Antiviral Efficacy and Acceptability of Therapeutic Antiseptic Mouth Rinses for Inactivation of COVID SARS-2 Virus
Phase: PHASE2
Conditions: Covid19, Coronavirus Infection, SARS-CoV-2 Infection
Sponsor: University of North Carolina, Chapel Hill

Mouth Rinses for Inactivation of COVID-19 
 
 
 
 
 
 
 
 
    NCT Number             NCT04584684 
Document Date             01/31/2024 
 
 
 

Clinical Intervention Study Protocol 
 
Antiviral Efficacy and Acceptability of Therapeutic 
Antiseptic Mouth Rinses for Inactivation of  
COVID SARS-2 Virus 
A randomized, placebo-controlled, double-masked clinical trial of antiseptic 
mouthwashes in the inactivation of COVID-19 SARS-2 virus in saliva 
 
Study Chairman or Principal Investigator:   
Laura Jacox, DMD, PhD, MS, Assistant Professor of Orthodontics, UNC Adams 
School of Dentistry 
 
Supported by: 
UNC Adams School of Dentistry 
 
Study Intervention Provided by: 
(Name of pharmaceutical company or device manufacturer, if any, providing support) 
 
 
 
 
Tool Revision History 
Version Number: 4 
Version Date: January 31, 2024 
 
 

 
TABLE OF CONTENTS 
 
Clinical Intervention Study Protocol Template 
PREFACE 
FULL PROTOCOL TITLE 
Tool Revision History 
TABLE OF CONTENTS 
STUDY TEAM ROSTER 
PARTICIPATING STUDY SITES 
PRÉCIS 
1. STUDY OBJECTIVES 
1.1    Primary Objective 
1.2    Secondary Objectives 
2. BACKGROUND AND RATIONALE 
2.1    Background on Condition, Disease, or Other Primary Study Focus 
2.2    Study Rationale 
3. STUDY DESIGN 
4. SELECTION AND ENROLLMENT OF PARTICIPANTS 
4.1    Inclusion Criteria 
4.2    Exclusion Criteria 
4.3    Study Enrollment Procedures 
5. STUDY INTERVENTIONS 
5.1    Interventions, Administration, and Duration 
5.2    Handling of Study Interventions 
5.3    Concomitant Interventions 
5.3.1 
Allowed Interventions 
5.3.2 
Required Interventions 

5.3.3 
Prohibited Interventions 
5.4    Adherence Assessment 
6. STUDY PROCEDURES 
6.1    Schedule of Evaluations 
6.2    Description of Evaluations 
6.2.1 
Screening Evaluation 
6.2.2 
Enrollment, Baseline, and/or Randomization 
6.2.3 
Blinding 
6.2.4 
Followup Visits 
6.2.5 
Completion/Final Evaluation 
7. SAFETY ASSESSMENTS 
7.1    Specification of Safety Parameters 
7.2    Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters 
7.3    Adverse Events and Serious Adverse Events 
7.4    Reporting Procedures 
7.5    Followup for Adverse Events 
7.6    Safety Monitoring 
8. INTERVENTION DISCONTINUATION 
9. STATISTICAL CONSIDERATIONS 
9.1    General Design Issues 
9.2    Sample Size and Randomization 
Treatment Assignment Procedures 
9.3    Definition of Populations 
9.4    Interim Analyses and Stopping Rules 
9.5    Outcomes 
9.5.1 
Primary Outcome 
9.5.2 
Secondary Outcomes 
9.6    Data Analyses 
10. DATA COLLECTION AND QUALITY ASSURANCE 
10.1  Data Collection Forms 

10.2  Data Management 
10.3  Quality Assurance 
10.3.1   
Training 
10.3.2   
Quality Control Committee 
10.3.3   
Metrics 
10.3.4   
Protocol Deviations 
10.3.5   
Monitoring 
11. PARTICIPANT RIGHTS AND CONFIDENTIALITY 
11.1  Institutional Review Board (IRB) Review 
11.2  Informed Consent Forms 
11.3  Participant Confidentiality 
11.4  Study Discontinuation 
12. COMMITTEES 
13. PUBLICATION OF RESEARCH FINDINGS 
14. REFERENCES 
 
 
 
 
 
 
 
 
 
 
 
 

