Toxicant Exposure Across Brands of Smokeless Tobacco
Conditions: Smokeless Tobacco Use, Tobacco Dependence
Sponsor: University of Minnesota

Novel Determinants and Measures of Smokeless Tobacco Use:   Study 1 
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Novel Determinants and Measures of Smokeless Tobacco Use 
 
Study 1:  Toxicant Exposure Across Brands of Smokeless Tobacco 
 
 
 
 
Principal Investigator: 
 
 
 
 
Dorothy K Hatsukami, PhD 
Department of Psychiatry 
 
UMN Co-investigators: 
 
 
Stephen S Hecht, PhD 
Masonic Cancer Center 
 
 
Sharon Allen, MD 
Family Medicine and Community Health 
 
 
Chap Le, PhD 
Biostatistics, School of Public Health 
 
Oregon Research Institute Co-investigators: 
 
 
 
Herb Severson, Ph.D. 
 
 
West Virginia University Co-investigators: 
 
 
Kimberly Horn, Ed.D. 
 
 
Robert Anderson 
 
 
Protocol #: 
Study 1 
IND #: 
NA 
IDS #: 
NA 
Funding Sponsor: 
NIH / NCI - R01 CA141531-01 
 
 
 
 
 
 
 
 
 
 
 
Original Date:  
 
5/19/2009 
Final Version Date: 
12/31/2009 

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REVISION HISTORY 
 
Version Date 
Revision summary 
Protocol 
Consent 
 Other 
Revised 
Version 
Date 
Revised 
Version 
Date 
Revised? 
Version 
Date 
5/25/2009 
original to IRB 
 
5/9/2009 
 
11/15/07 
 
11/15/07 
10/20/09 
Revisions from start-up meeting 
- eliminate visit 2 
 
Amend
ment 1 
10-20-
09 
 
10/29/09 
 
 
 
11/25/2009 
 
 
Final Protocol 
 
12/31/20
09 
 
 
 
 
 
4/5/2010 
Amendment 1 
Increase payment to $100; 
change advertisement 
Revised product list to change 
Skoal bandits straight to Skoal 
bandits wintergreen 
 
 
4/5/2010 
 
 
3/25/2010 
  
 
 
7/13/11 
 
 
Updated tobacco product list for 
eligibility and on page 9 
 
7/13/11 
NA 
 
NA 
 
 
 
 
PI CONTACT INFORMATION: 
 
Dorothy Hatsukami, PhD 
Forster Family Professor in Cancer Prevention 
Professor of Psychiatry 
University of Minnesota 
Minneapolis, MN 55454-1015 
Telephone (612) 627-1808 
Fax (612) 627-4899 
Email: hatsu001@umn.edu 

Novel Determinants and Measures of Smokeless Tobacco Use:   Study 1 
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A. Specific Aims 
Reducing toxicants in tobacco products and establishing performance standards for tobacco 
products have been discussed and recommended by the World Health Organization Tobacco 
Regulation Study Group and by tobacco control scientists and advocates [1, 2].  Although the focus to 
date has been primarily on cigarettes, reducing tobacco toxicants in smokeless tobacco should also be 
strongly considered and recommended [3].  The extent of toxicity varies considerably across different 
brands of smokeless tobacco (ST) products and data suggest that greater toxicity may result in greater 
health risks.  However, little is known about the actual extent of human exposure to toxicants from 
current ST products and factors that might moderate the extent of this exposure.  The overall goals of 
this study are to determine toxicant exposure across brands of ST that vary in levels of nicotine and 
TSNAs using a panel of biomarkers of exposure and effect, and to examine factors other than product 
toxicants that influence the extent of this exposure.   
 
B. Research Design and Methods  
 
B1. Overview 
Subjects using the brands listed in Table 2 will be recruited from three different regions in the U.S.  
They will undergo two assessment sessions.  During these sessions, urine and blood samples will be 
collected.  During the time of data collection, ST users will be recording the amount of tobacco products 
that they use on a daily basis using their diary cards.   
 
