Waterpipe Tobacco Additives and Their Effect on Human Puffing Behavior, Toxicant Exposures, Pulmonary Function, and Appeal
Phase: NA
Conditions: Tobacco-Related Carcinoma
Sponsor: Ohio State University Comprehensive Cancer Center

IRB Protocol Number: 2021C0039 
IRB Approved Original Date: 05.03.21 
Version: 3.0 
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RESEARCH PROTOCOL 
 
Waterpipe tobacco additives and their effect on human puffing behavior, 
toxicant exposures, pulmonary function, and appeal 
 
Table of Contents 
I. Objectives ............................................................................................................................... 3 
II. Background and Rationale ..................................................................................................... 4 
II.1. Who Smokes Waterpipe? ................................................................................................ 4 
II.2. How Do We Codify Harm? ............................................................................................... 4 
II.3. How Can Sweet WP Tobacco Additives (Sugars and Glycerol) Contribute to Direct 
Harm? .................................................................................................................................... 4 
II.4. How Can Sweet WP Tobacco Additives Contribute to Indirect Harm? ............................. 5 
II.5 Why Use Investigational Tobacco Products? .................................................................... 6 
II.6. Why Use a Research-Grade Waterpipe? ......................................................................... 8 
II.7. What will this study add? ................................................................................................. 9 
III. Procedures...........................................................................................................................11 
III.1. Research Design Overview ...........................................................................................11 
III.A1. Aim 1 Research Design ...........................................................................................11 
III.B1. Aim 1 Sample ..........................................................................................................11 
III.C1. Aim 1 Measurement/Instrumentation .......................................................................11 
III.D1. Aim 1 Detailed Study Procedures ............................................................................12 
III.A2. Aim 2 Research Design ...........................................................................................13 
III.B2. Aim 2 Sample ..........................................................................................................13 
III.C2. Aim 2 Measurement/Instrumentation .......................................................................13 
III.D2. Aim 2 Detailed Study Procedures ............................................................................13 
III.A3. Aim 3 Research Design ...........................................................................................14 
III.B3. Aim 3 Sample ..........................................................................................................15 
III.C3. Aim 3 Measurement/Instrumentation .......................................................................16 
IV. Detailed Study Procedures for the Human Study (Aim 3).....................................................16 
V. Risks, Benefits, Safety Plans ................................................................................................19 
V.1. Risks associated with the human study ..........................................................................19 
V.2. Benefits associated with the study..................................................................................20 

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VI. Data Storage .......................................................................................................................21 
VI.1. Paper materials .............................................................................................................21 
VI.2. Electronic data and subject identifying data ...................................................................21 
VI.3.Audio and video recordings .............................................. Error! Bookmark not defined. 
VII. Data Analysis ......................................................................................................................21 
VII.1 Analysis Plan.................................................................................................................21 
VII.2. Sample Size Justification..............................................................................................22 
VIII. Gender/Minority/Pedi-atric Inclusion for Research .............................................................22 
VIII.1 Inclusion of Women and Minorities ...............................................................................23 
VIII.2 Inclusion of Children .....................................................................................................23 
IX. Data and Safety Monitoring Plan .........................................................................................23 
X. References ...........................................................................................................................23 
Appendix A:  Construction and Verification of the Dual-Hose Research-Grade Waterpipe 
(RWP2) and Single-Hose Research-Grade Waterpipe (RWP) ................................................... 1 
Dual-Hose Research-Grade Waterpipe (RWP2) ..................................................................... 2 
RWP2 Construction ............................................................................................................ 2 
Topography Data Collection and Analysis ........................................................................... 3 
Single-Hose Research-Grade Waterpipe (RWP) .................................................................... 5 
RWP Construction .............................................................................................................. 5 
Topography Data Collection and Analysis ........................................................................... 6 
References ............................................................................................................................. 7 
Appendix B:  COVID-19 Related Procedures ............................................................................. 1 
Appendix C:  Example Safe Departure Checklist ....................................................................... 1 
Appendix D:  Data and Safety Monitoring Plan........................................................................... 1 
Responsibility of DSM ............................................................................................................ 2 
Adverse Events ................................................................................................................... 3 
Protocol Amendments/Changes .......................................................................................... 3 
Frequency of DSM.................................................................................................................. 3 
Content of DSM Report .......................................................................................................... 3 
 
 
 
 

IRB Protocol Number: 2021C0039 
IRB Approved Original Date: 05.03.21 
Version: 3.0 
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I. Objectives 
  
The study addresses the following FDA Scientific Interest Areas: Chemistry and Engineering, 
Toxicity, Behavior and Addiction; in that it will determine how specific WP tobacco additives 
affect users puffing behavior, liking, the quantities of toxicants they inhale and take up into their 
bodies, and acute pulmonary affects from WP tobacco smoking. These data will inform potential 
FDA product standards, or maximum limits for additives in WP tobacco, to curb the harm, 
addictiveness and appeal of WP tobacco smoking. Our multidisciplinary team includes experts in 
the conduct of clinical trials, WP smoking behavior, WP emissions testing, tobacco product 
analytical chemistry, psychophysical measurement, and tobacco use risk perception.  In the 
research conducted at The Ohio State University (OSU), we will conduct the study according to 
the following aims.  Note that Aim 3 is the only aim that involves the use of human participants. 
 
Aim 1: Characterize select harmful and potentially harmful constituents (HPHCs) and sugar 
content of four WP tobaccos (one brand prepared four different ways to vary glycerol and 
sugars). A commercial WP tobacco brand will be modified to produce four different 
tobaccos:  
(1) 
Medium glycerol (60 mg/g), Medium sugars (350 mg/g) 
(2)  
High glycerol (120 mg/g), Medium sugars (350 mg/g) 
(3) 
Medium glycerol (60 mg/g), High sugars (510 mg/g) 
(4) 
High glycerol (120 mg/g), High sugars (510 mg/g) 
 
Aim 2: Characterize select HPHCs and sugars yield in mainstream smoke generated from the 
four WP tobaccos characterized under Aim 1 using machine smoking of the research-
grade waterpipe, and a standardized WP puffing regimen. Hypothesis: Mainstream smoke 
generated from WP tobacco with higher concentrations of glycerol and sugars will have 
higher concentrations of HPHCs. 
 
Aim 3: Determine how WP tobacco content impacts puffing behaviors, CO biomarker, 
pulmonary function, nicotine uptake, and perceived sensory attributes and appeal of WP 
smoking. Experienced WP smokers will participate in four laboratory sessions to smoke 
the research-grade waterpipe(s) ad libitum using the four different WP tobacco 
preparations characterized under Aim 1. Hypotheses: Participants will inhale a greater 
total puff volume and report greater appeal when smoking tobacco with medium glycerol 
and high sugars; nicotine uptake will not differ across the four tobaccos. Participants will 
report lower risk perceptions and greater satisfaction when smoking tobacco with 
medium glycerol and high sugars. 
 
Aim 4: Determine the HPHC exposure ranges from the average puffing behaviors measured 
under Aim 2 for each tobacco. Topography data collected for all participants for each 
tobacco type will be averaged to produce four human-derived puffing regimens. Machine 
smoking will be conducted for each tobacco type, using the associated human-derived 
puffing regimens, to determine the range of HPHCs in the mainstream smoke. 
Hypothesis: Toxicant yields will be highest in the mainstream smoke generated from the 
medium glycerol and high sugars tobacco. 
 

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II. Background and Rationale  
 
The scientific premise of this study includes understanding who smokes waterpipe, how the 
harm of using a tobacco product is codified by the FDA, and how sweet WP tobacco additives 
may contribute to the harm of WP tobacco smoking.  
 
II.1. Who Smokes Waterpipe?  
Just within the past 16 years, the global spread of waterpipe (WP) tobacco smoking has exceeded 
predictions.1,29 This worldwide epidemic has increasingly spread among youth, primarily in the 
Middle East and North Africa.29 During 2011-2014, U.S. middle and high school student use 
substantially increased each year with an estimated 1.6 million adolescent WP users.30 
Longitudinal studies indicate the majority of experimentation begins in high school and early 
college,31,32 with up to 13.8% never-users initiating WP in the first month of college.33 Because 
of inconsistent indoor air legislation across the U.S., hookah lounges that support and thrive on 
early social experimentation with WP and other combustible tobacco products are prevalent near 
college campuses.34-36 The 1990s introduction and widespread availability of heavily sweetened 
WP tobacco,1,37 and the persistent perception that WP usage is a safer alternative to cigarette 
smoking,2,4,6-8 are thought to be two of the main contributing factors leading to the rise in 
popularity of WP tobacco smoking among youth. As cigarette smoking declines, more novel 
tobacco products that contain sweet flavors that mask the harshness of tobacco smoke are the 
products most often tried by youth.1,38  
 
II.2. How Do We Codify Harm? 
The U.S. Food and Drug Administration (FDA), through the Family Smoking Prevention & 
Tobacco Control Act,39,40 classifies harm from tobacco products in terms of the chemical 
constituents that can be inhaled, ingested, or absorbed into the bodies of users.10,11 These harmful 
and potentially harmful constituents (HPHCs) have been measured in WP tobacco smoke,12 and 
thus have the potential to cause direct harm via delivery of carcinogens, pulmonary, 
cardiovascular and developmental toxicants, and addictive chemicals to the body,12 or indirect 
harm by facilitating tobacco initiation,41,42 increasing progression to cigarette smoking,43-48 
impeding cessation of cigarette smoking,48,49 and/or increasing the intensity of tobacco product 
use.50  
 
II.3. How Can Sweet WP Tobacco Additives (Sugars and Glycerol) Contribute 
to Direct Harm? 
WP tobacco smoking is associated with increased risk for lung, oral, esophageal, and head and 
neck cancers, and cardiovascular and pulmonary disease.51-54 Despite its growth in popularity 
over the past 30 years, there is little data available on the ingredients in WP tobacco, as the bulk 
of research has focused on toxicant yields in the smoke that is inhaled.12 WP tobacco is very 
different from cigarette or even pipe tobacco15,16 in that initial reports show that sweet tasting 
additives such as sugars and glycerol can comprise up to 70% of WP tobacco by weight.13 

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Glycerol, which is not a sugar but still tastes sweet, can comprise 23–63% of the weight of the 
tobacco.13,22  Sugars such as sucrose, glucose, and fructose can also make up more than a third of 
WP tobacco weight.16 These same compounds are added to cigarettes, but at much lower levels 
(1-5%),55,56 to impart “smoothness” to the smoke and act as moisturizing agents, preservatives 
and solvents for flavor application.57,58  During the heating process, these sweet additives form 
volatile carbonyls, compounds that are known carcinogens and pulmonary toxicants such as 
acrolein, formaldehyde (IARC Group 1 carcinogen) and acetaldehyde (IARC Group 2B).59 
During smoking, glycerol can dehydrate to acrolein;60,61 thus it is not surprising that, given WP 
tobacco is heavily fortified with glycerol, acrolein levels in WP smoke exceed those in cigarette 
smoke by a factor of 15 (see Table 3).12 Acrolein is ciliatoxic and can inhibit both the immune 
system and lung clearance.62,63 When heated, glycerol can also produce formaldehyde and 
acetaldehyde,64,65 which are both contributors to lung cancer in cigarette smokers and chronic 
obstructive pulmonary disease.66 Moreover, acetaldehyde increases the abuse liability of 
nicotine, contributing to the addictiveness of tobacco smoke.67,68 Short-term effects from 
exposure to these carbonyls results in pulmonary irritation and edema.69,70 After their authorities 
were extended to more novel tobacco products like WP,40 FDA proposed adding glycerol to the 
HPHC list.11 
 
Semivolatile furans, including furfuryl alcohol (FFA), furfural, and 5-hydroxymethylfurfural 
(HMF), are another group of carcinogens and respiratory toxicants, that are substantially more 
abundant in mainstream WP smoke than cigarette smoke (see Table 3) due to the abundance of 
added sugars in WP tobacco. Simple sugars are comprised of molecules that contain six carbon 
atoms (hexoses) and five carbon atoms (pentoses).  Hexoses can be chemically transformed via 
thermal dehydration into 5-(hydroxymethyl)-2-furaldehyde (HMF).71 Similarly, pentoses can be 
thermally dehydrated into furfural.72 Concerningly, there are few data available regarding the 
toxicological implications of acute and chronic human inhalation of these compounds. In long-
term inhalation studies, FFA (IARC Group 2B) showed carcinogenic activity in the noses of 
male rats, and the kidneys (renal tubules) of male mice.22,73  Furfural shows carcinogenicity in 
experimental animals via oral administration, but is classified as an IARC Group 3 carcinogen 
due to the inadequacy of human evidence.74 However, FDA Center for Tobacco Products 
considered existing evidence strong enough to propose furfural also be added to the HPHC list.11 
Humans can metabolize both HMF and FFA to form genotoxic and mutagenic compounds (5-
sulfoxymethyfurfural75 and furfural sulphate,76 respectively) in the body via sulfotransferase 
enzymatic activity.77  For this reason, WHO has recommended HMF be given high priority for 
carcinogenic evaluation.78  
 
II.4. How Can Sweet WP Tobacco Additives Contribute to Indirect Harm? 
By masking the unpleasant, bitter taste of nicotine,79 sweet additives play a powerful role in 
initiation of and addiction to tobacco smoking.80 Tobacco industry documents reveal that 
cigarette manufacturers, in an attempt to maximize the appeal of cigarette smoke, conducted 
human studies to determine the optimal ratio of added sugars to nicotine content.58 Adding 
sugars increased the concentration of carbonyls in the smoke, which, as acids, reduced the smoke 
pH, resulting in nicotine being inhaled predominantly in its protonated form.58 Protonated 
nicotine is more appealing for first-time users because it is less harsh than freebase nicotine and 
is therefore easier to inhale.81,82 Nicotine is a highly addictive compound, and WP tobacco 

IRB Protocol Number: 2021C0039 
IRB Approved Original Date: 05.03.21 
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smoking delivers enough nicotine to support nicotine addiction.83 Moreover, glycerol can 
enhance the transfer of nicotine to the smoke increasing nicotine delivery to the user,17  and both 
glycerol and sugars reduce the harshness and increase the appeal of WP tobacco smoke.27,28 
There is evidence that WP tobacco smoking may function as a gateway for initiation45-48 and 
regular use of combustible cigarettes.43,84  
 
II.5 Why Use Investigational Tobacco Products? 
Some crossover studies have examined WP tobaccos that contain varying levels of glycerol and 
presence/absence of characterizing flavors and found differences in smokers puffing behaviors, 
toxicant exposure and appeal when using these products.27,85 However, the measured differences 
cannot be related to the level of specific additive in those studies because the WP tobacco brands 
tested had numerous other differences that were not quantified. To systematically examine the 
direct and indirect harm associated with specific additives it is necessary to precisely fortify the 
WP tobacco with specific levels of the additives. As the initial raw material for the manipulated 
tobacco, we will use a popular unflavored WP tobacco with low glycerol content.  
 
