
EDC 
Electronic data capture 
F 
Degree Fahrenheit 
FDA 
Food and Drug Administration 
FSH 
Follicle-stimulating hormone  
FTCD 
Fagerström Test for Cigarette Dependence 
GCP 
Good Clinical Practice 
GLP 
Good Laboratory Practice 
HbsAg 
Hepatitis B surface antigen 
HCV 
Hepatitis C virus 

VLN Cigarette Consumption Study 
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Celerion Project No.: CA24914 
 
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HIV 
Human immunodeficiency virus 
ICF 
Informed consent form 
ICH 
International Conference on Harmonization 
IRB 
Institutional Review Board 
ISO 
International Organization for Standardization 
ITT 
Intent to treat 
K2EDTA 
Dipotassium ethylenediaminetetraacetic acid 
MedDRA 
Medical Dictionary for Regulatory Activities 
mmHg 
Millimeters of mercury 
MNWS-R 
Minnesota Nicotine Withdrawal Scale - Revised 
n 
Sample size 
NNAL 
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol 
NNN 
total N-Nitrosonornicotine 
NNK 
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone 
PK 
Pharmacokinetic 
PP 
Per protocol 
ppm 
Parts per million 
QSU-brief 
Brief Questionnaire of Smoking Urges 
RCF 
Relative centrifugal force 
SAE 
Serious adverse event 
SAP 
Statistical analysis plan 
SD 
Standard deviation 
SEM 
Standard error of the mean 
SOP 
Standard operating procedure 
S-PMA 
S-phenylmercapturic acid 
Tmax 
Time to maximum plasma concentration 
Tneq 
Total nicotine equivalents 
UB 
Usual brand  
US 
United States of America 
VLN 
Very low nicotine 

VLN Cigarette Consumption Study 
22nd Century Group, Inc 
Celerion Project No.: CA24914 
 
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TABLE OF CONTENTS 
PROTOCOL HISTORY .................................................................................................................... 2 
SIGNATURE PAGE ......................................................................................................................... 3 
STUDY CONTACTS ........................................................................................................................ 4 
SYNOPSIS ....................................................................................................................................... 5 
SUMMARY OF STUDY EVENTS .................................................................................................... 9 
ABBREVIATIONS .......................................................................................................................... 13 
TABLE OF CONTENTS ................................................................................................................. 16 
1. 
INTRODUCTION AND BACKGROUND ........................................................................... 19 
1.1 
Background .......................................................................................................... 19 
1.2 
Study Purpose...................................................................................................... 19 
1.3 
Hypothesis ........................................................................................................... 19 
2. 
STUDY OBJECTIVES....................................................................................................... 20 
2.1 
Design and Procedures ....................................................................................... 20 
2.2 
Study Endpoints ................................................................................................... 21 
3. 
SUBJECT SELECTION .................................................................................................... 22 
3.1 
Inclusion Criteria .................................................................................................. 22 
3.2 
Exclusion Criteria ................................................................................................. 23 
3.3 
Study Restrictions ................................................................................................ 24 
3.3.1 
Concomitant Medications .................................................................... 24 
3.3.2 
Foods and Beverages .......................................................................... 25 
3.3.3 
Activity.................................................................................................. 25 
3.4 
Subject Numbering .............................................................................................. 25 
3.5 
Subject Early Discontinuation or Withdrawal ....................................................... 25 
4. 
STUDY PRODUCTS/MATERIALS ................................................................................... 26 
4.1 
Description of Study Products .............................................................................. 26 
4.2 
Study Product Accountability ............................................................................... 26 
4.3 
Labelling ............................................................................................................... 27 
4.4 
Blinding ................................................................................................................ 27 
4.5 
Randomization ..................................................................................................... 27 
4.6 
Product Handling.................................................................................................. 27 
5. 
STUDY PROCEDURES ................................................................................................... 28 
5.1 
Screening ............................................................................................................. 28 
5.2 
Baseline Visit (Start of Week -1) .......................................................................... 28 
5.3 
Ambulatory Periods Between Clinic Visits ........................................................... 29 
5.4 
Clinic Visits (End of Weeks -1, 2 and 6) .............................................................. 29 
5.5 
Clinic Visit (End of Week 4) ................................................................................. 30 
5.6 
End-of-Study (End of Week 6 Visit or on Early Discontinuation) ......................... 30 
5.7 
Follow-Up Call ...................................................................................................... 30 
5.8 
Confinement ......................................................................................................... 31 
5.9 
Tobacco Product Use .......................................................................................... 31 
5.10 
Product Trial ......................................................................................................... 31 
5.11 
Meals .................................................................................................................... 31 
5.12 
Study Assessments ............................................................................................. 31 
5.12.1 
Medical History/Demographic Data/Record of Concomitant 
Medication ........................................................................................... 31 
5.12.2 
Tobacco/Nicotine Product Use History ................................................ 32 

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5.12.3 
Urine collections for BoE Assessments ............................................... 32 
5.12.4 
Blood sampling for BoE assessments ................................................. 33 
5.12.5 
Puffing topography measurements ...................................................... 33 
5.12.6 
Blood sampling for nicotine PK assessments ..................................... 34 
5.12.7 
Product use recording .......................................................................... 35 
5.12.7.1 
Electronic diary for cigarette consumption .................... 35 
5.12.7.2 
Collection of used cigarette butts .................................. 35 
5.12.8 
Subjective measures ........................................................................... 35 
5.12.8.1 
Fagerström Test for Cigarette Dependence 
Questionnaire (FTCD)................................................... 35 
5.12.8.2 
Brief Questionnaire of Smoking Urges (QSU-
Brief).............................................................................. 35 
5.12.8.3 
Minnesota Nicotine Withdrawal Scale - Revised 
(MNWS-R) .................................................................... 35 
5.12.8.4 
Perceived health risk scale ........................................... 36 
5.12.9 
Exhaled Carbon Monoxide .................................................................. 36 
5.12.10 
Safety Assessments ............................................................................ 36 
5.12.10.1 
Physical Examination .................................................... 36 
5.12.10.2 
Height, Body Weight, and BMI ...................................... 36 
5.12.10.3 
Clinical Laboratory ........................................................ 36 
5.12.10.4 
Vital Signs ..................................................................... 37 
5.12.10.5 
Electrocardiogram ......................................................... 38 
5.12.11 
Adverse Events .................................................................................... 38 
5.12.11.1 
Monitoring ..................................................................... 38 
5.12.11.2 
Reporting ...................................................................... 38 
5.12.11.3 
Serious Adverse Events................................................ 38 
5.12.11.4 
Pregnancy ..................................................................... 39 
5.13 
Smoking Cessation Information ........................................................................... 39 
6. 
DATA ANALYSIS .............................................................................................................. 39 
6.1 
Sample Size Estimation ....................................................................................... 40 
6.1.1 
Cigarette consumption ......................................................................... 40 
6.1.2 
Puffing topography ............................................................................... 40 
6.2 
Analysis Populations ............................................................................................ 40 
6.3 
Data Analysis and Summarization ....................................................................... 41 
6.3.1 
Cigarette Consumption ........................................................................ 41 
6.3.2 
Urine Biomarkers of Exposure ............................................................. 41 
6.3.3 
Blood Biomarkers of Exposure ............................................................ 42 
6.3.4 
Nicotine pharmacokinetics ................................................................... 42 
6.3.5 
Puffing topography ............................................................................... 43 
6.3.6 
Subjective Effects Questionnaires ....................................................... 43 
6.3.7 
Statistical Methods for the Primary Endpoints ..................................... 43 
6.3.8 
Statistical Methods for the Secondary Endpoints ................................ 43 
6.4 
Safety ................................................................................................................... 43 
7. 
STUDY ADMINISTRATION .............................................................................................. 44 
7.1 
Ethics ................................................................................................................... 44 
7.1.1 
Ethical Conduct of the Study ............................................................... 44 
7.1.2 
Subject Information and Consent ........................................................ 44 
7.2 
Termination of the Study ...................................................................................... 44 
7.3 
Monitoring ............................................................................................................ 44 

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7.4 
Data Quality Assurance ....................................................................................... 45 
7.5 
Direct Access to Source Data/Documents ........................................................... 45 
7.6 
Data Handling and Record Keeping .................................................................... 45 
7.7 
Publication Policy ................................................................................................. 46 
8. 
DATA MANAGEMENT ...................................................................................................... 46 
8.1 
Database Design and Creation ............................................................................ 46 
8.2 
Data Coding ......................................................................................................... 46 
8.3 
Data Entry and Verification .................................................................................. 46 
8.4 
Study Results Data Transfer ................................................................................ 47 
8.5 
Data Validation ..................................................................................................... 47 
8.6 
Database Lock ..................................................................................................... 47 
9. 
REPORTING ..................................................................................................................... 47 
9.1 
Case Report Forms .............................................................................................. 47 
9.2 
Study Report ........................................................................................................ 47 
10. 
GENERAL ......................................................................................................................... 48 
10.1 
Confidentiality ....................................................................................................... 48 
10.2 
Responsibility of the Investigator ......................................................................... 48 
10.3 
Procedure for Amendments to Protocol ............................................................... 48 
10.4 
Institutional Review Board ................................................................................... 49 
10.5 
Study Record Retention ....................................................................................... 49 
11. 
REFERENCES .................................................................................................................. 50 
APPENDIX 1. 
Fagerström Test For Cigarette Dependence (FTCD) .......................... 51 
APPENDIX 2: 
Brief Questionnaire of Smoking Urges (QSU-Brief) ............................ 52 
APPENDIX 3: 
Minnesota Nicotine Withdrawal Scale - Revised (MNWS-R) .............. 54 
APPENDIX 4: 
Perceived Health Risk Scale ............................................................... 55 
 

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1. INTRODUCTION AND BACKGROUND 
1.1 
Background 
A gradual reduction of nicotine levels in cigarettes was first proposed by Benowitz and 
Henningfield in 1994 (1). In this paper, the authors proposed a long-term (over many 
years) reduction in cigarette nicotine levels as a measure to reduce smoking addiction and 
to aid cessation for smokers. The underlying assumption of this proposal was that there 
is a nicotine exposure threshold, below which reinforcing effects of nicotine are insufficient 
to cause or sustain addiction. The absolute level of such a threshold has not yet been 
determined. Hatsukami et al (2) published results of a study where subjects were switched 
to VLN cigarettes for six weeks. Compensatory smoking behavior, biomarkers of exposure 
(BoE), and tobacco dependence withdrawal were assessed. VLN cigarettes did not result 
in compensatory smoking behavior. Cigarettes per day and biomarkers of exposure were 
reduced after 6 weeks. VLN cigarettes were associated with a greater relief of withdrawal 
than nicotine lozenges.  
In March 2018, the Food and Drug Administration (FDA) announced through an Advanced 
Notice of Proposed Rule Making (ANPRM) that a tobacco standard for lowering nicotine 
in cigarettes is being considered (3). As new cigarettes with reduced amounts of nicotine 
in the tobacco blend are developed, clinical studies are required to assess whether they 
lead to reductions in cigarette consumption and whether this is associated with reductions 
in biomarkers of exposure to cigarette smoke toxicants and changes in cigarette 
dependence. Furthermore, subjective assessments of urges to smoke, withdrawal 
symptoms and perceived health risks are also useful indicators of whether low nicotine 
cigarettes will give rise to reduced addiction to cigarette smoking.  
 
