Examine the Effects of Novel E-cigarette Constituents and Determining Appropriate Concentrations Among Adults
Phase: NA
Conditions: E-Cig Use
Sponsor: Yale University

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CLINICAL STUDY PROTOCOL
Interventional Drug or Biologic
 
Examining Appeal and Addiction Potential 
of Novel E-cigarette Constituents among 
Adults
 Protocol Number
2000035551
Protocol Version
8-1-25
Version # 9
PRINCIPAL INVESTIGATOR:
Suchitra Krishnan-Sarin
Department of Psychiatry
203-974-7595
Suchitra.krishnan-sarin@yale.edu
IND Number: N/A
IND Holder: N/A
NCT Number: N/A
Study Phase: N/A
Confidentiality Statement: The phrases and circumstances of this Agreement are 
completely confidential between the parties and shall not be disclosed to anybody else. Any 
disclosure in violation shall be deemed a breach of this Agreement.
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REVISION HISTORY:
Revision #
Version Date
6
12/5/24
7
3/17/25
8
4/14/25
9
8/01/25
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Synopsis
Primary Objective
The primary objective of this study is to examine the appeal and addiction potential of e-cigs 
containing WS-3, WS-23 (synthetic coolants), with and without menthol, and nicotine. A pilot 
study will determine appropriate concentrations of WS-3, WS-23, and menthol. 
Secondary Objective
The secondary objective is to examine the interactive effects of different concentrations of WS-
3, WS-23, and menthol on appeal and addiction potential. Exploratory effects aim to examine 
the influence of predictors including sex, combustible tobacco use, and nicotine dependence.
Examine the feasibility and accuracy of a wearable smartband to detect e-cigarette puffing 
behavior. While in the lab for the e-cigarette use sessions, we will have participants wear a 
small smartband on their wrist, on the same hand they use to vape the e-cigarette. They will 
put on the smartband prior to starting the vaping session. The silicone smartband is small and 
lightweight, like a smartwatch or fitbit. The smartband collects movement data from geospatial 
sensors as the participant moves their arm and hand. The sensor data will be used to measure 
the movement during the directed e-cigarette puffing during the lab session. There are no 
changes to the lab procedure or puffing instructions.  
Study Duration
Approx. 3 years to complete entire study, approx. 2 weeks/participant for the pilot study, 4 
weeks/participant for the main study
Study Design 
Within subject’s human laboratory examination
Number of Study Sites
1; Yale School of Medicine 
Study Population
Adult e-cigarette with recent e-cig experience (past-month use), 50% with current cigarette use 
(equal number using menthol and non-menthol cigarettes). 
Number of Participants
30 participants for the pilot study, 66 participants for the main study. Up to 33 exclusive e-
cigarette users enrolled in the main study will be recruited for oral microbiome and 
inflammatory marker assessments.
Primary Outcome Variables
Liking/Wanting: Drug Effects Questionnaire
Labeled Hedonic Scale (LHS): Flavor/Sensory Perception
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Generalized Labeled Magnitude Scale (gLMS): Ratings of Flavor Intensity, Sweetness, 
Coolness, Irritation/Harshness, Bitterness
Secondary and Exploratory Outcome Variables (if applicable)
E-Cigarette Craving: The Yale Craving Scale
Salivary Nicotine/Cotinine Levels 
Oral microbiome, inflammatory markers, and self-reported oral health status in exclusive e-
cigarette users, compared by past-month e-cigarette flavor use.
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Abbreviations
Abbreviation 
Explanation
LHS
Labeled Hedonic Scale
gLMS
Generalized labeled magnitude scale
e-cig
Electronic cigarette
TCORS
Tobacco Center for Regulatory Science 
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Glossary of Terms
Glossary
Explanation
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Table of Contents
Synopsis ..................................................................................................................................2
Primary Objective.................................................................................................................2
Secondary Objectives ..........................................................................................................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 2.2 for relevant preclinical information.  
2.1.2
Clinical Experience
See 2.2 for relevant clinical information. 
2.2
Background/prevalence of research topic
The overall goal of this proposal is to investigate the impact of cooling components of WS-3, 
WS-23, and menthol on the appeal and addiction potential of nicotine-containing e-liquids 
among adults.
Menthol and Novel Cooling Constituents: There is a long history of including cooling 
flavorants like menthol in tobacco products.1 Menthol is characterized as a flavor constituent 
with a minty taste and aroma that produces cooling and analgesic sensory effects in the mouth 
and throat, and is one of the most common additives in nicotine and tobacco products.2 For 
example, most cigarettes, even those characterized as non-menthol, contain some amount of 
menthol.3 Menthol tobacco product advertising has targeted youth, young adults, and minority 
communities, and menthol’s sensory effects have been found to facilitate cigarette smoking 
initiation and nicotine intake.4-6 Further, use of menthol cigarettes is associated with greater 
dependence and lower rates of quitting smoking.7,8 Individuals from minority communities 
smoke more menthol cigarettes and also have a  greater risk for morbidity and mortality from 
smoking-related illness.9,10 In April 2022, the FDA announced a proposed rule to prohibit 
menthol as a characterizing flavor in combustible tobacco products. While this proposed rule 
does not extend to non-combustible tobacco products, it suggests that the FDA could utilize 
flavor restrictions to reduce the appeal of e-cigarettes to naïve users. In fact, they did ban 
flavors in cartridge-based e-cigarette devices based on their appeal to high school youth in 
February 2020. 
Menthol is commonly added to e-cigarettes, both in products advertised as menthol-flavored 
and those that not advertised as menthol-flavored.11,12 Menthol has concentration-dependent 
taste and olfactory effects. Menthol can also be harsh at high concentrations and therefore 
depending on the level of menthol added to e-cigarettes, menthol can reduce or enhance the 
sensory experience of irritation and harshness.13 In e-cigarettes, even very low concentrations 
of menthol have been shown to enhance sensory appeal,14 while also increasing the sensation 
of cooling in the mouth and throat.15 Laboratory examinations with youth and adults have 
shown that menthol can reduce the aversive sensory effects of nicotine.14,16,17 Importantly, 
nicotine and menthol in e-cigarettes can interact, such that high (but not low) concentrations 
of menthol may enhance e-cigarette liking/wanting for higher and harsher (but not lower) 
nicotine concentrations.14 In sum, menthol in e-cigarettes may enhance e-cigarette appeal via 
its cooling effects and via reducing harshness/irritation from nicotine and tobacco and altering 
reinforcement from nicotine. 
In line with findings from human laboratory studies, survey evidence from youth and young 
adult e-cigarette users observed that e-cigarettes that contain cooling components are popular 
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and that cooling may play a role in e-cigarette use behavior.18,19 Among youth, reporting use 
of cooling e-cigarettes was associated with more frequent e-cigarette use and greater 
likelihood of use of nicotine containing e-cigarettes. Recently, e-cigarettes that are labeled as 
ice-flavored, which typically indicates a combination of fruity/sweet and cooling flavors, have 
become very popular and among young adults were associated with more frequent e-cigarette 
use.19 Our TCORS Laboratory core analyzed a large number of e-cigarettes labeled as 
containing “ice” flavors and observed these e-liquids contained synthetic coolants called 
Wilkinson-Sword (WS)-3 or WS-23.20 These synthetic coolants, which are odorless and 
tasteless, were found in 13 of 14 Puffbar e-liquid flavors (i.e., a popular disposable e-cigarette 
brand) and 24 of 25 other e-liquids marketed as cooling. Many of the tested e-liquids contained 
WS-3 and/or WS-23 in combination with menthol, while others contained only WS-3 and/or 
WS-23. In addition to our findings, other researchers have also observed the presence of WS-
3 and WS-23 in Puffbar and JUUL products.21 The included concentrations of WS-3 and WS-
23 varied widely and had no discernable correlation with nicotine or menthol concentrations. 
Interestingly, our group found the menthol levels detected in these e-liquids were much lower 
(0.03-2.34%)22,23 than those detected in earlier examinations of commercial products that 
contained menthol (3.5%).13 This evidence suggests that as devices and nicotine formulations 
have evolved, manufacturers may also be changing the levels of menthol in e-liquids and 
adding other compounds like WS-3 or W-23 to produce cooling, perhaps with the intent of 
avoiding FDA regulations focused on menthol, or to address changes in sensory perceptions 
related to new nicotine formulations.    
WS (or Cyclohexanecarboxamide,N-Ethyl-5-Methyl-2-(1-Methylethyl)) compounds are non-
volatile, odorless and tasteless menthol derivatives which were initially synthesized to produce 
cooling in shaving products without a burning sensation in the eyes.24,25 These compounds 
are commonly used as cooling additives with WS-3 being the most popular.23 Importantly, WS-
3 evokes a sensory experience of cooling by activating the cold/menthol receptor TRPM8 in 
the oral cavity and upper airways, with greater efficacy than that of menthol.25-28 Notably, most 
of the evidence on synthetic coolants like WS-3 has examined their use in oral or skin 
applications. There are no investigations in humans isolating the effects of how synthetic 
coolants may influence sensory experience following inhalation via e-cigarettes, or what their 
effects are when they are combined with different concentrations of menthol, or with nicotine. 
Importantly, if these coolants are able to replicate the cooling and irritation-reduction 
experience of menthol without a characterizing flavor (i.e., odor) they could potentially be used 
as substitutes for menthol (in the event of a menthol or characterizing flavor ban) or used in 