To ensure the ITPs are ecologically valid, we will fortify the unflavored WP tobacco with the 
correct type and concentration of sugars. There is a common belief that WP tobacco is sweetened 
by adding honey, perhaps because “ma’assel” is taken from the Arabic “muassel,” which means 
“honeyed”.86  About 70% of honey is made up of two sugars, glucose and fructose, that are 
present in roughly equal amounts.87,88 Given the low cost of WP tobacco ($3 for a pack that has 
the same amount of tobacco as an $8 pack of cigarettes), and that the worldwide market for it 
exceeded $1.9B in 2017,89 it is more likely that inexpensive syrups are used.88,90 Table II.1 
shows the sweeteners that comprise honey and sugar, and less expensive syrups such as high 
fructose corn syrup (HFCS) and molasses, in comparison to the sugar content data we collected 
on flavored and unflavored WP tobacco.16 Based on these data, the unflavored WP tobacco may 
have been sweetened with a combination of sugar, honey and molasses; but molasses and HFCS 
55 are the more likely candidates given their availability and low cost. For the flavored WP 
tobacco, sucrose was not detected and thus neither molasses nor sugar were used. Molasses and 
HFCS 42 were most likely used to sweeten the flavored WP tobacco, given their availability and 
low cost. To more precisely isolate the additives of interest, we will fortify the unflavored 
tobacco with food grade (>99% purity) glycerol, glucose and fructose to produce sweetness and 
glycerol levels that do not exceed those measured in the flavored tobacco. 
 
 
 

IRB Protocol Number: 2021C0039 
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TABLE II.1. SUGAR CONTENT OF SWEETENERS, AND FLAVORED AND UNFLAVORED WP TOBACCO. 
Sweetener 
Hexoses, wt % 
Glycerol, wt % 
Sucrose 
Glucose  
Fructose 
Corn Syrup 
- 
100 
- 
- 
HFCS 55 
- 
45 
55 
- 
HFCS 42 
- 
58 
42 
- 
Honey 
 
32 
39 
- 
Molasses 
34 
8 
8 
- 
Sugar 
99.7 
- 
- 
 
Flavored WP Tobaccoa 
- 
30 
29 
38 
Unflavored WP Tobaccoa 
25 
34 
37 
4 
“ - ” = negligible; a = wt % expressed as a fraction of the sum of the primary sweeteners 
sucrose, glucose, fructose and glycerol. 
 
Not all sugars have the same sweetening power. A sweetness hierarchy for sugars has been 
determined empirically by asking participants to detect sweetness in progressively increasing 
concentrations of single sugars.91,92  Among the most highly fortified sweeteners added to WP 
tobacco, sucrose and fructose have the highest relative sweetness.92 A power function to predict 
the relative sweetness of sugars based on their concentration and individual sweetening power 
was determined empirically from human trials.92 Because the unflavored WP tobacco that is our 
starting material contains sucrose, it is important to tally the relative sweetness contributions of 
all the sugars and glycerol in the ITPs to produce medium and high sweetness levels, while also 
producing medium and high glycerol levels.  
 
Equally important to consider is the nicotine content of each of the proposed ITPs, not only 
because nicotine can drive use behavior, it also produces the bitterness that the sugars and 
glycerol are added to offset. Given that the additives can comprise more than half the weight of 
the WP tobacco, we must normalize the relative sweetness by the nicotine coming from the 
tobacco leaf itself. We define the term “Starter Product Index” (SPI) to assign a numerical 
quantity to the mass of sugars fortified in the tobacco to offset the bitter harshness of nicotine: 
 
SPI = sum of the relative sweetness of the sugars and glycerol / mass of nicotine 
 
The proposed additive concentrations in the ITPs are shown in Table II.2. For ecological 
validity, the SPI of the proposed ITPs are bracketed by the SPIs for the unflavored and flavored 
commercial WP tobaccos, also shown in Table II.2.  
 
 

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Table II.2. The proposed Investigational Tobacco Products (ITPs) are fortified with ecologically valid levels of sweet 
additives. 
 
WP Tobaccos 
Concentration (mg additive/g of tobacco) 
Relative 
Sweetness 
SPI 
Glycerol 
Sucrose 
Glucose 
Fructose 
Proposed 
ITPs 
Medium Glycerol 
Medium Sugars 
60 
90 
125 
135 
106 
64 
High Glycerol 
Medium Sugars 
120 
90 
125 
135 
101 
68 
Medium Glycerol 
High Sugars 
60 
90 
195 
225 
110 
91 
High Glycerol 
High Sugars 
120 
90 
195 
225 
103 
99 
Commercial 
Products 
Unflavored WP 
Tob 
13 
90 
125 
135 
106 
59 
Flavored WP Tob 
246 
0 
195 
190 
106 
103 
 
II.6. Why Use a Research-Grade Waterpipe? 
Commercially available WPs and their components vary widely in design and durability, 
including differences in fabrication materials used for stems, bases, bowls, and hoses; sealing 
joint designs and degree of leak-tight fit; and diameter of flow path. All of these components can 
affect puffing intensity, and exposure to nicotine, CO and other harmful toxicants.93-95 A rugged, 
reproducible WP is needed to reliably determine the variability in human WP puffing behavior 
(topography). Our team developed and qualified the use of standardized single- and dual-hose 
WPs (RWP and RWP2) equipped with puffing topography analyzers.96 The RWP2 is shown in 
Figure II.1.  The RWPs both operate with known precision and accuracy, and are well-accepted 
by established WP smokers in terms of satisfaction and reward.94-96 More information on the 
RWP and RWP2 is provided in Appendix A to this protocol. 
 
 

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II.7. What will this study add? 
 
The study design is highly innovative in 
several ways in that it 1) includes human 
and machine smoking of a manipulated, 
ecologically valid WP tobacco product to 
isolate the effects of glycerol and sugar 
additives, 2) it draws on a comprehensive 
set of chemical measurement techniques 
to estimate ranges of volatile and 
semivolatile HPHCs delivered to the 
smoker, and 3) it uses a validated,  
research grade waterpipe (RWP) of 
known precision and accuracy. More 
detail on these study design aspects is 
given below. 
 
Several studies13,96-98 have used 
behavioral data and machine smoking to 
characterize the chemical composition, 
toxicity, and carcinogenicity of popular 
WP tobaccos. To the best of our 
knowledge, this is the first human study 
to use a precisely manipulated WP 
tobacco in order to systematically 
examine the effects of the additives of 
greatest interest with respect to 
evaluating direct and indirect harm 
associated with WP smoking: glycerol, fructose and glucose. As the initial raw material for the 
manipulated tobacco, we will use a popular unflavored WP tobacco with low glycerol content 
(see Table II.2). Our data on the sweet additives measured in a popular flavored and a popular 
unflavored WP tobacco were used to ensure the identities and concentrations of additives in the 
ITPs are ecologically valid. 
 
The multi-analyte chemical characterization techniques for the measurement of established and 
proposed HPHCs have been developed and validated by MPI Brinkman for use in tobacco 
regulatory research on WPs.13, 16, 95, 96  Although the chemical composition of WP tobacco and 
emissions are important, other factors also contribute to the potential health impacts of WP 
smoking. The subjective data collection techniques include the application of a food industry-
established psychophysical measurement technique100-104 to quantify the perceived intensities of 
tobacco flavors. Moreover, general WP harm perceptions (absolute, relative) and specific health 
risk perceptions (e.g., addictiveness, lung cancer, oral cancer) will be captured using survey 
items adapted from the US Population Assessment of Tobacco and Health Study105 and  being 
used in MPI Brinkman’s current WP study (R01CA229306). We will also employ either the 
RWP or RWP2,96 novel, standardized devices developed by MPI Brinkman that operate with 
high precision and accuracy for the measurement of WP puffing behavior for one (RWP) or two 
 
Figure II.1. A research-grade waterpipe (RWP) equipped with 
human puffing topography data collection. 

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people (RWP2) smoking from the same WP. The research-grade waterpipe will also be used for 
the generation of machine-smoked constituent data using both human puffing topography 
collected in the proposed study, and an accepted standardized, multi-staged puffing regimen 
derived from a previously examined cohort of experienced WP smokers smoking the RWP and a 
flavored WP tobacco.95,96 The RWP2 was developed from the single hose research-grade 
waterpipe, the RWP, and is a tool with benchmarked performance metrics that was well-accepted 
by experienced WP smokers in a previously conducted clinical research.94-96 The RWP2 is 
fabricated from inert materials that do not scavenge, via adsorption to surfaces, nor generate via 
thermal degradation or off-gassing, the HPHCs of interest being measured in mainstream smoke. 
 
Currently there are no regulations governing the content of WP tobacco. Because mandated 
changes in tobacco content may lead to unintended consequences that ultimately result in public 
health declines, human use behaviors must be well understood prior to implementing regulatory 
product standards. Compensation, or the degree to which a smoker changes their intake of smoke 
to make up for the changes made to the tobacco product, is one possible unintended 
consequence. The proposed smoke HPHC yield measurements, made in smoke generated using 
both a standardized and a specific (to human-use of the tobacco) puffing regimen, along with the 
participants’ plasma nicotine boost from using the product, will allow us to determine if 
compensation, and thus greater HPHC exposures, is associated with the presence/absence of 
sweet additives.  
 
This study will be the first to combine powerful analytical chemistry techniques, CO and 
nicotine biomarkers, cutting edge psychophysical measurement tools, and risk perception 
instruments to map the relationship between sensory experiences and preferences of sweetness 
and flavor to specific additive content in WP tobacco that affect these experiences and 
preferences. The data from the proposed study will provide direct links between WP tobacco’s 
primary additives, CO and nicotine biomarkers, smoker preferences, perceptions of harm and 
puffing behaviors, and the subsequent range of HPHC exposures associated with these additives 
and behaviors. This work will inform the FDA regarding its regulatory authorities surrounding 
the manufacture, distribution and marketing of WPs, and inform the evaluation of product 
applications and development of product standards, such as glycerol and sugar content, to reduce 
the resulting public health toll from WP tobacco product use in the US. 
 
 
 

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III. Procedures 
 
III.1. Research Design Overview 
 
The proposed study will include preparing and characterizing the content  of 4 investigational 
tobacco products (ITPs) (Aim 1); characterizing the mainstream smoke toxicant emissions from 
machine smoking the 4 ITPs using a single, established puffing regimen (Aim 2); measuring 
human puffing behavior, general harm and specific health risk and flavor perceptions, lung 
function, and biomarkers of exposure in a group of established WP smokers smoking the 4 ITPs 
in the laboratory (Aim 3), and estimating toxicant exposure ranges using machine smoking and 
puffing regimens derived from the human laboratory testing (Aim 4). 
 
 
AIM 1 
 
III.A1. Aim 1 Research Design 
Characterize the HPHC and sugar content of four WP tobaccos (one brand prepared four 
different ways to vary glycerol and sugars). Aim 1 will focus on the preparation and 
subsequent chemical characterization of WP tobacco with high and medium levels of sugars and 
glycerol. A commercial WP tobacco will be manipulated to prepare a 2 x 2 matrix of 4 tobaccos 
with medium and high levels of glycerol and sugars. 
 
III.B1. Aim 1 Sample 
There are no human participants planned for Aim 1.  Sufficient quantities of the commercial WP 
tobacco for the grant will be purchased at the beginning of the study by a single vendor and 
stored in sealed containers at room temperature. 
 
III.C1. Aim 1 Measurement/Instrumentation 
Nicotine, humectants, sugars, carbonyls, and semivolatile furans, as detailed in Table III.3,  will 
be determined twice: once at the beginning and once at the end of the human study. Nicotine 
content of the unburned tobaccos will be determined using gas chromatography with mass 
spectral detection (GC-MSD).118 Humectant content will be determined using GC with flame 
ionization detection (FID),22 or GC/MS. Sugar content, including sucrose, glucose, and fructose, 
will be determined using high performance liquid chromatography (HPLC) techniques.155, 156 
Carbonyls will be determined in the tobacco as their 2,4-dinitrophenylhydrazine (DNPH) 
derivatives using liquid chromatography tandem mass spectrometry.22  Semivolatile furans will 
be determined in the tobacco using a liquid chromatography method previously used by our 
team.13 
 
Total aerobic microbial and total yeast and mold counts (TAMC and TYMC) will be determined 
according to USP 61,157 after the ITPs are prepared, midway through participant processing, and 
at the completion of participant processing. 
 