STUDY TEAM ROSTER 
Laura Jacox, Principal Investigator, 2090 First Dental, 385 S. Columbia St., Chapel 
Hill, NC 27599, (847) 702-0988, ljacox@live.unc.edu 
Christina Graves, Co-Investigator, 385 S. Columbia St., Chapel Hill, NC 27599, 
(919) 537-3725, cxg@email.unc.edu 
Shannon Wallet, Co-Investigator, 385 S. Columbia St., Chapel Hill, NC 27599, (919) 
537-3359, shannon_wallet@unc.edu 
Ralph Baric, Co-investigator, 3304 Michael Hooker Res Building CB #7435, Chapel 
Hill, NC 27599, (919) 966-3895, rbaric@email.unc.edu 
Natalie Bowman, Co-investigator, CB 7030 Bioinformatics Building, 130 Mason 
Farm Road, Chapel Hill, NC 27514, natalie_bowman@med.unc.edu 
S.T. Phillips, Study Coordinator, GO Health Clinic, 2040 Old Dental, 385 S. 
Columbia St., Chapel Hill, NC 27599, (919) 537-3424, sherrill_phillips@unc.edu 
Wendy Lamm, Research Assistant, GO Health Clinic, 2040 Old Dental, 385 S. 
Columbia St., Chapel Hill, NC 27599, (919) 537-3418, wendy_lamm@unc.edu 
Nare Ghaltakhchyan, Research Assistant, 2090 First Dental, 385 S. Columbia St., 
Chapel Hill, NC 27599, (919) 904-8493, nare@live.unc.edu 
Eleanor Nanney, Research Assistant, 2090 First Dental, 385 S. Columbia St., Chapel 
Hill, NC 27599, (704) 651-4579, ellien@ad.unc.edu 
Mandy Bush, Research Assistant, DELTA Core Lab Manager, 385 S. Columbia St., 
Chapel Hill, NC 27599, (919) 537-3968, mbush43@email.unc.edu  
 
 
 
 
 
 
 
 
 

PARTICIPATING STUDY SITES 
 
Study Site: UNC Adams School of Dentistry 
 
GO Health Clinic, Old Dental Building #2040 
 
CB# 7450 
 
385 S. Columbia St, Chapel Hill, NC 27599 
 
(919) 537-3424 (P), (919) 843-1798 (F) 
 
Laura Jacox, Principal Investigator, 2090 First Dental, 385 S. Columbia St., Chapel Hill, 
NC 27599, (847) 702-0988, ljacox@live.unc.edu 
Christina Graves, Co-Investigator, 385 S. Columbia St., Chapel Hill, NC 27599, (919) 
 
537-3725, cxg@email.unc.edu 
Shannon Wallet, Co-Investigator, 385 S. Columbia St., Chapel Hill, NC 27599, (919) 
537-3359, shannon_wallet@unc.edu 
Ralph Baric, Co-investigator, 3304 Michael Hooker Res Building CB #7435, Chapel 
Hill, NC 27599, (919) 966-3895, rbaric@email.unc.edu 
Natalie Bowman, Co-investigator, CB 7030 Bioinformatics Building, 130 Mason Farm 
Road, Chapel Hill, NC 27514, natalie_bowman@med.unc.edu 
S.T. Phillips, Study Coordinator, GO Health Clinic, 2040 Old Dental, 385 S. Columbia 
St., Chapel Hill, NC 27599, (919) 537-3424, sherrill_phillips@unc.edu 
Wendy Lamm, Research Assistant, GO Health Clinic, 2040 Old Dental, 385 S. Columbia 
St., Chapel Hill, NC 27599, (919) 537-3418, wendy_lamm@unc.edu  
Nare Ghaltakhchyan, Research Assistant, 2090 First Dental, 385 S. Columbia St., Chapel 
 
Hill, NC 27599, (919) 904-8493, nare@live.unc.edu 
Eleanor Nanney, Research Assistant, 2090 First Dental, 385 S. Columbia St., Chapel 
 
Hill, NC 27599, (704) 651-4579, ellien@ad.unc.edu 
Mandy Bush, Research Assistant, DELTA Core Lab Manager, 385 S. Columbia St., 
Chapel Hill, NC 27599, (919) 537-3968, mbush43@email.unc.edu  
 
 
 
 
 
 

