Water Content In E-Liquids
Phase: NA
Conditions: Nicotine E-Cigarette Use
Sponsor: Yale University

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CLINICAL STUDY PROTOCOL
Interventional Drug or Biologic
 
Examining the Effect of Varying Water 
Content of E-liquids on Sensory 
Experience    
 Protocol Number
2000031495
Protocol Version
5-25-22
Version # 3
PRINCIPAL INVESTIGATOR:
Name: Danielle Davis
Department: Psychiatry
Telephone Number: 678-983-1253
Email Address: Danielle.davis@yale.edu 
IND Number: 
IND Holder:
NCT Number: N/A
Study Phase (1, 2, 3, or 4-postmarketing):
Confidentiality Statement:
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REVISION HISTORY:
Revision #
Version Date
3
5-25-22
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Synopsis
Primary Objective
The primary objective of this study is to examine whether (a) sensory irritation and (b) appeal 
are decreased in e-liquids containing flavor aldehydes (i.e. vanilla, cherry, cinnamon) compared 
to e-liquids not containing flavor aldehydes (i.e. grape) when the water concentration is 
increased from the baseline water concentration to the following levels; 5% total water volume, 
10% total water volume, 20% total water volume.
Secondary Objective (if applicable)
At each water concentration, we will examine if there are differences in perception of sensory 
irritation and appeal across the distinct aldehyde containing flavors.
Study Duration
2 years to complete study from HIC submission; we anticipate the subject enrollment period will 
be 8 months (approximately 1 week/participant). Enrollment began in August 2022, so 
anticipated subject enrollment will end in March 2023. 
Study Design 
Within subjects’ human laboratory experiment 
Number of Study Sites
1; Yale School of Medicine. Yale sites include Connecticut Mental Health Center (intake visits) 
and John B. Pierce Laboratory (lab sessions).
Study Population
Population is regular e-cigarette users who report use of both nicotine and flavors in their e-
cigarettes. We are assessing response to alterations of water level in e-cigarettes among 
regular users as a potential regulatory tool, so current participants must have regular nicotine 
experience. 
Number of Participants
30; pilot study 
Primary Outcome Variables
(1) Change in irritation at each water level as measured by the Generalized Labeled 
Magnitude Scale (gLMS)
(2) Change in appeal at each water level as measured by the Labeled Hedonic Scale 
(LHS), Drug Effects Questionnaire (DEQ)
Secondary and Exploratory Outcome Variables (if applicable)
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(1) At each water level, differences in irritation (gLMS) and appeal  (LHS, DEQ) 
between flavors (to examine if there are differences in effect of water level on 
irritation/appeal by aldehyde flavor)
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Abbreviations
Abbreviation 
Explanation
LHS
Labeled Hedonic Scale
gLMS
Generalized Labeled Magnitude Scale
TCORS
Tobacco Centers for Regulatory Science 
e-cig
E-cigarette
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Glossary of Terms
Glossary
Explanation
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Table of Contents
Synopsis ..................................................................................................................................2
Primary Objective.................................................................................................................2
Secondary Objectives (if applicable)....................................................................................2
Study Duration......................................................................................................................2
Study Design........................................................................................................................2
Study Population ..................................................................................................................2
Number of Participants.........................................................................................................2
Number of Study Sites .........................................................................................................2
Primary Outcome Variables .................................................................................................2
Secondary and Exploratory Outcome Variables (if applicable)............................................2
Visit Schedule Table (Optional)............................................................................................2
Study Flow Chart (optional)..................................................................................................2
Abbreviations ...........................................................................................................................3
Glossary of Terms....................................................................................................................4
1
Introduction.......................................................................................................................6
1.1
Introductory Statement ..............................................................................................6
2
Background.......................................................................................................................7
2.1.1
Preclinical Experience........................................................................................7
2.1.2
Clinical Experience.............................................................................................7
2.2
Background/prevalence of research topic .................................................................7
3
Rationale/Significance ......................................................................................................8
3.1
Problem Statement....................................................................................................8
3.2
Purpose of Study/Potential Impact ............................................................................8
3.2.1
Potential Risks....................................................................................................8
3.2.2
Potential Benefits ...............................................................................................8
4
Study Objectives...............................................................................................................9
4.1
Hypothesis.................................................................................................................9
4.2
Primary Objective ......................................................................................................9
4.3
Secondary Objectives (if applicable) .........................................................................9
5
Study Design ..................................................................................................................10
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5.1
General Design Description.....................................................................................10
5.1.1
Study Date Range and Duration ......................................................................10
5.1.2
Number of Study Sites .....................................................................................10
5.2
Outcome Variables..................................................................................................10
5.2.1
Primary Outcome Variables .............................................................................10
5.2.2
Secondary and Exploratory Outcome Variables (if applicable)........................10
5.3
Study Population .....................................................................................................10
5.3.1
Number of Participants.....................................................................................10
5.3.2
Eligibility Criteria/Vulnerable Populations.........................................................10
6
Methods..........................................................................................................................12
6.1
Treatment - Drug .....................................................................................................12
6.1.1
Identity of Investigational Product/New Drug ...................................................12
6.1.2
Dosage, Admin, Schedule (if applicable) .........................................................12
6.1.3
Method of Assignment/Randomization (if applicable) ......................................12
6.1.4
Blinding and Procedures for Unblinding (if applicable).....................................12
6.1.5
Packaging/Labelling .........................................................................................12
6.1.6
Storage Conditions...........................................................................................12
6.1.7
Concomitant therapy ........................................................................................12
6.1.8
Restrictions.......................................................................................................12
6.2
Assessments ...........................................................................................................12
6.2.1
Efficacy.............................................................................................................12
6.2.2
Safety/Pregnancy-related policy.......................................................................13
6.2.3
Adverse Events Definition and Reporting.........................................................13
6.2.4
Pharmacokinetics (if applicable).......................................................................13
6.2.5
Biomarkers (if applicable).................................................................................13
6.3
Study Procedures....................................................................................................13
6.3.1
Study Schedule ................................................................................................14
6.3.2
Informed Consent.............................................................................................14
6.3.3
Screening .........................................................................................................14
6.3.4
Recruitment, Enrollment and Retention ...........................................................14
6.3.5
On Study Visits.................................................................................................14
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6.3.6
End of Study and Follow-up .............................................................................14
6.3.7
Removal of subjects.........................................................................................14
6.4
Statistical Method ....................................................................................................14
6.4.1
Statistical Design..............................................................................................14
6.4.2
Sample Size Considerations ............................................................................15
6.5
Planned Analyses....................................................................................................15
6.5.1
Primary Analyses .............................................................................................15
6.5.2
Secondary Objectives Analyses.......................................................................15
6.5.3
Safety/Pregnancy-related policy.......................................................................15
6.5.4
Analysis of Subject Characteristics ..................................................................15
6.5.5
Interim Analysis (if applicable)..........................................................................15
6.5.6
Health economic evaluation .............................................................................15
6.5.7
Other ................................................................................................................15
6.5.8
Subsets and Covariates ...................................................................................15
6.5.9
Handling of Missing Data .................................................................................15
7
Trial Administration.........................................................................................................16
7.1
Ethical Considerations: Informed Consent/Assent and HIPAA Authorization .........16
7.2
Institutional Review Board (IRB) Review.................................................................16
7.3
Subject Confidentiality.............................................................................................16
7.4
Deviations/Unanticipated Problems.........................................................................17
7.5
Data Collection ........................................................................................................17
7.6
Data Quality Assurance...........................................................................................17
7.7
Study Records.........................................................................................................17
7.8
Access to Source.....................................................................................................17
7.9
Data or Specimen Storage/Security ........................................................................17
7.10
Retention of Records...............................................................................................17
7.11
Study Monitoring......................................................................................................18
7.12
Data Safety Monitoring Plan....................................................................................18
7.13
Study Modification ...................................................................................................18
7.14
Study Discontinuation..............................................................................................18
7.15
Study Completion ....................................................................................................18
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7.16
Conflict of Interest Policy.........................................................................................18
7.17
Funding Source .......................................................................................................18
7.18
Publication Plan.......................................................................................................19
8
Appendices.....................................................................................................................20
9
List of Tables ..................................................................................................................21
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1 Introduction
1.1
Introductory Statement
This document is a protocol for a human research study. The purpose of this protocol is to 
ensure that this study is to be conducted according to ICH GCP guidelines, and according to 