B2. Procedures 
Subject recruitment.  Subjects (N=600) will be recruited from a) Minneapolis/St. Paul, MN, b) 
Eugene, OR, and c) Morgantown, WV.  Each site will recruit 200 subjects over the course of 2 years, 
with a total of 100 subjects recruited for each brand that is listed in Table 2.  We anticipate no problems 
with recruitment.  Surveys conducted in 2007or 2006 estimated Minnesota and Oregon male ST 
prevalence of 6.0% and 6.5%, respectively.  The 2006 West Virginia Adult Tobacco Survey estimated 
the male ST prevalence to be about 16%.  Multiple avenues of recruitment will be pursued to ensure as 
representative of a sample as possible.  These methods include radio, cable, newspapers (want ads 
and display ads), restroom stall ads, sporting good stores, WIC clinics, internet and press releases.  
The recruitment advertisements will state that daily ST users are needed for a study that examines the 
effects from ST use; blood (only at U of MN site), oral cells (only at U of MN site) and urine samples will 
be obtained.  It will further state that subjects will be paid for participation.  Potential adult subjects will 
be asked to call the research clinic number where the study will be explained, brief phone screening will 
be conducted, and interested and potentially eligible subjects will be asked to attend an orientation 
meeting.  The brands of ST will not be listed on the advertisements to ensure that we are recruiting 
actual users of the brands rather than subjects who falsely claim using one of the brands of interest in 
order to obtain money for study participation.  Subjects will be informed that the study determines the 
extent of exposure to toxicants and the effects of these exposures across different brands of ST.   
 
B2a. Subject inclusion/exclusion criteria  
Adult ST users (18 years or older) who are interested in the study will telephone the research 
clinics, be informed about the study, and initially screened in order to determine whether they meet 
specific inclusion/exclusion criteria.   
Inclusion criteria include:  
a) use of a consistent brand and daily amount of ST for the past 6 months;  
b) in good physical health (no unstable medical condition and no kidney or liver disease);  
c) stable, good mental health (e.g., no recent unstable or untreated psychiatric diagnosis, including 
 
substance abuse, as determined by the DSM-IV criteria).   
d) use of one of the following smokeless tobacco products:  

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(UPDATED BRANDS BY GROUP):  1)  Camel Snus, Marlboro Snus; 2) Skoal Long Cut Mint, 
Skoal Fine Cut Original, Copenhagen Long Cut Original, Kodiak Wintergreen, Skoal Bandits 
Wintergreen, Skoal Pouches Straight, Skoal Pouches Wintergreen; 3) Grizzly Long Cut 
Straight, Skoal Long Cut Straight, Grizzly Long Cut Mint, Grizzly Long Cut Wintergreen, 
Grizzly Pouches Wintergreen, Skoal Pouches Mint, Kodiak Pouches Wintergreen; 4) Grizzly 
Fine Cut Regular; 8) Copenhagen Fine Cut Original, Copenhagen Long Cut Straight, Grizzly 
Pouches Straight, Skoal Long Cut Classic, Skoal Long Cut Wintergreen, and Dual Users of 
Camel and Marlboro Snus and cigarettes. 
Exclusion criteria include: 
a) currently using other tobacco (>1 episode of smoking per week on average; no combustible 
tobacco use in the last 21 days and CO >8 ppm) or nicotine products;   
b) female subjects pregnant or nursing; 
c) use of alcohol within the last 24 hours of testing and no more than 21 drinks per week at phone 
 
screening; 
d) use of any inhalable drugs in the last 14 days and regular use >1 times per week;  
e) current infection or use of antibiotics; 
f)  active liver or kidney disease, or UTI which would interfere with biomarker measures. 
 
Subjects meeting these criteria will then be asked to come into the research clinic for an orientation 
visit, to provide informed consent, and to engage in more thorough screening.  Subjects will also be 
required to complete a tobacco use questionnaire, medical history form (which will be reviewed by a 
physician), and baseline measurements (see below).   
 
B2b.  Measures and Schedule of Administration   
The following provides the measures that will be administered. Table 1 describes the measures to 
be obtained and the timing of the measurement.  The majority of these measures have been used in 
our prior studies and have been found to be dose-sensitive to change in amount of smoking or ST use, 
or sensitive to switching products (e.g., conventional cigarettes to modified cigarettes, cigarettes to ST 
products). 
 