PRÉCIS 
Study Title 
Antiviral Efficacy and Acceptability of Therapeutic Antiseptic Mouth Rinses for 
Inactivation of COVID SARS-2 Virus 
Objectives 
The primary objectives are to quantify the impact of antiseptic mouthwashes on 
salivary protein antigen levels and viral RNA of the SARS-CoV-2 virus in COVID-
19+ patients. The tertiary objectives are to quantify the impact of antiseptic 
mouthwashes on salivary viral infectivity in COVID+ patients, and to determine 
patients’ acceptance of using antiseptic mouthwashes in healthcare settings. 
Design and Outcomes  
Randomized, double-blind prospective trial to test the efficacy and acceptability of 
therapeutic, antiseptic mouth rinses to inactivate the SARS-CoV-2 virus, and to 
reduce protein antigen levels and viral RNA in saliva of COVID-19 positive patients 
aged 18-65 years old. All mouthrinses are commercially available and will be used 
according to on-label instructions. Patients will be randomized to a mouthrinse and 
will be asked to give a saliva sample immediately before and after a one minute 
mouthwash. Saliva samples will be collected from patients at 15 minute intervals 
thereafter up to an hour (15, 30, 45 and 60 minutes). The samples will be stored and 
used for qRT-PCR detection of viral SARS-CoV-2 RNA, sandwich ELISA or rapid 
antigen tests for detection of SARS-CoV-2 protein antigens, and viral infectivity 
assays. Patients will also complete a short-survey on the taste and experience of using 
the mouthwash.  This study involves one, 75-90 minute visit. 
Interventions and Duration 
Subjects will be asked to rinse with an unlabeled/blinded antiviral mouth rinse for 60 
seconds and provide 5 mL of saliva prior to the rinse and 2 mL of saliva immediately 
post-rinse, 15 minutes post-rinse, 30 minutes post-rinse, 45 minutes post-rinse, and 60 
minutes post-rinse. Subjects will also be asked to complete a short survey about the 
rinsing experience. Subjects will be onsite for a 70-90 minute, single visit 
appointment.  
Sample Size and Population 
We will enroll 80 outpatient COVID+ subjects per mouth rinse, with 6 mouth rinses, 
requiring us to enroll 480 COVID+ patients. Patients will be randomized to their 
mouthrinse. These patients will have already had a confirmed COVID+ test prior to 
enrollment. There will be no stratification to our randomization. In addition to having 
a COVID+ test, patients will need to be at least 18 years of age and at most 65 years 
of age and in good oral health without any known allergies or reactions to commercial 

dental products, mouthrinses or iodine. Patients who are pregnant, nursing/lactating, 
who have a cognitive or developmental disability, who are taking lithium, who have 
or have ever had a thyroid problem, who have kidney dysfunction, who have an 
active oral herpes flare up, who have any oral viral infection and/or flare up, who 
have significant mucosal tears, growths or damage to their mouth, who have 
uncontrolled severe periodontal disease with bleeding gums, and those who have 
xerostomia will be excluded from the study. There is no exclusion criteria based on 
gender or race.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
1. STUDY OBJECTIVES 
1.1   
Primary Objectives 
The primary objectives of this study are to determine the efficacy of antiseptic 
mouthwashes on reducing SARS-CoV-2 viral RNA and protein antigen levels in 
COVID+ patient saliva. We hypothesize that COVID-19+ patients will have a >95% 
reduction in SARS-CoV-2 viral RNA and protein antigen levels following a 60 second 
oral rinse with an antiseptic mouthrinse and that this reduction will persist for at least 
30 minutes. These primary objectives will assess SARS-CoV-2 viral load based on viral 
RNA measured by quantitative real time PCR and viral protein antigen levels measured 
by rapid antigen or sandwich ELISA assays. These objectives provides urgently needed 
data to guide healthcare workers on the proper use of specific commercially available 
mouthwashes capable of reducing SARS-CoV-2 in saliva and potentially limiting 
COVID-19+ transmission.  
1.2  
Tertiary Objectives 
The first tertiary objective will focus on quantifying SARS-CoV-2 viral infectivity 
using an in vitro infectivity assay. We hypothesize that COVID-19+ patients will have 
a >95% reduction in SARS-CoV-2 viral infectivity following a 60 second oral rinse 
with an antiseptic mouthrinse and that this reduction will persist for at least 30 minutes. 
This objective provides urgently needed data to guide healthcare workers on the proper 
use of specific commercially available mouthwashes capable of reducing SARS-CoV-
2 viral infectivity and potentially limiting COVID-19+ transmission. Our second 
tertiary objective is to determine the feasibility and acceptability of using antiseptic 
mouthwashes by patients in healthcare settings. We hypothesize that patients will 
welcome the use of commercially available antiseptic mouthrinses into clinical 
practice.  Patients enrolled in the randomized mouthrinse study will be surveyed on the 
taste, appearance, sensation, and acceptability of using the mouthrinse in healthcare 
settings. Understanding acceptability of a mouthrinse protocol is critical to 
implementation and therefore the ability to reduce COVID-19 transmission risk in 
clinical settings. 
 