CFR 21 Part 312, other applicable government regulations and Institutional research policies 
and procedures.
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2 Background
2.1.1
Preclinical Experience
See background (2.2) for all relevant information.
2.1.2
Clinical Experience
See background (2.2) for all relevant information.
2.2
Background/prevalence of research topic
Most US tobacco products contain a wide variety of flavorants, with an estimated 15000+ 
flavors in e-cigarettes (e-cigs) alone.1 Flavors may enhance the appeal of tobacco products 
by their positive attributes (e.g. aroma/taste of sweet flavors) and/or by their ability to 
ameliorate nicotine/tobacco harshness (e.g. cool flavors)1-4. 
While the Family Tobacco Prevention and Tobacco Control Act (FSPTCA) banned the sale 
of cigarettes with added artificial and natural flavors in 2009, flavors are currently produced 
and available in electronic cigarette products. Even new FSPTCA regulations (introduced in 
May 2016) do not include any of the other assortments of flavors or sweeteners in any of the 
other tobacco products (e.g., e-cigs). One potential reason for the limited action on flavors is 
the limited scientific evidence on the influence on flavors and sweeteners on nicotine’s 
abuse potential. To support the regulation of tobacco products, the FDA requires rigorous 
scientific information on which to base their rulings and support these rulings in legal 
challenges. These regulatory decisions are further complicated by the multitude of flavors 
available in tobacco products and the constant innovation by the tobacco industry to use 
flavorant chemicals to enhance and maintain appeal for these products.
As flavors continue to be available in these products and used by e-cigarette users, one aim 
of researchers is to develop ways to reduce potential toxicity of these products. Flavor 
chemicals in e-liquids may expose users to respiratory harms.2,3 While many flavor 
chemicals used in e-liquids are designated as GRAS (generally recognized as safe), this 
designation applies to oral ingestion, not inhalation.4 Levels of flavor chemicals in e-
cigarettes vary widely, with flavors accounting for as much as 34% of the e-liquid.5 Currently, 
there are no regulations to mitigate risk associated with flavor chemicals in e-cigarettes. 
E-cigarettes contain many different ingredients to produce flavors. A common class of e-
liquid flavor chemicals are called aldehydes. Aldehyde flavor constituents are used in many 
popular flavors.5,6 For example vanillin produces a vanilla flavor and is added to many sweet 
and dessert e-liquids, benzaldehyde produces the popular fruit cherry flavor and is used in 
many fruit/sweet e-liquids, and cinnamaldehyde produces a cinnamon flavor, which is added 
to dessert flavored e-liquids, spice flavored e-liquids, and fruit e-liquids (e.g. cinnamon 
apple). 
Aldehydes are sensory and respiratory irritants and activate the sensory receptor TRPA1, an 
ion channel known to contribute to inflammation and cytotoxicity in airway tissue.7,8 
Aldehydes can undergo chemical reactions with the solvents that form the base on e-liquids; 
propylene glycol (PG) and vegetable glycerin (VG) to form chemical adducts named flavor 
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aldehyde PG/VG acetals (referred to as acetals henceforth). Recent studies have shown that 
acetals form under typical storage conditions9, are present in several commercial e-liquids 
including popular JUUL products,3,9-11 and can efficiently transfer to e-cigarette aerosols.9 
Acetals have been shown to cause both greater sensory irritation and cytotoxicity at the 
sensory receptor TRPA1 than their parent aldehydes in-vitro, suggesting that this chemical 
conversion can have negative health implications for users.3,9 One way to measure user 
experience of irritant effects would be to have users’ rate associated sensory irritation 
experienced. 
Preliminary (unpublished) evidence from our chemistry lab at the Yale Tobacco Center of 
Regulatory Science (TCORS) have indicated that it may be possible to reduce acetal 
formation simply by adding water to e-liquids. This reduces acetal formation by shifting the 
chemical equilibrium of the formation reaction. Water is physiologically innocuous to inhale 
via e-cigarette and adding water to e-liquids may be an elegant way to reduce the sensory 
irritation and cytotoxicity of e-liquids that contain flavor aldehydes. However, adding water 
also inherently dilutes the flavor and nicotine concentration of the product, which may reduce 
the appeal overall. This could be a negative outcome if trying to use flavors as a tool to shift 
combustible cigarette users to potential reduced harm e-cigarettes. However, if irritation is 
reduced and appeal is not significantly reduced, a regulation requiring a fixed level of water 
to e-liquids could be a simple and effective tool to reduce potential toxicity in e-liquids with 
these flavors. The goal of the proposed study is therefore to determine if the sensory 
irritation (a marker of potential toxicity) of commercial e-liquids, can be reduced by 
increasing their water content without adversely affecting flavor appeal in adult e-cigarette 
users.  
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3 Rationale/Significance
3.1
Problem Statement
Electronic cigarettes (e-cigarettes), once considered an emerging tobacco product, are 
now the most commonly used tobacco product among youth and growing in popularity 
among adults.12,13 Some data suggests that shifting combustible cigarette users to e-
cigarette use may have a net positive population benefit.14 An important facet of e-cigarette 
appeal is the availability of flavors,15,16 including non-tobacco flavors like fruit, dessert, 
candy, and mint.17 Non-tobacco flavors may be important for encouraging product switching 
among combustible tobacco users.18,19 However, flavor chemicals in e-liquids may expose 
users to respiratory harms.2,3 While many flavor chemicals used in e-liquids are designated 
as GRAS (generally recognized as safe), this designation applies to oral ingestion, not 
inhalation.4 Levels of flavor chemicals in e-cigarettes vary widely, with flavors accounting for 
as much as 34% of the e-liquid.5 Currently, there are no regulations to mitigate risk 
associated with flavor chemicals in e-cigarettes. 
A common class of e-liquid flavor chemicals are aldehydes (e.g. vanillin producing a 
vanilla flavor; benzaldehyde producing cherry flavor; cinnamaldehyde producing cinnamon 
flavor).5,6 Aldehydes are sensory and respiratory irritants and  activate the sensory receptor 
TRPA1, an ion channel known to contribute to inflammation and cytotoxicity in airway 
tissue.7,8 Aldehydes can undergo chemical reactions with the e-liquid solvents propylene 
glycol (PG) and vegetable glycerin (VG) to form chemical adducts named flavor aldehyde 
PG/VG acetals (referred to as acetals henceforth). Recent studies have shown that acetals 
form under typical storage conditions9, are present in several commercial e-liquids including 
popular JUUL products3,9-11 and can efficiently transfer to e-cigarette aerosols.9 Acetals have 
been shown to cause both greater sensory irritation and cytotoxicity at the sensory receptor 
TRPA1 than their parent aldehydes in-vitro, suggesting that this chemical conversion can 
have negative health implications for users.3,9
This study is based on preliminary evidence from our lab that adding water to e-liquids 
containing flavor aldehydes reduces acetal formation by shifting the chemical equilibrium of 
the formation reaction. Reducing acetal formation through the addition of physiologically 
innocuous water may be an elegant way to reduce the sensory irritation and cytotoxicity of e-
liquids that contain flavor aldehydes. However, it is possible that adding water might 
negatively impact the potential value of these flavors for switching from combustible 
cigarettes by diluting flavorant and nicotine concentrations”, potentially resulting in lower 
delivery per puff. The goal of the proposed study is therefore to determine if the sensory 
irritation (a marker of potential toxicity) of commercial e-liquids, can be reduced by 
increasing their water content without adversely affecting flavor appeal in adult e-cigarette 
users.  
3.2
Purpose of Study/Potential Impact
The goal of the proposed study is to determine if the sensory irritation (a marker of potential 
toxicity) of commercial e-liquids containing aldehydes compared to a control non-aldehyde 
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containing e-liquid, can be reduced by increasing their water content without adversely 
affecting flavor appeal in adult e-cigarette users. Given that adding water to e-liquid is an 
innocuous manipulation, if irritation is reduced and effect on appeal is limited, this could be a 
tool for regulation to reduce harm associated with e-cigarettes. 
3.2.1
Potential Risks
Participation in this study is thought to involve a minor increase over minimal risk. Potential 
risks and protections against risks are detailed below.
(1) E-cigarette Exposure: All participants will be required to have current regular experience 
with nicotine containing flavored e-cigarettes. Given this, the likelihood of adverse 
experience with study e-cigarettes/e-liquids is low. Additionally, we will only expose 
participants to two puffs per flavor x water level manipulation for a total of 32 puffs (16 
puffs/lab session) across the study. Participation is voluntary and participants can stop 
anytime they want and/or if they experience adverse events from nicotine or flavor 
exposure. 
There have been reported cases of severe pulmonary illness linked to vaping or e-
cigarette use (i.e. E-Cigarette or Vaping Associated Lung Injury; EVALI). These cases 
were first reported in August 2019. These cases included symptoms such as cough, 
shortness of breath, chest pain, nausea, vomiting, stomach pain, diarrhea, fever, chills, 
or weight loss. Some patients reported symptoms to have occurred over a few days and 
some reported to have occurred over a few weeks. Vaping-related disorders have 
ranged from mild to severe with hospitalization, intensive care with breathing machines 
and in some cases death. In most cases, but not all, people experiencing these 
symptoms were using cannabidiol (CBD) and marijuana (THC) e-liquids, and/or using e-
cigarette devices and e-liquids that were mixed at home or purchased off market (such 
as purchasing an e-liquid or device on the street, not from a licensed retailer). 
Laboratory data shows that Vitamin E Acetate, an additive in some THC-containing e-
cigarette or vaping products, is strongly linked to EVALI. 
Products used in our study are purchased only from a licensed retailer and do not 
contain CBD, THC, or Vitamin E Acetate, so the risks of developing EVALI as a result of 
study participation are highly unlikely. However, we will monitor for EVALI symptoms at 
intake and at each lab session using an assessment checklist developed in response to 
EVALI by our lab, the health assessment checklist (included in study measures). Any 
study participant reporting current THC/CBD vaping and report of mild or greater EVALI-
related symptoms (i.e. cough, shortness of breath, chest pain, nausea, vomiting, 
stomach pain, diarrhea, fever, chills, or weight loss) without non-EVALI reasonable and 
proximal cause will be ineligible or withdrawn and recommended. 
Additionally, e-cigarette liquids contain propylene glycol and vegetable glycerin, which 
some people may be allergic to. As participants are current e-cigarette users’, so all 
participants will have prior exposure to these constituents. Additionally, we will only 
allow participants with experience with flavored e-liquids. We will confirm at intake they 
do not have known allergies to either of these products or e-liquid flavors.
(2) E-Liquid Water Manipulation: Some of the e-liquids participants will use will have water 
added to liquid at levels, so total water level of the e-liquid is 5% total volume, 10% of 
total volume, 20% total volume. Unmanipulated e-liquids contain some level of water. 
There is no evidence that the addition of water will produce any additional harm to e-
cigarette users or that e-cigarette device will heat water to a level that produces harm.20 
Evidence suggests that addition of water may reduce irritation associated with e-
cigarette use. 
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(3) Nicotine Exposure: E-liquids will contain moderate levels of nicotine salts (36mg/ml). 
Common side effects of nicotine include nausea, vomiting, heartburn, and elevated 
heart rate and blood pressure. Toxic doses of nicotine may cause abdominal pain, 
hypersalivation, diarrhea, dizziness, confusion, hearing and vision problems, syncope, 
seizures, hypotension, irregular pulse, and death. However, these toxic effects occur at 
doses 40 to 50 times higher than those that will be used in our studies. Moreover, by 
recruiting adults who already have experience with e-cigarettes and nicotine, we will 
further mitigate the risk of these side effects.
(4) Flavor Exposure: We will expose participants to four different e-liquids flavors; cinnamon, 
cherry, vanilla, and grape e-liquid. All e-liquids are commercially available currently, 
except for the cinnamon e-liquid, which is currently only available for research use. This 
cinnamon e-liquid was developed to mirror commercially available cinnamon products.  
Cinnamon flavoring is known to have health impacts at levels much higher than 
participants will be exposed to in this study.21 We are not aware of any effects from the 
other flavors being used in this study but participants will be told that if they feel any 
adverse events or want to stop they are free to do so. Additionally, we will exclude 
anyone that reports allergies to any of the four flavors used. In the consent form, we will 
not state the flavors being used, but will note that one flavor is not currently 
commercially available.
(5) Urine/Saliva Collection: Urine/saliva collection at intake and during the study for females 
and should add no risks other than those normally associated with these procedures. 
(6) Rating Scales and Assessments:  These are all noninvasive and should add no risk.  
The major disadvantages are the time taken to complete them, and possible breach of 
confidentiality. Our past experience with these measures indicates that they are 
acceptable to subjects.  
(7) Limits to confidentiality: All participants will be specifically told that we will not reveal any 
personal information collected as part of the research procedures, including their 
reported use of e-cigarettes and other substance use history. However, there is always 
the possibility that participation in this study may make others, such as friends and 
family members, aware of their tobacco use status. They will be told that if they do not 
feel comfortable with this, then they should not participate in the project.  They will also 
be told that if they report any information to us about abuse or homicidal/suicidal 
behavior, we will be required to report this information to the appropriate authorities.
Protections Against Risk: We will reduce risks by:

Requiring participants to be regular nicotine & flavor e-cigarette users. 

Administering briefest possible exposure needed for our outcomes to each study product 
(2 e-cigarette puffs/product)

Using well-defined inclusion/exclusion criteria to rule out participants with pre-existing 
medical conditions

Providing participants with written information on reducing e-cigarette use at the end of 
study participation. 

Using study staff who have expertise conducting tobacco research and working with 
adults and who are sensitive to the issues that may arise in working with e-cigarette 
users.  

Protect right to privacy through coding of data and proper storage of research records.  

Obtain a certificate of confidentiality from NIH to further protect the research records of 
these participants.
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
 Use the Yale TCORS Independent Data Safety Monitoring Board including experts in 
the field of tobacco use behaviors and challenge studies (Dr. Tony George, FRCPC, 
Professor and Co-Director, Division of Brain and Therapeutics, Dept. of Psychiatry, U of 
Toronto; Dr. Thomas Brandon, Professor and Chair, Department of Health Outcomes & 
Behavior, H. Lee Moffitt Cancer Center & Research Institute) and a statistical expert (Dr. 
Hanga Galfalvy, Assistant Professor of Neurobiology, Columbia University) .
3.2.2
Potential Benefits
The proposed project will provide the FDA with important data regarding how adding an 
innocuous constituent, water, to e-cigarettes that contain flavors chemicals comprised of 
aldehydes, could have the ability to reduce user’ irritation. E-liquids with flavor chemicals 
containing aldehydes are popular and common but may have potential associated toxicity 
that produces user experienced irritation. Preliminary data from our chemistry lab suggests 
adding water may be one way to reduce potential toxicity. Regulating the level of water in e-
liquids is a relatively simple regulation for the FDA to implement. However, it is possible that 
adding water might negatively impact the potential value of these flavors for switching from 
combustible cigarettes by diluting flavorants and nicotine concentrations, potentially resulting 
in lower delivery (and thus appeal) per puff. Study outcomes could inform potential future 
legislation aiming at addressing certain health concerns of vaping products, such as a 
mandate for a minimum amount of water present in e-liquids.
An additional benefit in this study is to participants who are current THC/CBD vapers. They 
may be at additional risk for EVALI given their THC/CBD vaping, but are not currently being 
monitored. During the study, they will be monitored for EVALI related symptoms. 
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4 Study Objectives
4.1
Hypothesis
We hypothesized that as total water volume is increased in nicotine e-liquids containing 
flavor aldehydes, sensory irritation experienced by the e-cigarette user will decrease. We do 
not expect to see this decrease in the control e-liquid (grape e-liquid), which does not 
contain flavor aldehydes. 
4.2
Primary Objective
The primary objective of this study is to examine whether (a) sensory irritation and (b) appeal 
are decreased in e-liquids containing flavor aldehydes (i.e. vanilla, cherry, cinnamon) 
compared to e-liquids not containing flavor aldehydes (i.e. grape) when the water 
concentration is increased from the baseline water concentration to the following levels; 5% 
total water volume, 10% total water volume, 20% total water volume. 
4.3
Secondary Objectives (if applicable)
At each water concentration, we will examine if there are differences in perception of 
sensory irritation and appeal across the distinct aldehyde containing flavors.
4.4
Exploratory Objectives (if applicable)
N/A
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5 Study Design
5.1
General Design Description
The study is a within subjects’ laboratory study, in which participants will complete two 
laboratory sessions under double blind conditions. Across the study, participants will be 
exposed to four different e-liquid flavors at a single fixed nicotine concentration (36mg/ml 
nicotine salt formulation). Three of the e-liquids flavors will be aldehyde containing e-liquids 
and one flavor will be a non-aldehyde containing flavor and serve as a control. Each flavor 
will be presented with the follow manipulations to its water concentration; (1) flavor as 
commercially produced, (2) water added so 5% total water volume achieved, (3) water 
added so 10% total water volume achieved, (4) water added so 20% total water volume 
achieved. In each lab session, under double blind conditions, participants will sample two of 
the four flavors at each of the 4 water volumes (i.e. no change to water volume, 5% water 
volume, 10% water volume, 20% water volume). Participants will be randomized to flavor 
order and water concentration for each flavor will be presented from hypothesized least 
irritating to hypothesized most irritating, as is recommended with chemosensory studies. 
Figure 1. Diagram of Study Procedures 
Pre-screen potential participants via phone 
Intake Visit 
Informed Consent 
Medical History
Tobacco & E-Cigarette Use History
Urine Sample
Eligibility Checklist
Block Randomization to Assign Flavor 
Presentation Order
        