Table 1. Schedule of visits and measures Study 1 
 
 
Visit 
Orientation 
1 
Consent 
X  
 
Baseline and Moderator Measures 
Demographics 
Medical History and Concomitant meds 
Tobacco Use History and Exposure, 
Severson ST Dependency Scale 
Perceived Health Risk  
Stages of Change 
CES-D  
PANAS  
Perceived Stress Scale  
Michigan Alcohol Screening Test, 
NIAAA Alcohol Use Questionnaire  
 
X 
X 
X (brief) 
X 
X    
 
X 
 
 
X 
X 
X 
X 
X 
X 
Exposure and Lifestyle Measures 
Occupational Health History 
ETS and Social Influences 
Dietary – consumption of meats 
IPAQ 
Medical status (e.g., infections)  
 
 
 X 
X 
X 
X 
X 

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Daily Tobacco Use Diary 
X (start at screen) 
X  
Vitals (Blood pressure and heart rate) 
 
X  
Weight 
 
X 
Concomitant Medications 
 
X  
Carcinogen Exposure 
 
Urinary total NNAL  
 
Total NNN  
 
Nitrosoamino acids NPRO and MNPA 
  
Phenanthrene tetraol 
 
1-hydroxypyrene (1HOP) 
 
X  
Cardiovascular Measures (UMN only) 
 
Lipoproteins 
 
C-reactive protein 
 
Fibrinogen 
 
WBC 
 
 
X 
X 
X 
X  
1Buccal assay 
 
X  
2Nicotine and metabolites 
X  
X  
3Product Constituent Testing 
 
X  
4Weight per dip 
X 
X  
Carbon Monoxide 
 
X  
 
1Buccal assays to be placed in biorepository for future analyses (UMN only) 
2Urinary total nicotine equivalents and nicotine metabolite ratio (3-HC:cotinine) 
3Product constituent testing will conducted on a random sample of 30 tins for each brand of ST (10 brands from 
each site) 
4Estimated weight per dip will be calculated by averaging the weight of 3 typical dips 
 
B2bi. Subjective Measures 
 
1) Demographics such as age and gender, socioeconomic status and if possible, self-reported race 
or ethnicity. 
 
 2) Tobacco Dependence as assessed by the Severson Smokeless Tobacco Dependency Scale 
(SSTDS).   This scale consists of 8 items and a total score is calculated as the sum of items 2 through 
8.  The Cronbach coefficient alpha for the SSTDS was 0.69. Candidate items for this scale were drawn 
from the following sources: a version of the Fagerström scale used in previous studies [101, 102]; a 
version of the Cigarette Dependence Scale (CDS) with 5 items (CDS-5) [103] adapted for smokeless 
tobacco users. 
3) Tobacco Use History and Exposure Questionnaire is an interview with variables such as age of 
initiation of ST use, amount of use, brand used, duration of use, number of quit attempts, duration of 
quit attempts, and current and past other tobacco product use.  Tobacco use questions are taken from 
a national survey (Tobacco Use Supplement to the Current Population Survey) so that comparisons 
can be made with national samples.  
4) Centers for Epidemiological Studies-Depression (CES-D) is a 20-item scale which assesses 
current symptoms of depression that has been used in the general population [105]. 
5) Michigan Alcohol Screening Test (MAST) Short form assesses any negative consequences from 
alcohol use by self report [106] and the NIAAA Alcohol Use Questionnaire examines rate of alcohol 
use, in the last 3 months  
6) Positive Affect Negative Affect Scale (PANAS-X) [108] has been used in studies to assess 
negative affect prior to and after cessation [e.g., 109].  This scale contains two higher order scales, 
positive and negative, and 11 specific affects.  We will be focusing primarily on the negative affect 
scale, which measures subjective distress and unpleasant engagements (aversive mood states such as 
anger, disgust, guilt, fear and nervousness).   