1.2 
 Study Purpose 
The purpose of this study is to examine whether measures of cigarette consumption 
behavior (cigarettes per day and smoking topography) are changed when smokers switch 
from smoking conventional cigarettes to smoking cigarettes with a very low nicotine (VLN) 
content (0.4 mg nicotine per gram of tobacco). A secondary purpose is to examine 
changes in BoE, nicotine pharmacokinetics and subjective measures of dependence, 
smoking urges, withdrawal symptoms and perceived health risks, when subjects switch to 
smoking VLN cigarettes.  
 
1.3 
Hypothesis 
It is anticipated that when adult smokers switch to smoking VLN cigarettes, after habitually 
consuming their usual quantity and brand of cigarettes, their daily cigarette consumption 
will be reduced. It is further anticipated that puffing topography parameters will be 
unchanged following smoking VLN cigarettes for 6 weeks i.e. no smoking compensation 
will occur.   
 
 

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2. STUDY OBJECTIVES 
The primary objectives of this study are: 
1. To characterize cigarette consumption behavior (cigarettes per day and puffing 
topography) before, during and after a switch from UB to VLN cigarettes for 
6 weeks.  
The secondary objectives of this study are: 
1. To evaluate changes in tobacco-related BoE before, during and after a 6-week 
switch from UB to VLN cigarettes.  
2. To evaluate nicotine pharmacokinetics before, during and after a 6-week switch 
from UB to VLN cigarettes. 
3. To evaluate changes in subjective effects before, during and after a 6-week switch 
from UB cigarettes to VLN cigarettes.  
 
2.1 
 Design and Procedures 
This is an open-label, non-randomized, forced-switching study to be conducted at multiple 
study sites. Seventy (70) self-affirmed exclusive filtered king size non-mentholated 
cigarette smokers and 70 self-affirmed exclusive filtered king size mentholated cigarette 
smokers will be enrolled and begin the study at Week -1. 
All potential subjects will provide informed consent and successfully complete the 
Screening procedures prior to participation in the study. Subjects will also engage in a 
brief product trial with the VLN cigarettes. Subjects who react negatively (i.e., unwilling to 
use and/or cannot tolerate the product [experience adverse events (AEs) that will prevent 
them from continuing to use the product as judged by the Investigator]) to the VLN 
cigarettes during the product trial period will not continue in the study.  
At the start of Week -1, all subjects will be asked to smoke their UB cigarettes as per their 
usual daily consumption for the following week. Subjects will receive an electronic diary 
(e-diary) to record daily cigarette use (cigarettes per day [CPD]). Training in completion of 
the e-diary will be provided at the visit at the start of Week -1.  
Subjects will return at the end of Week -1, at the time indicated by the clinical research 
unit (CRU), for collection of blood and 24-hour urine samples for baseline BoE 
assessments. Subjective questionnaires for dependence, withdrawal symptoms, urges to 
smoke, and perceived health risk will also be completed at scheduled times. A subset of 
18 non-menthol and 18 menthol smoker subjects will complete an assessment of puffing 
topography with their UB cigarettes during this visit. A further subset of 12 of the non-
menthol and 12 of the menthol smoker subjects who complete the topography assessment 
will also complete a nicotine PK assessment at the end of this visit. Subjects who undergo 
topography and PK assessments will be assigned to switch to smoking VLN cigarettes.  
On Day 1 of Week 1, subjects will be randomly selected to either remain smoking their 
non-menthol (20 subjects) or menthol (20 subjects) UB cigarettes, or to switch to smoking 
non-menthol (50 subjects) or menthol (50 subjects) VLN cigarettes as per their UB 
cigarette flavor. Subjects will return at the end of Weeks 2 and 6, at the time indicated by 
the CRU, for collection of blood and 24-hour urine samples for BoE assessments. 
Subjective effects questionnaires will also be completed at scheduled times. Subjects will 
continue recording their CPD in their e-diaries. A subset of 18 non-menthol and 18 menthol 
smoker subjects will complete an assessment of puffing topography with the VLN 

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cigarettes at these visits, and a further subset of 12 non-menthol and 12 menthol smoker 
subjects will also complete an assessment of nicotine PK at the end of these visits. 
Subjects undergoing topography and PK assessments will have been assigned to switch 
to smoking VLN cigarettes. 
Additionally, all subjects will visit the clinic at the end of Week 4 to receive further supplies 
of cigarettes (if assigned to the VLN groups) and to complete subjective effects 
questionnaires.  
Subjects randomized to the VLN groups will be provided with a supply of VLN cigarettes 
at each visit, which will be 150 % of their usual daily consumption as reported during 
Week -1. If a subject runs out of cigarettes between clinic visits, they may visit the clinic 
to receive additional test cigarettes. All subjects will be asked to smoke their cigarettes ad 
libitum, recording their actual daily consumption in their e-diaries. Non-compliant nicotine 
product consumption should also be recorded. Used cigarette butts will also be collected 
during ambulatory periods to verify product use and/or assess compliance. During 
Week -1 (all subjects) and all subsequent weeks (subjects randomized to continue 
smoking UB cigarettes) subjects will be asked not to change their UB cigarette brand or 
flavor.  
The CRU will attempt to contact all subjects who participated in the study (including 
subjects who terminate the study early) using their standard procedures approximately 
7 days after the last contact to determine if any AE has occurred since the last study visit.  
 
2.2 
Study Endpoints 
Product use endpoints are: 
• 
Cigarettes smoked per day (CPD). Both compliant and non-compliant smoking 
should be captured in the e-diaries. For a definition of non-compliant smoking, see 
Section 5.12.7.1.  
• 
Number of collected used cigarette butts (apparent compliant, apparent non-
compliant, and total). 
• 
Puffing topography parameters (puff duration, puff volume, peak puff flow rate, 
average flow rate and inter-puff interval).  
 
Nicotine PK endpoints are: 
• 
Cmax 
• 
Tmax 
• 
AUC 
 
BoE endpoints are: 
• 
In urine  
o Total NNAL (NNK biomarker) 
o Total NNN 
o 3-HPMA (acrolein biomarker) 
o S-PMA (benzene biomarker) 

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o 1-OHP (hydroxypyrene biomarker) 
o Total nicotine equivalents (Tneq) 
o Creatinine concentration (for normalization of urinary BoE endpoints) 
• 
Carboxyhemoglobin in blood  
• 
Cotinine in plasma 
 
Subjective effects endpoints are:  
• 
Fagerström Test for Cigarette Dependence (FTCD) 
• 
Brief Questionnaire of Smoking Urges (QSU-Brief) 
• 
Minnesota Nicotine Withdrawal Scale - Revised (MNWS-R) 
• 
Perceived health risk scale  
 
Safety endpoints include: 
• Adverse events (AEs) 
 
3. SUBJECT SELECTION 
3.1 
Inclusion Criteria 
Potential subjects must fulfill all of the following inclusion criteria to be eligible for 
participation in the study. 
1. Is a healthy adult male or female adult smoker, 26 to 65 years of age, inclusive, at 
Screening. 
2. Has been a smoker for at least 5 years prior to Screening. Brief periods of 
non-smoking (e.g., up to 7 consecutive days due to illness, trying to quit, participation 
in a study where smoking was prohibited) during that time will be permitted at the 
discretion of the Investigator. 
3. Reports smoking an average of 10 or more manufactured combustible cigarettes per 
day at Screening.  
4. Usual brand (UB) of cigarette is a filtered king size cigarette (with an approximate 
length of 84 mm (± 3 mm)).  
5. Has a positive urine cotinine (≥ 500 ng/ml) at Screening. 
6. Has an exhaled CO > 10 ppm at Screening. 
7. If female, has a negative serum pregnancy test at Screening. 
8. A female subject of childbearing potential must have been using one of the following 
forms of contraception and agree to continue using it through completion of the study: 
• 
hormonal (e.g., oral, vaginal ring, transdermal patch, implant, or injection) 
consistently for at least 3 months prior to Screening; 
• 
double barrier method (e.g., condom with spermicide, diaphragm with spermicide) 
consistently for at least 14 days prior to Screening; 
• 
intrauterine device for at least 3 months prior to Screening;  
• 
Essure® or similar nonsurgical sterilization procedure at least 6 months prior to 
Screening 

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• 
a partner who has been vasectomized for at least 6 months prior to Screening; 
• 
abstinence beginning at least 14 days prior to Screening and through the End of 
Study.  
9. A female subject of non-childbearing potential must have undergone one of the 
following sterilization procedures at least 6 months prior to Screening: 
• 
hysteroscopic sterilization; 
• 
bilateral tubal ligation or bilateral salpingectomy; 
• 
hysterectomy; 
• 
bilateral oophorectomy;  
Or be postmenopausal with amenorrhea for at least 1 year prior to Day 1 and 
follicle-stimulating hormone (FSH) levels consistent with postmenopausal status. 
10. Willing to comply with the requirements of the study, including a willingness to use 
the test products. 
11. Voluntary consent to participate in this study documented on the signed informed 
consent form (ICF). 
12. Subject is not planning to leave the area during the course of the study. 
 