 

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III.D1. Aim 1 Detailed Study Procedures 
Selection of WP Tobacco. As the starting material for the preparation of the four test tobaccos, 
we will use a popularly smoked unflavored tobacco (see Unflavored Tobacco in Table II.2) 
claimed by the manufacturer to “present a plain molasses flavor.” This tobacco has a low 
glycerol content that is amenable to the proposed product manipulation.16 
 
Preparation of WP Tobacco. A popular, commercially available WP tobacco brand (Moassel 
Saloum) will be prepared four different ways:  
(1) 
Medium glycerol (60 mg/g), Medium Sugars (350 mg/g) 
(2) 
High glycerol (120 mg/g), Medium Sugars (350 mg/g) 
(3) 
Medium glycerol (60 mg/g), High sugars (510 mg/g) 
(4) 
High glycerol (120 mg/g), High sugars (510 mg/g)  
Several packages of the tobacco, purchased from the same source, will be combined, twigs and 
sticks removed, homogenized, and split into four equal batches. Food grade glycerol, glucose 
and fructose (>99% purity) will be added to the tobacco to obtain the concentrations shown for 
the ITPs in Table II.2.   
 
Determination of Nicotine Content. To aid in determining if humectant levels affect the transfer 
of nicotine from the tobacco into the mainstream smoke, the nicotine content of Batches 1-4 will 
be individually determined using a method previously qualified by our team.118 An aliquot (0.25 
g) of each tobacco sample will be extracted individually for four hours in a mixture of isopropyl 
alcohol, methyl tert-butyl ether, and sodium hydroxide using an orbital shaker table at 160 rpm. 
The extraction solvent mixture will contain a known quantity of quinoline as the internal 
standard (IS). An aliquot of the extract will be quantitated for total nicotine using gas 
chromatography with mass spectral detection (GC-MSD).  
 
Determination of Humectant Content. To aid in determining the fraction of humectants 
transferred to the mainstream smoke and verify the levels of added glycerol, the glycerol and 
propylene glycol content of each tobacco preparation will be determined using a Health Canada 
method adopted for use on WP tobacco by Schubert et al.22  
 
Determination of Sugars in the Four Tobacco Preparations. The sugar content of the four 
prepared batches will be quantified. An aliquot (1 g, 3 replicates) of each tobacco preparation 
will be extracted into HPLC grade water and analyzed using validated methods based on 
standard liquid chromatography (LC) techniques.155,156  
 
Determination of Carbonyl Content. To determine the fraction of carbonyls in the tobacco vs. 
those generated as a result of additives, carbonyls will be determined in the tobacco as their 2,4-
dinitrophenylhydrazine (DNPH) derivatives using liquid chromatography tandem mass 
spectrometry.22 
 
Determination of Semivolatile Furan Content. To determine the fraction of semivolatile furans in 
the tobacco vs. those generated as a result of additives, semivolatile furans will be determined in 
the tobacco using a liquid chromatography method previously used by our team.13 
 

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Determination of Aerobic Bacteria, Yeast and Mold. Because sugars and glycerol can function as 
preservatives in WP tobacco,17 it is important to document the storage stability of the ITPs. 
Therefore, the total aerobic microbial and total yeast and mold counts (TAMC and TYMC) will 
be determined according to USP 61,157 after the ITPs are prepared, midway through participant 
processing, and at the completion of participant processing. 
 
 
AIM 2 
 
III.A2. Aim 2 Research Design 
Aim 2: Characterize HPHC and sugars yield in mainstream smoke generated from the four 
WP tobaccos characterized under Aim 1 using machine smoking of the RWP2, and a 
standardized WP puffing regimen.  Aim 2 will focus on quantifying the yields of toxicants in 
the mainstream smoke from the four WP tobaccos. 
 
III.B2. Aim 2 Sample 
There are no human participants planned for Aim 2. Aliquots of the four tobacco preparations 
will be machine smoked and subsequent emissions will be collected/quantified for the toxicants 
of interest. 
 
III.C2. Aim 2 Measurement/Instrumentation 
Nicotine, sugars, glycerol, carbonyls, and semivolatile furans, as shown in Table III.3, will be 
quantified in the mainstream WP tobacco smoke for the four tobaccos. Additional sets of 
machine smoking will be individually conducted for each tobacco batch, the gases will be 
collected as their derivatives in impingers and bulk particles will be collected on glass fiber 
filters located at the mouth-end of the WP hose, and the resulting impinger solutions and filters 
will be extracted and analyzed according to the methods described in Aim 1. 
 
III.D2. Aim 2 Detailed Study Procedures 
Generation of WP Mainstream Smoke. The four tobacco preparations will be machine-smoked 
using a research-grade waterpipe, a calibrated smoking machine (Shisha Smoker, Borgwaldt), 
and an accepted standardized puffing regimen based on subjects smoking a popular flavored 
tobacco using the RWP in a laboratory environment.13,95,96 Particles and organic chemicals will 
be characterized and collected at the mouth-end of the WP hose. 
 
 
Determination of HPHCs in Mainstream WP Tobacco Smoke. Total particulate matter and the 
HPHC content of the volatile and semivolatile fractions of the mainstream WP tobacco smoke 
will be characterized. Selected HPHCs and other toxicants are shown, along with reported WP 
smoke yields and health effects, in Table II.3. Machine smoking will be conducted and total 
particulate matter will be collected at the mouth end of the WP hose. Particle samples will be 
chemically extracted and analyzed for the target semi-volatile HPHCs using GC/MS and LC-
MS/MS. Gas phase HPHCs will be collected in impingers and quantified as their DNPH 
derivatives. 

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AIM 3 
 
III.A3. Aim 3 Research Design 
Aim 3: Determine how WP tobacco content impacts puffing behaviors, CO biomarker, 
pulmonary function, nicotine uptake, and perceived sensory attributes and appeal of WP 
smoking. Aim 3 will focus on using a group of established adult and young adult WP smokers, a 
cross-over study design, CO and nicotine biomarkers, spirometry, cutting edge psychophysical 
measurement tools, and risk perception instruments to map the relationship between sensory 
experiences and preferences of sweetness and flavor to specific additive content in WP tobacco 
that affect these experiences, preferences, acute health effects, and toxicant exposures. 
 
Table III.3. Selected compounds for chemical characterization. 
Compound 
Mainstream Smoke Yield12 
Reason for Selection 
Waterpipe 
Cigarette 
Nicotine, TPM 
Nicotine (total), mg 
0.01 – 9.29 
0.1 – 3 
Major addictive constituent 
Total particulate matter, 
mg 
242 – 2,350 
1 – 27 
Cardiovascular, pulmonary toxicant 
Humectants, mg 
Glycerol 
423 
1.4 – 2.1158 
Pulmonary toxicants, potential for carcinogenic 
degradation products 
Propylene glycol 
211 
0.3 – 0.6159 
Semivolatile Furans, µg160 
5-(Hydroxymethyl)-2-
furaldehyde (HMF) 
2,420 – 
62,300 
1.3 – 7.4 
Potential for genotoxic and mutagenic metabolites 
in the lung 
Furfuryl alcohol (FFA) 
55.7 – 552 
18 – 65 
Nasal, kidney carcinogen; potential for genotoxic 
and mutagenic metabolites in the lung 
2-Furoic acid (2-FA) 
32.0 – 401 
na 
Pulmonary toxicant 
Furfural (2F) 
29.6 – 206 
0.71 – 27.5 
Potential human carcinogen; data gap 
2-Furyl methyl ketone 
(2-FMK) 
4.77 – 12.5 
na 
Pulmonary toxicant; data gap 
5-Methyl-2-furaldehyde 
(5-M2F) 
4.62 – 215 
na 
Pulmonary toxicant; data gap 
Carbonyl Compounds, µg 
Acetaldehyde 
120 – 2,520 
0.01 
Nasal mucosa, laryngeal carcinomas; enhances 
nicotine’s addictiveness 
Acetone 
20.2 – 118 
506 – 773161 
Pulmonary toxicant 
Acrolein 
10.1 – 892 
60 – 240 
Lung carcinomas; pulmonary toxicant 
Benzaldehyde 
<0.34 
0.4 – 6.4162 
Pulmonary toxicant 
Butyraldehyde 
10.9 – 70.6 
5.5 – 51.2163 
Respiratory and cardiovascular toxicant 
Formaldehyde 
36 – 360 
0.02 
Nasal sinus, nasopharyngeal carcinoma, leukemia 
Propionaldehyde 
5.71 – 403 
48.4 
Pulmonary and cardiovascular toxicant 
na = data gap, data not available; compounds in blue shaded rows are classified as HPHCs; compounds in orange shaded rows are 
proposed as additions to the HPHC list.11 
 
 

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III.B3. Aim 3 Sample 
We will recruit a total of 60 healthy adult men and women  with the goal of having complete 
data sets for 50 participants. Participants will meet the eligibility criteria  
shown in Table III.4. Established waterpipe smokers will be recruited from advertisements 
 
Table III.4. Participant inclusion and exclusion criteria. 
Inclusion 
Justification for Inclusion 
Sufficient understanding of consent form and study 
procedures 
Necessary to ensure that participants are 
adequately informed about the study 
Age 21-50 years old 
Legally of age to purchase, use, and possess 
tobacco and provide informed consent 
Experienced WP smokers defined as having smoked a WP 
at least 3 times per month in the last month 
To assure that participants are experienced 
WP smokers, and increase the probability of 
subjects being able to comfortably smoke 
Willing/able to abstain from nicotine product use for at 
least 12 hours prior to the laboratory visits  
Necessary to normalize participants’ 
willingness to smoke WP across visits 
Willing to attend four laboratory sessions at the same time 
of day lasting approximately 3 h each 
Necessary to complete all testing sessions 
and provide adequate power for data 
analysis 
Read and speak English. 
To ensure that participants are adequately 
informed about the study and can follow the 
Exclusion 
                       Justification for Exclusion 
Evident intoxication on any visit 
Intoxication would interfere with informed 
consent and study procedures 
Exhaled breath CO > 10 ppm 
Indicates recent combustible tobacco use. 
Pregnancy (known or suspected), trying to become 
pregnant, breastfeeding, unwillingness to use contraception 
for the duration of the study. 
Unethical to enroll pregnant or 
breastfeeding women for smoking research 
Significant smoking-related disease (by history) 
Unethical to enroll subjects with tobacco-
related diseases 
Any of the following in the past 30 days (self-report): 
• Uncontrolled asthma or asthma that is worse than usual. 
• Severe respiratory allergies, such as wheezing, coughing, 
shortness of breath when exposed to known allergens. 
• Acute upper or lower respiratory illnesses like the flu, 
common cold, or pneumonia. 
• Any other serious lung infection or disease, such as 
tuberculosis, cystic fibrosis, or lung cancer. 
• Hospitalization for difficulty breathing 
Unethical to enroll participants with 
significant pulmonary dysfunction or 
communicable disease 
Currently engaging in a WP tobacco smoking quit attempt Unethical to interfere with tobacco cessation 
 
 

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in college and community newspapers and through a variety of media outlets and the internet, 
including Study Search, as well as community events. Additionally, we will place flyers in WP 
cafés, and stores that cater to WP smokers, such as head shops and ethnic grocery stores. 
Participants from other studies who have agreed to be contacted regarding other study 
opportunities will also be contacted. Staff from those studies will prepare contact letters/emails 
and call participants on behalf of this study. Participants interested will be referred to this study 
for screening. Advertisements will direct interested potential participants to complete a secure, 
online screener. Participants will access the screening questionnaire using a public survey link 
generated by REDCap.  Information regarding eligibility and the participants’ email addresses 
and phone numbers will be uploaded into a secure database. Potential participants will then be 
further screened by phone. Further assessment will be completed in person in a private setting. 
We will facilitate visits by offering weekend appointments and using additional retention 
strategies (e.g., reminder calls/texts/emails) that have been successfully used in the past for WP 
studies of similar size, crossover design, and subject burden.  
 
 
III.C3. Aim 3 Measurement/Instrumentation 
Exhaled CO levels will be used to determine eligibility and CO boost (post- minus pre-smoking 
levels) from WP smoking using a handheld electrochemical sensor. The primary quantitative 
outcomes will be exhaled breath CO and plasma nicotine boost172 (post- subtracted from pre-
smoking levels), puffing topography, spirometry measures [e.g., forced vital 
capacity (FVC), forced expiratory volume in 1 second (FEV1), FEV1/FVC, peak expiratory flow 
(PEF) and forced expiratory flow at 25-75% of FVC; and height and weight will also be assessed 
to calculate expected spirometry measures for participants.169 In addition, data will be collected 
electronically on participants’ demographic characteristics, tobacco use history, nicotine 
dependence (Hooked on Nicotine Checklist,164 and Lebanese WP Dependence Scale165), general 
harm and specific health risk perceptions, and subjective effects, including Direct Effects of 
Tobacco scale,166 Direct Effects of Nicotine scale,167 and Questionnaire for Urges to Smoke.168 
 
The primary qualitative outcomes will be verbal and non-verbal communication regarding the 
WP smoking experience and flavors perception. To determine the relative intensity of specific 
flavors, e.g., sweetness, we will use the general version of the Labeled Magnitude Scale 
(gLMS),102-104 to obtain data that would permit ratio comparisons of perceived sensation 
magnitudes. To assess the degree of liking or disliking of flavors, we will use the Labeled 
Hedonic Scale (LHS) to collect ratio-level data on the magnitude of liking and disliking of 
sensation.134 Other themes that emerge from the discussions will also be coded. 
 