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7) Perceived Stress Scale is a 14-item scale that measures self-perceived stressful situations in the 
last month.  The items reflect the extent to which the respondents find that their lives are unpredictable, 
uncontrollable and overloading [110].  Each item is rated on a 0 to 4 scale that ranges from 0=never 
and 4=very often.  The Cronbach’s alpha observed in one study was 0.80 (Manning et al., 2005).  The 
scale has been related to smoking status and cessation [e.g., 111, 112, 113].  
8) Perceived Health Risk form assesses perceived health risk of the product for different disease 
states (heart disease, oral cancer, lung cancer, diabetes, etc.).  Subjects rate the product on a 0 to 10 
rating scale (0=no risk to 10=very high risk).  We have used this scale in a number of tobacco 
evaluation studies [e.g., 98].   
9) Stages of Change assessing intention of quitting in the next 30 days and 6 months. 
10) Environmental and Social Influences on Tobacco Use consists of a questions regarding: a) 
tobacco use (ST, cigarettes and other nicotine containing products) among the subject’s social network 
(e.g., significant other, friends, co-workers, siblings and parents); b) extent of social support for or 
against ST use; c) restrictions or bans on smokeless tobacco (and smoking) at work, school or in the 
home; and d) cost of smokeless tobacco products (unit price) and income.  These are variables found 
in studies to be associated with uptake and amount of tobacco use.  
11) Food frequency questionnaire for cooking practices assesses frequency of eating charred and 
processed meats within the last week. 
12) Modified International Physical Activity Questionnaire (IPAQ) interview assesses physical 
activity by recall 7 question assessment of moderate, hard and very hard physical activity engaged in 
week days and week ends modified to cover the last three months average activity.  
http://www.ipaq.ki.se/. 
13) Medical History and Concomitant Medications lists current diagnosis, symptoms, medications 
and past health problems, and a record of use of any over-the-counter and prescription medications. 
14) Environmental and Occupational Health History assesses environmental and occupational 
toxicant exposures, which might affect our biomarker measures [124]. 
15) Tobacco Use Status will be assessed using a Tobacco Use Questionnaire that asks about 
current tobacco use status (ST and other tobacco products).  This questionnaire will be administered at 
the clinic visit. The daily diary cards will collect similar information, but will be hand recorded and 
returned during the clinic visit.  The daily diary cards will be used in Study 1 to conserve costs.  We 
have successfully used daily diary cards in the past when examining the relationship between exposure 
measures and ST topography [41, 42, 44, 76]. 
 
B2bii. Biological measures 
 
Physiological Measures 
1) Heart rate and blood pressure.  After sitting for 5 minutes, the subject's radial pulse rate will be 
measured. After the heart rate measurements, blood pressure will be measured twice, 2 minutes apart 
for reliability. Measurements will be taken using a Critikon Dinamap (GE Medical Systems, Tampa, FL).   
Although acute increases in heart rate and blood pressure have been observed, most studies have 
shown a lack of sustained increases in heart rate and blood pressure from ST use [126].  On the other 
hand, a few studies have shown increased hypertension among ST users [127, 128] and one of the 
studies has implicated elevated blood pressure as the potential causal pathway for increased risk for 
fatal myocardial infarction observed  [129]. 
2) Body weight.  Subjects will remove their shoes, coats, sweaters, etc. and be weighed to the 
nearest 0.25 lb on an electronic scale. 
Nicotine metabolism 
3) Nicotine metabolite ratio. This ratio is an indicator of CYP2A6 enzyme activity and is the ratio 
between two nicotine metabolites, cotinine and trans-3’-hydroxycontinine (3-HC).  Because CYP2A6 is 
the primary enzyme associated with nicotine metabolism and also catalyzes the conversion of cotinine 
to 3-HC, the 3-HC:cotinine ratio is considered to reflect the enzymatic activity of CYP2A6 and reflect 
the extent of nicotine clearance rate (higher the ratio, the faster the clearance rate).  This metabolite 