3.2 
Exclusion Criteria 
Subjects may be excluded from the study if there is evidence of any of the following criteria 
at Screening, start of Week -1 (first clinic visit), or during the study as noted, in the opinion 
of the Investigator. 
1. History or presence of clinically significant gastrointestinal, renal, hepatic, 
neurologic, hematologic, endocrine, oncologic, urologic, pulmonary (especially 
bronchospastic diseases and asthma), immunologic, psychiatric, or cardiovascular 
disease, or any other condition that, in the opinion of the Investigator, would 
jeopardize the safety of the subject or impact the validity of the study results. 
2. Clinically significant abnormal findings on the physical examination, medical 
history, ECG, or clinical laboratory results, in the opinion of the Investigator. 
3. Positive test for human immunodeficiency virus (HIV), hepatitis B surface antigen 
(HBsAg), or hepatitis C virus (HCV). 
4. An acute illness (e.g., upper respiratory infection, viral infection) requiring 
treatment with prescription medication(s) within 14 days prior to Screening or first 
clinic visit.  
5. Fever (>100.5F) at Screening or first clinic visit. 
6. Body mass index (BMI) greater than 40.0 kg/m2 or less than 18.0 kg/m2 at 
Screening. 
7. History of drug or alcohol abuse or has used medical/recreational marijuana within 
12 months of Screening. 
8. Diabetes mellitus that is not controlled by diet/exercise alone, in the opinion of the 
Investigator. 
9. Seated heart rate is lower than 40 bpm or higher than 99 bpm at Screening, unless 
deemed not clinically significant by the PI.  

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10. Seated systolic blood pressure <90 mmHg or >150 mmHg, diastolic blood 
pressure <40 mmHg or >95 mmHg at Screening, unless deemed not clinically 
significant by the PI. 
11. Positive urine screen for drugs of abuse or alcohol at Screening or at the first clinic 
visit. 
12. Female subjects who are pregnant, lactating, or intend to become pregnant from 
Screening through the End of Study.  
13. Use of medications known to interact with cytochrome p450 2A6 (including, but not 
limited to, amiodarone, desipramine, isoniazid, ketoconazole, miconazole, 
phenobarbital, rifampin, tranylcypromine, methoxsalen) within 3 months prior to 
Screening and throughout the study.  
14. Use of inhalers to treat any medical condition within 3 months prior to Screening 
and throughout the study. 
15. Use of nicotine-containing products other than factory manufactured cigarettes 
[e.g., roll-your-own cigarettes, e-vapor products, bidis, snuff, nicotine inhaler, pipe, 
cigar, chewing tobacco, nicotine patch, nicotine spray, nicotine lozenge, or nicotine 
gum] within 28 days prior to Screening. 
16. Use of any prescription smoking cessation treatments, including, but not limited to, 
varenicline (Chantix®) or buproprion (Zyban®) within 3 months prior to Screening. 
17. Self-reported puffer/non-inhaler (i.e., a smoker who draws smoke from the 
cigarette into the mouth and throat but does not inhale). 
18. Planning to quit smoking during the study period or postponing a quit attempt in 
order to participate in the study. 
19. Plasma donation within 7 days prior to Screening or at any time during the study.  
20. Donation of blood or blood products (with the exception of plasma as noted above), 
had significant blood loss, or received whole blood or a blood product transfusion 
within 56 days prior to Screening. 
21. Participation in a previous clinical study for an investigational drug, device, 
biologic, or tobacco product within 30 days prior to Screening. 
22. Subject or a first-degree relative (i.e., parent, sibling, child) is a current or former 
employee of the tobacco industry or a named party or class representative in 
litigation with the tobacco industry. 
23. Subject or a first-degree relative (i.e., parent, sibling, child) is a current employee 
of the clinic sites. 
 
3.3 
Study Restrictions 
3.3.1 Concomitant Medications 
Except for those medications noted in the exclusion criteria (See Section 3.2), stable 
doses (i.e., no dosage adjustments within approximately 30 days prior to Check-in) of 
prescription or over-the-counter medications required to treat an Investigator-approved 
disease or condition (e.g., hypertension) are permitted at the discretion of the Investigator. 
Hormonal contraceptives (e.g., oral, vaginal ring, transdermal patch, implant, injection) 
and hormonal replacement therapy are permitted. Occasional use of over-the-counter 
analgesics (e.g., acetaminophen, ibuprofen), antihistamines, nasal decongestants, and 

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dietary supplements are permitted. Exceptions may be permitted at the discretion of the 
Investigator in consultation with the Sponsor, providing the medication in question would 
have no impact on the study. Any exceptions will be documented. 
All concomitant medications (and reasons for their use) taken by subjects during the study 
will be recorded and listed.  
 
3.3.2 Foods and Beverages 
Consumption of foods and beverages containing the following substances should not be 
consumed as indicated below. 
• 
Xanthines/caffeine: throughout each clinic visit, apart from one approximately 250ml 
caffeinated beverage with each meal.  
• 
Alcohol: 48 hours prior to and throughout each clinic visit. 
Exceptions may be permitted at the discretion of the Investigator. 
 
3.3.3 Activity 
Subjects will be instructed to refrain from strenuous physical activity outside of their usual 
routine for the whole study period between Screening and the end of Week 6.  
 
3.4 
Subject Numbering 
Subjects will be assigned a unique screening number. Upon enrollment into the study, a 
definitive subject number will be assigned. Replacement subjects may be used at the 
discretion of the Sponsor.  
 
3.5 
Subject Early Discontinuation or Withdrawal 
Subjects will be advised that they are free to withdraw from the study at any time. In 
addition, subject participation in this trial may be discontinued for any of the following 
reasons. 
• Adverse event 
• Lost to follow-up 
• Non-compliance with study procedures 
• Protocol violation 
• Pregnancy for female subjects 
• Study terminated by Sponsor, US Food and Drug Administration (FDA), or other 
regulatory authorities 
• Withdrawal of consent 
• Investigator’s discretion, including a severe laboratory abnormality or other 
medical condition that indicates to the Investigator that continued participation is 
not in the best interest of the subject 
 

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Subject’s e-diary completion rates will be checked at each study visit. If the percentage of 
days on which the e-diary was completed is below 70%, subjects may be withdrawn from 
the study.  
Other protocol deviations/violations should not lead to subject withdrawal unless they 
indicate a significant risk to the subject’s safety or jeopardize the scientific integrity of the 
study. 
If premature withdrawal from the study occurs for any reason, the Investigator must 
determine the primary reason and record this information in the case report form (CRF). 
Additionally, subjects withdrawing after study product use will undergo all end-of-study 
safety procedures as feasible and as deemed necessary by the Investigator. 
A subject withdrawn from the study due to any AE or clinically significant abnormal 
laboratory test values will be evaluated by the Investigator or other monitoring physician 
and will be treated and/or followed up until the symptoms or values return to normal or 
acceptable levels or until lost to follow-up, as appropriate in the opinion of the Investigator. 
Subjects withdrawing or removed from this study cannot re-enter. 
 
4. STUDY PRODUCTS/MATERIALS 
4.1 
Description of Study Products 
The following products will be used during this study. 
• 
Subjects UB non-mentholated filtered king size cigarettes sourced by the subjects 
• 
Subjects UB mentholated filtered king size cigarettes sourced by the subjects 
• 
Non-mentholated very low nicotine (VLN) cigarettes sourced by 22nd Century 
Group, Inc 
• 
Mentholated VLN cigarettes sourced by 22nd Century Group, Inc 
• 
 
VLN cigarettes contain 0.4 mg nicotine per gram of tobacco. Subjects will be provided with 
a supply of VLN cigarettes, up to 150 % of their usual daily consumption as reported during 
Week -1.  
 
4.2 
Study Product Accountability 
Usual brand (UB) cigarettes will be provided by the study subjects. All VLN study products 
will be provided by the Sponsor. The pharmacy staff at the site will coordinate receipt of 
products from the subjects and the Sponsor. The pharmacy will document the date on 
which products were received and record this in the inventory records. The site pharmacy 
will document and reconcile the total number of products received at the site, the total 
number of products dispensed during the study, and the total number of products 
remaining at the end of clinical conduct.  
Subjects will be instructed to not bring any tobacco- or nicotine-containing products into 
the clinic, other than UB cigarettes at the end of Week -1 visit or VLN cigarettes at the end 
of Weeks 2 and 6 visits. After their Week -1 visit, subjects assigned to the VLN groups will 
be requested only to smoke VLN cigarettes during all the clinic visits and the ambulatory 
periods.  

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All investigational products will be stored in locked, limited-access areas in the clinic site’s 
pharmacies at controlled room temperature (defined as 20˚-25˚C [68˚-77˚F], with 
excursions permitted to 15˚-30˚C [59˚-86˚F]). 
Individual study product dispensing records will be maintained by the site pharmacy for 
each subject. During clinic visits, used cigarette butts will be returned to the staff following 
each product use for accountability purposes and will be disposed of following 
accountability by the pharmacy staff. During ambulatory periods, used cigarette butts will 
be collected by subjects for the purposes of assessing cigarette consumption, checking 
compliance and study product accountability, and will be disposed of following 
accountability checking.  
Unused VLN cigarettes will be disposed of as directed by the Sponsor. Unused UB 
cigarettes may be returned to the study subjects at the end of the study, according to site 
policy.  All returns of products will be documented. Subjects will not return unused UB.  
 
4.3 
Labelling 
The containers housing the VLN cigarettes will be labelled to include the following 
information: 
• 
Study number  
• 
Product identifier 
• 
Administration requirements/handling directions 
• 
Storage conditions  
• 
Expiry date  
• 
Sponsor's name and address  
• 
Name of PI  
• 
“For Clinical Trials use only”  
• 
“Keep out of reach of children”  
 
4.4 
Blinding 
This is an open-label study.    
 
4.5 
Randomization 
A randomization scheme will be provided by Celerion for the clinical sites. A suitably 
qualified person will monitor the randomization to ensure the study groups are balanced 
in terms of age and gender, as far as practicable. Each study group will include no fewer 
than 10 subjects of either gender and no fewer than 10 subjects from each age group 
(≤ 40 years old and > 40 years old).  
 
 
4.6 
Product Handling 
While at the sites, products used during the study will be kept in their packages until use 
and stored under temperature controlled conditions (See Section 4.2). All VLN products 
will be securely stored at the clinical sites, in areas which are locked and with restricted 
access. No special procedures are required for the safe handling of the UB products.  

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During ambulatory periods, subjects will be asked to keep the study products at room 
temperature.  
The VLN cigarettes in this study do not have a specified shelf life. They were manufactured 
in March 2018.  
 