 
 
 

 
2. BACKGROUND AND RATIONALE 
2.1   
Background on Condition, Disease, or Other Primary Study Focus 
The COVID-19 global pandemic, caused by SARS-CoV-2 virus, represents a public 
health emergency with severe societal and economic impacts. Evaluating the potential for 
antiviral mouthrinses to inactivate SARS-CoV2 holds great potential for quelling the 
virus’ spread among healthcare workers, who comprise up to 10% of COVID-19 cases.1,2 
Healthcare providers like dentists, who deliver care in and near the oropharynx, suffer the 
highest risk of occupational exposure.3 This is because SARS-CoV-2 is transmitted 
primarily through aerosol and respiratory droplets and salivary glands are a site of early 
SARS-CoV-2 viral replication and transmission.4,5 Dentists work in close proximity to 
the mouth and nasal passages with many procedures producing salivary airborne 
particles, particularly those involving ultrasonic and rotary instruments (hand pieces, 
“drills”) and 3-way syringes.5–8  High rates of SARS-CoV-2 transmission suggest that the 
minimum infectious dose is low compared to other viral diseases, and recent data suggest 
even one viral particle may be sufficient for infection.9 Working in close proximity to the 
oropharynx and generating salivary aerosols increases viral load exposure and therefore 
the risk of infection among dentists, oral surgeons, and other interventional doctors of the 
face.9 Furthermore, the proclivity of aerosols to remain airborne for up to 3 hours puts 
other patients, providers and staff at risk of exposure during aerosol-generating 
procedures, particularly in open bay clinics common to dental schools and large 
practices.4–6,10 
One promising strategy to reduce clinical spread is use of antiviral mouthrinses to 
inactivate SARS-CoV-2 infectability in saliva. Several commercially available 
mouthrinses have promising in vitro data with SARS-CoV-2 and in vivo data with other 
enveloped viruses, with a proposed mechanism of membrane disruption.3,11 Ethanol 
solutions (21% EtOH) have been shown to reduce SARS-CoV-2 viral infectivity levels in 
vitro, and in patients suffering Herpes Simplex Virus-1 flare-ups, a 30 second oral rinse 
significantly reduced salivary infectivity for over 30 minutes.12 Povidone-iodine (PVP), is 
a fast-acting, broad-spectrum antiseptic with antiviral activity in vitro and is commonly 
encountered in dental settings outside the US.13 PVP is effective in reducing SARS-CoV-
2 viral titers by 99.99%, lasting up to 20 minutes in vitro, suggesting it could be effective 
in reducing transmission during dental procedures.14,15 Chlorhexidine Gluconate (0.12%) 
has been widely adopted by practices despite discouraging in vitro data.3,11 Finally, 
hydrogen peroxide mouthrinse (e.g. 1.5% w/v H2O2) is a widely available oral antiseptic 
capable of hydroxylating membranes, and is the only preprocedural mouthrinse currently 
recommended by the ADA to prevent transmission, despite no studies evaluating its 

efficacy against SARS-CoV-2.16 No in vivo clinical data exists to-date for PVP or H2O2 
mouthrinses.  
Despite promising in vitro data, there is a dearth of in vivo clinical trials interrogating the 
efficacy of mouthrinses on reducing salivary SARS-CoV-2 viral infectivity. Given the 
rapid transmission and widespread distribution of the COVID-19 pandemic, the 
development of protocols and treatments mitigating transmission represent a major and 
urgent unmet public health need; this study will rigorously address the in vivo utility of 
widely available oral rinses in limiting SARS-CoV-2 viral infectivity and their 
acceptability in the dental healthcare setting. 5–8 
 