IV. Detailed Study Procedures for the Human Study (Aim 3) 
 
 
Study subjects will participate in a series of 4 laboratory sessions, each visit separated by at least 
a week, in which they smoke one of 4 tobacco preparations (blinded) using the RWP2, shown in 
Figure II.1, in a randomly assigned sequence, using the block randomization method.174 Smoking 
sessions will be carefully standardized: they will take place in a controlled setting, individual 
roomsdesignated for smoking research with sufficient ventilation rate at the Center for Tobacco 

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Research) within one hour of the same time-of-day after overnight nicotine abstinence (≥8 hours, 
exhaled CO ≤ 10 ppm173).  
 
Research staff will light and add the charcoal and record tobacco and charcoal weights. The 
research-grade waterpipes will be prepared by the laboratory staff, and participants will smoke 
ad lib to satiation. Participants will be limited to one bowl (15 g) of tobacco and one quick-light 
charcoal (~10 g).  
 
Prior to the start of the first laboratory visit, a research assistant will explain the study to the 
participant and answer any questions they may have.  The participants will give an exhaled 
breath sample into the handheld CO monitor to determine if they are eligible to participate in the 
laboratory smoking session. If the participant’s exhaled CO concentration is less than or equal to 
10 ppm) they are deemed eligible to participate further, and will be asked to read the informed 
consent form.  Once all participant questions have been answered, the participants will each be 
asked to sign their consent to participate in the study.  Each female participant of child-bearing 
age will be asked to provide a urine sample for use in conjunction with a “dip-stick” pregnancy 
test to determine participation eligibility.  Female participants with a negative urine pregnancy 
will be eligible to participate if they provide written agreement (via signing the consent form) to 
use reliable contraception throughout their participation in the study.  
 
Each participant will then be asked to provide a pre-smoking blood sample by venipuncture. If 
the phlebotomist is unable to take a sufficient blood sample (10 mL) from the participant, the 
participant will be deemed ineligible to participate further in the study.  Participants will then 
answer demographic and tobacco use questions.   
 
After a successful blood sample collection, all participants will provide responses electronically 
to a series of questionnaires gathering information on nicotine dependence, and other subjective 
ratings. Participants will complete a practice session to learn how to record flavor perceptions.  
Height and weight will be measured for each participant. 
 
Smoking will take place in a negative pressure room with a stable ventilation rate sufficient to 
keep ambient CO levels < 30 ppmv. Participants will be seated in comfortable chairs designed 
for ease of blood collection by venipuncture.  Using warm bottled water (body temperature), 
participants will clean their mouth and spit into a cup three times.  Then, for 20 seconds, they 
will smell the unburned tobacco that they are assigned to smoke during that visit, and complete 
the questionnaire on their flavor perceptions.  Participant lung function data will be collect using 
a handheld spirometry device.  Exhaled breath CO levels will be collected for each participant 
just prior to the smoking session. 
 
Participants will be encouraged to go to the bathroom prior to the start of the smoking session. 
The participant will be provided with a research-grade waterpipe that has been prepared with 15 
g of one of the four tobacco preparations and one lit, quick-light charcoal.  The participants will 
be instructed to smoke as they normally would or at a rate that is natural for them, through the 
hose/mouthpiece for up to 60 minutes. Puff topography parameters, including puff volume, 
duration, average and peak flow rate, and interpuff interval, will be measured continuously 
during the smoking session using the research-grade waterpipe.96 After the first 10 minutes of 

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smoking, and at the end of each smoking session, participants will be asked to rate the perceived 
intensities of relevant sensory attributes, including “sweetness,” “harshness,” “bitterness,” 
“smoothness,” and overall intensity, using the general version of the Labeled Magnitude Scale 
(gLMS). We will also collect data on liking/disliking by using the Labeled Hedonic Scale (LHS).  
If participants choose to smoke for 45 minutes, they will be asked to provide exhaled breath CO 
levels.  If any participants breath CO levels exceed 50 ppmv, the smoking session will be 
stopped.  Otherwise, participants may smoke ad libitum for as long as they choose, up to 60 total 
minutes.  At the end of the smoking session, a second exhaled breath sample will be collected, 
and participants will then provide answers to questions regarding flavor perception.  They will 
give a second blood sample and perform another lung function test, as before.  Afterwards they 
will answer questions regarding nicotine dependence and their waterpipe smoking experience.  
 
At the end of each smoking session, participants will respond to all subjective questionnaires 
including the gLMS and LHS. For each test tobacco, participants will be asked to rate perceived 
intensities of all relevant flavor qualities (i.e., sweetness, harshness, bitterness, smoothness, 
overall intensity, and specific flavor) as well as their liking/disliking on gLMS and LHS. 
 
Sensory Intensity and Hedonic Scales. To determine the relative intensity including sweetness, 
harshness, and bitterness, and specific attributes of added flavors (e.g., apple) as well as the  
degree of liking/disliking for the four tobacco preparations, a psychophysical measurement 
technique will be employed to quantify the relationship between the tobacco flavors and their 
perceived intensity. Specifically, we will use the general version of the Labeled Magnitude Scale 
(gLMS),102-104 to obtain data that would permit ratio comparisons of perceived sensation 
magnitudes. The gLMS is a category-ratio scale bounded by “no sensation” at the bottom and 
“strongest imaginable sensation of any kind” at the top, with its intermediate intensity labels 
(weak, moderate, strong, and very strong) spaced logarithmically according to the empirically 
determined semantic magnitude.102,103 To assess the degree of liking or disliking of flavors, we 
will use the Labeled Hedonic Scale (LHS) to collect ratio-level data on the magnitude of liking 
and disliking of sensation.134 The properties of LHS are similar to the properties of the gLMS 
where the bipolar gLMS has ‘‘neutral’’ at its midpoint with negative descriptors to the left 
bounded by “strongest imaginable displeasure of any kind,’’ and positive descriptors to the right 
bounded by ‘‘strongest imaginable pleasure of any kind.”   
 
 
Biological Sample Collection.  Exhaled breath CO (via handheld electrochemical cell) and whole 
blood will be collected before and immediately after the WP smoking sessions according to 
previously developed protocols. Exhaled CO will also be measured after 45 minutes of ad 
libitum smoking, should participants choose to smoke that long. If after smoking for 45 minutes, 
any participant’s exhaled breath is greater than 50 ppm, the smoking session will be stopped. 
Within one hour of collection, the blood will be separated using centrifugation (1200 xg for 15 
minutes), and the plasma layer will be transferred into a cryotube and stored at -80 °C until 
analysis for nicotine according to validated methods using LC-MS/MS.172 
 
Spirometry. Before and after smoking at each clinic visit, subjects will be asked to take a deep 
breath and then instructed to exhale forcefully into a handheld spirometer [NDD EasyOne Air 
Spirometer (NDD Medical Technologies, Andover, MA),169 or similar spirometer] according to 
American Thoracic Society Guidelines.  This maneuver will be repeated up to 5 times in order to 

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ensure reproducible results. This non-invasive procedure will take up to 15 minutes to perform. 
Participants’ height and weight will also be measured to calculate their expected spirometry 
measures as thresholds to use for determining potential impairment in lung function.  
 
Safe Departure and Remuneration. At the end of each clinic session, research assistants will 
assess each of the participants for safe departure using a standardized checklist.  An example 
checklist is provided in Appendix C. Visits 2-4 will be similar to the first visit, but the tobacco 
and use history data collection and the urine pregnancy test (women only) will not be repeated.  
Participants will receive $50 for each laboratory visit and a bonus payment of $50 for completing 
all four visits, for a total of $250 at the conclusion of the last laboratory visit. 
 
V. Risks, Benefits, Safety Plans 
 
V.1. Risks associated with the human study 
Potential risks are minimal for the human participant study. Potential risks associated with the 
procedures and equipment that will be used to collect the exhaled breath CO, topography, and 
pulmonary data are minimal. Potential risks resulting from collecting blood samples include being 
unable to provide blood samples because forearm veins are not adequate, feeling light-headed or 
faint, nausea, dizziness, hematoma or bruising at or near the needle site. We minimize this risk by 
using certified technicians with extensive experience in collecting blood samples. However, if the 
participant does not feel comfortable for any reason and wants to end their participation in the 
study, he/she can do so at any time without penalty. Some of the survey questions may upset 
individuals; however, we have used these questions and procedures in many previous studies 
without upsetting participants.  
 
There are no potential risks involved in the collection of waterpipe smoking topography, as the 
device does not change the resistance to draw or toxicity of the smoke inhaled.  The data are 
acquired continuously to an electronic file, and may be displayed on a laptop screen that is not 
viewable by either participant. Therefore the possibility of the participant amplifying their puffing 
rate in order to see the on-screen response is eliminated. 
 
Participants may feel hungry because they will not be permitted to eat until after their post-
smoking lung function test is completed. Pulmonary function studies involve forceful respiratory 
maneuvers and occasionally people develop light-headedness during the procedures or soreness of 
the chest for a few days.  Subjects will be seated and closely monitored by trained/certified 
personnel during those procedures.   
 
The RWP that participants will smoke at the clinic is specially designed for investigational 
research only, and as such may have unknown health risks.  Both the single- and dual-hose 
versions of the RWP have been used by participants in several previous studies. 
 
A potential risk is loss of confidentiality. We have rigid procedures in place to protect against 
loss of confidentiality. Participant identifiers will be kept in a locked file cabinet. This study is not 
unlike our previous work investigating the efficacy of WP warning labels; we collect the 

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information and keep it confidential and anonymous through the use of IDs only and not 
personally identifying information. 
 
De-identified, coded participant data from the general version of the Labeled Magnitude Scale 
(gLMS) and the Labeled Hedonic Scale (LHS) will be analyzed by Dr. Juyun Lim, Associate 
Professor, Department of Food Science and Technology, Oregon State University. Dr. Lim will 
receive all scale ratings, matched with participant identification codes, date of participant 
laboratory visit, period data was collected (e.g., pre-smoking, post-smoking, etc.) and select 
demographic and tobacco use history data such as sex, age, and how many cigarettes smoked per 
day.  All data sent to Dr. Lim will be stripped of participant identifying information.    
 
During an ongoing virus pandemic (such as COVID-19), we will follow strict safety procedures 
to reduce risk of spread of the disease among the participants and staff. Depending on currently 
recommended guidelines, these procedures may entail: 
• Collecting data on forehead temperature and assessing symptoms of viral infection at each 
visit 
• Wearing personal protective equipment and sanitizing hands 
• Routine cleaning of surfaces and equipment 
• Implementing physical distancing 
 
V.2. Benefits associated with the study 
There are no direct benefits to be gained by participants. However, the anticipated societal 
benefit resulting from this study is considerable, given the increasing prevalence of WP 
tobacco smoking. Societal benefits include increased public health resulting from this study 
providing evidence that directly informs and strengthens FDAs tobacco control efforts. Few 
studies have been conducted on WP tobacco additives and at the same time, there is mounting 
evidence that young adults, a vulnerable population from a tobacco control perspective, are 
largely misinformed of the risks of this form of tobacco use.  
 

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VI. Data Storage 
 
VI.1. Paper materials 
Informed consent forms, receipts, and paper surveys will be stored in locked file cabinets only 
accessible by study staff. Paper surveys may be shredded after they have been entered in an 
electronic database. 
 
VI.2. Electronic data and subject identifying data 
REDCap will be used for entry of paper surveys and collection of survey data. Protection against 
loss of confidentiality and privacy will be maintained by numerically coding all data, disguising 
identifying information, and keeping data locked in file drawers or in a secure, password-
protected database. Only study research assistants and the PIs will have the information that 
connects participant’s name and ID number. All electronic data will be numerically coded and 
stored in a password protected database, on a password protected computer in a secure research 
space. Participant information will be accessible only to research staff, who are pledged to 
confidentiality and complete training in the ethical conduct of research (i.e., both HIPAA and 
CITI trainings). Identifying information will not be reported in any publication. 
 
 
 
VII. Data Analysis 
 
VII.1 Analysis Plan 
The main research questions from the four Aims in the proposed study have the same structure 
for data analysis. That is, there are differences attributable to the four tobaccos in the means of 
the outcome measures of interest. All relevant outcome variables will be first examined with 
exploratory/descriptive statistical analyses that focus on describing and understanding patterns 
and distributions for all relevant measures (e.g., tobacco chemical content, mainstream smoke 
constituents, spirometry measures, topography measures, psychophysical ratings) by the four 
tobaccos. We will identify any potential outliers, assess distributional assumptions, and apply 
appropriate transformations to the outcome variables (e.g., log-transformation) if the normality 
assumption is violated.  
 
For each of the four aims, the following pairwise comparisons will be performed:  
H0i j: μi = μj vs. Hai j : μi ≠ μj 
where μi = mean target outcome for tobacco type i, and μj = mean target outcome for tobacco 
type j. In Aims 1, 2, and 4, the data will be analyzed using one-way ANOVA models for 
independent outcome data and Tukey’s method will be used to control type-I error rate across the 
six multiple comparisons. A different analysis strategy will be used in Aim 3 due to the clustered 
study design: participants will complete four different smoking sessions. Thus, in order to 
account for the hierarchical design, the data in Aim 3 will be analyzed using linear mixed models 
containing random subject effects. Since linear mixed models allow for incomplete data (e.g., 

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due to dropout), data from all completed smoking sessions will be used in the analysis. As in the 
other aims, Tukey’s method will be used to adjust for multiple comparisons of the tobacco 
treatments in Aim 3.  In addition, for Aim 3, we will use linear mixed models to examine the 
relationships between the perceived sensory intensity ratings and the liking/disliking ratings for a 
given tobacco preparation.  
 
Integrating results from Aims 1, 2 and 4 with Aim 3 of the study, the absence or presence, or 
exposure level of certain HPHCs and other chemical compounds across the different tobacco 
preparations will be mapped with the data on perceived sensory intensity such as sweetness and 
liking/disliking ratings.  
 