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5.1.1
Study Date Range and Duration
The expected duration of the total study from recruitment 2 years to complete study from 
HIC submission; we anticipate the subject enrollment period will be 8 months (approximately 
1 week/participant). Enrollment began in August 2022, so anticipated subject enrollment will 
end in March 2023. The expected duration/participant is approximately 1-2 weeks to 
complete intake appointment(s), lab 1 and lab 2. 
5.1.2
Number of Study Sites
The study will take place at Yale University School of Medicine. Study sites will include the 
Connecticut Mental Health Center (Rm S201) for intake appointments and Pierce 
Laboratories (290 Congress Avenue, New Haven, CT), which are equipped with ventilated 
research chambers utilized for smoking and vaping research studies. 
5.2
Outcome Variables
The outcome variables used in this study are irritation, as measured by the generalized 
Labeled Magnitude Scale (gLMS)22,23 and appeal, as measured by the Revised Drug Effects 
Questionnaire24 (measuring liking & stimulation), the Labeled Hedonic Scale (LHS) to 
measure overall liking, the gLMS to measure sweetness, and the Multiple Choice 
Questionnaire (MCQ; e-cig value).25 
5.2.1
Primary Outcome Variables
(3) Change in irritation at each water level 
(4) Change in appeal at each water level 
5.2.2
Secondary Outcome Variables (if applicable)
(2) At each water level, differences in irritation and appeal between flavors (to 
examine if there are differences in effect of water level on irritation/appeal by 
aldehyde flavor)
5.2.3
Exploratory Outcome Variables (if applicable)
N/A
5.3
Study Population
Participant will be healthy adult (21 or older) regular (at least 4x/week) e-cigarette users who 
are users of nicotine containing e-cigarettes (urine cotinine >200 ng/ml). 
5.3.1
Number of Participants
The current study is a pilot study and we aim to enroll 30 subjects. Based on previous pilot 
experimental work with e-cigarettes in our group, 30 has been sufficient for by our 
biostatistical staff for determining large effects by water level. 
5.3.2
Eligibility Criteria/Vulnerable Populations
In order to eligible to participate in the study, individuals must meet all of the follow criteria:
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
At least 21 years old (legal e-cigarette use age)

Able to read and write 

Report at least 6 months of e-cigarette use 

Report e-cigarette use at least on average 4 days/week in the past month

Report use of flavored e-cigarettes (i.e. cannot be using unflavored e-cigarettes)

Report use of nicotine containing e-cigarettes 

Urine cotinine levels of at least 200ng/mL

Do not plan to discontinue e-cigarette use or other current tobacco product use (e.g. 
cigarettes) during study duration

Spirometer reading with an FEV1 of ≥70%.
An individual who meets any of the following criteria will be excluded from study 
participation: 

Currently pregnant, breastfeeding, or trying to become pregnant 

Medical conditions including chronic and untreated acute pulmonary conditions, that 
in the investigators view will increase risk of respiratory problems among participants 

Known hypersensitivity to propylene glycol, vegetable glycerin, and/or e-liquid 
flavorants

Current use of illegal substances, not including cannabis, as determined by both self-
report and a urine drug screen at intake. 

For current cannabis vapers: Any report of mild or great EVALI-related symptoms 
(i.e. cough, shortness of breath, chest pain, nausea, vomiting, stomach pain, 
diarrhea, fever, chills, or weight loss) without non-EVALI reasonable and proximal 
cause

Report of greater than “without any difficulty” on any item of the PROMIS Physical 
Function Short Form

Any significant current medical condition such as neurological, cardiovascular, 
endocrine, renal, or hepatic pathology that would increase risk or would interfere 
with/mimic tobacco abstinence.
o
Medical conditions increasing risk of respiratory problems in nonpatient 
populations: Chronic Pulmonary Conditions;
o
Untreated, unresolved, acute pulmonary conditions (recurring bronchitis and 
Reactive airway disorder, as examples).