5. STUDY PROCEDURES 
5.1 
Screening 
Potential subjects will undergo Screening procedures to ensure that they meet the 
requirements for inclusion in the study within 28 days prior to their first study visit at the 
start of Week -1. 
Screening procedures will include the following: 
• 
Sign ICF 
• 
Demographics 
• 
Smoking/tobacco/nicotine product use history 
• 
UB cigarette and flavor documentation, including taking a color photocopy of the 
front and back of the subject’s UB cigarette pack along with a ruler 
• 
Medical history  
• 
Review of Concomitant Medications 
• 
Standard physical examination including oral cavity and oropharynx 
• 
Review of Inclusion/Exclusion criteria 
• 
Clinical laboratory tests (clinical chemistry, hematology, urinalysis) 
• 
HIV, HBsAg, and HCV serology 
• 
Urine drug and alcohol screens 
• 
Exhaled breath CO measurement 
• 
Urine cotinine screen 
• 
Vital signs 
• 
Height, body weight, and BMI 
• 
12-lead ECG 
• 
Serum pregnancy test for females 
• 
Serum FSH for postmenopausal females 
• 
Review of smoking and nicotine product restrictions 
• 
VLN cigarette trial 
• 
Begin AE recording after product trial 
• 
Provide smoking cessation information 
 
5.2 
Baseline Visit (Start of Week -1) 
Subjects will visit the clinic at the start of Week -1 at a time specified by the clinic. The 
following procedures will take place during this visit: 

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• 
Verification that inclusion and exclusion criteria have not been violated since 
Screening 
• 
UB cigarette documentation 
• 
Review of AEs and concomitant medication use 
• 
Urine drug and alcohol screens 
• 
Urine cotinine screen and eCO measurement 
• 
Vital signs 
• 
Symptom-driven physical examination, if applicable 
• 
Body weight 
• 
Serum pregnancy test for females 
• 
Training on e-diary use 
• 
Dispense canisters for used cigarette butt collection 
• 
Review smoking and nicotine product use restrictions 
 
5.3 
Ambulatory Periods Between Clinic Visits 
Subjects will be required to record their daily cigarette consumption in electronic diaries 
and to collect all used (both compliant and non-compliant) cigarette butts in the provided 
canisters. Subjects will receive reminder contact to return to the CRU. At each visit, 
concomitant medications and AEs will be reviewed. 
 
5.4 
Clinic Visits (End of Weeks -1, 2 and 6) 
Subjects will check in to the clinic at the end of Weeks, -1, 2 and 6 at times specified by 
the clinic. Subjects will be housed in the clinic until after the 24-hour urine collection and 
other study procedures. The following procedures will take place during this visit:  
• 
Verification that inclusion and exclusion criteria have not been violated since last visit 
• 
UB cigarette documentation for subjects assigned to continue smoking UB cigarettes 
• 
Urine drug and alcohol screens 
• 
Vital signs 
• 
Urine pregnancy test for females 
• 
Review of AEs and concomitant medication use. 
• 
Usual brand cigarette documentation (only at Week -1).  
• 
Return canister with used cigarette butts for counting 
• 
24-hour urine collection for BoE analysis 
• 
Plasma sample for cotinine analysis 
• 
Blood sample for carboxyhemoglobin (COHb) analysis  
• 
Daily recording of CPD in the e-diary. 
• 
Completion of subjective effects questionnaires  
• 
Puffing topography assessment (subset of subjects) 
• 
Nicotine PK assessment (subset of subjects) 

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• 
Dispense new canister for collecting used cigarette butts during next ambulatory 
period, except at Week 6 
• 
Review smoking and nicotine product use restrictions 
• 
Test product dispensing for the following ambulatory period; except at Week 6 
 
All subjects will have their personal belongings thoroughly checked at the start of these 
clinic visits.   
 
5.5 
Clinic Visit (End of Week 4) 
Subjects will visit the clinic at the end of Week 4 at a time specified by the clinic. The 
following procedures will take place during this visit:  
• 
Verification that inclusion and exclusion criteria have not been violated since last visit 
• 
UB cigarette documentation for subjects assigned to continue smoking UB cigarettes 
• 
Urine drug and alcohol screens 
• 
Vital signs 
• 
Urine pregnancy test for females 
• 
Review of AEs and concomitant medication use. 
• 
Return canister with used cigarette butts for counting 
• 
Completion of subjective effects questionnaires  
• 
Dispense new canister for collecting used cigarette butts during next ambulatory 
period 
• 
Review smoking and nicotine product use restrictions 
• 
Test product dispensing for the following ambulatory period 
 
5.6 
End-of-Study (End of Week 6 Visit or on Early Discontinuation) 
At the end of the study, the following procedures will be performed: 
• 
Review of AEs and concomitant medication use. 
• 
Vital signs 
• 
Body weight 
• 
12-lead ECG 
• 
Clinical chemistry, hematology and urinalysis 
• 
Symptom-driven physical examination, if applicable 
• 
Provide smoking cessation information  
 
5.7 
Follow-Up Call 
The CRU will attempt to contact all subjects who participated in the study (including 
subjects who terminate the study early) using their standard procedures approximately 
7 days after the last contact. This will be to: 
• 
Review AEs and concomitant medication use 

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• 
Provide smoking cessation information 
A symptom-driven physical examination may also be required, in which case subjects will 
return to the clinic for this to take place.  
 
5.8 
Confinement 
Subjects will check in to the clinic at the end of Weeks, -1, 2 and 6, at times specified by 
the clinic. Subjects will be housed until after the 24-hour urine collection, nicotine PK 
assessment, puffing topography and other study procedures.  
 
5.9 
Tobacco Product Use 
Except as required by the protocol, consumption of tobacco- or nicotine-containing 
products will not be permitted during the entire duration of the study from the start of Week 
-1 through discharge from the study. During the baseline period, subjects will be asked to 
confine their smoking to one brand which will be deemed their UB. While in the CRU during 
24-hour confinement periods, cigarette consumption will be tracked by the number of 
products dispensed.  
 
5.10 Product Trial 
At screening, subjects will have an opportunity to smoke 2-3 VLN cigarettes over a 1-hour 
period to ensure that they can tolerate smoking those cigarettes for a period of 6 weeks. 
The number of cigarettes smoked will be documented, as will any abnormal events (e.g., 
excessive coughing) during the product trial. Subjects who react negatively (i.e., unwilling 
to use and/or cannot tolerate the product [experience adverse events (AEs) that will 
prevent them from continuing to use the product as judged by the Investigator]) to the VLN 
cigarettes during the product trial period will not continue in the study. 
 
5.11 Meals 
Standard meals and snacks will be served at appropriate times as determined by the clinic 
during confinement. Meals each day should be provided at similar times. One 
approximately 250 ml, caffeinated beverage will be allowed to be taken with each meal. 
During confinement, water will be allowed ad libitum and subjects will be encouraged to 
maintain their usual hydration habits.   
 
5.12 Study Assessments 
All study assessments will take place at the times noted in the Summary of Study Events 
tables above unless otherwise noted below. 
 
5.12.1 Medical History/Demographic Data/Record of Concomitant Medication 
Self-reported medical history and socio-demographic data, including name, sex, age 
(each subject must show proof of age with government-issued identification [e.g., driver’s 
license or Verified Clinical Trials]), race, ethnicity, address, and phone number will be 
recorded at Screening for each subject.  

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Medical history will include any clinically significant neurological, gastrointestinal, renal, 
hepatic, cardiovascular, psychiatric, respiratory, metabolic, endocrine, hematological or 
other major disorders. Medical history is defined as any condition that started prior to 
Screening and ended prior to the product trial. A concomitant disease is any disease that 
started prior to, and which was still ongoing at Screening. 
Any concomitant medications taken from 30 days prior to Screening through discharge 
will be recorded in the CRF and source documents. 
 
5.12.2 Tobacco/Nicotine Product Use History 
Subjects will be required to report previous tobacco- and nicotine-product use histories to 
satisfy the study inclusion and exclusion criteria. In addition, the subject will be asked if 
he/she is planning to quit smoking within the next 3 months. Those planning to quit will be 
excluded from participating in the study.  
The subject’s tobacco use and smoking history (including a color photocopy of their UB 
cigarette package along with a ruler) will be recorded at Screening. Verification of the 
subject’s UB cigarette brand will be made at the end of Week -1 clinic visit. 
 
5.12.3 Urine collections for BoE Assessments 
Urine will be collected over 24-hour periods for urine BoE measurements, during the clinic 
visits at the end of Weeks, -1, 2 and 6. Creatinine in urine will also be measured and used 
to report BoE as amount per unit of creatinine.  
Prior to the first urine sample collection, each subject will be instructed as to the urine 
collection methods. All urine during an interval is to be collected and subjects will be asked 
to empty their bladder before the start of the urine collection period.  
The urine samples collected during a single 24-hour period will be pooled together and 
weighed. The pooled samples will be thoroughly mixed before providing aliquots for 
analyses. A separate document will be provided for handling and processing of the 
samples. 
The total weight of all urine collected over each 24-hour period will be documented. Urine 
aliquots will be prepared within 60 minutes from removal of the pooled collection from the 
refrigerator and then stored at approximately -20°C pending analysis, according to the 
table below: 
 
 
 
 
 
 
Analyte 
Volume per 
aliquot 
Number of 
aliquots 
Total NNAL 
10ml 
2 
Total NNN 
10ml 
2 
Tneq 
10ml 
2 

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5.12.4 Blood sampling for BoE assessments 
Blood samples for COHb in whole blood and cotinine in plasma will be collected via direct 
venipuncture at the scheduled visits as delineated in the Summary of Study Events 
Tables. Samples will be collected at approximately 13:00, following the subject’s lunchtime 
meal, and will be preceded by at least a 30-minute abstention from study product use.  
Samples for COHb will be collected into two 4 ml sodium heparin vacutainers. Immediately 
following collection, the blood samples will be inverted gently to mix between 8 and 10 
times. The whole blood samples will be stored at 5C ( 3C) until analyzed by the Celerion 
Bioanalytical Laboratory (Lincoln, Nebraska).  
Samples for plasma cotinine will be collected into a 4 ml plastic K2EDTA (lavender top) 
vacutainer tube. Immediately following the collection, blood samples will be gently mixed 
by inverting the tubes 8 times. The samples may be kept at ambient temperature prior to 
centrifuge, and will be centrifuged at 1000 - 1300 RCF for 10 minutes at 5C within 
60 minutes of collection. After centrifugation, approximately 1 ml of plasma will be 
transferred using into 2 appropriately labeled 3.5 ml polypropylene screw top tubes.  
Samples will then be stored at approximately -20C within 120 minutes of collection and 
until analyzed by Celerion Bioanalytical Services (Lincoln, Nebraska). Samples will be 
analyzed using a validated analytical method with the appropriate quality controls in 
accordance with FDA Good Laboratory Practice (GLP) regulations (Title 21 Code of 
Federal Regulations [CFR] Part 58). 
Approximately 36 ml of blood will be required for blood BoE assessments during the entire 
study. 
Additional instructions for collection, processing, and shipping of blood BoE samples will 
be provided separately.  
 