VII.2. Sample Size Justification.  
Since Aims 1, 2, and 4 do not involve human subjects, we expect low variability across 
replicates. Thus, assuming a somewhat conservative coefficient of variation (CV) of 20% and 
log-normal outcomes, a total of 4 replicates per tobacco preparation will provide 80% power 
to detect a two-fold change in the geometric means of two groups at a conservative two-sided 
Bonferroni-corrected type-I error rate of 0.0083. 
 
Our sample size for Aim 3 was selected to ensure that we have adequate power to detect 
moderate effect sizes in our comparisons of topography outcomes. We made the following 
assumptions for the power analysis:  
• Based on the intravariability data analysis for the RWP,94 correlation coefficients within 
subjects (r) were assumed to be 0.6, 0.7 or 0.8. 
• Small, medium, and large effect sizes (d) of 0.2, 0.5, and 0.8 standard deviations. 
• A two-sided, Bonferroni corrected type-
I error rate of 0.0083. 
Our power analysis, summarized in Table 
VII-5, revealed that a sample size of   43 
subjects will provide over 80% power to 
detect a moderate effect size unless the 
within pair correlation is high (0.2 or 0.3) 
and the within participant correlation is 
modest (0.6). However, since this power 
analysis assumes complete follow-up, we 
will recruit 60 participants to account for a 
possible attrition rate of 20%., and ensure 
the Latin square ordiering is balanced across 
the four tobacco types.  
 
 
VIII. Gender/Minority/Pedi-
atric Inclusion for Research  
 
Table VII.5.  Estimated % power to for pairwise 
comparisons of tobacco preparations based N = 25 pairs 
(50 subjects). 
  
  
Power 
r 
ρ 
d=0.2 
d=0.5 
d=0.8 
0.6 
0.1 
0.108 
0.803 
0.998 
 
0.2 
0.097 
0.759 
0.996 
 
0.3 
0.088 
0.717 
0.994 
0.7 
0.1 
0.153 
0.918 
> 0.999 
 
0.2 
0.138 
0.889 
> 0.999 
 
0.3 
0.125 
0.857 
0.999 
0.8 
0.1 
0.255 
0.989 
>0.999 
 
0.2 
0.228 
0.981 
>0.999 
 
0.3 
0.207 
0.971 
>0.999 

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VIII.1 Inclusion of Women and Minorities 
Inclusion of Women and Minorities: According to U.S. Census Data, 51.3% of Columbus, OH 
residents are female. In our previous studies with smokers, 55-62% of participants were female. 
According to U.S. Census data, the racial composition of individuals living in Columbus is 61% 
White, 28% Black or African American, 5% Asian, 0.2% American Indian/Alaska Native, 0% 
Native Hawaiian/Other Pacific Islander, and 4% two or more races. The ethnic composition of 
individuals living in Columbus is 6% Hispanic/Latino. We expect that our distributions will be 
similar to these but we may potentially have a larger distribution of ethnic and racial minorities, 
based on our previous studies. However, we will continuously monitor enrollment in order to 
ensure that we are meeting recruitment goals to avoid under-recruiting minorities. If the targeted 
enrollment for minorities is not met because they do not respond to the advertisements, we will 
make special efforts to solicit their participation by advertising in community newspapers, local 
church organizations, and community centers. 
VIII.2 Inclusion of Children 
This study will be restricted to individuals 21 years of age and older, which is the legal age to 
purchase tobacco products. 
 
IX. Data and Safety Monitoring Plan 
 
The Data and Safety Monitoring Plan can be found in Appendix D to this Research Protocol.   
 
All data and safety monitoring will adhere to the policies and procedures of the OSU 
Comprehensive Cancer Center’s Data and Safety Monitoring Plan which has been approved by 
the National Cancer Institute 
 
X. References 
 
1 Maziak W, Ward KD, Soweid RA, Eissenberg T. Tobacco smoking using a waterpipe: a re-
emerging strain in a global epidemic. Tob Control. 2004;13:327-333. 
doi:10.1136/tc.2004.008169. 
 
2 Jamal A, Gentzke A, Hu SS, Cullen KA, Apelberg BJ, Homa DM, King BA. Tobacco use 
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126 James S, Meier E, Wagener T, Smith K, Neas B, Beebe L. E-cigarettes for immediate 
smoking substitution in women diagnosed with cervical dysplasia and associated disorders. 
International journal of environmental research and public health. 2016;13(3):288. 
 
127 Busch AM, Leavens EL, Wagener TL, Buckley ML, Tooley EM. Prevalence, reasons for use, 
and risk perception of electronic cigarettes among post-acute coronary syndrome smokers. 
Journal of cardiopulmonary rehabilitation and prevention. 2016;36(5):352. 
 
128 Wagener TL, Floyd EL, Stepanov I, et al. Have combustible cigarettes met their match? The 
nicotine delivery profiles and harmful constituent exposures of second-generation and third-
generation electronic cigarette users. Tobacco control. 2017;26(e1):e23-e28. 
 
129 Leavens EL, Brett EI, Morgan TL, Lopez SV, Shaikh RA, Leffingwell TR, Wagener TL. 
Descriptive and injunctive norms of waterpipe smoking among college students. Addictive 
behaviors. 2018 Feb 1;77:59-62. 
 
130 Stevens EM, Kim S, Leshner G, Leavens ELS, Vidrine JI, Frank-Pearce SG, Hale JJ, 
Wagener TL. The Attitudinal and Motivational Effects of Anti-waterpipe Messages. American 
Journal of Health Behavior. 2019;43: 478-489. 
 
131 Leavens EL, Morgan TL, Brett EI, Patzkowsky K, Son J, Molina N, Eissenberg T, Shihadeh 
A, Leffingwell TR, Wagener TL. Concurrent alcohol use and waterpipe tobacco smoking: 
smoking topography, toxicant exposure, and abuse liability. Nicotine & Tobacco Research. 
2019 Mar 1. 
 
132 Leavens EL, Meier E, Tackett AP, Miller MB, Tahirkheli NN, Brett EI, Carroll DM, Driskill LM, 
Anderson MP, Wagener TL. The impact of a brief cessation induction intervention for 
waterpipe tobacco smoking: a pilot randomized clinical trial. Addictive behaviors. 2018 Mar 
1;78:94-100. 
 
133 Leavens EL, Brett EI, Frank S, Shaikh RA, Leffingwell TR, Croff JM, Wagener TL. 
Association between breath alcohol concentration and waterpipe lounge patrons’ carbon 
monoxide exposure: A field investigation. Drug and alcohol dependence. 2017 Jan 
1;170:152-5. 
 
134 Lim J, Wood A, Green BG. Derivation and evaluation of a labeled hedonic scale. Chemical 
Senses. 34: 739-51, 2009. PMCID: PMC2762053. 
 
135 Lim J. Hedonic scaling: A review of methods and theory. Food Quality and Preference. 22: 
733-747, 2011. 
 
136 Lim J, Fujimaru T. Evaluation of the Labeled Hedonic Scale under different experimental 
conditions. Food Quality and Preference. 21: 521-530, 2010. 
 
137 Roberts, M.E., Ferketich, A.K. (2019). Hookah susceptibility and transitions over the first year 
of college. In press at Journal of Studies on Alcohol and Drugs. 
 
138 Roberts, M.E., Klein, E.G., Berman, M.L., Berhane, B., & Ferketich, A.K. Young adult 
perceptions surrounding hookah use (2017). Health Behavior Policy and Review, 4(6), 593-
600.  

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139 Evans, A.T., Peters, E., Keller-Hamilton B., Loiewski, C., Slater, M.D., Lu B., Roberts, M.E., 
& Ferketich, A.K. (2018). Warning Size Affects What Adolescents Recall from Tobacco 
Advertisements. Tobacco Regulatory Science, 4(3), 79-87. 
 
140 Keller-Hamilton, B., Roberts, M.E., Slater, M.D., Berman, M., & Ferketich, A.K. (2019). 
Adolescent males’ responses to blu’s fake warnings. Tobacco Control. 
 
141 Keller-Hamilton, B., Roberts, M.E., Slater, M.D., & Ferketich, A.K. (2019). Memorability of 
cigarette advertisements making “natural” claims among adolescents. Tobacco Regulatory 
Science, 5(4), 326-331. 
 
142 Paravisini, L. and Peterson, D.G. 2019.  Reactive Carbonyl Species as a Control Point of 
Reaction Flavors.  Food Chem.  274: 71-78. 
 
143 Paravisini, L. and Peterson, D.G. 2018.  Role of Reactive Carbonyl Species on Non-
Enzymatic Browning of Apple Juice During Storage. Food Chem.  245: 1010-1017. 
 
144 Kokkinidou, S. Peterson, D.G.  2016.  Identification of compounds that contribute to 
trigeminal burn in aqueous ethanol solutions. Food Chem. 211: 757-762. 
 
145 Zhang, L., Xia, Y. and Peterson, D.G.  2014.  Identification of bitter modulating Maillard-
catechin reaction products. J. Agric. Food Chem. 62, 10092−10100. 
 
146 Brinkman MC, Keller-Hamilton B, Carney-Knisely G, Somogyi A, Roberts ME, Ferketich A, 
Stepanov I, Wagener TL, Shields PG, Halquist MS, Clark PI. Effect of pH of the nicotine 
liquid on JUUL puffing behavior and nicotine uptake. Scheduled for podium presentation, 
March 14, 2020, Society for Research on Nicotine and Tobacco annual meeting. 
 
147 Knobloch, T.J.; Ryan, N.M.; Brushweiler, L.; Wang, C.; Bernier, M.C.; Somogyi, Á.; Yan, 
S.P.; Copperstone, J.; Mo, X.; Bruschweiler, R.P.; Weghorst, C.M.; Qghumu, S. 
“Metabolomic Regulation of Glycolysis and AMP Activated Protein Kinase Pathways during 
Black Raspberry-Mediated Oral Cancer Chemoprevention” Metabolites 2019, 9, 140. 
 
148 Maltz, R.M.; Keirsey, J.; Kim, S.C.; Mackos, A.R.; Gharaibeh, R.Z.; Morre, C.C.; Somogyi, Á.; 
Bailey, M.T. Social Stress Affects Colonic Inflammation, the Gut Microbiome, and Short 
Chain Fatty Acid Levels and Receptors J. Pediatr Gastroenterol Nutr. 2019, 68, 533-540. 
 
149 Leggett, A.; Wang, C.; Li, D.W.; Somogyi, Á.; Bruschweiler-Li, L.; Bruschweiler, R.P. 
“Identification of Unknown Metabolomics Mixture Compounds by Combining NMR, MS and 
Chemoinformatics” Methods in Enzymology 2019, 615, 407-422. 
 
150 Somogyi, Á." Probing Peptide Fragment Ion Structures by Combining Sustained Off-
Resonance Collision-Induced Dissociation and Gas-Phase H/D Exchange (SORI-HDX) in 
Fourier Transform Ion-Cyclotron Resonance (FT-ICR) Instruments", J. Am. Soc. Mass 
Spectrom. 2008, 19, 1771-1775. 
 
151 Russomanno J, Patterson, JG, Jabson JM. Food Security and Transgender and Gender 
Non-conforming Individuals in the Southeast United States: A Qualitative Study. 
Transgender Health 2019; 4(1), 1-11. doi: 10.1089/trgh.2018.0024. 
 

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152 Jabson Tree JM, Patterson JG (2019). A Test of Feasibility and Acceptability of Online 
Mindfulness-Based Stress Reduction for Lesbian, Gay, and Bisexual Women and Men at 
Risk for High Stress: Pilot Study. JMIR Mental Health, 6(8), e15048. doi: 10.2196/15048 
 
153 Patterson JG, Jabson Tree JM, Kamen C. 2019. Cultural competency and microaggressions 
in the provision of care to LGBT patients in rural and Appalachian Tennessee. Patient 
Education Counseling 2019; doi: 10.1016/j.pec.2019.06.003 
 
154 Macisco JM, Patterson JG, Nemeth JM. Lapse to Relapse: Homeless Youth Return to 
Smoking When Supportive Structures and Self-Regulation Are Weakened. (accepted) 
Society for Behavioral Medicine, San Francisco, CA, April 1-4, 2020. 
 
155 Shaw, P. E. Handbook of Sugar Separations in Foods by HPLC; CRC Press: Boca Raton, 
FL, 1988. 
 
156 Yang D-J, Chen B. Simultaneous determination of nonnutritive sweeteners in foods by 
HPLC/ESI-MS. J. Agric. Food. Chem 57:3022-3027 (2009). 
 
157 United States Pharmacopeia (USP). <61> Microbiological examination of nonsterile 
products: microbial enumeration tests. Available from 
https://hmc.usp.org/sites/default/files/documents/HMC/GCs-Pdfs/c61.pdf 
 
158 Liu C. Glycerol transfer in cigarette mainstream smoke. Beiträge zur Tabakforschung 
International/Contributions to Tobacco Research. 2004 Jul 1;21(2):111-6. 
 
159 Laurene AH, Cundiff RH, Greene GH. Determination of glycerol and propylene glycol in 
cigarette smoke. Research Department, R.J. Reynolds Tobacco Company, 1965. Bates 
number 510552130-510552133. 
 
160 Brinkman MC, Wagener TL, Kassem NOF. COMMENTS REGARDING DOCKET NO. FDA-
2012-N-0-0143, “Harmful and Potentially Harmful Constituents in Tobacco Products; 
Established List; Proposed Additions; Request for Comments. Comment Tracking Number: 
1k3-9cj9-o3vi.   
 
161 Ding YS, Yan X, Wong J, Chan M, Watson CH. In situ derivatization and quantification of 
seven carbonyls in cigarette mainstream smoke. Chemical research in toxicology. 2016 Jan 
19;29(1):125-31. 
 