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ratio has been observed to be moderately correlated to CYP2A6 genotype and the rate of nicotine 
metabolism [20, 21, 130-133]. 
Biomarkers.   
4) Biomarkers for tobacco exposure measures.  Urinary total nicotine equivalents--total nicotine 
(free plus nicotine N-glucuronide) and its main metabolites, total cotinine (free plus cotinine N-
glucuronide), total trans-ƍ-hydroxycotinine (3-HC) (free plus 3-HC N- and O-glucuronide)--which 
together account for 85-90% of the nicotine dose and is a useful measure of daily nicotine exposure, 
will be assessed by the Hecht laboratory at the Masonic Cancer Center, University of Minnesota.  
Carbon monoxide, which is rapidly absorbed into the blood stream during smoke inhalation and binds 
with hemoglobin to form carboxyhemoglobin (COHb), will be measured using the Bedfont Micro 
Smokerlyzer® (Bedfont Scientific Limited, Kent, UK) device to verify abstinence from smoking. A spot 
urine samples will be collected at the orientation visit and at the clinic visit.  
5) Biomarkers for carcinogen exposure and effect.  The following urinary biomarkers of carcinogen 
exposure will be assessed: a) NNAL and NNAL-glucuronides (total NNAL), metabolites of the tobacco-
specific carcinogen NNK; b) N’-nitrosonornicotine (NNN) and its glucuronides in urine (total NNN), 
reflecting the uptake of the tobacco-specific carcinogen NNN; c) the nitrosamino acids NPRO, a widely 
accepted biomarker of endogenous nitrosation, and MNPA, a biomarker of exposure to the carcinogen 
MNPA found in smokeless tobacco products; d) 1-HOP, a well established biomarker of exposure to 
PAH; e) phenanthrene tetraol, a marker for PAH.  First urine void in the morning will be collected for the 
urine analyses [64].  The analyses will be carried out in the Hecht laboratory using well established 
methods described in our previous publications [62, 134-136]. Results will be expressed per mg urinary 
creatinine.   
For the sample collection at the other sites, staff will be trained on how to collect, process and send 
the urine samples.  Urine samples will be collected, frozen and shipped on dry ice to Minnesota for 
analysis.  The names and other identifying information will be stripped from the data before 
transmission and the file connecting the data with the subject number will be transferred via secure 
VPN in a separate file to Minnesota project staff.  The file connecting the data to the identifying 
information of the participant will be stored at each site in a locked file cabinet and will be accessible 
only to approved study staff. 
6) Cardiovascular Disease (CVD) Risk Factors.  We will collect samples to measure CVD risk 
factors only at the Minnesota site to save costs. CVD risk factors will be analyzed at the University or 
Minnesota Medical Center Fairview, Collaborative Studies Clinical Laboratory, Minneapolis, MN.  To 
date, most of the existing studies on CVD risk factors in ST users have been conducted in Sweden. A 
review of these studies shows that exclusive ST users do not demonstrate higher CVD risk factors 
(e.g., lipoproteins, biomarkers of oxidative stress and inflammation, endothelial function and platelet 
activation, carotid intima media thickness) compared to non-users among Swedish ST users and 
similar results have been observed with healthy young US snuff users [126, 138].  On the other hand, a 
few studies conducted with ST users in India have shown elevation in lipoproteins and triglycerides 
[139, 140].  Because of the limited knowledge of US products and a meta-analyses that shows an 
increased risk in CVD and stroke observed in the US population of smokeless tobacco users (although 
not the Swedish population [33] with the exception of fatal cardiovascular disease [128, 141]), the 
following CVD risk factors, which have been predominantly observed to be increased in cigarette 
smokers, will be assessed in our study: a) Lipoproteins, such as total plasma cholesterol, high and low 
density lipoproteins (HDL and LDL, respectively), and triglycerides.  These markers were found to be 
dose-sensitive to cigarette exposure in our prior studies.  Subjects will fast for 10 hours prior to blood 
draws.  Preferably sampling will be done in early morning, but samples can be done mid-day according 
to work schedules of subjects as long as the subjects are fasting.  b) C-reactive protein is considered to 
be an inflammatory marker or mediator and its release by the liver is stimulated by pro-inflammatory 
cytokines, including interleukin 6 (IL-6).  Increases in C-reactive protein have been predictive of future 
vascular events and course of pre-existing vascular disease, and smoking has been found to modulate 
levels of this protein [e.g., 142, 143]. Several mechanisms by which C-reactive protein contributes to 
the risks of cardiac disease have been described [143, 144].  c) Fibrinogen is a marker for thrombosis 