5.12.5 Puffing topography measurements 
At clinic visits at the end of Weeks -1, 2 and 6, a randomly-selected subset of 18 non-
menthol and 18 menthol smoker subjects will complete a puffing topography evaluation 
session with either their UB (Week -1) or the VLN (Weeks 2 and 6) cigarettes. These 
subjects will be/have been assigned to switch to smoking VLN cigarettes. These 
assessments will only be performed at a single clinical site (Lincoln) and will take place 
during the 24-hour confinement period.  
3-HPMA 
10ml 
2 
S-PMA 
10ml 
2 
1-OHP 
10ml 
2 
Creatinine 
10ml 
2 

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During the puffing topography assessment, subjects will engage in a 1-hour ad libitum 
smoking session with their UB (Week -1) or the VLN (Weeks 2 and 6) cigarettes. In these 
sessions, subjects will smoke cigarettes with the mobile smoking puff analyzer (SPA-M; 
Sodim). The topography device will be monitored to ensure the device is actively recording 
during each session.  
Topography assessment will be started at least one hour following either the subject’s 
breakfast or lunch. The puffing topography session for each subject at Weeks 2 and 6 
should be within  2 hours of the time of their Week -1 session. 
Additional details and instructions for puffing topography procedures will be provided 
separately. 
The following topography parameters will be assessed: 
• 
Puff duration 
• 
Puff volume 
• 
Peak puff flow rate 
• 
Average flow rate 
• 
Inter-puff interval 
 
5.12.6 Blood sampling for nicotine PK assessments 
At clinic visits at the end of Weeks -1, 2 and 6, a randomly-selected subset of 12 non-
menthol and 12 menthol smoker subjects will complete a nicotine pharmacokinetic 
assessment session with either their UB (Week -1) or the VLN (Weeks 2 and 6) cigarettes. 
These subjects will be/have been assigned to switch to smoking VLN cigarettes and will 
be selected from the subjects who undergo topography assessment. These assessments 
will only be performed at a single clinical site (Lincoln) and will take place on the morning 
following the 24-h confinement period.  
Following the completion of the 24-hour urine collection period, selected subjects will 
remain in the clinic for a further night, in which they will abstain from smoking any 
cigarettes/using any nicotine-containing products for at least 12 hours prior to the PK 
assessment session. During this session, subjects will smoke a single cigarette ad libitum 
during a 5-minute period. A 4 ml blood sample for plasma nicotine analysis will be drawn 
into a plastic K2-EDTA (lavender top) vacutainer tube at -5, 2, 5, 7, 10, 12, 15, 30, 45, 60, 
90, 120, 150 and 180 minutes relative to the start of cigarette smoking, as delineated in 
the Summary of Study Events Tables. In total, approximately 144 ml of blood will be drawn 
from each of the selected subjects during the entire study for nicotine pharmacokinetic 
analysis.  
The blood samples collected for plasma nicotine analysis may be kept at room 
temperature prior to centrifugation, and will be centrifuged at approximately 
1000-1300 RCF at ~5°C for approximately 10 minutes, within 60 minutes from collection. 
After centrifugation, the plasma will be transferred to two methanol prewashed 3.5 ml 
polypropylene screw cap tubes. Samples will then be stored at approximately -20C within 
120 minutes of collection and until analyzed by Celerion Bioanalytical Services (Lincoln, 
Nebraska). 
Samples will be analyzed using a validated liquid chromatography coupled to tandem 
mass spectrometry detection analytical method with the appropriate quality controls in 

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accordance with FDA Good Laboratory Practice (GLP) regulations (Title 21 Code of 
Federal Regulations [CFR] Part 58). 
Additional instructions for collection, processing, and shipping of samples for nicotine PK 
assessments will be provided separately.  
 
5.12.7 Product use recording 
5.12.7.1 Electronic diary for cigarette consumption 
Subjects will be provided with e-diaries to record their daily cigarette consumption, both 
when in clinical confinement during study visits and during ambulatory periods.  Subjects 
will be instructed to record both compliant and non-compliant (if any) daily numbers of 
cigarettes smoked.  Non-compliant consumption in the subjects randomized to smoke 
VLN cigarettes is defined as nicotine product consumption other than their UB cigarettes 
during the baseline period (Week -1) or VLN cigarettes following switching (Weeks 1 to 
6). Non-compliant consumption in the subjects randomized to continue to smoke their UB 
cigarettes is defined as nicotine product consumption other than their UB cigarettes 
between Week -1 and Week 6.  
For the purposes of data analysis, the e-diary record of cigarette consumption will be used 
as the primary variable.  
 
5.12.7.2 Collection of used cigarette butts 
Subjects will be provided with metal canisters in which to store their used cigarette butts 
during the ambulatory study periods. This is for the purposes of assessing both 
compliance with the study requirements and the accuracy of their self-reported (electronic 
diary) cigarette consumption.  Subjects will be requested to collect butts of all cigarettes 
smoked, both compliant and non-compliant.  
 
5.12.8 Subjective measures 
Questionnaires examining subjective effects will be completed using an electronic data 
collection system (Clinical Ink).  
 
5.12.8.1 Fagerström Test for Cigarette Dependence Questionnaire (FTCD) 
The FTCD (Appendix 1) will be completed at the end of each clinic visit, at the end of 
Weeks, -1, 2, 4 and 6, as delineated in the Summary of Study Events Tables.  
 
5.12.8.2 Brief Questionnaire of Smoking Urges (QSU-Brief) 
The QSU-Brief (Appendix 2) will be completed at the end of each clinic visit, at the end of 
Weeks, -1, 2, 4 and 6, as delineated in the Summary of Study Events Tables. 
 
5.12.8.3 Minnesota Nicotine Withdrawal Scale - Revised (MNWS-R) 
The MNWS-R (Appendix 3) will be completed at the end of each clinic visit, at the end of 
Weeks, -1, 2, 4 and 6, as delineated in the Summary of Study Events Tables. 
 

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5.12.8.4 Perceived health risk scale 
The perceived health risk scale (Appendix 4) will be completed at the end of each clinic 
visit, at the end of Weeks, -1, 2, 4 and 6, as delineated in the Summary of Study Events 
Tables. 
 
5.12.9 Exhaled Carbon Monoxide 
Exhaled CO levels will be measured using a Bedfont Micro+ Smokerlyzer, or similar 
device, as delineated in the Summary of Study Events Tables. 
 
5.12.10 
Safety Assessments 
Safety assessments in addition to those below may be obtained as necessary at the 
discretion of the Investigator. In the case of an early subject withdrawal, discharge safety 
assessments should be collected to the extent possible. 
 
5.12.10.1 
Physical Examination 
A standard physical examination (including oral cavity and oropharynx) assessing the 
general physical well-being will be completed at Screening by a qualified member of the 
clinic staff. A symptom-driven physical examination may be conducted at discharge (or 
upon early discontinuation), if required, as delineated in the Summary of Study Events 
Tables.  
 
5.12.10.2 Height, Body Weight, and BMI 
Body height will be measured in cm and body weight will be measured in kilograms. BMI 
will be recorded as kg/m2. 
 
5.12.10.3 Clinical Laboratory 
Collection and processing of samples for these purposes will be performed using standard 
clinical laboratory procedures at an approved laboratory or using a CLIA approved kit on 
site. Approximately 25 ml of blood will be required for clinical laboratory purposes. Values 
for the clinical laboratory parameters are to be within the laboratory normal ranges or 
deemed not clinically significant in the opinion of the Investigator.   
 

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Clinical Chemistry1 
Urinalysis2  
Additional Tests 
• 
Albumin 
• 
Alkaline phosphatase 
• 
Alanine 
aminotransferase 
• 
Aspartate 
aminotransferase 
• 
Bicarbonate 
• 
Blood urea nitrogen 
• 
Creatinine 
• 
Glucose (fasting) 
• 
Potassium 
• 
Sodium 
• 
Total bilirubin 
• 
Total protein 
• 
Uric acid  
 
Hematology 
• 
Hematocrit 
• 
Hemoglobin 
• 
Platelet count 
• 
Red blood cell count 
• 
White blood cell count 
with differential 
 
• 
Bilirubin 
• 
Blood 
• 
Glucose 
• 
Ketones 
• 
Leukocyte esterase 
• 
Nitrite 
• 
pH 
• 
Protein 
• 
Specific gravity 
• 
Urobilinogen 
• 
Serology 
o HIV 
o HBsAg 
o HCV 
• 
Serum pregnancy test3  
• 
Serum FSH4 
• 
Urine cotinine 
• 
Urine drug screen 
o Amphetamines 
o Cannabinoids 
o Cocaine 
o Opiates 
o Alcohol 
• 
Urine pregnancy test5 
 
1Clinical chemistry tests will be performed after at least an 8-hour fast; however, in case of dropouts or 
rechecks, subjects may not have fasted for 8 hours prior to the clinical chemistry sample being taken. 
2A microscopic examination for red blood cells, white blood cells, bacteria, and casts will be performed if an 
abnormality is noted in leukocyte esterase, protein, blood, or nitrite. 
3At Screening and baseline clinic visit (start of Week -1); females only.  
4For postmenopausal females; at Screening only.  
5At start of each clinic visit (end of Weeks -1, 2, 4 and 6); females only.  
 
 
5.12.10.4 Vital Signs 
Vital signs (respiratory rate, heart rate, blood pressure, and oral temperature) will be 
measured following a rest period of at least 10 minutes in a seated position, and at least 
15 minutes after the subject has smoked a cigarette. 
 

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5.12.10.5 Electrocardiogram 
12-lead ECGs will be taken following resting in the supine position for at least 10 minutes. 
ECGs will be interpreted, signed, and dated by the Investigator or a qualified designee. 
 
5.12.11 
Adverse Events 
An AE is any untoward medical occurrence associated with the use of the study product, 
whether or not considered study product-related. An AE can therefore be any unfavorable 
and unintended sign (including an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of a study product, whether or not related to the study 
product. 
 
5.12.11.1 Monitoring 
The subjects will be instructed to inform the study physician or staff of any AEs and 
intercurrent illnesses experienced during the study. Additionally, a specific inquiry 
regarding AEs will be conducted during clinic visits, at End-of-Study (or upon early 
discontinuation), or during the follow-up telephone call. The inquiry will be posed in a non-
specific manner using open-ended questions so as not to bias the response (e.g., How 
are you feeling today?). 
A subject who has any clinically significant AE or clinically significant abnormal laboratory 
test value will be evaluated by the Investigator or other monitoring physician and will be 
treated and/or followed up until the symptoms or values return to normal or acceptable 
levels (as appropriate in the opinion of the Investigator), or until the subject is lost to follow-
up. Where appropriate, medical tests and examinations will be performed to document 
resolution of the event(s). 
 