162 Pang X, Lewis AC. Carbonyl compounds in gas and particle phases of mainstream cigarette 
smoke. Science of the total environment. 2011 Nov 1;409(23):5000-9. 
 
163 Gregg E, Hill C, Hollywood M, Kearney M, McAdam K, McLaughlin D, Purkis S, Williams M: 
The UK Smoke Constituents Testing Study. Summary of Results and Comparison with 
Other Studies. Beitr zur Tabakforsch Int. 2004, 21: 117-138. 
 
164 DiFranza JR, Savageau JA, Fletcher K, et al. Measuring the loss of autonomy over nicotine 
use in adolescents: the DANDY (Development and Assessment of Nicotine Dependence in 
Youths) study. Arch Pediatr Adolesc Med. 2002;156(4):397-403. 
 
165 Salameh P, Waked M, Aoun Z. Waterpipe smoking: construction and validation of the 
Lebanon Waterpipe Dependence Scale (LWDS-11). Nicotine Tob Res. 2008;10(1):149-158. 

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166 Foulds J Stapleton J Feyerabend C Vesey C Jarvis M Russell MA . Effect of transdermal 
nicotine patches on cigarette smoking: a double blind crossover study . 
Psychopharmacology (Berl)  . 1992 ; 106 : 421 – 427. 
 
167 Evans SE Blank M Sams C Weaver MF Eissenberg T . Transdermal nicotine-induced 
tobacco abstinence symptom suppression: nicotine dose and smokers’ gender . Exp Clin 
Psychopharmacol. 2006;14:121-135 . 
 
168 Tiffany ST, Drobes DJ. The development and initial validation of a questionnaire on smoking 
urges. Br J Addict. 1991;86(11):1467-1476. 
 
169 Pérez-Padilla R, Vázquez-García JC, Márquez MN, Jardim JR, Pertuzé J, Lisboa C, Muino 
A, López MV, Tálamo C, De Oca MM, Valdivia G. The long-term stability of portable 
spirometers used in a multinational study of the prevalence of chronic obstructive pulmonary 
disease. Respiratory care. 2006 Oct 1;51(10):1167-71. 
 
170 Pals SL, Murray DM, Alfano CM, Shadish WR, Hannan PJ, Baker WL. Individually 
randomized group treatment trials: a critical appraisal of frequently used design and analytic 
approaches. Am J Public Health. 2008 Aug;98(8):1418-24 
 
171 Donner A, Klar N. Design and Analysis of Cluster Randomization Trials in Health Research. 
New York, NY: Wiley; 2000. 
 
172 Breland, A. B., Kleykamp, B. A., & Eissenberg, T. (2006). Clinical laboratory evaluation of 
potential reduced exposure products for smokers. Nicotine & Tobacco Research, 8,727–
738. 
 
173 Cobb CO, Blank MD, Morlett A, Shihadeh A, Jaroudi E, Karaoghlanian N, et al. Comparison 
of puff topography, toxicant exposure, and subjective effects in low- and high-frequency 
waterpipe users: a double-blind, placebo-contro... - PubMed - NCBI. Nicotine Tob Res. 
2015;17(6):667. 
 
174 Altman DG, Bland JM. How to randomise. BMJ. 1999 Sep 11;319(7211):703-4. 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix A 
 
A-1 
 
Appendix A:  Construction and Verification of the Dual-Hose 
Research-Grade Waterpipe (RWP2) and Single-Hose Research-
Grade Waterpipe (RWP) 
 
 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix A 
 
A-2 
 
Dual-Hose Research-Grade Waterpipe (RWP2) 
RWP2 Construction 
The RWP2, pictured in Figure A-1, is constructed from the same materials as the single-hose 
research-grade waterpipe (RWP) we developed and validated previously.1-3 Briefly, all wetted 
materials have inert surfaces to reduce contamination, guard against memory effects and 
degradation over time, and facilitate easy cleaning between smoking sessions.2   
Modifications that were made to the RWP to create the dual-hose RWP2 included changes to the 
cap design and the addition of a second hose, as shown in Figure 1.  Both hoses are equipped 
with inline topography systems 
consisting of polycarbonate 
pneumotachometers (2 total) that 
are connected to an amplifier as 
described previously.2  Two sets of 
pressure transducers inside the 
amplifier convert the pressure drop 
across the restriction inside each 
pneumotachometer to an electrical 
response.  These data are now 
recorded electronically for each 
hose using LabView 2017.  Data 
files are processed manually or 
automatically in batches using the 
same algorithm for peak recognition 
and analysis (MatLab R2018a).  
The result is a spreadsheet report 
that describes the volume, duration, 
average and peak flow rates of each 
puff, and interpuff intervals for each 
of the two participants smoking the 
RWP2.   
To increase ruggedness, the design 
of the neck of the glass flask was 
changed to eliminate the force 
associated with the cap’s o-ring seal 
against the inside of the neck. 
Because the flasks are now being 
manufactured (by Kimax) with 
 
Figure A-1. Photo showing the main components of the 
dual-hose research-grade waterpipe (RWP2); data 
acquisition system not shown. 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix A 
 
A-3 
 
thinner walls, the neck could no longer 
safely support the old design.  The new 
design includes a thicker mouth on the 
flask which supports the weight of the 
cap.  The cap is now sealed to the top of 
the mouth of the flask using an o-ring 
and a clamp, as shown in Figure A-2.  
The dimensions and materials of the 
head, body, and one-way valve of the 
RWP2 are unchanged from the original 
RWP. 
Topography Data Collection and 
Analysis 
The RWP2 has an inline topography 
system that allows real-time 
measurement and recording of the flow rate of smoke drawn through each of the two hoses.  This 
allows the separate measurement and reporting of two individual’s puffing behaviors (puff 
volume, duration, flow rate, and interpuff interval) when smoking a single, shared waterpipe in 
our controlled clinical research facility. The pneumotachometers introduce minimal draw 
resistance to the waterpipe (<5% at 27 L/min), and perform reliably, with known precision and 
accuracy, when measuring flows of waterpipe tobacco smoke.1-3 
As with the RWP, the relationship between pneumotachometer response and flow through hoses 
1 and 2 of the RWP2 is best described using a quadratic curve fit, as shown in Figure A-3.  These 
data agree well with what was previously reported for the RWP.2  To simulate normal waterpipe 
“smoking etiquette,” these data were collected in series, first for Hose #1 while Hose #2 was 
plugged, then for Hose #2 while Hose #1 was plugged.  As with all commercial dual-hose 
waterpipes, smokers of the RWP2 must put their thumb over their mouthpiece when the other 
person is puffing, otherwise the person will only be sucking in room air.  Study participants will 
be reminded to follow normal waterpipe “smoking etiquette,” during the clinic smoking sessions: 
• Do not puff at the same time as your partner.  
• Unless you are in the act of puffing, plug your hose end with your thumb so that your 
partner isn’t just sucking air from their hose. 
• Do not blow or exhale into your hose. 
Figure A-2. The neck of the RWP2 was modified with a thicker 
upper lip and clamp to create a safer seal with the cap, given 
the manufacturer has reduced the flask’s wall thickness. 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix A 
 
A-4 
 
 
 
 
 
Hose #1
Flow through Hose 1, Research-Grade Waterpipe (L/min)
0
5
10
15
20
25
30
Pneumotach Response (Vdc)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Quadratic fit, R2 = 0.997
95% Confidence band
95% prediction band
 
 
Hose #2
Flow through Hose 2 of Research-Grade Waterpipe (L/min)
0
5
10
15
20
25
30
Pneumotach Response (V)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Quadratic fit, R2 = 0.997
95% Confidence Band 
95% Prediction Band 
 
Figure A-3. Relationship between flow rate through each hose of the dual-hose research-
grade waterpipe and the topography measurement device response. 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix A 
 
A-5 
 
Single-Hose Research-Grade Waterpipe (RWP) 
RWP Construction 
The RWP, pictured in Figure A-4, was developed and validated previously.1-3 Briefly, all wetted 
materials have inert surfaces to reduce contamination, guard against memory effects and 
degradation over time, and facilitate easy cleaning between smoking sessions.2   
For this study, modifications were made to the RWP including the use of new data acquisition 
hardware and software, and a change to how the cap seals to the flask, as shown in Figures A-4 
and A-5.  The hose is equipped with an inline topography system consisting of a polycarbonate 
pneumotachometer that is 
connected to an amplifier as 
described previously.2  The 
amplifier can take inputs from two 
separate RWPs. Two sets of 
pressure transducers inside the 
amplifier convert the pressure drop 
across the restriction inside the 
pneumotachometer to an electrical 
response.  These data are now 
recorded electronically for each 
RWP using LabView 2017.  Data 
files are processed manually or 
automatically in batches using the 
same algorithm for peak recognition 
and analysis (MatLab R2018a).  
The result is a spreadsheet report 
that describes the volume, duration, 
average and peak flow rates of each 
puff, and interpuff intervals for each 
of the two participants separately 
smoking their own RWP.   
To increase ruggedness, the design 
of the neck of the glass flask was 
changed to eliminate the force 
associated with the cap’s o-ring seal against the inside of the neck. Because the flasks are now 
 
Figure A-4. Photo showing the main components of the dual-
hose research-grade waterpipe (RWP); data acquisition 
system not shown. 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix A 
 
A-6 
 
being manufactured (by 
Kimax) with thinner 
walls, the neck could no 
longer safely support 
the old design.  The new 
design includes a 
thicker mouth on the 
flask which supports the 
weight of the cap.  The 
cap is now sealed to the 
top of the mouth of the 
flask using an o-ring 
and a clamp, as shown 
in Figure A-6.  The 
dimensions and 
materials of the head, 
body, and one-way 
valve are unchanged 
from the original RWP. 
Topography Data 
Collection and 
Analysis 
Each RWP has an inline 
topography system that 
allows real-time 
measurement and recording of the flow rate of smoke drawn through the hose.  The two RWPs 
are connected to the same signal amplifier. This allows the separate measurement and reporting 
of two individual’s puffing behaviors (puff volume, duration, flow rate, and interpuff interval) 
when each are smoking separate RWPs in separate, well-ventilated rooms in our controlled 
clinical research facility. The pneumotachometer introduces minimal draw resistance to the RWP 
(<5% at 27 L/min), and performs reliably, with known precision and accuracy, when measuring 
flows of waterpipe tobacco smoke.1-3 The relationship between pneumotachometer response and 
flow through the hose of each RWP is best described using a quadratic curve fit, as shown 
previously in Figure A-3.2   
 
 
 
Figure A-6. The neck of the RWP was modified with a thicker upper lip and 
clamp to create a safer seal with the cap, given the manufacturer has 
reduced the flask’s wall thickness. 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix A 
 
A-7 
 
References 
1. Brinkman MC, Kim H, Buehler SS, Adetona AM, Gordon SM, Clark PI. Evidence of 
compensation among waterpipe smokers using harm reduction components. Tob Control. 
2018 Oct 30:tobaccocontrol-2018. 
2. Brinkman MC, Kim H, Gordon SM, Kroeger RR, Reyes IL, ... & Clark PI. Design and 
Validation of a Research-Grade Waterpipe Equipped with Puff Topography Analyzer. 
Nicotine Tob Res. 2015;18(5):785-93. 
3. Kim H, Brinkman MC, Sharma E, Gordon SM, Clark PI. 2016. Variability in puff 
topography and exhaled CO in waterpipe tobacco smoking. Tobacco Regulatory Science, 
2(4), pp.301-8. 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix B 
 
B-1 
 
Appendix B:  COVID-19 Related Procedures 
 
 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix B 
 
B-2 
 
Due to the COVID-19 pandemic, processes and procedures have been implemented to help 
protect participants and research staff. These processes and procedures will be followed as long 
as social distancing requirements are necessary for conducting study visits.. All study 
participants will be provided with a facemask upon entry. The research assistant will meet the 
participant outside the building for a temperature check, direct the participant into the building, 
and the two of them will ride the elevator to the 4th floor physically distanced at least 6 feet apart, 
both wearing masks.  No more than 2 persons may ride the elevator at any given time. The 
participant will immediately be escorted to a private exam/draw room. Therefore, there will be 
no waiting in open lobby/waiting areas. 
 
When in the exam room, the research assistant will stand at least 6 feet away from the study 
participant to give instructions.  Afterwards, the study coordinator will leave the exam room to 
allow the study participant to conduct the instructed procedures. The research assistant and study 
participant will be at least 6 feet away from one another and wearing protective masks at all 
times during each visit.   
 
Each research assistant will have a designated exam/draw room and smoking room in which to 
conduct their designated research study. Each smoking room is separated from the staff control 
station in the hallway by its own door and contains a large window for the research assistant to 
be able to see in and monitor study participant activity within the room. There is also a speaker 
and microphone system within each smoking room along with the Genetec software system on 
the outside of each room at the computer stations. Therefore, the research assistant and study 
participant can communicate with one another without being in the room together.   
 
For study measures which cannot be physically distanced, appropriate PPE will be worn at all 
times by research staff during these procedures including goggles, face masks, gloves, and 
isolation gowns or lab coats.  
After each participant visit is complete, there will be at least a 45-minute period for cleaning and 
air exchange in the negative pressure rooms, and for cleaning exam rooms and equipment before 
the next participant visit. All smoking rooms are under negative pressure with a ventilation rate 
of 36.8 – 44.1 air changes per hour (ACH). 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
 
RESEARCH PROTOCOL – Appendix C 
 
C-1 
 
Appendix C:  Example Safe Departure Checklist 
 
 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
 
RESEARCH PROTOCOL – Appendix C 
 
C-2 
 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix D 
 
D-1 
 
Appendix D:  Data and Safety Monitoring Plan 
 
 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix D 
 
D-2 
 
Responsibility of DSM 
Because a Data and Safety Monitoring Board (DSMB) is only required for higher risk and/or 
multisite studies, and the proposed research is a single site pilot study with minimal risk, we do 
not include a formal DSMB. However, the Center for Tobacco Research in conjunction with the 
OSU Comprehensive Cancer Center does have a standing DSMB that we can utilize if the 
funding agency or the reviewers request one.  
 