addition to menthol to enhance menthol’s cooling and analgesic effects. Therefore, it is critical 
to understand user experiences to these synthetic coolants. We therefore propose to conduct 
the first study of the influence of multiple commercially available concentrations of the 
synthetic coolant WS-3 on the appeal and addictive potential of e-liquids when used with and 
without commercial levels of menthol, among adults who use e-cigarettes alone or with 
combustible tobacco products.
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3 Rationale/Significance
3.1
Problem Statement
There is little data on how the synthetic coolants WS-3 and WS-23 affect appeal and 
sensory perceptions of e-cigarettes and how its effects compare to menthol. 
3.2
Purpose of Study/Potential Impact
The overall goal of this proposal is to investigate the impact of synthetic cooling components 
from comparable concentrations of WS-3, WS-23, and menthol, on the appeal and addiction 
potential of nicotine-containing e-liquids among adults. This proposal will provide valuable 
evidence to support the FDA's nicotine and flavor regulatory frameworks and generate 
additional scientific data to inform future research and regulations concerning these 
synthetic constituents. We propose to conduct two experiments. Firstly, a pilot sensory 
experiment will examine cooling from comparable concentrations of WS-3, WS-23, and 
menthol to determine appropriate concentrations to use in the main study. Secondly, the 
main study will characterize the appeal and addiction potential of nicotine-containing e-
liquids that contain WS-3, WS-23, and menthol. This proposal will be the first to examine the 
influence of multiple commercially available concentrations of the synthetic coolants WS-3 
and WS-23 on the appeal and addictive potential of e-liquids when used with and without 
commercial levels of menthol, among adults who use e-cigarettes alone or with combustible 
tobacco products.
3.2.1
Potential Risks
Participation in this study is thought to involve a greater than minimal risk. Potential risks and 
protections against risks are detailed below:
(1) E-cigarette Exposure: All participants will be asked to use e-cigarettes in human 
laboratory sessions and will use study e-cigarettes only in the laboratory (i.e., will not 
take them outside of the laboratory). All participants will be required to have experience 
with e-cigarettes. Given this, the likelihood of adverse experience with study e-
cigarettes/e-liquids is low. 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). 
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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. We will confirm at intake they 
do not have known allergies to either of these products or e-liquid flavors.
(2) Flavor Exposure: We will expose participants to three different e-liquids flavorants; 
synthetic coolants WS-3, WS-23 and menthol. All products are widely available 
commercially and commonly added to popular e-liquids. Menthol, WS-3 and WS-23 
administration is reported to produce cooling sensory effects in the mouth and throat.25-
28 In the current study, we will be examining doses of menthol, WS-3 and WS-23 in e-
cigarettes that are in line with commercially available doses.23 In spite of ubiquitous use 
of menthol in a wide range of products, only few cases of menthol poisoning have been 
described in the literature in case studies, none related to tobacco or e-cigarette use 
and an extremely high level of menthol would need to be ingested in a short amount of 
time to risk toxic effects.29 Menthol poisoning reported to cause ataxia, confusion, coma, 
nausea, and vomiting. We are not aware of any cases of poisoning reported for WS-3 
and WS-23. Participants will be told that if they feel any adverse effects during the 
laboratory sessions or want to stop for any reason, they are free to do so. Additionally, 
we will exclude anyone that reports allergies to any of the flavors used (menthol, WS-3, 
WS-23, other synthetic coolants). We will also monitor for adverse events. 
(3) Nicotine Exposure (applicable for the main study only): E-liquids will contain 
nicotine in nicotine salt formulation (59mg/ml) that is commonly available in popular 
commercial e-cigarettes. Common side effects of nicotine include irritation/harshness 
experienced in the throat, 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 much 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) Urine & Saliva Collection: Urine and saliva collection will occur at intake and during 
laboratory sessions and should add no risks other than the time taken to complete them. 
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(5) 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.  
(6) 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 report this information to the appropriate authorities.
(7) COVID & Tobacco Product Use: The following information will be listed in the consent. 
Although the science on the relationship between getting COVID and using tobacco 
products like cigarettes and e-cigarettes is still not clear, tobacco products can lead to 
respiratory conditions like asthma. Therefore, if participants use tobacco products and 
become infected with COVID-19, there is a potential that participants could have worse 
health outcomes. Although scientific evidence is incomplete, some studies have 
suggested that use of e-cigarettes may add to the risk of getting COVID -19 and may 
contribute to the severity of illness if infected by the virus. We recommend that 
participants follow the CDC guidelines for the most up to date information about ways to 
reduce their risk for exposure to COVID-19. https://www.cdc.gov/coronavirus/2019-
ncov/index.html. If participants develop symptoms for COVID-19 (fever, cough, 
shortness of breath, difficulty breathing, muscle aches, sore throat, congestion or runny 
nose, nausea or vomiting, diarrhea, new loss of taste or smell) or have known or 
suspected exposure, participants will be instructed to self-isolate and contact their 
healthcare provider to discuss obtaining a test for COVID-19. Participants will be 
instructed to inform research staff if they develop any of these symptoms or have 
known/suspected exposure to COVID-19. If participants become infected with COVID-
19, we recommend they stop using all tobacco products.
Protections Against Risk: We will reduce risks by:
(1) Risk of E-cigarette, Nicotine, and Flavor Administration: All participants will self-
report on their physical and mental health and we will continue to monitor their health 
closely during the study. We will monitor heart rate, blood pressure, lung spirometry 
(measures pulmonary function) and pulse oximetry (measurement of the oxygen in the 
blood). Participants will also fill out a health assessment checklist to monitor health 
symptoms (e.g., headaches, dizziness, fainting, nausea, diarrhea) and severity of these 
symptoms that could be related to nicotine exposure/vaping illness. The e-cigarette lab 
sessions will be conducted by trained research staff who are sensitive to the smoking 
population and trained to monitor any potential adverse effects. 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. We will also provide 
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participants with referral sources to quit vaping or smoking (if applicable) if they are 
interested. We have used similar procedures in prior vaping studies. Subjects will be 
told that participation is voluntary, and they can stop anytime if they want to and/or if 
they experience adverse events from nicotine or flavor exposure. The participants are 
current e-cigarette users, and are, therefore, already self-administering nicotine at doses 
similar to what is administered in the current studies. The e-cigarettes and e-liquid pods 
that we will use in the current study are commercially available and the e-liquids were 
mixed at Yale Pharmacy and do not contain CBD or THC. At this time, we don’t know 
what the risks associated with the use of the e-cigarettes and e-liquids, flavors, etc. that 
we use in this study are, and who might develop symptoms. The e-liquids we are giving 
participants contain nicotine, solvents, and flavorings. 
(2) Risks associated with rating scales and questionnaires: Careful efforts aimed at 
maintaining confidentiality will be made and subjects will be reminded that they can 
choose not to answer any questions they do not feel comfortable answering.
(3) Limits to Confidentiality: Research data will be collected using in-person interviews, 
assessments, objective measures of smoking behavior, and self-reports. All identifiable 
information (names and demographic information) collected on paper will be stored in a 
locked file cabinet and all identifiable information housed online will be stored on 
secured password protected servers (e.g., Redcap, Yale Shared Drive) only accessible 
to research staff. All participants will be assigned a study participant ID made up of 
numbers and letters. A list of IDs and the corresponding names will be maintained by 
the Principal Investigator and stored in a locked research cabinet. All other research 
data (interviews, assessments, objective measures of smoking behavior, and self-
reports) will not contain identifiable information and will be labeled only with the subjects' 
unique numerical indicator. Several steps will be taken to safeguard the confidentiality of 
subjects and their data. Right to privacy for participation in this research will be 
protected through coding of data and proper storage of research records. Any 
information published because of the study will be in aggregate and such that it will not 
permit identification of any participant. We are not directly assessing incidents of child 
abuse or elderly abuse. However, if this information is disclosed by a participant or 
volunteer in the context of this research, a report will be made to the Department of 
Child and Families Services or other agency as required by law. Subjects will be 
informed of this limit to confidentiality as it is stated in the informed consent document. 
Individually identifiable health information will be protected in accordance with the 
Health Insurance Portability and Accountability Act of 1996 and by additional protections 
of substance abuse treatment records afforded under Code of Federal Regulations 
(CFR) Part 2, Subpart E. All research personnel will be trained on human subjects' 
protection and HIPAA procedures.
(4) In general, to minimize risk in this study we will:

Obtain consent

Recruit adult e-cigarette users 

Use well-defined inclusion/exclusion criteria
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
Use study staff who have extensive training conducting tobacco and substance use 
research and working with adults and who are sensitive to the issues that may arise 
in working with tobacco 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.
3.2.2
Potential Benefit
We expect that the results of the study will benefit science and others through increasing our 
knowledge of how flavor constituents alter the appeal and addiction potential.  
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4 Study Objectives
4.1
Hypothesis
We aim to examine the impact of cooling components of WS-3, WS-23, and menthol on the 
appeal and addiction potential of nicotine-containing e-liquids among adults. We hypothesize 
that any addition (low, high) of WS-3, WS-23, or menthol will result in similar increases in 
addiction potential, and that when WS-3, WS-23, and menthol are combined they will 
produce additive effects relative to menthol alone. We also hypothesize similar changes in 
appeal outcomes. 
4.2
Primary Objective
Examine addiction potential (liking/wanting) and appeal (overall flavor appeal, intensity, 
sweetness, bitterness, irritation, cooling), following a single exposure (5 puffs) to nicotine-
containing e-cigs (~59 mg/ml nicotine salt, commonly used in commercial e-cigs), with one 
of three WS-3 concentrations (none, low, high) and one of the three WS-23 concentrations 
(low, high, 4%) in combination with one of three menthol concentrations (none, low, high). 
4.3
Secondary Objectives (if applicable)
Examine the interactive effects of different concentrations of WS-3, WS-23, and menthol on 
appeal and addiction potential.
4.4
Exploratory Objectives (if applicable)
An exploratory objective of this study is to examine the influence of predictors such as sex, 
combustible tobacco use, and nicotine dependence on study outcomes.
Another exploratory objective is to examine oral microbiome, salivary inflammatory markers 
and self-reported oral health status in exclusive e-cigarette users and compare the 
differences among users of different e-cigarette flavor groups (1. Mint/menthol, 2. fruit/sweet, 
3. tobacco). We hypothesize that users of mint/menthol or fruit/sweet flavors will have higher 
levels of anaerobic bacteria in their oral microbiome, greater inflammatory markers in saliva, 
and poorer oral health compared to tobacco flavor users.
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5 Study Design
5.1
General Design Description
Pilot study: This pilot study will establish low and high concentrations of WS-3, WS-23, and 
menthol concentrations to test in the main study. 30 adult e-cigarette users will participate in 
one laboratory session during which they will be exposed to e-liquids containing all the 
conditions in Table 1 without nicotine. Participants will receive a two-puff exposure to each 
condition; each exposure will be separated by 10 mins to allow the flavor to dissipate, similar 
to our prior work on menthol.13,14 Each session will begin with familiarization with the sensory 
rating scales used in the current study30,31 and training in how to puff the lab e-cig in a directed 
and standardized way. WS-3, WS-23 and menthol will be presented in blocks (block order will 
be counterbalanced) with each block presenting increasing menthol/WS-3/WS-23 
concentrations as in our earlier work.13  Participants will rate “coolness” using the generalized 
Labeled Magnitude Scale (gLMS), an established category ratio scale with 7 semantic labels: 
“no sensation”, “barely detectable”, “weak”, “moderate”, “strong”, “very strong”, and “strongest 
imaginable sensation of any kind”, positioned quasi-logarithmically based on their semantic 
magnitude.32,33 Using averages of participant ratings, we will select a low concentration of 
each constituent that is rated as more than “barely detectable” but less than “moderate” to 
ensure that cooling is weak but clearly perceptible. For the high concentration of each 
constituent, we will select a concentration that on average falls between “strong” and “very 
strong” to identify a strong cooling sensation while avoiding sensations that would also be 
uncomfortable. We will also determine overall flavor intensity, sweetness, and 
harshness/irritation using the gLMS. During the lab sessions, we may have participants wear 
a small smartband on their wrist, on the same hand they use to vape the e-cigarette. The 
smartband collects movement data from geospatial sensors as the participant moves their 
arm and hand. The sensor data will be used to measure the movement during the directed e-
cigarette puffing during the lab session. The smartband is only being used for data collection 
and is not being evaluated/used as a medical device. There are no changes to the lab 
procedure or puffing instructions. 
Table 2. Main Study Experimental Design
No 
WS-3
Low (0.1%) 
WS-3 
Concentration 
High (2.0%) 
WS-3 
Concentration 
Low (0.1%) 
WS-23
Concentration
High (2.0%) 
WS-23 
Concentration
4% WS-23 
Concentration
Low (0.1%)  
Menthol  
S1-S66
S1-S66
S1-S66
S1-S66
S1-S66
S1-S66
High (2.0%) 
Menthol  
S1-S66
S1-S66
S1-S66
S1-S66
S1-S66
S1-S66
No Menthol
S1-S66
S1-S66
S1-S66
S1-S66
S1-S66
S1-S66
S=Subject. All subjects (1-66) will be exposed to all conditions in the adjacent table. 
    Table 1. Pilot Study Design
Concentration 1
Concentration 2
Concentration 3
Concentration 4
Concentration 5
Concentration 6
WS-3 Block
0.05%
0.1%
0.5%
1.0 %
2.0 %
-
WS-23 Block
0.05%
0.1%
0.5%
1.0 %
2.0 %
4.0%
Menthol Block
0.05%
0.1%
0.5%
1.0 %
2.0 %
-
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Main Study: The purpose of the main study will be to determine sensory responses following 
exposure to e-liquids containing nicotine and WS-3, WS-23, and menthol. This study will 
recruit 66 adult e-cigarette users (50% with current cigarette use, of which 50% will use 
menthol cigarettes), to participate in 3 laboratory sessions, separated by at least 24 hrs. At 
the intake visit, participation in the oral microbiome and inflammatory marker testing will be 
optional for exclusive e-cigarette users (up to 33). Specifically, they will be asked to provide 
saliva and urine samples for oral microbiome and inflammatory marker testing and complete 
a brief survey about their oral health status. Additionally, participants will be asked to submit 
their most used e-cigarette device/pods from the past-month for analysis. These will be used 
as exploratory assessments. During the lab visits, participants will receive all eighteen 
conditions shown in Table 2 below and will be asked to abstain from tobacco use for two hours 
prior to session start (to control for time since last tobacco exposure). Participants will be 
randomized to receive one of the 3 menthol conditions (no-menthol, low menthol, high 
menthol, levels determined by the pilot study above) in Lab 1 and will receive the alternative 
conditions in randomized order in Lab 2 and Lab 3. During each lab session the menthol 
condition will be paired with three WS-3 concentrations: no WS-3, low (0.1%) WS-3, or high 
(2.0%) WS-3 and three WS-23 concentrations: low (0.1%) WS-23, high (2.0%) WS-23, or 4% 
WS-23 (concentration determined by pilot study) in randomized order. All e-cigarettes will 
contain nicotine salt at a nicotine concentration of ~59mg/ml, commensurate with levels in 
popular commercially available nicotine salt e-cigarettes.34-36 Each exposure will consist of a 
5-puff bout (each puff 3-sec long and separated by 30 sec.) collected via a puff topography 
device (an apparatus used to measure puffing behavior) and will be separated by 30 mins to 
allow any sensory effects to dissipate between exposures. To precisely measure puffing 
behavior, we will utilize a validated electronic cigarette topography device (eTop). This device 
connects to a computer with software designed to measure puffing behavior. An e-cigarette is 
inserted into a silicone mouthpiece and the mouthpiece is connected to tubing which feeds 
into the device. As a participant inhales this device measure puffing behavior including number 
of puffs, puff volume, puff duration, puff flow rate (a calculation of volume/time). These indices 
help us understand vaping behavior and make it possible to compare e-cigarette exposure 
across participants. Topography mouthpieces are sanitized in between subjects and inhaling 
e-cigarettes via topography devices present no increased risk to participants.  At the end of 
each of the three lab sessions, participants will choose between the 6 e-liquids they sampled 
in the lab using a free-choice paradigm (~30 mins). 
Each session will begin with administration of baseline questionnaires assessing withdrawal 
and craving, collection of samples to assess nicotine levels, familiarization with the sensory 
and hedonic rating scales30,31 and training in how to puff the lab e-cig. Training will be 
conducted using e-cigs that contain the base liquid of 50% propylene glycol (PG)/50% 
vegetable glycerin (VG) with no nicotine. Similar to our earlier trials14,16, after each exposure, 
participants will rate addiction potential (liking/wanting: Drug Effects Questionnaire (DEQ)14; 
and appeal (overall flavor appeal: Labeled Hedonic Scale (LHS)31; flavor intensity: overall 
flavor intensity, irritation, coolness, sweetness, bitterness: generalized Labeled Magnitude 
Scale (gLMS30,32). Salivary nicotine samples will be collected pre-sessions and after each 
flavor exposure to assess potential impact of nicotine levels on responses, with participants 
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rinsing their mouths before providing each sample. A brief description of the e-cigarette device 
and all ingredients that will be used for the pilot and main studies is included in Table 3.
Table 3. Description of all investigational tobacco products.
Ingredients or products
Manufacturer name
Concentrations (mg/ml or %)
E-cigarette device
Suorin iShareTM
-
PG
JT Baker: Propylene Glycol, 
U.S.P. - F.C.C.
50%
GL
Fisher Scientific: Glycerol, 
USP/FCC
50%
Nicotine Salt
Nicotine River: PurNic Smooth 
Nicotine Salt 100mg/ml in 
50PG/50GL
0 or 59mg/ml
Menthol
Sigma-Aldrich: L-Menthol
0.05%, 0.1%, 0.5%, 1.0%, and 
2.0%
WS-3
Sigma-Aldrich: WS-3
0.05%, 0.1%, 0.5%, 1.0%, and 
2.0%
WS-23
Vigon: 503872
0.05%, 0.1%, 0.5%, 1.0%, 
2.0%, and 4.0%
5.1.1
Study Date Range and Duration
The expected duration of this study is 09/2023 – 09/2026 (3 years). The expected 
duration/participant is 3 weeks to complete the intake and single laboratory session for the 
pilot study and 5 weeks to complete the intake + 3 laboratory sessions for the main study. 
Recruitment and enrollment for the main study will start after the completion of the pilot study 
(tentatively by the end of year 2024), meaning after all 30 pilot subjects have completed their 
lab sessions and the arm is closed.
5.1.2
Number of Study Sites
The study will take place at the Yale University School of Medicine. Study sites will include the 
Connecticut Mental Health Center (CMHC; 34 Park Street, New Haven, CT) or the Substance 
Abuse Treatment Unit (SATU;1 Long Wharf Dr., New Haven) for intake appointments and 
Pierce Laboratories (290 Congress Avenue, New Haven, CT) or West Campus Research Unit 
(WCRU; 500 West Campus Dr., West Haven, CT), which are equipped with ventilated 
research chambers utilized for smoking and vaping research studies. 
5.2
Outcome Variables
The outcome variables in this study are appeal, sensory effects, and reinforcing value (detailed 
below). 
5.2.1
Primary Outcome Variables
Liking/Wanting: Drug Effects Questionnaire
Labeled Hedonic Scale (LHS): Flavor/Sensory Perception
Generalized Labeled Magnitude Scale (gLMS): Ratings of Flavor Intensity, Sweetness, 
Coolness, Irritation/Harshness, Bitterness
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5.2.2
Secondary Outcome Variables (if applicable)
E-Cigarette Craving: The Yale Craving Scale
Salivary Nicotine/Cotinine Levels
5.2.3
Exploratory Outcome Variables (if applicable)
E-cig Appeal: E-cigarette Evaluation Questionnaire (exploratory)
Examine impact of sex, combustible tobacco use and nicotine dependence on experiences 
from WS-3, WS-23, menthol and nicotine. 
Comparative difference among exclusive e-cigarette users of different flavor groups (menthol 
or mint, fruit /sweet, tobacco), between oral microbiome constitution and inflammatory 
markers and self-reported oral health.
5.3
Study Population
Participants will be females and male adult e-cigarette users aged 18 years or older. 
5.3.1
Number of Participants
We aim to recruit 30 adults for the pilot study and 66 adults for the main study (33 with 
current cigarette use, 16 reporting menthol cigarette use). In the main study, we will recruit a 
subset of eligible exclusive e-cigarette users (up to 33) for oral microbiome and inflammation 
assessment.
5.3.2
Eligibility Criteria/Vulnerable Populations
For the pilot study: 
Inclusion Criteria:

18 years or older

Able to read and write 

Use e-cigarettes containing nicotine

Have used e-cigs at least 30% of days/past month (at least 8 days on 28-day timeline 
followback)

No strong dislike of the menthol flavor

At least 50% have used only e-cigs with no combustible tobacco products (e.g., 
cigarettes, cigars, cigarillos) in the past month

Urine cotinine ≥200ng/ml 

Willing to abstain from tobacco/nicotine use 2 hrs prior to sessions 

Not currently quitting/trying to quit nicotine/tobacco use
Exclusion Criteria: 

Current criteria for moderate or severe cannabis and alcohol use disorder as per 
DSM-V criteria

Current criteria for mild, moderate or severe substance use disorder on any other 
psychoactive substances as per DSM-V criteria except for tobacco use disorder

Use of psychoactive drugs including anxiolytics, antidepressants, and other 
psychostimulants unless prescribed and stable for two months
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
Current diagnosis of any severe and untreated psychiatric disorder

Any significant current medical condition such as neurological, cardiovascular, 
endocrine, renal, or hepatic pathology that would increase study risk

Any blood pressure values with systolic <100 or >160 mmHg or diastolic <65 or >100 
mmHg

Allergy to menthol, PG/VG, WS-3, or WS-23

If of childbearing potential: pregnant, trying to become pregnant or breastfeeding

Uncontrolled asthma (defined as <20 on Asthma Control Test AND/OR endorsement 
of “yes” to environmentally induced bronchospasm that requires prescription Epipen 
Medical conditions, including chronic and untreated acute pulmonary conditions, that 
in the investigators view will increase risk of respiratory problems among participants

Report of greater than “without any difficulty” on any item of the PROMIS Physical 
Function Short Form 

For current (past-month use) 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

Dislike of menthol flavor 
For the main study:
Inclusion Criteria:

18 years or older

Able to read and write 

Use e-cigarettes containing nicotine

Have used e-cigs at least 30% of days/past month (at least 8 days on 28-day timeline 
followback)

Urine cotinine ≥200ng/ml 

Willing to abstain from tobacco/nicotine use 2 hrs prior to sessions 

Not currently quitting/trying to quit nicotine/tobacco use

For cigarette using participants only: use of cigarettes at least 30% of days/past 
month (at least 8 days on a 28-day timeline followback)

A subset of eligible participants (exclusive e-cigarette users only) for oral microbiome 
assessment must be willing to refrain from eating, drinking (except water) or brushing 
their teeth for 2 hour before saliva collection.
Exclusion Criteria: 

Current criteria for moderate or severe cannabis and alcohol use disorder as per 
DSM-V criteria

Current criteria for mild, moderate or severe substance use disorder on any other 
psychoactive substances as per DSM-V criteria except for tobacco use disorder

Use of psychoactive drugs including anxiolytics, antidepressants, and other 
psychostimulants unless prescribed and stable for two months
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
Current diagnosis of any severe and untreated psychiatric disorder

Any significant current medical condition such as neurological, cardiovascular, 
endocrine, renal, or hepatic pathology that would increase study risk

Any blood pressure values with systolic <100 or >160 mmHg or diastolic <65 or >100 
mmHg

Allergy to menthol, PG/VG, WS-3, or WS-23

If of childbearing potential: pregnant, trying to become pregnant or breastfeeding