5.12.11.2 Reporting 
All AEs occurring during this clinical trial after the subject has used study products 
(including the product trial at Screening) must be recorded on the CRF, including the date 
and time of onset, action taken, outcome (resolved, improved, unchanged, worse, fatal, 
or unknown/lost to follow-up), duration, relationship to product administration, and severity 
for each event. AEs will be coded using most current version of the Medical Dictionary for 
Regulatory Activities (MedDRA®) available at Celerion. 
Events captured between Screening until the first product use (Product Trial) will be 
documented as baseline signs and symptoms. 
The Investigator will review each event and assess its relationship to product 
administration as unrelated, unlikely, possibly, probably, or likely. 
In addition, each sign or symptom reported will be graded on a 3-point severity scale using 
mild, moderate, or severe. 
 
5.12.11.3 Serious Adverse Events 
A serious adverse event (SAE) is any AE that in the view of either the Investigator (or 
designee) or Sponsor, results in any of the following outcomes: death, a life threatening 
AE, inpatient hospitalization or prolongation of existing hospitalization, a persistent or 
significant incapacity or substantial disruption of the ability to conduct normal life functions, 

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or a congenital anomaly/birth defect. Important medical events that may not result in 
death, be life threatening, or require hospitalization may be considered serious when, 
based upon appropriate medical judgment, they may jeopardize the subject and may 
require medical or surgical intervention to prevent one of the outcomes listed in the above 
definition. 
Life threatening is defined as an AE that in the view of the Investigator (or designee) or 
Sponsor, its occurrence places the subject at immediate risk of death. It does not include 
an AE that, had it occurred in a more severe form, might have caused death. 
Unexpected is defined as an AE that is not consistent with the known risk information 
associated with the study product. 
All SAEs, whether or not considered study-related, must be reported by telephone and by 
fax or e-mail to the Sponsor within 24 hours of the site’s learning of the SAE or, at the 
latest, on the following workday. The Sponsor’s representative to contact about this study 
is provided in the list of study contacts. The Investigator must also inform the IRB, in 
compliance with GCP reporting guidelines, and the site monitor of any SAE. 
 
5.12.11.4 Pregnancy 
A pregnancy occurring in a female study subject during the study will be documented as 
an unanticipated problem to the Institutional Review Board (IRB). Pregnancy itself is not 
a SAE. The Investigator or designee will discontinue the pregnant subject from the study 
and will advise her to seek prenatal care and counseling from her primary care provider. 
Advice given will be documented in the subject’s source document. 
The site clinical staff will request the pregnant subject to notify the site of the outcome of 
the pregnancy (i.e., birth, loss, or termination). To help ensure this, the site clinical staff 
will follow up with the subject until the end of pregnancy, if in compliance with the site’s 
standard operating procedures (SOPs) and with the subject’s consent. This request and 
the subject’s response will be documented in the subject’s source documents. 
 
5.13 Smoking Cessation Information 
At Screening and prior to discharge (or upon early withdrawal) all subjects will be advised 
that to reduce the health effects of smoking, the best thing to do is to quit. Subjects will be 
directed towards further information on the QuitAssist website (www.quitassist.com). 
 
6. DATA ANALYSIS 
Data will be handled and processed according to Celerion SOPs, which are written based 
on the principles of GCP. A brief description of the statistical analysis is included below; 
detailed methodology for all summary and statistical analyses of the data collected in this 
trial will be documented in a Statistical Analysis Plan (SAP) prepared by Celerion and 
agreed upon by the Sponsor. The SAP may modify the plans outlined in the protocol; 
however, any major modifications of the primary endpoints and/or their analysis will also 
be reflected in a protocol amendment. If deemed appropriate, additional statistical 
analyses other than those described in this section may be performed and included in the 
plan. 
 

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6.1 
Sample Size Estimation 
6.1.1 Cigarette consumption 
An intended sample size of 50 subjects in each of the two groups who switch from smoking 
usual brand to VLN cigarettes has been set for this study. This is based on powering the 
primary objective of a within-group comparison of cigarette consumption at baseline and 
end-of-study. The calculation was based on the number of pairs required to perform a two-
tailed paired t-test with 80% power and an alpha level of 0.05 for a decrease in cigarette 
consumption of 3 CPD compared to available historical consumption data (2, 4). The 
sample size was determined to be adequate based on potential non-compliance with the 
use of VLN cigarettes (2). ). A lower number (20) of smokers will be randomized to the 
continue-to-smoke usual brand groups, given the lower variability in consumption data 
and lower non-compliance anticipated in these groups. This will provide sufficient power 
for the secondary objective of between-group comparisons in cigarette consumption. 
 
6.1.2 Puffing topography 
An intended sample size of 12 subjects in each of the two groups who switch from smoking 
usual brand to VLN cigarettes has been set for this study. This is based on powering the 
primary objective of a within-group comparison of puffing topography parameters at 
baseline and end-of-study. The calculation was based on the number of pairs required to 
perform a two-tailed paired t-test with 80% power and an alpha level of 0.05 to detect 
significant differences between UB and VLN cigarettes and is based on available puffing 
topography data (5). This sample size will also provide sufficient power to detect 
differences in nicotine PK between UB and VLN cigarettes.  
 
6.2 
Analysis Populations 
The statistical analysis will be based on separate, hierarchically organised, analysis 
populations defined as the following: 
Safety population. All subjects who smoked at least one VLN cigarette 
Intent-to-treat (ITT) population. All subjects who had at least one valid recording of 
cigarette consumption (a period of one week of >70% completion of their e-diary).  
Per-protocol (PP) population. All subjects who had valid recording of cigarette 
consumption and completed the study according to the protocol. Data will be excluded 
from this population: 
• 
If a subject self-reports smoking a significant number of cigarettes other than those 
which they have been assigned according to the protocol, or if this is apparent 
following checking of their collected used cigarette butts 
• 
If there is a significant discrepancy between the self-reported number of cigarettes 
smoked per day and the number of used cigarette butts collected 
• 
Based on a published method of assessing compliance (6). After switching, 
subjects will be deemed non-compliant if their ratio of [plasma cotinine/CPD 
VLN]/[plasma cotinine/CPD baseline] exceeds 0.2.  
If it is determined that a subject is pregnant during the study, the pregnant subject’s data 
will be listed but excluded from all PK parameter summaries. 
 

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6.3 
Data Analysis and Summarization 
SAS software (version 9.3 or higher, Cary, North Carolina) will be used for all data 
presentation and summarization including statistical analyses, summary tables, graphs, 
and data listings. In general, all data will be listed by subject and time point and 
summarized by group and time point using descriptive statistics appropriate for the 
endpoint. Absolute and percent change from baseline values will also be listed and 
summarized where appropriate. Figures will be used to display the data graphically. 
Missing data will not be imputed. Where individual data points are missing because of 
dropouts or other reasons, the data will be considered missing at random and summarized 
based on reduced denominators.  
Within-group comparisons will be made for all groups for endpoints that have baseline 
(end of Week -1) and post-baseline measurements.   
 
6.3.1 Cigarette Consumption 
Study product use will be listed and summarized by subject using descriptive statistics 
(n, arithmetic mean, standard deviation (SD), coefficient of variation (CV%), standard 
error of the mean (SEM), minimum, median, and maximum). 
 
6.3.2 Urine Biomarkers of Exposure  
The urine biomarker data will be presented as follows for the assessments at the end of 
Weeks -1, 2 and 6: 
• 
Measured concentration  
• 
Total mass excreted per 24 hours  
• 
Total mass excreted per 24 hours absolute change from baseline  
• 
Total mass excreted per 24 hours percent change from baseline  
• 
Creatinine-normalized excretion level  
• 
Creatinine-normalized change from baseline  
• 
Creatinine-normalized percent change from baseline 
Note: the appropriate total mass excreted variables will be used in the statistical analyses. 
The amount excreted data will be reported for subjects who have an incomplete urine 
collection during a sampling interval but will not be included in the summary statistics or 
analysis for any of the urine biomarkers in that interval. Such subjects will be included in 
the summarization of the creatinine-normalized data. 
The measured urine biomarker concentrations will be converted to total mass excreted 
per 24 hours by multiplying the concentration by the total urine volume of the interval. All 
values reported as below the limit of quantitation (BLQ) will be set to one-half of the limit 
of quantitation (LOQ) for summarization and analysis. 
Nicotine equivalents will be calculated as the molar sum of nicotine, cotinine, 
trans-3’hydroxycotinine and their glucuronides excreted in urine over 24 hours: 
 
Nicotine  
= (free nicotine [mg] + nicotine gluc [mg]) + (free  

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Equivalents (mg)  
cotinine [mg] + cotinine gluc [mg]) + (free trans-3’-
hydroxycotinine [mg] + trans-3’-hydroxycotinine gluc [mg]) 
 
The concentration of each metabolite will first be adjusted by the 24-hour urine volume to 
obtain the total amount excreted in 24 hours, then divided by the molecular weight of the 
metabolite to obtain the total amount of each in moles. The sum in moles will then be 
converted to mass of nicotine equivalents by multiplying by the molecular weight of 
nicotine: 
 
Nicotine  
 
=  [nicotine (mg/162.23 [mg/mmol] + nicotine- 
equivalents (mg/24h)   
gluc (mg/338.36 [mg/mmol] + cotinine (mg/176.22 
[mg/mmol] + cotinine-gluc (mg/352.34 [mg/mmol] + 
trans-3'-hydroxycotinine (mg/192.22 [mg/mmol] + trans-
3'-hydroxycotinine-gluc 
(mg/368.34 
[mg/mmol]) 
x 
162.23 (mg/mmol) 
 
Urine creatinine will also be measured in each 24-hour collection and used to adjust the 
biomarker concentration values as appropriate according to the following formula: 
Biomarker 
 
= 
Biomarker (mass/ml) x 100 
(mass/mg creatinine)  
       creatinine (mg/dl) 
 
All data will be listed by subject and summarized by time point using descriptive statistics. 
Change from baseline will be calculated for each post-baseline time point. 
 
6.3.3 Blood Biomarkers of Exposure  
All data will be listed by subject and summarized by product and time point. Change from 
baseline will be calculated for each post-baseline time point.  
 