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and is a glycoprotein involved in the coagulation response to vascular and tissue injury. Fibrinogen is 
also considered to contribute to vascular disease in other ways besides its role in thrombosis such as 
stimulating smooth muscle cell proliferation, promoting platelet aggregation and increasing blood 
viscosity [145]. Elevated fibrinogen levels have been found to be strongly predictive of cardiovascular 
morbidity and mortality [143].  Prior studies have shown a dose-response relationship between 
fibrinogen levels and number of cigarettes smoked [146].  d) White blood count, also an inflammatory 
marker, has been found to be higher in smokers than non-smokers [147, 148] and associated with 
cardiovascular disease [142] and in our studies was observed to be the most dose-related marker for 
cigarette reduction [84].  
7) Buccal cells biomarkers.  Although the grant proposal will not be able to bear the cost for 
analyses of buccal biomarkers, we will be collecting buccal cell samples to be placed in a biorepository 
with the hopes of obtaining future funding for their analysis.  Buccal cells will be obtained through two 
methods (oral check swab and mouth wash for DNA extraction).  These buccal cells will be frozen, 
stored and labeled in accordance to the procedures used in our NCI contract (N01-PC-64402) and will 
be made available to investigators associated with the contract or outside of this contract upon review 
of their proposal and approval from their respective IRBs.  All samples will be de-indentified.  Examples 
of potential biomarkers include cellular autoflorescence (a method being tested by Dr. John Pauly at 
Roswell Park Cancer Institute), mitochondrial mutations in oral cavity cells (an analyses being tested by 
Dr. Peter Shields at Georgetown University) and nf Kappa B p65 staining (an analyses conducted for 
NCI contract N01- CN-15000 by Dr. Frank Ondrey at the University of Minnesota).  
 
B2c. Product constituent testing   
In collaboration with the Center for Disease Control and Prevention, we will analyze the 
constituents of the smokeless tobacco products (un-ionized nicotine, tobacco specific 
nitrosamines and pyrene) used by our participants.  This information will be related to the 
biomarker data.  A random sample of tins of unused ST from 10 subjects for each brand of ST 
at each site will be analyzed.  For Study 1, 180 subject samples (10 random subjects x 6 brands 
x 3 sites) will be analyzed.  The tins will be labeled with the subject number, site identification, 
date of collection, brand of ST and tin lot number.  Samples will be stored in the freezer until a 
batch is shipped to CDC on dry ice.  Subjects will be reimbursed for their tin of ST.  The major 
advantage of taking tins from the subjects is that they may more accurately reflect actual levels 
of toxicants.  Prior studies have shown change in TSNAs based on storage conditions.  CDC 
will analyze samples for NNAL, NNN, NAT, NAB, PAHs, Nitrosamino acids  (MNPA & NIS will 
be added to CDC usual analyses). 
 
B2d.  Tobacco Products 
For Study 1, usual brand of ST products will be used.  We chose products that had substantial 
market share and showed a range in nicotine level and in NNK and NNN levels [4, 46, 75, 151].  Table 
2 shows the brands that were chosen for testing.  Free/unprotonated nicotine levels were considered 
high (H) if they were > 5 mg/g, medium (M) if they were between 2-5 mg/g and low if they were < 2 
mg/g [3].  TSNA levels were based on a classification that was developed by Stephen Hecht (Hecht, 
report for WHO, TobReg).  High (H) levels were NNN plus NNK > than 10 µg/g, Medium (M) levels 
were NNN plus NNK from 2-10 µg/g, and Low (L) levels were NNN plus NNK < 2 µg/g.  Products that 
are in the Low range for both nicotine and TSNAs tend not be manufactured. 
 
 
 
 
 
 
 
 

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Table 2 
 
 
 
B2e:  Clinic Visits 
 
B2ei. Orientation meeting.  Subjects will attend an orientation meeting to be further informed about 
the study, to obtain consent, and to determine eligibility.  Once determined to be eligible, subjects will 
provide a spot urine sample for nicotine levels, and then they will be scheduled for a clinic visits and will 
keep a daily diary to monitor their ST intake (time of dip onset and time when dip was expectorated).  
We have used daily diaries with ST users for up to a period of 8 weeks with good compliance. 
 