Co-PD/PIs Brinkman and Wagener will be responsible for monitoring the safety and integrity of 
the research project, executing the DSM plan for the projects, and complying with the reporting 
requirements. They will also provide annual and interim progress reports to NCI and FDA.   
 
With this project, the DSM will begin by reviewing the revised protocol and establishing 
guidelines for data and safety monitoring. This will include reviewing the standard procedures 
for day-to-day monitoring by the internal monitors, principal and co-investigators and study staff. 
DSM will include evaluating the progress of the trial; reviewing data quality, participant 
recruitment, and study retention; and examining other factors that may affect study outcome. 
The PIs will review major proposed modifications to the study prior to their implementation. They 
will also review the participants’ ability to achieve the study requirements, and the rates of 
adverse events to determine whether there has been any change in participant risk. Their 
review will ensure that subject risk does not outweigh the study benefits. Following each DSMB 
meeting, a written report of their findings will be generated for the study record and provided to 
NCI and each site’s Institutional Review Boards (IRB). 
 
The specific DSM responsibilities will include the following: 
• 
Review the research protocol, informed consent documents, and plans for data and 
safety monitoring; 
• 
Evaluate the progress of the trial, including periodic assessments of data quality and 
timeliness, participant recruitment, accrual and retention, participant risk versus benefit, 
performance of the trial sites, and other factors that can affect study outcome; 
• 
Consider factors external to the study that may be relevant, such as scientific or 
therapeutic developments, which could have an impact on the safety of the participants 
or the ethics of the study; 
• 
Protect the safety of the study participants and report on the safety and progress of the 
trial; 
• 
Make recommendations to the research team and sponsor, as required, concerning 
continuation, termination or other modification(s) to the trial. 
• 
Ensure the confidentiality of the trial data and the results of monitoring; 
• 
Assist on any problems with study conduct, enrollment, sample size, and/or data 
collection. 
• 
Review data for accuracy and quality 
 
Study progress reports will be prepared by the research team. These reports will include 
updates on the protocol, including revisions, enrollment progress and projections, retention, and 
safety data (description, severity, attribution, response, reporting and resolution of those events 
as well as if any events are unanticipated. If more than 10% of the participants experience an 

 
 THE OHIO STATE UNIVERSITY 
 
 
 
 
RESEARCH PROTOCOL – Appendix D 
 
D-3 
 
adverse event during the course of the study then the PIs will consider suspension or 
discontinuation of the study.  
 
Adverse Events 
Adverse events will be assessed by study staff at each follow-up visit via participant self-report 
and managed immediately. All adverse events will be reported to the OSU IRB. We will monitor 
for risk of smoking waterpipe by screening participants for general medical precautions 
(pregnancy, respiratory and cardiovascular disease). Participants will be given contact numbers 
of study personnel and Co-PD/PIs Brinkman and Wagener. Any adverse events, breaks of 
confidentiality, or any other data or safety issues that arise will be discussed immediately 
between study personnel and the Co-PD/PIs.  
 
Co-PD/PI Brinkman will be responsible for completing an Adverse Events Form should an event 
occur. She will report Serious Adverse Events to the OSU IRB within 24 hours of having 
received notice of the event. She will gather any information needed to investigate the event 
and to determine subsequent action. Any subsequent action will be documented and reported to 
the OSU IRB and the Program Officer at NIH. Adverse event reports will be reviewed annually 
with the OSU IRB to ensure participant safety.  
 
Protocol Amendments/Changes 
Any changes or amendments to the protocol made in response to adverse events/SAEs (or 
independent of AEs/SAEs) will be requested in writing to the IRB, which will then grant or deny 
permission to make the requested change in protocol.  Approved changes and amendments will 
be reported to the NIH. 
Frequency of DSM 
On a regular basis (e.g., weekly), investigators and key personnel will review data quality, 
recruitment, retention, and examine other factors that may affect outcomes. They will also 
review any adverse events to determine if there are any significant or unanticipated participant 
risks. The Project Leaders (M. Brinkman & T. Wagener) and the Project Manager will be 
available to meet outside of the scheduled meetings if concerns regarding any adverse trends 
or other major problems should arise. 
Content of DSM Report 
The DSM annual report will include enrollment information, demographics and characteristics of 
the participants, the expected versus actual recruitment rates, quality assurance or regulatory 
issues that may have occurred during the year, a summary of adverse events and SAEs, 
protocol violations, and any actions or changes to the protocol. Also included will be any and all 
actions by the IRB as they occur. 
 

CONSENT 
Biomedical/Cancer 
 
 
 
IRB Protocol Number: 
2021C0039 
IRB Approval date:  05.23.21 
Version:  3.0 
 
 
Page 1 of 11 
Form date: 
05/17/2019  
 
 
1 
      
2 
The Ohio State University Consent to Participate in Research 
3 
 
4 
Study Title: 
Evaluating waterpipe tobacco additives 
Principal Investigator: 
Marielle C. Brinkman and Theodore L. Wagener 
Sponsor:  
National Cancer Institute, National Institutes of Health; Food and 
Drug Administration Center for Tobacco Research 
 
5 
• This is a consent form for research participation.  It contains important information 
6 
about this study and what to expect if you decide to participate.  Please consider the 
7 
information carefully. Feel free to discuss the study with your friends and family and 
8 
to ask questions before making your decision whether or not to participate. 
9 
• Your participation is voluntary.  You may refuse to participate in this study.  If you 
10 
decide to take part in the study, you may leave the study at any time.  No matter what 
11 
decision you make, there will be no penalty to you and you will not lose any of your 
12 
usual benefits.  Your decision will not affect your future relationship with The Ohio 
13 
State University.  If you are a student or employee at Ohio State, your decision will 
14 
not affect your grades or employment status. 
15 
• You may or may not benefit as a result of participating in this study.  Also, as 
16 
explained below, your participation may result in unintended or harmful effects for 
17 
you that may be minor or may be serious depending on the nature of the research. 
18 
• You will be provided with any new information that develops during the study 
19 
that may affect your decision whether or not to continue to participate.  If you 
20 
decide to participate, you will be asked to sign this form and will receive a copy of the 
21 
form.  You are being asked to consider participating in this study for the reasons 
22 
explained below.  
23 
 
24 
Key Information About This Study 
25 
The following is a short summary to help you decide whether or not to be a part of this study. 
26 
More detailed information is listed later in this form.  
27 
 
28 
The purpose of this study is to evaluate hookah (waterpipe) tobacco additives and their effect 
29 
on puffing behaviors, lung function, and the appeal of hookah smoking.  Eligible participants 
30 
will attend four clinic visits to smoke hookah, give exhaled breath and blood samples, and 
31 
answer questions about your smoking experience at the clinic and your tobacco use history.  
32 
Clinic visits will be roughly one week apart and take place at roughly the same time of day.  
33 
The clinic is near Riverside Hospital and has ample free parking.  Each clinic visit will take 
34 
2.5-3 hours, so participants can expect to spend up to 12 hours over the course of 4 weeks 
35 
participating in the study.  The biggest risk and discomfort may be dizziness or nausea from 
36 

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IRB Approval date:  05.23.21 
Version:  3.0 
 
 
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giving two blood samples at every clinic visit, for a total of eight blood samples.  Another risk 
37 
is loss of confidentiality if other people find out about your participation in the study.  All 
38 
efforts are made to keep your information confidential, but confidentiality is not absolute.  
39 
There are no personal benefits from being in the study; however, society stands to benefit 
40 
from your providing valuable information for public health research on tobacco products. 
41 
 
42 
1.   Why is this study being done? 
43 
 
44 
The goal of this study is to evaluate hookah (waterpipe) tobacco additives and their effect 
45 
on puffing behaviors, lung function, and the appeal of hookah smoking.   
46 
 
47 
2.   How many people will take part in this study? 
48 
 
49 
Up to 60 people will participate in this study.  
50 
 
51 
3.   What will happen if I take part in this study? 
52 
 
53 
If you decide to take part in the study, you will make a series of 4 visits to our clinic, at about 
54 
the same time each day, each visit separated by at least a week.  Each visit will last about 2.5-
55 
3 hours, and include the activities listed in Table 1.  For each visit, you must come to the 
56 
clinic after not using any tobacco- or nicotine-containing products after 10 pm the previous 
57 
day.  We also encourage you to eat two hours before coming to your scheduled appointment 
58 
because we will be drawing your blood. In addition, we suggest that you drink plenty of water 
59 
before your scheduled appointment and refrain from drinking alcohol after 10 pm the previous 
60 
day.  Doing these things will make it easier to draw your blood.  During your session, you 
61 
will be instructed to smoke a hookah as you normally would, and we will explain everything 
62 
in detail to you.  On arrival at the laboratory you will be asked briefly about your tobacco and 
63 
hookah use history and patterns. 
64 
 
65 
Please note: you will be asked not to eat or drink anything other than water during your clinic 
66 
visits.  For this reason, please make sure to have eaten regularly prior to attending your clinic 
67 
visits.  
68 
 
69 
In addition, at each visit, we will require you to follow safety procedures in place to enter 
70 
the clinic and engage as a participant to reduce your risk of acquiring or spreading 
71 
COVID-19, or a similar pandemic disease. You will be asked to do the following things if 
72 
you agree to participate in this study:  
73 
 
74 
 
 
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IRB Protocol Number: 
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IRB Approval date:  05.23.21 
Version:  3.0 
 
 
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Table 1. Sequence of experimental procedures and durations for laboratory visits. 
76 
Experimental Procedures 
Length of Time 
Needed 
Visit 1 
Visits 2-4 
Informed Consent and Pre-Smoking Questionnaires 
30 min 
10 min 
Complete or review informed consent + consent addendum 
X 
X 
Confirm eligibility (exhaled breath sample and questionnaires) 
X 
X 
Confirm eligibility (urine pregnancy test, women only) 
X 
 
Give blood sample 
X 
X 
Eligibility status will be communicated to you 
X 
 
Complete pre-smoking subjective effects questionnaires 
X 
X 
Complete practice session for flavor perceptions questionnaires 
X 
 
Measure height and weight 
X 
 
Pre-Smoking Sample Collection and Smelling of the 
Hookah Tobacco 
30 min 
30 min 
Cleanse mouth with bottled water 3 times 
X 
X 
Smell assigned tobacco type for 20 seconds, then give flavor 
perceptions 
X 
X 
Complete lung function test 
X 
X 
Give exhaled breath sample 
X 
X 
Hookah Smoking 
30-90 
min 
30-90 
min 
Smoke hookah with assigned tobacco type as you normally 
would for 10 minutes, then give flavor perceptions 
X 
X 
After smoking for 45 minutes give exhaled breath sample 
X 
X 
Continue to smoke hookah until satiated 
X 
X 
Puffing behavior is recorded automatically 
X 
X 
Post-Smoking Sample Collection 
20 min 
20 min 
Give exhaled breath sample 
X 
X 
Give flavor perceptions 
X 
X 
Give blood sample 
X 
X 
Complete lung function test 
X 
X 
Give answers to other post-smoking questionnaires 
X 
X 
Complete demographic/tobacco product use questionnaires 
X 
 
 
77 
 
78 
First clinic visit 
79 
You will arrive at the clinic at your scheduled time.  You will be asked to conduct all of 
80 
the study activities.  First you will breathe into a handheld carbon monoxide monitor to 
81 
confirm that you have not smoked tobacco in the last 12 hours.  The study design will be 
82 
explained to you, and you will have the opportunity to ask questions before making the 
83 
decision to participate further.  If you decide to participate further, you will sign your 
84 
name giving your informed consent to participate.  If you are biologically able to become 
85 

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IRB Protocol Number: 
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IRB Approval date:  05.23.21 
Version:  3.0 
 
 
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pregnant, you will also be asked to take a urine pregnancy test to confirm that you are not 
86 
pregnant.  If the result of the test is positive, this will be communicated to you privately 
87 
and you will not be eligible to participate in the study. 
88 
 
89 
A blood sample will be collected by a trained and certified phlebotomist technician using 
90 
a needle and syringe to draw up to 2 teaspoons or 10 mL on each stick.  A second sample, 
91 
for a total of 20 mL per needle stick, may be collected for quality control (QC) purposes if 
92 
the subject agrees.  Only one QC sample will be collected for any subject at any given 
93 
clinic visit.  If we have difficulty drawing your blood, there will be a maximum of 3 
94 
needle sticks per draw.  If you are unable to give a sufficient blood sample, you will be 
95 
ineligible to participate further. 
96 
 
97 
You will be asked to answer some general questions about yourself, like your age, gender, 
98 
and your history of tobacco product use.  If your survey responses and/or pregnancy test 
99 
(if applicable) and/or ability to give a sufficient blood sample indicate that you are eligible 
100 
to continue participation you will proceed with participation; otherwise you will not 
101 
participate further. 
102 
 
103 
We will collect data on your height and weight.  Prior to smoking hookah, you will 
104 
participate in a training session to learn how to complete liking/disliking and flavor and 
105 
answer some questions about hookah smoking.  If there is an active pandemic, such as 
106 
COVID-19, you will be put into a ventilated room designed to vent tobacco smoke from 
107 
the room.  You will be provided with warm (body temperature) bottled water and asked to 
108 
rinse out your mouth and spit three separate times into a cup.  You will be provided with a 
109 
small container of the waterpipe tobacco that you will smoke that day.  You will be asked 
110 
to smell the tobacco and then provide your responses to the questionnaires describing your 
111 
liking/disliking and flavor perceptions. 
112 
 