Uncontrolled asthma (defined as <20 on Asthma Control Test AND/OR endorsement 
of “yes” to environmentally induced bronchospasm that requires prescription Epipen 
Medical conditions, including chronic and untreated acute pulmonary conditions, that 
in the investigators view will increase risk of respiratory problems among participants

Report of greater than “without any difficulty” on any item of the PROMIS Physical 
Function Short Form 

For current (past-month use) 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

For oral microbiome and inflammation assessment, participants who report a medical 
condition such as diabetes, uncontrolled hypertension, renal and hepatic disorders, 
antibiotics use in the past 3 months, oral cancer diagnosis, or who are undergoing 
active treatment for any medical condition
6 Methods
6.1
Treatment 
6.1.1
Identity of Investigational Product
Products used in this study are investigational tobacco products including 16 different e-
liquids and 1 e-cigarette device for the pilot study and 18 different e-liquids and 1 e-cigarette 
device for the main study. The e-cig device to be used will be the Suorin iShareTM pod 
device. iShare characteristics resemble the popular JUUL device in terms of power output, 
shape, and size, but with the benefit of refillable cartridges. The Yale TCORS lab core has 
demonstrated its near 100%-effectiveness in delivering nicotine to users using a custom-
made vaping machine. Nicotine concentrations will be 0 mg/ml in the pilot study and will be 
59 mg/ml nicotine salt (nicotine benzoate) in the main study, a widely used and well tolerated 
nicotine salt formulation and concentration, similar in strength to a 5% JUUL product. PG/VG 
levels for the study e-liquids will be 50:50 PG/VG, a ratio typical of nicotine salt e-liquids and 
used in previous studies by our group. 
E-liquids will be created upon review of the (Investigational Tobacco Product) ITP application 
by the FDA; Dr. Krishnan-Sarin leads Yale TCORS ITP applications and will oversee this 
process. All e-liquids will be created via the Yale Investigational Drug Service (IDS) 
Pharmacy under the direction and supervision of Dr. Patel Prashant (Manager, 
Investigational Drug Service Pharmacy). E-liquid recipes have been developed via the Yale 
Laboratory Core and with consultation of Dr. Tony Pace, a chemist with American E-
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liquids/Vaping Direct and collaborator in research with our group with years of experience 
creating e-liquids for research.27 E-liquid ingredients will be purchased by our group, then 
shipped to our research team and upon receipt be transported to and stored in the Yale IDS 
Pharmacy (see 6.1.6) and created in 30mL batches as needed by Yale IDS. A research 
assistant/associate/fellow will document the receipt from IDS. E-liquid ingredients all meet 
the requirements of United States Pharmacopeia (USP) or Flavor and Extract Manufacturers 
Association of the United States (FEMA) “GRAS” (generally recognized as safe) have been 
reviewed by Yale IDS and the Yale Tobacco Center of Regulatory Science. Additionally, 
FDA has reviewed the proposed ITP of all e-liquids prior to study start. The e-liquids being 
created will be produced with three flavors; (1) menthol, (2) WS-3 and 3) WS-23 to create 16 
e-liquids for the pilot and 18 e-liquids for the main study. Pilot e-liquids will contain no 
nicotine and main study e-liquid will contain a fixed amount of nicotine. E-liquid nicotine and 
flavor profiles will be confirmed and characterized by the Yale TCORS lab core.38 
6.1.2
Dosage, Administration, Schedule 
Regarding the pilot study, participants will receive two puff exposures to each condition; 
each exposure will be separated by 10 mins to allow the flavor to dissipate. All e-cigarette 
pods will be filled to 0.5mL of e-liquid. 
Regarding the main study, e-cigarette pods will be filled with approximately 0.9mL of e-liquid, 
which in previous trials has produced at least 30 puffs. Participants will be randomized to 
receive one of the 3 menthol conditions (no-menthol, low menthol, high menthol, levels 
determined by the pilot study above) in Lab 1 and will receive the alternative conditions in 
randomized order in Lab 2 and Lab 3. During each lab session the menthol condition will be 
paired with three WS-3 concentrations: no WS-3, low (0.1%) WS-3, or high (2.0%) WS-3 or 
three WS-23 concentrations: low (0.1%) WS-23, high (2.0%) WS-23, or 4% WS-3 
(concentration determined by pilot study) in randomized order. All e-cigarettes will contain 
nicotine salt at a nicotine concentration of ~59mg/ml, commensurate with levels in popular 
commercially available nicotine salt e-cigarettes.55-57 Each exposure will consist of a 5-puff 
bout (each puff 3-sec long and separated by 30 sec) via a puff topography a device (an 
apparatus used to measure puffing behavior) and will be separated by 30 mins to allow any 
sensory effects to dissipate between exposures. The e-cig pods containing e-liquids will be 
weighed before and after the session. 
Participants will be told they can discontinue puffing behavior at any time if they experience 
discomfort. The lab sessions will be conducted by trained research staff who are sensitive to 
the 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. Blood 
pressure will be taken before and after each e-cigarette exposure during laboratory sessions. 
A blood pressure and heart monitoring form will be used to assess if blood pressure or heart 
rate is above or below the following threshold (>180, <100 systolic, >110, <60 diastolic) 
sessions will be paused and potentially discontinued if blood pressure is unable to return to in 
range values. If blood pressure is above or below these thresholds, blood pressure will be 
repeated 2 times to confirm value. Additionally, we will assess blood pressure across 
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laboratory sessions and contact the study physician if necessary if any concerning trends (with 
or without threshold blood pressure occur). 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. Suchitra Krishnan-
Sarin), will consult with Yale Tobacco Center for Regulatory Science (TCORS) co-Is, 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 if this occurs. 
6.1.3
Method of Assignment/Randomization 
Participants will be randomized to order of menthol conditions and WS-3 or WS-23 
conditions. 
6.1.4
Blinding and Procedures for Unblinding 
E-cig menthol conditions, WS-3, and WS-23 conditions will be double-blinded and letter 
coded. Letter codes will differ by participant. 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.
In the event of a serious adverse event, the PI (Dr. Suchitra Krishnan-Sarin), will consult with 
the project Co-Is and DSMB 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 if this 
occurs. 
6.1.5
Packaging/Labelling
All e-liquids will be created by the Yale IDS Pharmacy as described in 6.1.1. All ingredients 
sourced will be USP or FEMA GRAS, assessed by the Yale TCORS Lab Core and Yale IDS 
Pharmacy. All e-liquids will contain nicotine, propylene glycol and vegetable glycerin. The e-
liquid mixtures (propylene glycol [PG], vegetable glycerin [VG] and, menthol, WS-3, and WS-
23 conditions will consist of concentrated flavor liquids 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. The Yale IDS Pharmacy has consulted with PI on this project. 
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.
E-liquid nicotine and flavor profiles will be confirmed and characterized by the Yale TCORS 
lab core.38 These products do not have an expiration date but will be tested at regular 
intervals to confirm not change in product composition, as is done in HIC 200023077. Once 
purchased, if unopened, ingredients will be stored for 3 years or until expiration dates 
(whichever is sooner, per Yale IDS Pharmacy). Raw ingredients will only be opened at the 
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Yale IDS Pharmacy. Once opened, ingredients will be disposed of after 1 year (per Yale IDS 
Pharmacy). Once mixed and dispensed, e-liquids will be disposed of after 90 days of use 
(per Yale IDS Pharmacy). 
6.1.6
Storage Conditions
In order to ensure that we have adequate amount of e-liquid, of the same constituency, we 
will purchase large enough quantities of ingredients to create e-liquids for the entire study 
prior to starting these experiments which will be stored at and dispensed at Yale IDS 
Pharmacy. All raw ingredients (nicotine, propylene glycol, vegetable glycerin, flavorants) will 
be stored unopened in a locked cool and dry place onsite until recruitment begins. 
Ingredients will be transported to Yale IDS Pharmacy prior to recruitment start. Once 
recruitment begins and e-liquids are needed, Yale Pharmacy will dispense e-liquid in 30mL 
batches. Once complete, e-liquids will be immediately transported to Pierce Labs, which is a 
secure building accessible only by key code, where they will be delivered and stored in 
appropriate light/temperature conditions. E-liquids will be delivered and stored in a controlled 
and dark laboratory environment housed in John Pierce Labs and/or West Campus 
Research Center. Pods will be filled with e-liquid mixtures at John Pierce Labs.  
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 menthol, WS-3, and WS-23 condition orders for 
lab sessions will be generated by study biostatisticians prior to study start. This information 
is stored on a randomization sheet that has each participant’s information. The individual 
filling the pods will be notified of which subject ID number needs to be filled. Each 30mL 
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 0.9ml into the pipette from the e-juice 
bottle and carefully transfer it into the pod for the main study (0.5ml will be loaded to the 
pods for the pilot study). 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 container until the lab is set to begin. Once 
the research assistant is ready to begin each lab, they will open the 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 box. 
A research assistant/associate/fellow will maintain complete and accurate records to account 
for the receipt, use, and disposition of these pods with e-liquids, as well as the e-cigarette 
devices, to ensure that these investigational tobacco products (ITPs) are not 
commercialized. All post-study ITPs from the lab will be collected, properly transferred, and 
disposed of to the head of Plant Operations at Connecticut Mental Health Center (CMHC; 34 
Park Street, New Haven, CT) at the end of the study, with the PI signing off to ensure 
compliance and prevent commercialization. Regarding the remaining e-liquids stored but not 
used in the Yale IDS Pharmacy, they will be disposed of by the Yale IDS Pharmacy following 
the policy on the use of controlled substances in research. This commitment underscores 
our dedication to adhering to FDA guidelines and maintaining the integrity of our research.
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6.1.7
Concomitant therapy
N/A
6.1.8
Restrictions
There are no restrictions. 
6.2
Assessments
6.2.1
Efficacy