Current diagnosis of an untreated psychiatric condition

Not fully vaccinated for COVID-19
6 Methods
6.1
Treatment 
6.1.1
Identity of Investigational Product
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The investigational tobacco products being used in the study are the following e-liquids in 
36mg nicotine salt concentration: (1) DuraSmoke Red Label (50/50 propylene 
glycol/vegetable glycerin base) Cherry flavor liquid, (2) DuraSmoke Red Label ((50/50 
propylene glycol/vegetable glycerin base) Vanilla flavor liquid, (3) DuraSmoke Red Label 
((50/50 propylene glycol/vegetable glycerin base) Grape flavor liquid, and (4) American E-
liquids Store (50/50 propylene glycol/vegetable glycerin base) Cinnamon Flavor Liquid. All of 
these products will be administered in a Suorin iShare Pod System E-cigarette. All of these 
products are currently commercially available, except American E-liquids Store (50/50 base) 
Cinnamon Flavor Liquid, which is currently only available as a research product. However, 
this flavor was a previously commercially available e-liquid and was developed the 
formulation has not changed. It is not currently sold by American E-Liquids due to lack of 
popularity. In the current study, these e-liquids will be manipulated by adding water to each 
product, so that water concentration in each of the four flavors equals the following amounts; 
(1) 5% of the total e-liquid concentration, (2) 10% of the total e-liquid concentration, (3) 20% 
of the total e-liquid concentration. Additionally, e-liquids will be presented without added 
water. Participants will sample (i.e. take 2 directed puffs of each) the four flavors unaltered 
and in each of the listed water dilutions in randomized order. FDA has reviewed the
proposed use of the products and does not have concerns with the use of the investigational
tobacco products as described in the protocol. FDA issued a No Concerns letter to 
Investigators on 2/3/22. 
6.1.2
Dosage, Administration, Schedule 
6.1.3
Water Concentration Manipulation: 
E-liquid water content of each e-liquid container will be quantified by Karl Fischer (KF)-
titration or by gas chromatography/thermal conductivity detection (GC-TCD). Once the water 
content of the unaltered product has been determined, the difference to reach 5%, 10%, or 
20% water content will be added to the e-liquid in question. For example, if the water content 
was measured at 1%, 4% will be added (all percent values are weight-based). To do so, 
weighed amounts of e-liquids will be added to sterile, amber glass bottles (30mL), and the 
appropriate amount of sterile, deionized water will be added to reach the desired water 
content level (5%, 10%, 20%). This preparation will take place at the Center for Green 
Chemistry and Green Engineering (Co-PI Hanno Erythropel’s lab location). The resulting 4 
bottles per flavor (native water content, 5%, 10%, 20%) will then be transferred to the testing 
location and stored in a cool and dry location until use. For subject testing, the needed 
quantity of approx. 0.5mL of e-liquid will be pipetted from the storage container to the e-
cigarette device for testing. 30mL of each e-liquid manipulation is sufficient to conduct 
procedures with all 30 anticipated pilot participants.
E-liquid Administration/Participant Instructions/Pausing due to AEs: Participants will be 
administered e-liquid in an observed laboratory setting in which they are instructed to take 
two directed puffs of each e-liquid at each water concentration. Between 2-puff puff bouts, 
participants will be required to wait 10-minutes before engaging in next puff bout. 
Participants are not allowed to leave the site with product. There will no changes in nicotine 
concentration (only flavor administered) and all participants will sample all flavor/water 
concentration manipulations. The lab sessions will be conducted by trained research staff 
who are sensitive to the adolescent and smoking population and trained to monitor any 
potential adverse effects. Heart rate, blood pressure, and pulse oximetry will be assessed at 
baseline and every in-person visit for safety. If they experience any side effects or concerns 
during the study, we ask them to let us know. Participants will be told that they are able to 
stop the study at any point. If they feel any discomfort or need to stop for any reason, they 
can let the research team know. In the event of a serious adverse event, the PI (Dr. Danielle 
Davis), will consult with Yale Tobacco Center for Regulatory Science (TCORS; the funder of 
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this pilot) co-Is, Dr. Stephanie O’Malley and Dr. Suchitra Krishnan-Sarin, along with center 
chemosensory expert Dr. Barry Green to determine if blind needs to be broken. Given all 
study participants are administered all e-liquids, the study will be paused during this period.
6.1.4
Method of Assignment/Randomization 
All participants will receive every flavor/water concentration (4 e-liquids flavors at 4 water 
concentrations = 16 total manipulations). Participants and research assistants will be blinded 
to flavors administered. We will use block randomization to determine order flavors are 
presented in. Water concentration will be presented from hypothesized least irritating to most 
irritating (20% water, 10% water, 5% water, no alteration). 
6.1.5
Blinding and Procedures for Unblinding 
A researcher not administering lab session procedures will be responsible for filling e-liquids 
pods. Our biostatistician, Ralitza Gueorguieva, will produce a randomization chart that will be 
given to the designated researcher and stored in a locked cabinet. Pods will be numbered 
from 1-4 for each participant corresponding to the participant. Water concentration (which is 
not blinded for researchers) will be labeled A, B, C, D, with A representing “No water 
concentration change”, B representing “5% water volume”, C representing “10% water 
volume”, and D representing “20% water volume”. Participants will be blinded to water 
concentration. In the event of a serious adverse event, the PI, will consult with Yale Tobacco 
Center for Regulatory Science (TCORS; the funder of this pilot) co-Is, Dr. Stephanie 
O’Malley and Dr. Suchitra Krishnan-Sarin, along with center chemosensory expert Dr. Barry 
Green to determine if blind needs to be broken. Given all study participants are administered 
all e-liquids, the study will be paused during this period. 
6.1.6
Packaging/Labelling
We obtain all of our e-liquids from American e-Liquid Store, which manufactures all its 
products in Wauwatosa, WI and reports being the first e-liquid manufacturer in the US to 
obtain the International Organization for Standardization (ISO) 9001:2008 and Current Good 
Manufacturing practices (cGMP) certification. The e-liquid mixtures (propylene glycol [PG], 
vegetable glycerin [VG] and either cinnamon, grape, vanilla, or cherry flavorings will consist 
of concentrated flavorings added to a base liquid which will consist of a commonly used 
ration of 50% PG and 50% VG. PG is the original base liquid and is believed to produce a 
throat sensation (“throat hit”) which mimics the feel of smoking a cigarette. VG is included to 
enhance the volume of vapor production, giving a greater sensory illusion of smoking. 
E-cigarette device to be used is the Suorin iShare auto battery and Suorin iShare 
replacement pods will be used. This e-cigarette is similar in size and function to other 
commercially available more popular pod style devices. A new e-cigarette device will be 
used for each subject, and new pods will be used for each experiment.
6.1.7
Storage Conditions
In order to ensure that we have adequate amount of e-liquid, of the same constituency, we 
will purchase large quantities of these solutions prior to starting these experiments which will 
be stored at and dispensed at John Pierce Labs. Pods will be filled with e-liquid mixtures at 
John Pierce Labs.  
After the e-liquids are received, they will be delivered and stored in a controlled and dark 
laboratory environment housed in the analytical core of the Yale TCORS (PI: Zimmerman), 
which is a secured building. The liquids will be kept in a locked storage container in a drawer 
within the lab room before manipulation. Dr. Erythropel, chemist on the analytical core of the 
TCORS, and co-PI of this pilot (on a non-human project arm not detailed in the current 
protocol), will perform water manipulations (see above 6.1.2) to e-liquid containers. Once 
complete, e-liquids will be immediately transported to Pierce Labs, which is a secure 
building, where they will be delivered and stored in appropriate light/temperature conditions.  
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On the day prior to the lab sessions, a research assistant/associate/fellow who is not 
involved in the actual conduct of the lab sessions will fill the e-cig pods with the appropriate 
doses. The information on randomizations to flavor order for lab sessions will be generated 
by study biostatisticians prior to study start. This information is stored for each participant in 
an individual envelope and a printed study ID on the cover. These envelopes are stored at 
Pierce lab along with the purchased e-liquids. The individual filling the pods will be notified of 
which subject ID number needs to be filled and they will then go to Pierce labs and open the 
envelope and fill the sixteen pods with the appropriate e-liquids. First, they will label each 
pod with the study ID number and 1A, 1B, 1C, 1D, 2A, 2B, 2C, 2D if Lab 1 or if 3A, 3B, 3C, 
3D, 4A, 4B, 4C, 4D if Lab 2.  Each bottle of e-juice will be shaken vigorously prior to filling 
the pods.  Next, they will open the top of the pod and using a mechanical pipette will load 
.9ml into the pipette from the e-juice bottle and carefully transfer it into the pod. Each pod is 
then stored in a separate airtight bag to avoid cross contamination of flavors.  The individual 
bags are then stored together in a larger bag labeled with the study ID number and stored 
back in the locked container until the lab is set to begin.  Once the research assistant is 
ready to begin each lab, they will open the locked container, take the appropriate pod(s) out 
and leave the others.  At the end of the lab, they will return the pod(s) used that day to their 
proper bag and again return it to the lock box.  None of the e-juice or pods will be 
transported outside of Pierce lab.  The research staff will have the group randomization list 
and will dispense the appropriate e-liquid needed for each subject.
6.1.8
Concomitant therapy
N/A
6.1.9
Restrictions
There are no restrictions. 
6.2
Assessments
6.2.1
Efficacy
To evaluate the efficacy of manipulating water content on irritation reduction and its potential 
impact on appeal, we will use the following assessments:
1. General Labeled Magnitude Scale (gLMS): gLMS ratings will be obtain following 
each 2-puff puff bout. Participants will rate overall intensity, sweetness, coolness, 
irritation using the gLMS which is a category ratio scale with 7 semantic labels: “no 
sensation”, “barely detectable”, “weak”, “moderate”, “strong”, “very strong”, and 
“strongest imaginable”. The labels are positioned quasi-logarithmically according to their 
empirically determined semantic magnitudes22. The gLMS will be displayed on a monitor 
via a custom LabView® program, and participants will make their ratings using a mouse. 
Ratings are numerically transformed and then used for statistical analyses. gLMS 
irritation will be used to assessed change in irritation (primary outcome), and 