B2eii. Clinic visit.  At the clinic visit, subjects will be asked to complete questionnaires on 
demographic and tobacco use history, on ST dependence, on psychiatric, medical and alcohol use 
history, and on factors that might moderate the extent of tobacco exposure.  Subjects will also be asked 
to complete a series of questionnaires during each of the orientation and clinic visits.  These 
questionnaires will assess for tobacco use status and any factors that might confound our biomarker 
measures.  These factors include exposure to environmental tobacco smoke, dietary intake, level of 
physical activity, medical status (e.g., infections), alcohol intake prior to visit and use of any other 
nicotine containing products.  First morning urine sample will be collected to measure carcinogen 
biomarkers. Two spot urines (one at orientation and one at the clinic visit) total nicotine equivalents and 
nicotine metabolite ratio.  Vital sign measurements and CO will be obtained and at the U of MN site, 
blood samples taken (23 ml of blood obtained by a nurse).  In addition, subjects will provide us with at 
least 3 samples (usual size dips) of their smokeless tobacco product at each of the two visits.  Each of 
these dips will be weighed to obtain an estimate of the mean weight per dip.  Clinic visits will be 
scheduled on Tuesdays, Wednesdays or Thursdays.   At each site, tins of unused ST will be obtained 
from a random sample of 10 subjects from each of the 6 brands of ST and sent to CDC for toxicant 

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analysis.  At the end of the study, all subjects will be encouraged to quit ST use.  Cessation materials 
and referrals will be provided to all participants at the conclusion of the second visit. 
 
B2f. Subject payments.   
‘ 
Subjects will be paid $100 for participation, $25 for the Orientation and $75 for the clinic visit. 
 
B2g. Adherence to protocol procedures and data integrity.   
 
Standard operating procedures will be developed.  Face-to face training for research personnel will 
occur prior to the study, where the protocol and procedures will be carefully described.  Case report 
books will be made for the different sites to maximize parallel recording of data.  Each visit will have a 
checklist of all the measures that need to be taken and the order by which these measures are 
administered.   
Weekly conference calls will occur among the research coordinators to trouble shoot and to make 
sure that all protocol procedures are followed.  On a once a month basis, the PI and co-investigators 
will participate in this conference call.  During these conference calls, the number of subjects enrolled, 
the data collection process, the results from data monitoring and other issues of concern will be 
discussed.  As another measure to ensure data collection integrity, the project manager from the 
University of Minnesota will make a visit to the various sites after they have enrolled 10 subjects to 
make sure that the protocol is being followed carefully and all the data is being collected properly.  
Another on site visit will occur 3 months from the initiation of the study.  An additional visit will be made 
in Year 1 if necessary, and 2 visits in Year 2.  
Human subject consent forms, pen and paper and electronic subjective forms, and containers for 
the urine biosamples and for ST collections will be given bar codes individualized for each subject.  
Each site will receive a separate box for each subject, packaged at the University of Minnesota, that 
contains the bar coded paper and pencil subjective forms and collection containers.  Most of the 
subjective measures will be administered via computer in electronic forms on a secured website.  
Subject numbers, represented on the bar codes, will be entered into the computer prior to subject data 
entry.  Forms will include programming features to ensure valid data (i.e., input masks, validation 
criteria, skipout logic) and data will be exported to Microsoft Excel files that are de-identified of any 
personal health identifiers.  All other data not captured directly in the website at the other sites (e.g., 
pencil-and-paper forms) will be sent to the University of Minnesota on a weekly basis (with photocopies 
of originals maintained at the other sites).  A data monitor will be available at the University of 
Minnesota site and irregularities (e.g., wrong date on the forms, inconsistent data) or missing data will 
be flagged by the monitor and sent to the site for comment or correction to ensure the integrity of the 
data.  
All de-identified biosamples will be sent to the University of Minnesota on a monthly basis after both 
biosamples have been collected for each subject.  A list of subject number and biosamples sent will be 
recorded by each site and when the samples are received, the researcher at the University of 
Minnesota will note each subject number and biosample that was received.  The samples-received list 
will be posted on a secure website so that each site can check it against the samples-sent list.   
 
C. Statistical analyses 
 
C1. Analytic Approach 
Primary Aims 
1. To determine the toxicant exposure associated with currently marketed oral tobacco products with 
varying levels of toxicants and nicotine. 
Subject characteristics will be examined across sites and ST users at each site will be compared to 
the characteristics of the respective state population of ST users. The outcomes for this study are the 
uptake of toxicants, as measured by biomarkers of exposure including NNAL, NNN, nitrosoamino acids, 

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1-HOP and total nicotine equivalents and biomarkers of effect such as CVD risk factors. The 
distribution of each biomarker will be examined to determine if a data transformation is necessary.  
From our previous experience, the distributions of many biomarkers are often skewed with a long right 
tail and need to be analyzed in the natural log scale.  The two biomarker assessments for nicotine from 
each subject will be used to evaluate the reliability, as measured by the intraclass correlation.  The two 
readings will then be averaged for the subsequent analysis.  The geometric mean and the 
corresponding 95% confidence interval for the biomarkers will be calculated for each brand.  The one-
way analysis of variance (ANOVA) will be used to compare the mean toxicant levels between all six 
brands.  If this global test is significant, then the Tukey’s studentized range procedure will be used for 
pair-wise comparisons between brands, while maintaining an overall significance level of 5%.   
 