113 
To measure lung function before and after smoking, you will be asked to take a deep 
114 
breath and then breathe out forcefully into a handheld spirometer to measure your lung 
115 
function. You may be asked to repeat this procedure up to five times before and after 
116 
smoking.  You will then be provided with a prepared research-grade hookah and asked to 
117 
smoke as you normally would until you feel satiated. 
118 
 
119 
The hookah will be connected to a device that measures how you are smoking it. You will 
120 
be restricted to one bowl of tobacco and one charcoal.   
121 
 
122 
After 10 minutes, we will ask you to stop smoking to provide your responses to the 
123 
questionnaires describing your liking/disliking and flavor perceptions.  After smoking for 
124 
45 minutes you will again be asked to exhale into the same handheld carbon monoxide 
125 
monitor as before; at that time, we may stop the smoking session.  If we do not stop the 
126 
smoking session, you will then proceed to smoke the hookah as you normally would until 
127 
you feel satiated.   
128 
 
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Biomedical/Cancer 
 
 
 
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2021C0039 
IRB Approval date:  05.23.21 
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When you feel you are done smoking, you will notify us and we will end the smoking 
130 
session.  Immediately after your smoking session, you will be asked to exhale into the 
131 
carbon monoxide monitor and provide your responses about liking/disliking and flavor 
132 
perceptions on questionnaires.  We will collect blood samples from you and repeat the 
133 
lung function measurement.  You will be asked to answer some more questions about your 
134 
hookah smoking experience.  You will schedule a second clinic visit to take place in one 
135 
week at approximately the same time of day. 
136 
 
137 
Clinic visits 2-4 
138 
 
139 
If you are female and able to get pregnant, you will be asked to use a birth control method 
140 
for the duration of your participation in the study (e.g., birth control pills, implants, IUD, 
141 
Depo-Provera, or condoms) to prevent pregnancy.  All participants will be asked to 
142 
abstain from smoking hookah or other tobacco products for at least 12 hours prior to their 
143 
next clinic visit.  
144 
 
145 
It will take you up to 3 hours from the time you check in with the research staff until the 
146 
time you complete all the procedures for the first visit.  The remaining 3 visits will take 
147 
about 2.5 hours each.   
148 
 
149 
4.   How long will I be in the study? 
150 
 
151 
Each clinic visit will last up to three hours and be separated by at least a week.  Therefore 
152 
participants can expect to be in the study for about four weeks. 
153 
 
154 
5. Can I stop being in the study? 
155 
 
156 
You may leave the study at any time.  If you decide to stop participating in the study, 
157 
there will be no penalty to you, and you will not lose any benefits to which you are 
158 
otherwise entitled.  Your decision will not affect your future relationship with The Ohio 
159 
State University.  
160 
 
 
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Biomedical/Cancer 
 
 
 
IRB Protocol Number: 
2021C0039 
IRB Approval date:  05.23.21 
Version:  3.0 
 
 
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6.   What risks, side effects or discomforts can I expect from being in the study? 
162 
 
163 
While completing questionnaires, there is a small risk of mental discomfort. You can 
164 
refuse to answer any question(s) if you feel uncomfortable.  
165 
 
166 
Although many hookah smokers think it is less harmful, hookah smoking has many of the 
167 
same health risks as cigarette smoking. Carbon monoxide is produced through hookah 
168 
smoking, therefore sufficient ventilation is used to ensure safe levels in the room. 
169 
However, if at any time you feel faint, light-headed, dizzy, headache, or shortness of 
170 
breath please notify us immediately and the smoking session will be stopped at that time. 
171 
 
172 
You will give 20 mL of blood before and at the end of each smoking session, for a total of 
173 
40 mL for each laboratory visit.  The amount of blood you give during each laboratory 
174 
visit is approximately 8 teaspoons, which is about ten times less than would be taken in a 
175 
routine blood donation at the American Red Cross (about 450 mL).  You may feel some 
176 
pain, like a slight sting or “pinch” in your arm, when the blood is drawn.  You may also 
177 
get a small bruise as a result of the blood draw.  Some people get dizzy, feel nauseous, or 
178 
faint when their blood is drawn, but this is somewhat rare.  There is a very small chance 
179 
that you might get an infection as a result of giving the blood sample.  Our professional 
180 
staff will follow a standardized procedure and take any necessary steps to ensure your 
181 
safety.  Each needle used in the procedure is sterile and is disposed of after a single use.  If 
182 
you have a history of problems resulting from blood donations or blood draws, you should 
183 
not take part in this study. 
184 
 
185 
The hookah you will be smoking is specially designed for investigational research only, 
186 
and as such may have unknown health risks.  This hookah has been used by participants in 
187 
three previous studies. 
188 
 
189 
You may feel hungry during the clinic visit because you will not be permitted to eat until 
190 
after your last lung function test is completed. 
191 
 
192 
Lung function tests involve forceful breathing out. Sometimes, people become light-
193 
headed during the tests or have chest soreness for a few days.  You will be seated and 
194 
closely monitored during these tests. 
195 
 
196 
There is a risk of breach of confidentiality or a loss of privacy if other people find out 
197 
about your participation. All efforts are made to keep your information confidential, but 
198 
confidentiality is not absolute. 
199 
 
 
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Biomedical/Cancer 
 
 
 
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IRB Approval date:  05.23.21 
Version:  3.0 
 
 
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7.   What benefits can I expect from being in the study? 
201 
 
202 
You will not experience any personal benefits from being in the study. However, you will 
203 
be providing valuable information for the researchers to use in future phases of the study. 
204 
 
205 
8.   What other choices do I have if I do not take part in the study? 
206 
 
207 
You may choose not to participate without penalty or loss of benefits to which you are 
208 
otherwise entitled. 
209 
 
210 
9.   What are the costs of taking part in this study? 
211 
 
212 
There are no costs to you for participating in this study. 
213 
 
214 
10. Will I be paid for taking part in this study? 
215 
 
216 
By law, payments to participants are considered taxable income. You will be paid up to 
217 
$250 for taking part in the study clinic visits, as outlined below. You will receive your 
218 
payments at the end of your last clinic visit.  Payments will be made using the Greenphire 
219 
ClinCard to increase accountability and facilitate ease of payment.  If you withdraw from 
220 
the study before your last clinic visit, you may receive payment for the visit(s) you 
221 
attended. 
222 
• Visit 1: $50 
223 
• Visit 2: $50 
224 
• Visit 3: $50 
225 
• Visit 4: $50 
226 
• Bonus payment for completing all four visits: $50 
227 
 
228 
 
229 
11. What happens if I am injured because I took part in this study? 
230 
 
231 
If you suffer an injury from participating in this study, you should notify the researcher or 
232 
study doctor immediately, who will determine if you should obtain medical treatment at 
233 
The Ohio State University Wexner Medical Center.   
234 
 
235 
The cost for this treatment will be billed to you or your medical or hospital insurance. The 
236 
Ohio State University has no funds set aside for the payment of health care expenses for 
237 
this study.  
238 
 
 
239 

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Biomedical/Cancer 
 
 
 
IRB Protocol Number: 
2021C0039 
IRB Approval date:  05.23.21 
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12. What are my rights if I take part in this study? 
240 
 
241 
If you choose to participate in the study, you may discontinue participation at any time 
242 
without penalty or loss of benefits.  By signing this form, you do not give up any personal 
243 
legal rights you may have as a participant in this study. 
244 
 
245 
You will be provided with any new information that develops during the course of the 
246 
research that may affect your decision whether or not to continue participation in the 
247 
study. 
248 
 
249 
You may refuse to participate in this study without penalty or loss of benefits to which 
250 
you are otherwise entitled. 
251 
 
252 
An Institutional Review Board responsible for human subjects research at The Ohio State 
253 
University reviewed this research project and found it to be acceptable, according to 
254 
applicable state and federal regulations and University policies designed to protect the 
255 
rights and welfare of research participants. 
256 
 
257 
13. Will my de-identified information (and bio-specimens) be used or shared for 
258 
future research?   
259 
 
260 
No. 
261 
 
262 
14. Will my study-related information be kept confidential? 
263 
 
264 
Efforts will be made to keep your study-related information confidential.  However, there 
265 
may be circumstances where this information must be released.  For example, personal 
266 
information regarding your participation in this study may be disclosed if required by state 
267 
law.   
268 
 
269 
Also, your records may be reviewed by the following groups (as applicable to the 
270 
research): 
271 
• 
Office for Human Research Protections or other federal, state, or international 
272 
regulatory agencies; 
273 
• 
U.S. Food and Drug Administration; 
274 
• 
The Ohio State University Institutional Review Board or Office of Responsible 
275 
Research Practices; 
276 
• 
The sponsor supporting the study, their agents or study monitors; and 
277 
• 
Your insurance company (if charges are billed to insurance). 
278 
 
279 
If this study is related to your medical care, your study-related information may be placed 
280 
in your permanent hospital, clinic, or physician’s office records. Authorized Ohio State 
281 
University staff not involved in the study may be aware that you are participating in a 
282 
research study and have access to your information.  
283 

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Biomedical/Cancer 
 
 
 
IRB Protocol Number: 
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IRB Approval date:  05.23.21 
Version:  3.0 
 
 
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284 
The NIH has issued a Certificate of Confidentiality for this study. This Certificate 
285 
provides extra protection for you and your study information, documents, or samples 
286 
(cheek cells, etc.). The Certificates are issued so that we cannot be required to disclose 
287 
any identifiable, sensitive information collected about you as a part of this study in a 
288 
lawsuit or legal proceeding. We are also prevented from releasing your study information 
289 
without your consent. This is a layer of protection over and above the already existing 
290 
protections in place for you and your information, documents, or samples. 
291 
 
292 
However, these protections do not apply in some situations. For example, we may have to 
293 
release your information if a law requires us to do so, the Agency that is funding this 
294 
study requests the information, or if the FDA tells us to release this information. We may 
295 
also use your information to conduct other scientific research as allowed by federal 
296 
regulations. 
297 
 
298 
Study information that has health implications may be placed in your medical record 
299 
where authorized employees may see the information. Further, authorized requests for 
300 
your records (medical record release for continuity of care) may result in research-related 
301 
information being released. 
302 
 
303 
Please talk to your study team, or contact the Office of Responsible Research Practices at 
304 
614-688-8641, if you have questions.  You may also visit the NIH website at 
305 
https://humansubjects.nih.gov/coc/faqs to learn more. 
306 
 
307 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as 
308 
required by U.S. law.  This website will not include information that can identify you.  At 
309 
most, the website will include a summary of the results.  You can search the website at 
310 
any time. 
311 
  
312 
You may also be asked to sign a separate Health Insurance Portability and Accountability 
313 
Act (HIPAA) research authorization form if the study involves the use of your protected 
314 
health information. 
315 
 
316 
15. Who can answer my questions about the study? 
317 
 
318 
For questions, concerns, or complaints about the study you may contact Marielle 
319 
Brinkman at 614-688-3226, or Additive Study staff by email at Additive-
320 
Study@osumc.edu. 
321 
 
322 
For questions about your rights as a participant in this study or to discuss other study-
323 
related concerns or complaints with someone who is not part of the research team, you 
324 
may contact the Office of Responsible Research Practices at 1-800-678-6251. 
325 
 
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Biomedical/Cancer 
 
 
 
IRB Protocol Number: 
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IRB Approval date:  05.23.21 
Version:  3.0 
 
 
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If you are injured as a result of participating in this study or for questions about a study-
327 
related injury, you may contact Marielle Brinkman at 614-688-3226. If, after you leave the 
328 
clinic, you are unsure if you are having an adverse or negative reaction, please call 
329 
Marielle Brinkman as soon as possible. 
330 
 
331 
Signing the consent form 
332 
 
333 
 
334 
 
335 
I have read (or someone has read to me) this form and I am aware that I am being asked to 
336 
participate in a research study.  I have had the opportunity to ask questions and have had them 
337 
answered to my satisfaction.  I voluntarily agree to participate in this study.  
338 
 
339 
I am not giving up any legal rights by signing this form.  I will be given a copy of this form. 
340 
 
341 
 
 
 
 
Printed name of participant 
 
Signature of participant 
 
 
 
 
 
AM/PM 
 
 
Date and time 
 
 
 
 
 
 
 
 
 
Printed name of person authorized to consent for 
participant (when applicable) 
 
Signature of person authorized to consent for participant  
(when applicable) 
 
 
 
 
 
AM/PM 
Relationship to the participant 
 
Date and time 
 
 
342 
 
343 
 
344 
Investigator/Research Staff 
345 
 
346 
I have explained the research to the participant or his/her representative before requesting the 
347 
signature(s) above.  There are no blanks in this document.  A copy of this form has been given 
348 
to the participant or his/her representative. 
349 
 
350 
 
 
 
 
Printed name of person obtaining consent 
 
Signature of person obtaining consent 

CONSENT 
Biomedical/Cancer 
 
 
 
IRB Protocol Number: 
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IRB Approval date:  05.23.21 
Version:  3.0 
 
 
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AM/PM 
 
 
Date and time 
 
 
351 
Witness(es) - May be left blank if not required by the IRB 
352 
 
353 
 
 
 
 
Printed name of witness 
 
Signature of witness 
 
 
 
 
 
AM/PM 
 
 
Date and time 
 
 
 
 
 
Printed name of witness 
 
Signature of witness 
 
 
 
 
 
 
AM/PM 
 
 
Date and time 
 
 
354