intensity, sweetness, and coolness will be used to assess change in overall appeal 
(primary outcome). 
2. Labeled Hedonic Scale (LHS)23 : Liking will be assessed after following each 2-puff 
puff bout using the LHS scale. The LHS is a bipolar scale with “neutral” in the middle and 
5 symmetrical semantic labels: “like/dislike slightly”, “like/dislike moderately”, “like/dislike 
very much”, “like/dislike extremely”, “most liked/disliked imaginable”, positioned on the 
scale according to their semantic magnitude. The LHS yields ratio-level data on the 
magnitude of liking/disliking of sensation equivalent to that produced by magnitude 
estimation (ME). This scale will also be displayed on a computer screen as above and 
numerically transformed. LHS will be used to assess change in overall appeal 
(primary outcome).
3. E-cig Effects (Adapted from Drug Effects Questionnaire (DEQ26): A modified 
version of the Drug Effects Questionnaire 24,27 will be used in which participants will rate 
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acute responses to the e-cig on a 0 to 100 mm scale, from “not at all” to “extremely.” 
Following each 10-puff bout, we will assess E-cig Liking/Wanting (average of “I feel good 
e-cig effects”, “I want more of that e-cig I received”, “I feel the e-cig strength” and “I like 
the e-cig effect”). At baseline and following each fixed-dose bout, we will also assess 
Stimulant effects (average of “I feel energized” and “I feel high”), and Nicotine 
Withdrawal (average of “I feel sleepy”, “I feel angry”, “I feel irritable”, “I am having 
difficulty concentrating”, “I feel restless” and “I feel hungry”). E-cig Effects will be used 
to assess change in overall appeal (primary outcome).
4. Multiple Choice Questionnaire to assess e-cig value (Adapted from Multiple 
Choice Procedure (MCP)25,26: At the end of each 2-puff puff bout, participants will be 
asked to make discrete hypothetical choices between 10-puffs of the e-cig they had just 
used or a series of 44 monetary values ($0.25-$15.06). The minimum monetary value at 
which money is chosen over the e-cig puffs is a contingency-based estimate of e-cig 
value. At the end of each lab session, participants will be given a choice between 
another 10 e-cig puffs or one of the monetary amounts they picked in the MCPs 
completed earlier. MCP will be used to assess change in overall appeal (primary 
outcome).
6.2.2
Safety and Pregnancy-related policy
Women who report pregnancy, trying to become or breastfeeding will be excluded from the 
study. All participants will be informed if pregnancy status changes to inform staff. 
Participants will be given a urine pregnancy test at every visit. 
6.2.3
Adverse Events Definition and Reporting
During screening, participants will undergo a review of their medical history and a brief 
physical (blood pressure, spirometry, heart rate, pulse oximetry, urine drug and pregnancy 
test) to assess health. Participants with chronic and untreated acute pulmonary conditions, 
current, untreated psychiatric conditions, or any other condition that could interfere with 
study participation/meets exclusion criteria will not be enrolled. Special attention will be 
placed on serious mental health issues not being treated and those at risk will be 
encouraged to seek mental health treatment. Participants will be terminated from 
participation if the investigator feels that their health or well-being may be threatened by 
continuation in the study. All participants will receive educational material of tobacco product 
use at the end of their study participation. We will measure health status throughout the 
study by using self-report questionnaires to assess fatigue, dyspnea, asthma and overall 
health with a checklist of symptoms related to the reported vaping illnesses. These 
assessments will be administered at every in-person visit. We will also assess heart rate, 
blood pressure, and pulse oximetry at baseline and every in-person visit.
This protocol presents a minor increase over minimal risk to the subjects and adverse events 
are not anticipated. In the unlikely event that such events occur, serious and unanticipated 
and related adverse events will be reported in writing within 48 hours to the Yale IRB and 
NIDA. The initial SAE report will be followed by submission of a completed SAE report to 
both institutions. In the event that a participant either withdraws from the study or the 
investigator decides to discontinue a participant due to an SAE, the participant will be 
monitored by the investigator via ongoing status assessment until 1) a resolution is reached 
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i.e., the problem requiring hospitalization has resolved or stabilized with no further changes 
expected 2) the SAE is determined to be clearly unrelated to the study intervention, or 3) the 
SAE results in death. Outcome of SAEs will be periodically reported to NIDA. A summary of 
the SAEs that occurred during the previous year will be included in the annual progress 
report to NIDA.
The principal investigator, Dr. Davis, will be responsible for evaluating the adverse events 
and study data at regular intervals and determine whether the adverse event affects the 
Risk/Benefit ratio of the study and whether modifications to the protocol (at Risks to 
Subjects) or consent form (at Risks and Inconveniences) are required.  During the review 
process, the PI will evaluate whether the study should continue unchanged, require 
modification/amendment, continue or close to enrollment. If necessary, PI will consult with 
study physician. Either the PI or the IRB or the DSMB have the authority to stop or suspend 
the study or require modifications.  The review of all adverse events by the PIs will determine 
the attribution and grade of severity of the adverse event by using the following scales:
Attribution of Risk Categories:
Definite: There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. The clinical event, including an abnormal 
laboratory test result, occurs in a plausible time relationship to study intervention 
administration and cannot be explained by concurrent disease or other drugs or 
chemicals. The response to withdrawal of the study intervention (dechallenge) should 
be clinically plausible. The event must be pharmacologically or phenomenologically 
definitive, with use of a satisfactory rechallenge procedure if necessary.
Probable: There is evidence to suggest a causal relationship, and the influence of 
other factors is unlikely. The clinical event, including an abnormal laboratory test 
result, occurs within a reasonable time after administration of the study intervention, 
is unlikely to be attributed to concurrent disease or other drugs or chemicals, and 
follows a clinically reasonable response on withdrawal (dechallenge). Rechallenge 
information is not required to fulfill this definition.
Possible/Potential: There is some evidence to suggest a causal relationship (e.g., the 
event occurred within a reasonable time after administration of the trial medication). 
However, other factors may have contributed to the event (e.g., the participant’s 
clinical condition, other concomitant events). Although an AE may rate only as 
“possibly related” soon after discovery, it can be flagged as requiring more 
information and later be upgraded to “probably related” or “definitely related”, as 
appropriate. 
Unlikely: 
A clinical event, including an abnormal laboratory test result, whose 
temporal relationship to study intervention administration makes a causal relationship 
improbable (e.g., the event did not occur within a reasonable time after administration 
of the study intervention) and in which other drugs or chemicals or underlying 
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disease provides plausible explanations (e.g., the participant’s clinical condition, 
other concomitant treatments).
Unrelated: The AE is completely independent of study intervention administration, 
and/or evidence exists that the event is definitely related to another etiology. There 
must be an alternative, definitive etiology documented by the clinician.
Grades of Risk:
0:
No adverse event or within normal limits
1: Mild adverse event; Events require minimal or no treatment and do not interfere 
with the participant’s daily activities.
2: Moderate adverse event; Events result in a low level of inconvenience or concern 
with the therapeutic measures. Moderate events may cause some interference with 
functioning.
3: Severe adverse event resulting in hospitalization or prolongation of existing 
hospitalization, a persistent or significant disability/incapacity, or a congenital 
anomaly/birth defect; Events interrupt a participant’s usual daily activity and may 
require systemic drug therapy or other treatment. Severe events are usually 
potentially life-threatening or incapacitating.  Of note, the term “severe” does not 
necessarily equate to “serious”.]
4: Life-threatening or disabling adverse event
5: Fatal adverse event
Expectedness:
The Principal Investigator will be responsible for determining whether an AE is expected or 
unexpected.  An AE will be considered unexpected if the nature, severity, or frequency of the 
event is not consistent with the risk information previously described for the study 
intervention.
6.2.4
Pharmacokinetics (if applicable)
N/A
6.2.5
Biomarkers (if applicable)
Urine collection will be done at study intake to assess cotinine levels, illicit drug use, and 
pregnancy status. Urine collection will be done at Lab 1 and Lab 2 for females to determine 
pregnancy status. Saliva collection will be done at intake to assess nicotine/cotinine levels. 
6.3
Study Procedures
Prescreening
Potential participants will be recruited via flyers, online recruitment (e.g. Yale Daily News, 
YCCI website, Craigslist), and/or materials with study information and phone number. PI 
and/or study research staff will be responsible for contacting participants. Potential 
participants will be directed to a voluntary link containing brief screening questions including 
information for research team to contact and/or phone number for participants to contact 
research staff directly. If interested participants will be asked if they regularly use nicotine 
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containing e-cigarettes, if those e-cigarettes are flavored (including flavors like tobacco), and 
if they are over 21. If interested, potential participants will be invited to take part in an intake 
visit to determine eligibility. Intake visits will be completed in person, as a urine sample is 
needed. 
Intake Visit 
Participants will be asked to complete intake in two parts; the first remotely (via Zoom) and 
an in person session to collect biochemical specimens. If participants do not have the 
necessary technology to complete the first part of the intake remotely, the full intake will be 
done in person. All intakes will conducted at CMHC (34 Park Street). At intake, participants 
will be consented via Redcap consent. Informed consent will be obtained while participant is 
in Zoom or in person. If on Zoom, participant will be instructed to be in a private setting away 
from others. Research staff will administer consent from a private room. If in person, consent 
will be obtained in a private room. Participants will have as much time as needed to consider 
consent. 
If participant provides consent, they will provide identifying information (to be kept separately 
from deidentified study materials in a locked cabinet). At intake participants will complete the 
following assessments:

30-day Timeline Follow Back assessing e-cigarette use, other tobacco product use, 
marijuana use, and alcohol use, 

E-cigarette/Tobacco Use History assessing e-cigarette use history and tobacco 
use history, products used, flavors tried

Demographic Questionnaire

E-Cigarette Dependence Scale (EDS)28 

Medical History assessing psychiatric and medical conditions

Asthma Control Test,29 participants will be asked to rate their asthma symptoms on 
a 1-5 scale, from “all of the time” to “none of the time”. This five-question health 
survey is used to measure asthma control in individuals 12 years and older.

FACIT cough questionnaire, a single item assessing cough severity 

PROMIS Fatigue,30 we will use the 7-item PROMIS measure of fatigue which rates 
fatigue from 1=” never” to 5=” always”.

PROMIS Dyspnea,31 We will use the 5-item PROMIS measure of dyspnea which 
rates shortness of breath from 1-10. Items include: shortness of breath in general, 
intensity of shortness of breath, frequency of shortness of breath and duration of 
shortness of breath. 

EVALI Health Assessment checklist, a checklist of health symptoms and severity 
of these symptoms that could be related to nicotine exposure/vaping illness 
Additionally, at the in-person portion of the intake, we will collect a urine sample and test 
cotinine level (a byproduct of nicotine) using a dichotomous dipstick (Countrywide 
Testing, 1 panel quickscreen cotinine/nicotine test dipcard – (COT200) DCT-114), illicit 
drug use, and if female, pregnancy. We will do a brief physical and collect blood 
pressure, spirometry, pulse oximetry, heart rate, and breath carbon monoxide (CO)
Lab Visits 
See list of assessments in Table 1. Participants will be asked to abstain from e-cigarette use 
and other tobacco product use two hours prior to the session, which will be assessed via 
self-report. At session arrival, participants will complete baseline questionnaires (see Visit 
Schedule below), heart rate, blood pressure, spirometry, and pulse oximetry. After 
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completion of baseline assessments, at Lab 1, participants will be familiarized with the 
sensory and hedonic scales and trained in how to puff the e-cigarette (using non-nicotine 
and non-flavored e-liquid). Participants will then begin testing procedures for their assigned 
Flavor 1, which will be administered under double-blinded randomized conditions (see 
above). For each flavor, participants will take two puffs of the flavor when diluted by four 
levels of water concentration. Participants will be instructed to take two 3-second puffs with a 
30 second inter puff interval. Each two-puff bout will be separated by 10-minutes. These 
procedures are adapted from our recent work (HIC #2000023077), with the number of puffs 
is reduced as we are not examining the effects of nicotine and only sensory effects of flavor. 
Per our current practice we will record timed instructions for each puff bout and participants 
will be trained to follow these directions. Following each puff exposure participants will 
complete the General Labeled Magnitude Scale (gLMS) for measures of overall intensity, 
irritation, coolness and sweetness, the Labeled Hedonic Scale (LHS) for measures of liking, 
the E-Cig Effects Questionnaire for measures of e-cigarette drug effects, and the Multiple-
Choice Questionnaire to assess e-cigarette value. Participants will complete the above 
procedures for two flavors and four water concentrations for each flavor during each lab 
session. For each flavor, water concentration order will not be randomized and will be 
presented from hypothesize least irritating (20% water concentration) to hypothesized least 
irritation (e-liquid as commercially available with no water dilution) to a) maintain consistency 
of presentation of water concentration across flavors and b) to avoid potential irritation 
effects from hypothesized more irritating products to less irritating products. Following at 
least 24 hours, participants will repeat procedures with the remaining two flavors. Each lab 
session will last approximately two hours.
Debrief  
At the end of Lab 2, participants will be thanked for their participation and ask to evaluate the 
four flavors they sampled (i.e. guess what flavors they tried, what nicotine concentration they 
e-liquids were) and will be given information from the CDC on e-cigarette cessation.
Table 1. List of Study Assessments 
Pre-screening
(Pre-consent)
Intake 
Lab 1 Baseline 
Assessments 
Lab 
1Assessments 
Following each 
E-Liquid 
Administration 
Lab 2 Baseline 
Assessments 
Lab 2 
Assessments 
Following each 
E-Liquid 
Administration 
Contact Info & Eligibility Review
X
Informed Consent
X
Identifying Information
X
Demographics
X
30-day Timeline Follow-back 
X
Tobacco & E-Cigarette Use 
History 
X
Medical History 
X
E-Cigarette Dependence Scale
X
Cotinine & Urine Drug Screen
X
Asthma Control Test
X
X
X
PROMIS Fatigue
X
X
X
PROMIS Dyspnea 
X
X
X
FACIT Cough Question
X
X
X
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6.3.1
Study Schedule
Participants will participate in an intake visit (that will be completed in two parts; remote and 
in-person as possible). Following intake, they will complete two lab sessions approximately 2 
hours each that occur at least 24 hours apart.  
6.3.2
Informed Consent
At intake, participants will be read informed consent and any questions will be discussed 
with researcher. Participants will sign informed consent (via Redcap) and consent will be 
administered by trained research staff. 
6.3.3
Screening
Prescreening
Potential participants will be recruited via flyers, online recruitment (e.g. Yale Daily News, 
YCCI website, Craigslist), and/or materials with study information and phone number. 
Potential participants will be directed to a voluntary link and/or phone number. If interested, 
participants will be asked if they regularly use nicotine containing e-cigarettes, if those 
cigarettes are flavored (including flavors like tobacco), and if they are over 21. If interested, 
potential participants will be invited to take part in an intake visit to determine eligibility. 
6.3.4
Enrollment 
If eligibility criteria is met, participant will be enrolled. PI (Danielle Davis) will deem subjects 
eligible following review and will assign enrolled subject ID. 
6.3.5
On Study Visits
See 6.3 “Study Procedures” above for detailed description of on study (Lab 1 & 2) 
procedures and assessments administered in Table 1. 
6.3.6
End of Study and Follow-up
After study completion, participants will be given written material for cessation. Given low 
study risk and brief e-cigarette administrations, no follow up sessions will occur. 
6.3.7
Removal of subjects
Subjects may withdraw at time voluntarily for any reason. Once participants have signed 
consent, they may withdraw at any time by communicating to any member of the research 
team. Any data that has already been collected can still be used, as necessary to ensure the 
integrity of the study and/or study oversight. Additionally, the research team may decide to 
withdraw participants if necessary including if participants do not follow directions of the 
study team, the study team decides the study is not in a participant’s best interest, or if a 
participant becomes pregnant, intends to become pregnant, or begins nursing a child. 
 No anticipated adverse events are expected that would require participant withdrawal. 
Adverse events are not anticipated. In the event that a participant either withdraws from the 
EVALI Health Assessment 
X
X
X
General Labeled Magnitude 
Scale (gLMS) 
X
X
Labeled Hedonic Scale (LHS)
X
X
E-Cigarette Drug Effects
X
X
Multiple Choice Questionnaire
X
X
Heart Rate, Blood Pressure, 
Pulse Oximetry
X
X
X
Pregnancy Test 
X
X
X
Debrief
X
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study or the investigator decides to discontinue a participant due to an adverse event, the 
participant will be monitored by the investigator via ongoing status assessment until 1) a 
resolution is reached i.e., the problem requiring hospitalization has resolved or stabilized 
with no further changes expected 2) the SAE is determined to be clearly unrelated to the 
study intervention, or 3) the SAE results in death. Outcome of SAEs will be periodically 
reported to NIDA. A summary of the SAEs that occurred during the previous year will be 
included in the annual progress report to NIDA. Withdrawal will be documented in a study 
participant tracker. 
6.4
Statistical Method
6.4.1
Statistical Design
To examine the primary objective of the study, which is to examine how increasing the 
concentration of e-liquids with aldehyde flavor chemicals impacts sensory perception of 
irritation and appeal among current adult e-cigarette users, we will use mixed effects models 
with water concentration as the within subject factor and each of the measure (e.g. irritation, 
appeal) as outcomes in separate models. Outcome analyses will be intent-to-treat and using 
mixed-effects models. These models allow for different numbers of observations per subject, 
use all available data, and can handle missing-at-random data. If model assumptions will 
appear to be violated, we will transform the data or fit more flexible generalized linear or 
nonparametric mixed models. A significant main effect of condition will be considered 
supportive of our hypothesis. We will construct confidence intervals for least square means 
and contrasts between least square means to estimate effect sizes and to inform future 
studies.
For the secondary aim, which is to examine differences by flavor for outcomes at each water 
concentration, we will examine at each water concentration, flavor as the within subject 
factor and each of the measure (e.g. irritation, appeal) as outcomes in separate models. We 
will construct confidence intervals for least square means and contrasts between least 
square means to estimate effect sizes and to inform future studies.
6.4.2
Sample Size Considerations
Given this is a pilot study, formal power analyses were not conducted. However, based on 
prior pilot data in which brief lab exposure to e-cigarettes were conducted by our group (HIC 
2000025687) determined that 30 participants is sufficient to detect large differences of 
irritation and appeal by water concentration at two sided alpha level of 0.05.
6.5
Planned Analyses
6.5.1
Primary Objective Analysis
To examine the primary objective of the study, which is to examine how increasing the 
concentration of e-liquids with aldehyde flavor chemicals impacts sensory perception of 
irritation and appeal among current adult e-cigarette users, we will use mixed effects models 
with water concentration as the within subject factor and each of the measure (e.g. irritation, 
gLMS and appeal, LHS) as outcomes in separate models. A significant main effect of 
condition will be considered supportive of our hypothesis. We will construct confidence 
intervals for least square means and contrasts between least square means to estimate 
effect sizes and to inform future studies.
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6.5.2
Secondary Objectives Analyses
For the secondary aim, which is to examine differences by flavor for outcomes at each water 
concentration, we will examine at each water concentration, flavor as the within subject 
factor and each of the measure (e.g. irritation, appeal) as outcomes in separate models. We 
will construct confidence intervals for least square means and contrasts between least 
square means to estimate effect sizes and to inform future studies.
6.5.3
Exploratory Objectives Analyses (if applicable)
N/A
6.5.4
Safety 
See 6.1.2 for treatment of adverse events.
6.5.5
Analysis of Subject Characteristics
Participant demographics, tobacco product history, e-cigarette history, and e-cigarette 
dependence level will be collected at intake and used to characterize population. All 
participants will receive the same treatment (within subject design).
6.5.6
Interim Analysis (if applicable)
N/A
6.5.7
Health economic evaluation
N/A
6.5.8
Other
N/A
6.5.9
Subsets and Covariates
Given this is a pilot trial, we do not have apriori defined associations between subject 
characteristics and response to change in water concentration. Additionally, as this is a 
within subject design subject characteristics that influence e-cigarette perception (i.e. 
menthol status, heaviness of e-cigarette use, prior flavor experience) are likely not to impact 
analyses. 
6.5.10 Handling of Missing Data
Outcome analyses will be intent-to-treat and using mixed-effects models. These models 
allow for different numbers of observations per subject, use all available data, and can 
handle missing-at-random data. If model assumptions will appear to be violated, we will 
transform the data or fit more flexible generalized linear or nonparametric mixed models.
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7  Trial Administration
7.1
 Ethical Considerations: Informed Consent/Assent and HIPAA Authorization
The study will be conducted in accordance with the conducted in accordance with the ethical 
principles that have their origin in the Declaration of Helsinki.
Participants will not be told of the flavors administered, the water concentration administer, 