Generalized Labeled Magnitude Scale (gLMS)30,32, 33: Participants will rate overall 
intensity, sweetness, bitterness, coolness, irritation, mintiness, and smell using the 
gLMS, a category ratio scale with 7 semantic labels: “no sensation”, “barely 
detectable”, “weak”, “moderate”, “strong”, “very strong”, and “strongest imaginable 
sensation of any kind”. Labels are positioned quasi-logarithmically according to their 
empirically determined semantic magnitudes. The gLMS will be displayed on a 
monitor via a custom program. The scale produces ratio-level data equivalent to that 
produced by the method of magnitude estimation.30 Intensity ratings are typically 
normalized via log-transformation prior to parametric statistical analyses.

The Yale Craving Scale39: Based on the gLMS and displayed in the same way, this 
scale will be used to measure e-cigarette craving. Ratings will be evaluated for 
normality and transformed as needed.

Labeled Hedonic Scale (LHS)31: 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 per their semantic magnitude. The LHS, also displayed on a 
computer monitor, yields ratio-level data on liking/disliking and typically does not 
require log-transformation.

Drug Effects Questionnaire (DEQ): A modified version of the DEQ14,37,40 will be 
used to assess Liking/Wanting (average of “I feel good e-cig/pouch effects”, “I want 
more of that e-cig/pouch I received”, “I feel the e-cig/pouch strength” and “I like the e-
cig/pouch effect”), and 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”), on 
a 0-100 mm scale, from “not at all” to “extremely.”

Urine Cotinine and Nicotine levels: Urine cotinine levels are accurate indicators of 
tobacco use41,42 and will be obtained at intake to verify eligibility and measure 
baseline nicotine and cotinine levels. Additionally, urine samples collected at intake 
will be used for total nicotine equivalents assessment for oral microbiome and 
inflammation analysis for up to 33 exclusive e-cigarette users enrolled in the main 
study. Participation in the oral microbiome and inflammation assessments is optional.

Nicotine Equivalents: Salivary samples for nicotine and cotinine levels will be 
obtained at baseline and following exposures. To avoid buccal nicotine 
contamination, participants will rinse their mouths prior to providing each sample. 
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
Demographic Information (at Intake): Age, race, socioeconomic status, marital 
status, educational and occupational levels, and medical history will be assessed 
with interviews and self-report forms.

Tobacco Use History Questionnaire (at Intake): Self reports questions used in 
Population Assessment of Tobacco and Health (PATH) Study and other standardized 
measures.

Timeline Follow Back Interview43,44 (at Intake): Use of e-cigarettes, cigarettes, and 
other tobacco products will be examined using a 28-day TLFB at intake, and follow-
up appointments to determine eligibility. Test-retest reliability is high in adult tobacco 
users.38

Nicotine Dependence (at Intake): The 8-item PROMIS measure developed for 
cigarettes45 and adapted for e-cigs,46 with good internal consistency & measurement 
invariance across sex and race will be used.

Self-reported Oral Health Status (at intake): Exclusive e-cigarette users (up to 33)  
will answer questions related to oral health, such as ‘In the past-month, did you 
observe any bleeding after brushing or flossing, or due to other conditions in your 
mouth. (Yes/No)’, ‘In the past-month, did you notice pain in your mouth and how 
often (if yes)’, ‘In the past-month, did you notice sores inside your mouth’, ‘In the 
past-month did you notice dark spots on your teeth’. The responses to self-perceived 
oral health will be examined to explore differences based on flavor of e-cigarette 
used in the past. Participation in this assessment is optional for this subset of 
participants.

Oral Microbiome (at intake): Participation in this assessment is optional. Three 
saliva samples from exclusive e-cigarette users will be collected at intake and 
analyzed for oral microbiome (bacteria, viruses, and fungi) and markers of 
inflammation. 

Puff Topography (at lab): Puff topography will be measured using a device that was 
developed for e-cigarettes47,48 and adapted for the disposable e-cigarette device to 
be used in this study during the three lab sessions. This device includes a 
mouthpiece in which the e-cigarette device is inserted into and the participant uses 
the mouthpiece to inhale via the e-cigarette. It does not change the puffing or 
introduce an additional risk. For each vaping session, the puff topography device 
uses computer software to determine the parameters of each puff. The software 
converts signals to airflow (ml/sec) and integrates the flow data, producing measures 
of puff volume, duration, number, and interpuff interval (IPI). Puff topography will be 
collected during the directed puff procedures to assess consistency in puffing 
behavior across participants. Topography mouthpieces are sanitized in between 
subjects and inhaling e-cigarettes via topography devices present no increased risk 
to participants. 
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
Cardiovascular Measures (at intake and lab): During intake, participants’ blood 
pressure will be recorded using a general home Omron device. To monitor 
participants’ vital signs during directed puffing bouts in the lab session, physiological 
measures such as heart rate and blood pressure will be recorded at the beginning (6 
times) and end of (6 times) of each lab session using the Omron Professional 
IntelliSense Blood Pressure Monitor, which is clinically validated for professional use 
and offers enhanced precision. The device will automatically measure triplicate 
readings (separated by 30-sec breaks) to accurately monitor participants’ heart rate 
and blood pressure. 