2.To determine if toxicant and nicotine exposure is related to product factors such as the levels of these 
constituents (nicotine, NNK and NNN) in the products and patterns of use such as tins per week, dips 
per day, duration per dip, total dip duration (dip per day x duration per dip), weight per dip. 
 
Pearson or Spearman correlations will assess the relationship between toxicant and nicotine 
exposure and the level of toxicants in the tobacco products.  Furthermore, regression models will 
determine the degree of association between exposure and patterns of use, while controlling for the 
different ST brands.  Specifically, total nicotine equivalents will be correlated with the free nicotine 
levels of the products and patterns of use; NNAL will be correlated with the NNK levels of the product 
and patterns of use; NNN exposure will be correlated with the amount of NNN in the product and 
patterns of use; and 1-HOP exposure will be correlated with the amount of pyrene and patterns of use. 
 
Secondary Aims 
3. To determine if exposure is also moderated by individual factors such as degree of dependence, 
nicotine metabolism, age of initiation and duration of use, negative affect and stress, amount of alcohol 
use and perception of ST harm and environmental and social factors such as use among social 
network, cost of the product, bans on tobacco use. 
 
Analysis of covariance (ANCOVA) and regression methods will measure the impact of level 
of tobacco constituents (e.g., free nicotine, NNK, NNN, pyrene), ST Dependency score, nicotine 
metabolite ratio, age of initiation, duration of use, negative affect and depressed mood (PANAS 
negative affect score and CESD), stress (Perceived Stress Scale), amount of alcohol use (NIAAA 
Alcohol Use Questionnaire), perception of ST harm (Perceived Health Risk), social network (percent of 
social network who uses ST and who smokes), price of product and income, and tobacco bans 
(smoking and ST bans at work, school or home) on exposure measures (total nicotine equivalents, total 
NNAL, total NNN).  Potential confounding factors such as site and as assessed by the Exposure and 
Lifestyle measures will be examined and controlled.  The ST brand will be included in the model to 
access the main effects adjusted for product brand and to explore interactions between individual, 
environmental and social factors and brands on exposure measurements.  For example, does a high 
ST Dependency score increase exposure to toxicants for some brands more than others?   
 
4. To estimate the overall toxicant burden in the population by examining the extent of carcinogen 
exposure of the products that have the greatest market shares. 
 
The analysis for this aim is an extension of primary aim 1.  The market share for each of the 
six ST products will be represented as the percent of the total.  Market share information will be 
obtained from the Maxwell Report.  Using the mean toxicant exposure level for the six brands from the 
one way ANOVA, a weighted mean value will be calculated across the brands, with the percent of the 
market as the weight.  This will be done for each biomarker.  Thus, the more popular brands will have 
more influence on the overall exposure level. 
 
C2. Power Considerations: 
 
The goal is to enroll 100 subjects for each of the six brands listed in Table 2.  The mean 
toxicant uptake level is expected to vary between brands; however, the standard deviation (SD) should 

Novel Determinants and Measures of Smokeless Tobacco Use:   Study 1 
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12.31.09_rev7/13/11 
 
be similar for all brands.  This is the common SD and is equivalent to the root of the Mean Square Error 
(MSE) from the one-way ANOVA.  With a total of 600 subjects, this F-test will have 92% power to 
detect a significant difference at the 0.05 level if the brands with the largest and smallest means differ 
by at least one-half the common SD.  Therefore, if the common SD for the biomarker was 10 units, then 
there would be a 92% chance that we find a significant difference between brands if the biggest and 
smallest means were at least 5 units apart.  This analysis was done using Power Analysis and Sample 
Size (PASS) 2005 by NCSS, Inc. 
 
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