or the nicotine concentration of the product. They will be told that products containing 
moderate levels of nicotine, flavorings that do or do not contain components considered 
“flavor aldehydes” and that we will be adding water to some of the formulations they will be 
trying. We will inform participants of this in the consent and indicate that we do not plan to 
tell them as we do not want prior perceptions of nicotine content or flavors to influence their 
experience with the product. Participants will be asked not to sign consent if they are 
uncomfortable with this. 
Participants will be compensated $50 for completing both parts of intake ($30 for remote 
portion, $20 for in person portion). This is commensurate to other e-cigarette studies in our 
labs. Participants will be compensated $75 for each laboratory session that is approximately 
2 hours in length. Additionally, participants are asked to abstain from vaping for 2 hours prior 
to each lab session, so this compensation is included. Additionally, at in person 
appointments, participants will be compensated for travel with $25. This is the equivalent of 
the cost of a rideshare in the greater New Haven area to and from our appointment space. 
Given the short duration of the study and participant preference in previous vaping studies, 
all compensation will be provided in cash. 
The only potential unknown condition identified by the study team that could be discovered 
as a result of study procedures would be EVALI, due to increased health monitoring for 
EVALI symptoms. This is included as an added study benefit. During the study, they will be 
monitored for EVALI related symptoms. Any endorsement of EVALI symptoms that does not 
have a clear related and proximal cause will result in withdrawal and participants will be 
referred to a primary care provider. 
No information from this study will be added to participant medical records.
7.2
Institutional Review Board (IRB) Review
The protocol will be submitted to the IRB for review and approval. Approval of the protocol 
must be obtained before initiating any research activity. Any change to the protocol or study 
team will require an approved IRB amendment before implementation.  The IRB will 
determine whether informed consent and HIPAA authorization are required.  
The IRB will conduct continuing review at intervals appropriate to the degree of risk, but not 
less than once per year.
A study closure report will be submitted to the IRB after all research activities have been 
completed.   
Other study events (e.g. data breaches, protocol deviations) will be submitted per Yale’s 
IRB's policies.
7.3
Subject Confidentiality
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Subject confidentiality is held in strict trust by the research team.  Subject medical record 
review will be limited to the just the elements needed to complete the study.  Only authorized 
HIPAA and GCP trained study team members will be allowed to collect data for this project. 
No direct subject identifiers will be entered into study tracker. 
Each subject will be assigned a unique study number that is not linked to their identifying 
information. A master list linking the unique study number to the human subject will be 
maintained in a locked drawer in PI’s office and on an encrypted document on a secure 
server. Only study staff will have access to this document.
7.4
Deviations/Unanticipated Problems
If the study team becomes aware of an anticipated problem (e.g. data breach, protocol 
deviation), the event will be reported to the IRB by PI within 24 hours of being aware of 
event if necessary. Protocol deviations will documented in Study Deviation Tracker. 
7.5
Data Collection
Data will be collected via surveys and questionnaires listed in the Visit Schedule table 
above. No licensure is required for any administration and all questionnaires will be 
administered by research assistants who have been trained in administration of sensory 
tasks. All data will be collected either via paper forms or via Redcap (CRF 11 compliant) 
with the exception of the gLMS and LHS, which will be collected on custom software for 
quantification of subjective responses. The data will downloaded on a monthly basis from 
Redcap and stored on a secure server. Any paper data will be stored in a locked file 
cabinet. Data will be stored this way for 7 years after the final data is collected. After this 
point, the Principal Investigator will oversee the process in which data is destroyed or de-
identified.
7.6
Data Quality Assurance
All data forms will be identified with the study ID of the participant and include date/time and 
study visit. The codes that link the name of the participant and the study ID will be kept 
confidential by the Principal Investigator in a secured cabinet. Online data will downloaded 
on a monthly basis from Redcap and stored on a secure server. Error checking and data 
validation will occur weekly and any problems will be queried and resolved immediately.  Dr. 
Davis will receive monthly data quality reports to check for completeness and accuracy of 
key demographic and prognostic variables, as well as rates of recruitment and retention.. 
Multiple measures are in place to ensure the validity and integrity of the data.  First, all 
research staff receive Human Subjects Protection and Good Clinical Practice training. 
Second, formal training in clinical assessments and procedures will be conducted to ensure 
quality of the data and help implement and improve recruitment strategies so that enrollment 
goals are achieved. Third, weekly research staff meetings take place, as a forum for in-
service training as well as to discuss questions regarding issues that arise in the research 
protocols.  Lastly, adherence to assessment administration will be monitored and if research 
drift is observed, the research assistants will be re-trained. 
7.7
Study Records
Study records are considered protocol, consents, and survey/questionnaires collected as 
part of the study procedures. No medical records are collected as part of this study. 
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7.8
Access to Source Documents
Source documents include paper and Redcap data collected as a part of the study. Online 
data will downloaded on a monthly basis from Redcap and stored on a secure server. Paper 
data will be stored in a locked cabinet. 
7.9
Data or Specimen Storage/Security
The majority of data for this study will be collected, recorded and stored using REDCap 
(Research Electronic Data Capture). REDCap is a secure, web application designed to 
support data capture for research studies. It includes features for HIPAA compliance 
including real-time data entry validation (e.g. for data types and range checks), a full audit 
trail, user-based privileges, de-identified data export mechanism to statistical packages 
(SPSS, SAS, Stata and R), and integration with the institutional Active Directory. Access to 
study data in REDCap will be restricted to the members of the study team with 
authentication through University NetID credentials.
The REDCap@Yale database and web server are housed on secure platforms that are 
backed up daily. REDCap@Yale meets the security standards for use with high risk data as 
set forth by the Yale Information Security Office.
Identifying information (consent, contact information) will be collected on paper documents 
and stored in a locked cabinet and/or uploaded to secure server and encrypted. 
All participants will be assigned a Study ID following consent and all study documents will 
use that ID. 
7.10 Retention of Records
The data will downloaded on a monthly basis from Redcap and stored on a secure server. 
Any paper data will be stored in a locked file cabinet. Data will be stored this way for 7 
years after the final data is collected. After this point, the Principal Investigator will oversee 
the process in which data is destroyed or de-identified.
7.11 Study Monitoring
The principal investigator and study team are responsible for monitoring the data, assuring
protocol compliance and conducting the safety reviews. Study monitoring will occur weekly. 
The study team, in consultation with the Yale TCORS Independent Data Safety Monitoring 
Board (DSMB), will evaluate adverse events and determine whether the adverse event 
affects the Risk/Benefit ratio of the study and whether modifications to the protocol or 
consent form are needed. Reviews will be held every 6 months. 
7.12 Data Safety Monitoring Plan
The study is consider a minor increase over minimal risk as participants are all current e-
cigarette users using nicotine and flavors. It is unlikely brief exposure to e-cigarettes with or 
without added water would present additional risk to this population. 
The study team, in consultation with the Yale TCORS Independent Data Safety Monitoring
Board (DSMB), will evaluate adverse events and determine whether the adverse event 
affects Reviews will be held every six months. Following each DSMB meeting written 
minutes will be prepared and distributed summarizing any recommendations. These written 
reports will insure timely communication with the PI with preparation of any protocol 
amendments necessary. After each DSMB meeting, this written report will describe all 
recommendations including additional safety steps.
a. DMSB Members and affiliation
The Yale TCORS Independent Data Safety Monitoring Board includes experts in the field of
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tobacco use behaviors and challenge studies (Chair: Dr. Tony George, FRCPC, Professor 
and Co-Director, Division of Brain and Therapeutics, Dept. of Psychiatry, U of Toronto; Dr. 
Thomas Brandon, Professor and Chair, Department of Health Outcomes & Behavior, H. Lee 
Moffitt Cancer Center & Research Institute) and a statistician (Dr. Hanga Galfalvy, Assistant 
Professor of Neurobiology, Columbia University).
b. Frequency of meetings
Meeting will be held twice a year or more often if requested by the Board.
c. Conflict of interest
The members of the DSMB and all study Investigators have completed Conflict of Interest 
forms created by Yale’s IRB in accordance with NIH guidelines.
d. Protection of confidentiality
Members of the DSMB agree to maintain the confidentiality of all information discussed in 
the meetings and reports of the DSMB.
e. Monitoring activities (initial and ongoing study review)
The DSMB will review the study information and the plans for review prior to the initiation of 
the studies. They will provide an ongoing review every six months thereafter based on the 
attached DSMP report.
f. Communication plan to IRB, NIDA, and FDA (if applicable)
The summary of the DSMB meeting will be submitted to the IRB and to NIDA following each 
meeting.
7.13 Study Modification
All study modifications will be submitted to the HIC and will not be implemented before 
receiving approval. Old versions of the protocol will be saved for reference.
7.14 Study Discontinuation
There are no anticipated circumstances that would warrant termination. If the study is 
prematurely terminated or suspended, the Principal Investigator (PI) will promptly inform 
study participants, the Institutional Review Board (IRB), and sponsor and will provide the 
reason(s) for the termination or suspension.  Study participants will be contacted, as 
applicable, and be informed of changes to study visit schedule.
7.15 Study Completion
Study completion will be when 30 participants have completed both lab sessions of the 
study. Once study enrollment is closed, the IRB and FDA will be notified.
7.16 Conflict of Interest Policy
No persons with a conflict of interest will be have any role in study design, conduct, analysis, 
or publication. 
7.17 Funding Source
This study is funded through the Yale Tobacco Center of Regulatory Science (TCORS) pilot 
program. All pilots including this one receive FDA/NIH approval before receiving funding. 
FDA/NIH approval to conduct this pilot was awarded. 
7.18 Publication Plan
The principal investigator holds the primary responsibility for publishing study results. The PI 
will seek to publish the results expeditiously after data analysis. 
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8 Appendices
Appendix #
Title
Section
Topic
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9 List of Tables
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Zimmerman JB, Jordt SE. Chemical Adducts 
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12. Dai H, Leventhal AM. Prevalence of e-
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17. Krusemann EJZ, Boesveldt S, de Graaf K, 
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18. Gendall P, Hoek J. Role of flavours in vaping 
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smokers and non-smokers: a cross-sectional 
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24. Morean ME, de Wit H, King AC, Sofuoglu M, 
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questionnaire: psychometric support across 
three drug types. Psychopharmacology (Berl). 
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25. Griffiths RR, Troisi JR, Silverman K, Mumford 
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