Spirometry Lung Function Measures (at intake and lab): We will collect 
participants’ spirometry at intake and all lab sessions using the Vitalograph Model 
4000 Respiratory Monitor Spirometer (Smoking Motivator Edition). This procedure 
follows standardized guidelines to ensure accurate and reliable data collection, 
assessing participants’ respiratory function throughout the study.
6.2.2
Safety and Pregnancy-related Policy
For participants of childbearing potential, pregnancy tests will be administered at intake and 
each experimental session. Persons reporting pregnancy, breastfeeding, or trying to become 
pregnant will not be enrolled. If a participant becomes pregnant during the study, they will be 
withdrawn and if desired, provided with local pregnancy resources. 
6.2.3
Adverse Events Definition and Reporting
During screening, participants will undergo a review of their medical history, antibiotic use, a 
brief physical check (e.g., blood pressure, spirometry, heart rate, pulse oximetry, urine drug 
and pregnancy test) and a list of EVALI-related symptoms check (i.e., cough, shortness of 
breath, chest pain, nausea, vomiting, stomach pain, diarrhea, fever, chills, or weight loss) 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. Additionally, participants with any report symptoms that resemble 
EVALI without another reasonable and proximal cause will not be enrolled. Participants who 
experience these symptoms during the lab sessions will seek consultation with study 
medical staff and determine whether to complete the lab session. 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 greater than minimal risk to the subjects and adverse events are not 
anticipated. In the unlikely event that such events occur, serious and unanticipated and 
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related adverse events will be reported in writing within 48 hours to the Yale IRB and NIDA. 
The initial serious adverse event (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 
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. Krishnan-Sarin, 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. 
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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 
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 Cotinine/Nicotine/Other Drug Use/Pregnancy: Urine samples to test 
nicotine/cotinine will be collected at intake. Urine samples will be tested for pregnancy at 
each lab session and at intake. A urine drug screen will be conducted at intake. If any 
positive drug test results (aside from marijuana) are obtained, participants will have their 
intake appointment rescheduled to conduct another follow-up drug test prior to the lab 
session. Urine samples from exclusive e-cigarette users enrolled in the main study will be 
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tested for total nicotine equivalents and used for oral microbiome and inflammation 
assessments.
Saliva Samples: Salivary samples for nicotine and cotinine levels will be obtained at baseline 
and following each exposure. Salivary samples for the oral microbiome will only be collected 
at intake. To avoid buccal nicotine contamination, participants will rinse their mouths prior to 
providing each sample. 
6.3
Study Procedures
Prescreening: We aim to recruit 66 adult participants (33 female, 33 male) using online 
advertisements, community flyering, and referrals. We will use the centralized recruitment 
services provided by the Yale TCORS Administrative Core which involves the use of various 
recruitment techniques including advertising on social media websites (e.g., Facebook, 
Instagram, Snapchat, Reddit), at local colleges and jobsites and through Craigslist and flyers 
in the community, and a clinical trials recruitment company, BuildClinical. BuildClinical is a 
data-driven platform that helps academic researchers recruit participants for research 
studies more efficiently using social media, software, and machine learning. They have 
worked with IRBs in the US to ensure we adhere to all the appropriate guidelines and 
procedures. They utilize study-specific advertisements to engage participants on digital 
platforms such as Facebook, Google, WebMD, etc. and redirect them to a study-specific 
landing page should they click it. On the landing page, the person can complete an online 
pre-screen questionnaire that gets routed into BuildClinical's platform. BuildClinical's Secure 
Socket Layer (SSL) software encrypts all inputted information and keeps your information 
private and HIPAA compliant. Their backend servers are stored in the US at some of the 
most secure data centers in the world. The 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 containing e-cigarettes, and if they are at least 18. 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. The Yale 
TCORS centralized repository protocol (HIC# 2000037658) may refer potential participants 
for other future study recruitment.
Intake Visit: Participants will complete an initial intake appointment to sign informed consent 
and complete assessments to determine eligibility. Demographic information (age, biological 
sex, gender identity, race/ethnicity, marital status, socioeconomic status), self-reported 
medical and substance use history, urine drug, cotinine and pregnancy tests, heart rate, 
blood pressure, spirometry, and pulse oximetry will be collected. The Structured Clinical 
Interview for DSM-V (SCID) will be used to assess SUDs, as used in similar trials led by Dr. 
Krishnan-Sarin (HIC # 200023077). Tobacco use history assessments will assess 
lifetime/current tobacco product and flavor use, use frequency, nicotine dependence 
(Fagerstrom Test for Nicotine Dependence70), e-cig dependence (E-cig Dependence 
Scale71), perceptions of tobacco product use and risk, measures of usual brand cigarette/e-
cig appeal and reinforcing efficacy, and cessation attempt history. 
Saliva samples will be collected from exclusive e-cigarette users only (up to 33) at intake 
from the main study for testing oral microbiome and inflammatory markers. Additionally, 
participants will be asked to bring their most frequently used e-liquid/device in the past-
month to the study visit, for collection and testing by the study team. The e-liquids will be 
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tested in our lab for flavor and nicotine strength. Participants will receive an additional $10 
as compensation for turning in their device/pods which will NOT be returned to the 
participants. Urine samples will be collected for testing total nicotine equivalents. The saliva 
and urine samples collected at intake visit will only be used for oral microbiome and 
inflammation assessment if the participant is deemed eligible for the main study after intake. 
If not eligible, the samples will be discarded.
Lab Visits: See list of assessments in Table 4. We will collect urine pregnancy test, heart 
rate, blood pressure, pulse oximetry, spirometry, a health assessment checklist and health 
changes questionnaire, a craving measure.
The pilot study will establish low and high concentrations of WS-3, WS-23, and menthol 
concentrations to test in the main study. Participants will attend one lab session during which 
they will be exposed to e-liquids containing 16 conditions of menthol, WS-3, and WS-23 
without nicotine. Participants will receive two puff exposures to each condition; each 
exposure will be separated by 10 mins to allow the flavor to dissipate. Each session will 
begin with familiarization with the sensory rating scales and training in how to puff the lab e-
cig. WS-3, WS-23, and menthol will be presented in blocks (block order will be 
counterbalanced) with each block presenting increasing menthol/WS-3/WS-23 
concentrations. E-cig pods containing e-liquids will be weighed before and after the session. 
The TCORS Laboratory Core will prepare the e-liquids and analyze the amount transferred 
into the vape aerosol using the “vaping/lung machine” developed by the core. 
In the main study session, participants will engage in a 15-min training session of the 
psychophysical tasks they will use to report sensory experience at Lab 1. For Labs 1-3, 
participants will engage in six directed puffing bouts (5 puffs/3 sec puffs/30 second interpuff 
intervals) will be separated by 30 mins to allow any sensory effects to dissipate between 
exposures, a procedure adapted from Dr. Krishnan-Sarin’s previous studies. Puffing will 
occur using a puff topography device (i.e., an established device in which the e-cigarette is 
attached to a mouthpiece) to measure the parameters of each puff taken (puff duration, 
volume, etc.). During the 15-minute training session at Lab 1, participants will familiarize 
themselves with vaping using a puff topography device. Following bout, participants will 
complete in the following order measures of overall liking/disliking, intensities of sweetness, 
bitterness, cooling, and irritation/harshness, and where irritation/harshness is felt (i.e., 
mouth, throat, both). Sensory measures will be followed by collection of general appeal and 
reinforcing efficacy measures, and a post-exposure measure of craving (collected 15-min 
after exposure). Cardiovascular measures, including blood pressure and heart rate, will be 
recorded both before and after each bout session. Participants will be randomized to receive 
one of the 3 menthol conditions (no-menthol, low menthol, high menthol, levels determined 
by the pilot study above) in Lab 1 and will receive the alternative conditions in randomized 
order in Lab 2 and Lab 3. During each lab session the menthol condition will be paired with 
three WS-3 concentrations: no WS-3, low WS-3, or high WS-3 and three WS-23 
concentrations: low WS-23, high WS-23, or 4% WS-23 (concentration determined by pilot 
study) in randomized order. E-cig pods containing e-liquids will be weighed before and after 
the session. Lab sessions will occur no more frequently than every 24-hrs. At the end of 
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each of the three lab sessions, participants will choose between the 6 e-liquids they sampled 
in the lab using a free-choice paradigm.
Debrief: Following study completion, participants will be thanked for their participation and 
will be given information from the CDC on cigarette smoking and e-cigarette cessation.
Table 4. List of Study Assessments 
Pre-
screening 
(
Intake (pilot)
Lab 1 (Pilot)
Intake (main)
Lab 1 (main)
Lab 2 (main)
Lab 3 (main)
Contact Info & Eligibility 
Review
X
Informed Consent
X
X
Identifying Information
X
X
Demographic 
Information
X
X
Medical History
X
X
Timeline Follow Back
X
X
Urine Drug Screen
X
X
Urine Nicotine/Cotinine 
Sample
X
X
Saliva Sample
X
X
X
X
Urine Pregnancy Screen 
X
X
X
X
X
X
Blood Pressure/Heart 
Rate/Pulse Oximetry
X
X
X
X
X
X
Spirometry
X
X
X
X
X
X
Puff Topography
X
X
X
Tobacco Use 
History/Dependence 
Measures
X
X
SCID IV: Substance 
Section
X
X
Asthma Control Test
X
X
X
X
X
X
PROMIS Dyspnea
X
X
X
X
X
X
FACIT Cough Item
X
X
X
X
X
X
PROMIS Fatigue
X
X
X
X
X
X
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EVALI Health 
Assessment
X
X
X
X
X
X
gLMS Questionnaire
X
X
X
X
LHS Questionnaire
X
X
X
Drug Effects 
Questionnaire
X
X
X
Questionnaire of Vaping 
Craving 
X
X
X
Oral health 
questionnaire
X
Urine for nicotine 
equivalents testing for 
oral microbiome and 
inflammatory marker 
assessment (from up to 
33 exclusive e-cigarette 
users only)
X