6.3.4 Nicotine pharmacokinetics 
Nicotine PK parameters will be determined from the individual plasma concentrations 
applying a non-compartmental approach using appropriate validated PK software (e.g., 
Phoenix® WinNonlin® version 6.3 or higher).  
For each study product, the following PK parameters will be calculated from the nicotine 
concentration-time data: 
• 
Cmax Maximum measured plasma concentration over the duration of the 
measurement interval. 
• 
Tmax Time to reach the maximum measured plasma concentration over the 
duration of the measurement interval. If the maximum value occurs at more than 
one time point, Tmax is defined as the first time point with this value.  
• 
AUC0-120 
Area under the nicotine concentration-time curve from time zero 
(defined as the start of product use) to 120 minutes (or the last quantifiable 
concentration during that interval) as calculated by the linear trapezoidal method. 

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Baseline adjustments will be performed. Concentration values below the lower limit of 
quantitation (LOQ) will be set to one-half of the LOQ for the calculation of the descriptive 
statistics of unadjusted plasma nicotine concentrations. Data will be listed by subject and 
summarized by product and time point using descriptive statistics. 
 
6.3.5 Puffing topography 
Data will be listed by subject and summarized by product and time point using descriptive 
statistics. 
 
6.3.6 Subjective Effects Questionnaires  
Data will be listed by subject and summarized by product and time point. Change from 
baseline will be calculated for each post-baseline time point. 
 
6.3.7 Statistical Methods for the Primary Endpoints 
Paired t-tests will be used to compare cigarette consumption (CPD) and puffing 
topography parameters at the Week 2 and 6 time points with the baseline (Week -1) 
values. This analysis will be conducted on the evaluable subjects in the PP population. If 
data are unexpectedly found not to be normally distributed, an appropriate non-parametric 
test will be performed. Differences will be considered statistically significant at an alpha 
level of 0.05. 
In addition, a linear mixed model analysis of variance will be used to compare the 
between-cohort differences in the absolute change from baseline values of the 
consumption and puffing topography parameters. 
 
6.3.8 Statistical Methods for the Secondary Endpoints 
Paired t-tests will be used to compare urinary NNAL, NNN, 3-HPMA, S-PMA, 1-OHP, Tneq 
mass excreted, blood COHb, nicotine PK parameters, and subjective effects measures at 
the Week 2 and 6 time points with the baseline (Week -1) values. This analysis will be 
conducted on the evaluable subjects in the PP population. If data are unexpectedly found 
not to be normally distributed, an appropriate non-parametric test will be performed. 
Differences will be considered statistically significant at an alpha level of 0.05. 
In addition, a linear mixed model analysis of variance will be used to compare the 
between-cohort differences in the absolute change from baseline values of each of the 
urinary and blood BoEs.   
 
 
6.4 
Safety 
All clinical safety data will be listed by subject and time point as appropriate. The following 
items will be summarized using n, arithmetic mean, SD, median, minimum, and maximum 
for continuous data and frequency counts for categorical data. 
• 
Subject disposition 
• 
Subject demographics 
• 
Tobacco product use history 

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• 
Vital signs 
All events captured in the database will be listed in by-subject data listings. However, only 
study product use-emergent AEs will be summarized. A study product use-emergent AE 
is defined as an AE that is starting or worsening at the time of or after the first study product 
use (i.e., Product Trial). 
Frequencies of subjects with study product use-emergent AEs, regardless of relationship 
to study product will be summarized and sorted by system organ class. Frequencies of 
subjects with study product use-emergent serious AEs will be likewise summarized. 
Frequencies of study product use-emergent AEs will be summarized by severity and 
relationship to study product. 
Changes in physical examinations (if any) will be described in the text of the final report. 
All concomitant medications recorded during the study will be listed by subject. 
 
7. STUDY ADMINISTRATION 
7.1 
Ethics 
7.1.1 Ethical Conduct of the Study 
This protocol will be reviewed by an IRB and the study will not start until the Board has 
approved the protocol or a modification thereof. The Board is constituted and operates in 
accordance with the principles and requirements described in the US Code of Federal 
Regulations (21 CFR Part 56). The Board is compliant with the International Conference 
on Harmonization (ICH). 
This research will be carried out in accordance with the protocol, the US CFR (21 CFR 
Parts 50, 56, and 312), the ICH harmonized tripartite guideline regarding GCP (E6 
Consolidated Guidance, April 1996), and the ethical princliples set forth in the Declaration 
of Helsinki. 
 
7.1.2 Subject Information and Consent 
All prospective subjects will have the study explained by the PI or his/her designee and 
will be required to read, sign, and date an IRB-approved ICF prior to undergoing any 
screening or other study procedures. This consent form will provide the subjects in non-
technical terms with the purpose of the study, procedures to be carried out, and potential 
hazards. The subjects will be assured that they may withdraw from the study at any time 
without jeopardizing medical care related to or required as a result of study participation. 
Subjects will be given a signed and dated copy of their ICF. Subjects will be given a copy 
of their signed ICF. 
 
7.2 
Termination of the Study 
The Sponsor reserves the right to discontinue this study at any time. The Investigator, in 
collaboration with the Sponsor, reserves the right to discontinue the study for safety 
reasons at any time. 
 
7.3 
Monitoring  
The responsible study monitor(s) will contact and visit the study sites as necessary, and 
he/she will be allowed, upon request, to inspect and verify all records of the study (e.g., 

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source document, ICFs, CRFs, regulatory documents) in a manner consistent with GCP 
and all other applicable state and federal law. 
It will be the study monitor’s responsibility to inspect the source documents to verify the 
adherence to the protocol and the completeness, consistency, and accuracy of the data 
being entered in the CRF. The monitor will verify that each subject has consented in writing 
prior to any study procedures being performed. Where the terms of the Informed Consent, 
GCP, and all other applicable state and federal law permit, the monitor should have access 
to laboratory test reports and other subject records needed to verify the entries on the 
CRF. The Investigator (or his/her designee) agrees to cooperate with the monitor to 
ensure that any issues detected in the course of these monitoring visits are resolved. 
In addition, the Sponsor’s internal auditors (or designee) and government inspectors may 
evaluate the study and must be allowed access to CRFs, source documents, and other 
study files. 
The Investigator must notify the Sponsor (or designee) promptly of any inspections of the 
study or activities related to the study scheduled by regulatory authorities, allow the 
Sponsor (or designee) to be present, and promptly forward copies of inspection reports to 
the Sponsor (or designee).  
 
7.4 
Data Quality Assurance 
SOPs are available for all activities performed at Celerion relevant to the quality of this 
study. Designated personnel of Celerion will be responsible for maintaining quality 
assurance (QA) and quality control to ensure that the trial is conducted, and that data are 
generated, documented and reported in compliance with the study protocol, GCP, and 
GLP requirements as well as applicable regulatory requirements. All activities performed 
will be reported in the QA certificate and will be included in the study report.  
All clinical data will undergo a 100% quality control check prior to clinical database lock. 
Edit checks are then performed for appropriate databases as a validation routine using 
SAS to check for missing data, data inconsistencies, data ranges etc. Corrections are 
made prior to database lock. 
 
7.5 
Direct Access to Source Data/Documents 
The Investigator will prepare and maintain adequate and accurate source documents and 
agree that the Sponsor, its representatives, the IRB, and representatives from worldwide 
regulatory agencies will have the right to review and inspect pertinent records related to 
the clinical trial both during and after the clinical trial. Examples of source documents may 
include the study product inventory, study product label records, CRFs, recorded data 
from automated instruments, and other primary records that are relevant to this clinical 
trial. These documents are designed to record all observations and other pertinent data 
for each subject enrolled in this clinical trial. 
 
7.6 
Data Handling and Record Keeping 
Each CRF will be reviewed and signed off by the Investigator within the electronic data 
capture system. 
All raw data generated in connection with this study, together with the original copy of the 
final report, will be retained by the sites until at least 5 years after the last approval of a 

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marketing application in an ICH region and until there are no pending or contemplated 
marketing applications in an ICH region or at least 5 years have elapsed since the formal 
discontinuation of clinical development of the investigational product. These documents 
should be retained for a longer period, however, if required by the applicable regulatory 
requirements or by an agreement with the Sponsor. It is the responsibility of the Sponsor 
to inform the Investigator/Institution as to when these documents no longer need to be 
retained. 
 
7.7 
Publication Policy 
All unpublished information given to Celerion by the Sponsor shall not be published or 
disclosed to a third party without the prior written consent of the Sponsor.   
The data generated by this study are considered confidential information and the property 
of the Sponsor. This confidential information may be published only in collaboration with 
participating personnel from the Sponsor or upon the Sponsor’s written consent to publish 
the information.  
 
8. DATA MANAGEMENT 
Data management activities will be detailed in the Data Management Plan (DMP). Each 
vendor involved with this study will adhere to Good Documentation Practices and their 
standard operating procedures covering their respective activities relevant to participation 
in this study. The Investigator will ensure that all data related to the conduct of this study 
at his/her site is attributable, legible, contemporaneous, original, accurate, enduring, and 
readily accessible. 
 
8.1 
Database Design and Creation 
An appropriate database will be designed and created within a validated Clinical Data 
Management System (CDMS). Electronic data capture will be used for this study and 
electronic case report form (eCRFs) will be developed according to the study protocol 
specifications. Clinical and analytical laboratory data will be collected external to CDMS 
as external data files. 
 
8.2 
Data Coding 
AEs and medical history coding will be undertaken using most current version of 
MedDRA®. Concomitant medications coding will be undertaken using the WHO Drug 
Dictionary. Each dictionary version will remain the same throughout the trial. Coding will 
be completed by qualified members of the Celerion staff. 
 
8.3 
Data Entry and Verification 
Data will be transcribed from original sources by the Investigator or Investigator’s staff into 
the eCRF. External data received from clinical and analytical laboratories (clinical 
laboratory results) will be integrated into the Clinical Data Interchange Standards 
Consortium study data tabulation model datasets. 
 

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8.4 
Study Results Data Transfer 
Study data transfers will be sent to the Sponsor or their designee, electronically on a 
schedule and in a format mutually agreed upon by the Sponsor or their designee, and 
Celerion for the analysis of these study data. No personally identifiable information will be 
transferred to the Sponsor at any point in the study. 
 
8.5 
Data Validation 
After the data have been entered and source data verified by the monitor, various edit 
checks (including manual review of listings) will be performed to ensure the accuracy, 
integrity and validation of the database against the eCRF as described in the DMP. 
Inconsistencies that arise from these edit checks will be resolved with the Investigator or 
designee. 
 