Saliva for oral 
microbiome and 
inflammatory marker 
assessment (from up to 
33 exclusive e-cigarette 
users only)
X
6.3.1
Study Schedule
For each study, participants will complete an intake visit (approx. 2 hrs). Following intake 
and eligibility determination, participants will complete the pilot study with a single lab 
session and the main study with 3 labs sessions, 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
For the pilot study, we aim to recruit 30 adult participants and for the main study, we aim to 
recruit 66 adult participants (33 female, 33 male) using online advertisements, community 
flyering, and referrals. Potential participants will be recruited via the centralized recruitment 
services provided by the Yale TCORS Administrative Core which involves the use of various 
recruitment techniques including advertising on social media websites (e.g., Facebook, 
Instagram, Snapchat, Reddit), at local colleges and jobsites and through Craigslist and flyers 
in the community, and a clinical trials recruitment company, BuildClinical. 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 
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directly. If interested, participants will be asked if they regularly use nicotine containing e-
cigarettes, and if they are at least 18. If interested, potential participants will be invited to 
take part in an intake visit to determine eligibility. Intake visits will be completed in person 
and should take approximately 2 hours. 
Participants who fail to be eligible for current medical reasons (e.g., just started a new 
medication, have an upcoming procedure, or have an ailment that has resolved) or have 
another temporary ineligibility may be rescreened at a later date. PI will assess in concert 
with study nurse to determine if they are ok to rescreen. Participants will not be told at any 
intake why they are not eligible to protect inclusion/exclusion criteria and avoid any 
deceptive tactics for enrollment.
6.3.4
Enrollment 
If eligibility criteria are met, participant will be enrolled. PI will deem subjects eligible 
following review and will assign enrolled subject ID. 
6.3.5
On Study Visits
See 6.3 “Study Procedures” above and Table 1. 
6.3.6
End of Study and Follow-up
After completion, participants will be provided with written materials on e-cigarette cessation. 
Brief follow-up phone calls will take place at 1 month and 6 months after study completion to 
assess their current tobacco use behavior. 
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 
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
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Preliminary analyses will provide an overview of the data (e.g., randomization success, 
outcome distribution, missingness patterns). Outcome analyses will be intent-to-treat and 
use mixed-effects models which allow for different numbers of observations/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. For the pilot study, separate mixed-effects models will examine the influence 
of different concentrations of menthol, WS-23 and WS-3 on coolness and overall intensity 
and will be used to select the concentrations of menthol and WS-3 that product the weak but 
detectable coolness and the maximum coolness. For the main study, separate mixed-effects 
model will include menthol (0, Concentration 1, Concentration 2), WS-3 (0, Concentration 1, 
Concentration 2), WS-23 (0, Concentration 1, Concentration 2, Concentration 3) and time 
within lab as factors, all interactions among those factors and adjust for stratification 
variables. A combination of random effects and structured variance-covariance matrices will 
be used to account for within-subject correlations. The best-fitting structure will be selected 
based on Akaike information criterion (AIC). For the main study, higher liking/wanting for 
WS-3 (at Concentration 1 or 2) or WS-23 (at Concentration 1 or 2 or 3) combined with 
menthol (at Concentration 1 or 2), compared to menthol alone, will be supportive of the 
primary hypothesis. Similar models will examine the other outcomes and interactions of 
different concentration combinations of WS-3, WS-23 and menthol with nicotine. Exploratory 
analyses will also consider the impact of sex, combustible tobacco use and nicotine 
dependence on experiences from WS-3, menthol and nicotine.
6.4.2
Sample Size Considerations
For our pilot study, we will recruit 30 subjects. While we did not conduct a power calculation 
for this pilot study, we conducted a similar pilot study with 10 participants in our earlier work 
with multiple menthol concentrations, and do not anticipate any problems with identifying 
differences in coolness between the WS-3 and menthol concentrations. For the main study, 
we will recruit 66 subjects. With an anticipated dropout of 10%, 60 subjects are expected to 
have complete data. Because we have 4 primary hypotheses (each WS-3 concentration X 
each menthol concentration > menthol concentration alone), we used a Bonferroni-corrected 
alpha=0.1. With N=60 completers and a SD of paired differences of 25 as observed in our 
prior data, we have 80% power to detect a difference of 11.4 (moderate effect size d’=.45) in 
liking/wanting (0-100 scale) between each WS-3 concentration X each menthol 
concentration and menthol alone using a paired t-test at alpha=0.01.
6.5
Planned Analyses
6.5.1
Primary, Secondary, and Exploratory Objective Analysis
For the pilot study, separate mixed-effects models will examine the influence of 5 different 
concentrations of menthol and WS-3 on coolness and overall intensity and will be used to 
select the concentrations of menthol and WS-3 that product the weak but detectable 
coolness and the maximum coolness. For the main study, separate mixed-effects model will 
include menthol (0, Concentration 1, Concentration 2), WS-3 (0, Concentration 1, 
Concentration 2), WS-23 (0, Concentration 1, Concentration 2, Concentration 3), and time 
within lab as factors, all interactions among those factors and adjust for stratification 
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variables. A combination of random effects and structured variance-covariance matrices will 
be used to account for within-subject correlations. The best-fitting structure will be selected 
based on Akaike information criterion (AIC). For the main study, higher liking/wanting for 
WS-3 (at Concentrations 1 or 2) or WS-23 (at Concentrations 1 or 2 or 3) combined with 
menthol (at Concentration 1 or 2), compared to menthol alone, will be supportive of the 
primary hypothesis. Similar models will examine the other outcomes and interactions of 
different concentration combinations of WS-3/WS-23 and menthol with nicotine. Exploratory 
analyses will also consider the impact of sex, combustible tobacco use and nicotine 
dependence on experiences from WS-3/WS-23, menthol and nicotine. 
Oral microbiome and inflammatory marker assessment will serve as another exploratory 
objective in this study, Microbial testing, including DNA extraction, library preparation and 
sequencing methods, will be performed at the CosmosID lab. The analysis will evaluate 
alpha and beta diversity across different flavor-use groups, with the significance of group-
wise clustering determined using a permutational multivariate analysis of variance 
(MANOVA). Statistical methods such as ANOVA, t-tests and chi-square will assess 
between-group differences, while controlling for nicotine concentration based on total 
nicotine equivalents measured in urine samples from participants.
6.5.2
Safety 
See 6.1.2 for treatment of adverse events. Additionally, a health assessment will be 
collected at every visit.
6.5.3
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. 
6.5.4
Interim Analysis (if applicable)
N/A
6.5.5
Health economic evaluation
N/A
6.5.6
Other
N/A
6.5.7
Subsets and Covariates
As noted, we will stratify by menthol cigarette status in randomization and include as a 
covariate in analyses as that is a known confound to ratings of cooling e-cigs. If necessary, 
we will also adjust for participant abstinence compliance. 
6.5.8
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 menthol or WS-3 or WS-23 conditions administered or the 
nicotine concentration of the product. They will be told that products containing levels of 
nicotine and flavorings similar to commercial flavors of products they are using. 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. 
For the pilot, participants will be compensated up to $150: $50 for the intake. For the lab, 
participants will be compensated $90 in addition to a $10 travel payment if they provided 
their own travel. 
For the main study, participants will be compensated up to $510: $50 for the intake, $115 for 
Lab 1, $115 for Lab 2, and $115 for Lab 3 in addition to $10 travel payments to the lab 
sessions when they use their own transportation. Participants can also earn a $35 
completion bonus for completing all parts of the study. The above compensation will be 
provided by cash. Those eligible for the oral microbiome and inflammation assessment who 
submit their e-cigarette device/pods will receive an additional $10 (cash payment).
For the 1-month and 6-month follow-up phone calls (for the main study), participants will be 
paid $20 in the form of an Amazon gift card or cash. 
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
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. 
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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 be 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 be 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 be 
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. Krishnan-Sarin 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, biosamples, and survey/questionnaires 
collected as part of the study procedures, as well as investigational tobacco products (ITPs), 
including records showing the receipt, shipment, or other disposition of the e-liquids, pods, 
and e-cigarette devices. 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 be 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. Saliva for 
inflammatory marker testing and urine samples will be stored in tubes in -80 degree C 
freezer designated for study samples. Samples for oral microbiome analysis will be stored at 
room temperature for 30 days. If required to store for longer duration, the samples will be 
moved to freezer at -80 degree C. All specimens will be labelled only using study ID and no 
identifying information. Only PI or trained research staff will have access to the specimens.
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
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The study is considering a greater than minimal risk as participants are all current tobacco 
users using nicotine and flavors. It is unlikely brief exposure to e-cigarettes 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
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
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Study completion will be when 66 participants have completed all 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 have any role in study design, conduct, analysis, or 
publication. 
7.17 Funding Source
The study is funded through the Center for Tobacco Products and the National Institute on 
Drug Abuse as part of a Grant #U54DA036151. 
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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