8.6 
Database Lock 
On study completion, after data entry is complete, the data has been pronounced clean, 
and the Investigators have reviewed and provided approval via signature, the database 
will be locked and final write access will be removed. 
The Sponsor will be required to provide database lock approval. 
Any changes to the data following database lock will be documented and approved by the 
Sponsor prior to unlocking the database to make changes to the data. 
The final transfer of all study data (without subject personally identifying information) to 
the Sponsor will be in SAS format with supporting SAS documentation according to the 
specifications of the Sponsor. Subject initials, serology results, date of birth (except year), 
and other personal identifiers will be removed from this data transfer file; any such 
information removed will be documented at the time of transfer. 
 
9. REPORTING  
9.1 
Case Report Forms 
CRFs will be used for each screened subject whether or not he/she has completed the 
study. The Investigator will assure complete and accurate entries on the forms. All CRFs 
will be reviewed and signed by the Investigator. 
 
9.2 
Study Report 
According to the ICH Harmonized Tripartite Guideline (Organization of the Common 
Technical Document for the Registration of Pharmaceuticals for Human Use M4 and the 
ICH M2 Expert Working Group), the final report will be written according to the 
ICH E3 Guideline (Structure and Content of Clinical Study Reports). 
The report will be provided by Celerion to the Sponsor. The report will include a description 
of the clinical conduct of the study, safety evaluation, analytical methods and results, and 
the statistical analysis described in the statistical methodology section of the protocol and 
the SAP.  

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At the time the draft study report is completed, Celerion’s QA unit will audit the report 
against the SAS data and the raw data. At the completion of the audit, a QA report will be 
issued internally allowing any findings to be addressed before report finalization. 
 
10. GENERAL 
10.1 Confidentiality 
All members of the Investigator’s staff and vendors will have signed confidentiality 
agreements with Celerion. Celerion will regard all information provided to the Investigators 
dealing with the study and information obtained during the course of the study as 
confidential. 
The CRUs will not supply to the Sponsor any subject names, initials, date of birth (except 
year), or other personal identifiers. 7 All such information appearing on any study 
document must be redacted before a copy of the document is supplied to the Sponsor. 
Study findings stored on a computer will be stored in accordance with local data protection 
laws. The subjects will be informed during the consenting process that representatives of 
the Sponsor, IRB, or regulatory authorities may inspect their medical records to verify the 
information collected, and that all personal information made available for inspection will 
be handled in the strictest confidence and in accordance with local data protection laws. 
 
10.2 Responsibility of the Investigator 
The Investigator is responsible for ensuring that the clinical study is performed in 
accordance with GCP based on the current ICH Guideline for GCP, the corresponding 
sections of the US CFR governing Protection of Human Subjects (Title 21 CFR Part 50), 
Institutional Review Boards (Title 21 CFR Part 56), the Basic Principles of the Declaration 
of Helsinki, and other applicable legal and regulatory requirements. 
The Investigator should ensure that all persons assisting with the study are qualified for 
the duties assigned, adequately informed and trained on the protocol and amendments to 
the protocol, the study products, and their study-related duties and functions. 
The Investigator will maintain a list, including signatures, of sub-investigators and other 
appropriately qualified persons to whom significant study-related duties are delegated. 
Any personnel change in this list during the course of the study will be documented. All 
study related training will be documented. 
 
10.3 Procedure for Amendments to Protocol 
No deviations from this protocol will be permitted, except in a medical emergency. The 
Investigator and the Sponsor will discuss any amendment to this study. If agreement is 
reached concerning the need for modification, this agreement will be made in a formal 
amendment to the protocol. 
All revisions and/or amendments to the protocol must be approved, if applicable, in writing 
by Chesapeake IRB. 
All persons who are affected by the amendment to the protocol will be retrained if deemed 
necessary. 
 

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10.4 Institutional Review Board 
This protocol and ICFs will be reviewed and approved in writing by the IRB prior to 
commencement of the study. The study will not be initiated without the approval from the 
IRB. Any amendments after protocol approval, if applicable, will be reviewed and approved 
by the IRB prior to implementation. The IRB operations are in compliance with Title 21 
CFR Part 56. Notice that the IRB approved protocol, ICF and any amendments to the 
protocol and ICF will be in the final study report. 
 
10.5 Study Record Retention 
Investigator-specific essential documents and all primary data and copies thereof (e.g., 
CRFs, laboratory records, data sheets, correspondence, photographs, computer 
records,), which are a result of the original observations and activities of the study and are 
necessary for the reconstruction and evaluation of any study report will be retained in the 
investigative site’s archives for a minimum of 20 years after the completion or termination 
of the study. It is the responsibility of the Sponsor to inform the Investigator as to when 
these documents no longer need to be retained. The study report and final database will 
be retained in Celerion’s archives for a minimum of 20 years after the completion or 
termination of the study and will be available for inspection at any time by the Sponsor. At 
completion of the study (i.e., at issuance of final study report), the final database will be 
transferred to the Sponsor. Subject initials, serology results, date of birth (except year), 
and other personal identifiers) will be redacted from this data transfer file; any such 
information removed will be documented at the time of transfer.  
 
 
 

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11. REFERENCES 
 
1. Benowitz NL, Henningfield JE. Establishing a nicotine threshold for addiction. The 
implications for tobacco regulation. New England Journal of Medicine, 1994, 331, 
123-125.  
 
2. Hatsukami DK, Kotlyar M, Hertsgaard LA, Zhang Y, Carmella SG, Jensen JA, Allen 
SS, Shields PG, Murphy SE, Stepanov I, Hecht SS. Reduced nicotine content 
cigarettes: effects on toxicant exposure, dependence and cessation. Addiction. 2010, 
105, 343-355.   
 
3. https://www.federalregister.gov/documents/2018/03/16/2018-05345/tobacco-
product-standard-for-nicotine-level-of-combusted-cigarettes. Accesses on 28th Match 
2018.  
 
4. Tidey JW, Pacek LR, Koopmeiners JS, Vandrey R, Nardone N, Drobes DJ, Benowitz 
NL, Dermody SS, Lemieux A, Denlinger RL, Cassidy R, al'Absi M, Hatsukami DK, 
Donny EC. Effects of 6-Week Use of Reduced-Nicotine Content Cigarettes in 
Smokers With and Without Elevated Depressive Symptoms. Nicotine and Tobacco 
Research, 2017, 19, 59-67.  
 
5. Watson CV, Richter P, de Castro BR, Sosnoff C, Potts J, Clark P, McCraw J, Yan X, 
Chambers D, Watson C. Smoking Behavior and Exposure: Results of a Menthol 
Cigarette Cross-over Study. American Journal of Health Behavior, 2017, 41, 309-319.  
 
6. Benowitz NL, Nardone N, Hatsukami DK, Donny EC. Biochemical estimation of 
noncompliance with smoking of very low nicotine content cigarettes. Cancer 
Epidemiology Biomarkers and Prevention, 2015, 24, 331-335.  
 
 
 

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APPENDIX 1. Fagerström Test For Cigarette Dependence (FTCD) 
Please answer each question below by checking () your answer in the box (). 
 
1. How soon after you wake up do you smoke your first cigarette? 
□ Within 5 minutes 
  
□ 6 - 30 minutes 
  
□ 31 - 60 minutes 
  
□ After 60 minutes 
  
 
2. Do you find it difficult to refrain from smoking in places where it is forbidden 
(e.g., in church, at the library, in the cinema, etc.)? 
□ Yes 
  
□ No 
  
 
3. Which cigarette would you hate most to give up? 
□ The first one in the morning 
  
□ Any other 
  
 
4. How many cigarettes per day do you smoke? 
□ 31 or more 
  
□ 21 - 30 
  
□ 11 - 20 
  
□ 10 or less 
  
 
5. Do you smoke more frequently during the first hours after waking than during 
the rest of the day? 
□ Yes 
  
□ No 
  
 
6. Do you smoke if you are so ill that you are in bed most of the day? 
□ Yes 
  
□ No 
  
 

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APPENDIX 2: Brief Questionnaire of Smoking Urges (QSU-Brief) 
 
For each item, please indicate how you feel RIGHT NOW. 
 
1. I have a desire to smoke right now. 
 
Strongly 
 
Strongly 
 
Disagree
 
Agree 
 
2. Nothing would be better than smoking right now. 
 
Strongly 
 
Strongly 
 
Disagree 
 
Agree 
 
3. If it were possible, I probably would smoke right now. 
 
Strongly 
 
Strongly 
 
Disagree 
 
Agree 
 
4. I could control things better right now if I could smoke. 
 
Strongly 
 
Strongly 
 
Disagree 
 
Agree 
 
5. All I want right now is a cigarette. 
 
Strongly 
 
Strongly 
 
Disagree
 
Agree 
 
6. I have an urge for a cigarette. 
 
Strongly 
 
Strongly 
 
Disagree 
 
Agree 
 
7. A cigarette would taste good now. 
 
Strongly 
 
Strongly 
 
Disagree 
 
Agree 
 
8. I would do almost anything for a cigarette now. 

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Strongly 
 
Strongly 
 
Disagree 
 
Agree 
 
9. Smoking would make me less depressed. 
 
Strongly 
 
Strongly 
 
Disagree 
 
Agree 
 
10. I am going to smoke as soon as possible. 
 
Strongly 
 
Strongly 
 
Disagree 
 
Agree 
 

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APPENDIX 3: Minnesota Nicotine Withdrawal Scale - Revised (MNWS-R) 
 
Please rate yourself for the last day 
Scale: 0 = none, 1 = slight, 2 = mild, 3 = moderate, 4 = severe 
 
1. Angry, irritable, frustrated  
0    1    2    3    4 
2. Anxious, nervous  
0    1    2    3    4 
3. Depressed mood, sad  
0    1    2    3    4 
4. Desire or craving to smoke  
0    1    2    3    4 
5. Difficulty concentrating  
0    1    2    3    4 
6. Increased appetite, hungry, weight gain  
0    1    2    3    4 
7. Insomnia, sleep problems, awakening at night  
0    1    2    3    4 
8. Restless  
0    1    2    3    4 
9. Impatient  
0    1    2    3    4 
10. Constipation  
0    1    2    3    4 
11. Dizziness  
0    1    2    3    4 
12. Coughing  
0    1    2    3    4 
13. Dreaming or nightmares  
0    1    2    3    4 
14. Nausea  
0    1    2    3    4 
15. Sore throat  
0    1    2    3    4 
 
 
 

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APPENDIX 4: Perceived Health Risk Scale 
 
On the scale below, indicate your perception of the risk of becoming addicted to the 
cigarette you are currently using. 
 
 
Very 
low 
risk 
 
 
 
 
 
 
 
 
Very 
high 
risk 
 
 
 
 
 
 
 
 
 