1.        State Officials Announce Latest COVID-19 Facts Including New Data on Racial 
Demographics and Expanded Health Care Worker Data. California Department of Public 
Health(2020). 
2.        Sorveglianza Integrata COVID-19 in Italia. Instituto Superiore di 
Sanitahttps://portale.fnomceo.it/wp-content/uploads/2020/04/Infografica_9aprile-ITA.pdf 
(2020). 
3.        O’Donnell, V. B. et al.Potential Role of Oral Rinses Targeting the Viral Lipid 
Envelope in SARS-CoV-2 Infection. Function 1, (2020). 
4.        Harrel, S. K. & Molinari, J. Aerosols and splatter in dentistry: A brief review of 
the literature and infection control implications. J. Am. Dent. Assoc.(2004) 
doi:10.14219/jada.archive.2004.0207. 
5.        Izzetti, R., Nisi, M., Gabriele, M. & Graziani, F. COVID-19 Transmission in 
Dental Practice: Brief Review of Preventive Measures in Italy. Journal of Dental 
Research(2020) doi:10.1177/0022034520920580. 
6.        Ather, A., Patel, B. & Ruparel, N. Coronavirus Disease 19 (COVID-19): 
Implications for Clinical Dental Care. J. Endod.(2020). 
7.        Baghizadeh Fini, M. What dentists need to know about COVID-19. Oral 
Oncology(2020) doi:10.1016/j.oraloncology.2020.104741. 
8.        Cleveland, J. L. et al.Transmission of blood-borne pathogens in US dental health 
care settings: 2016 update. J. Am. Dent. Assoc.(2016) doi:10.1016/j.adaj.2016.03.020. 
9.        Pfefferle, S. et al.Low and high infection dose transmission of SARS-CoV-2 in the 
first COVID-19 clusters in Northern Germany. medRxiv(2020) 
doi:10.1101/2020.06.11.20127332. 

10.      Ge, Z. yu, Yang, L. ming, Xia, J. jia, Fu, X. hui & Zhang, Y. zhen. Possible 
aerosol transmission of COVID-19 and special precautions in dentistry. Journal of 
Zhejiang University: Science B(2020) doi:10.1631/jzus.B2010010. 
11.      Carrouel, F. et al.COVID-19: A Recommendation to Examine the Effect of 
Mouthrinses with ß-Cyclodextrin Combined with Citrox in Preventing Infection and 
Progression. J. Clin. Med.(2020) doi:10.3390/jcm9041126. 
12.      Dennison, D. K., Meredith, G. M., Shillitoe, E. J. & Caffesse, R. G. The antiviral 
spectrum of Listerine antiseptic. Oral Surgery, Oral Med. Oral Pathol. Oral 
Radiol.(1995) doi:10.1016/S1079-2104(05)80124-6. 
13.      Hasheminia, D. et al.The efficacy of 1% betadine mouthwash on the incidence of 
dry socket after mandibular third molar surgery. J. Clin. Exp. Dent.(2018) 
doi:10.4317/jced.54444. 
14.      Challacombe, S. J., Kirk-Bayley, J., Sunkaraneni, V. S. & Combes, J. Povidone 
iodine. British Dental Journal(2020) doi:10.1038/s41415-020-1589-4. 
15.      Kirk-Bayley, J., Challacombe, S., Sunkaraneni, V. & Combes, J. The Use of 
Povidone Iodine Nasal Spray and Mouthwash During the Current COVID-19 Pandemic 
May Protect Healthcare Workers and Reduce Cross Infection. SSRN Electron. J.(2020) 
doi:10.2139/ssrn.3563092. 
16.      COVID-19 Frequently Asked Questions. American Dental Assocation: Center for 
Professional Sucess(2020). 
17.      Sethuraman, N., Jeremiah, S. S. & Ryo, A. Interpreting Diagnostic Tests for 
SARS-CoV-2. JAMA - Journal of the American Medical Association(2020) 
doi:10.1001/jama.2020.8259. 
 
2.2   
Study Rationale 
One promising strategy to reduce clinical spread is use of antiviral mouthrinses to 
inactivate SARS-CoV-2 infectability in saliva. Several commercially available 
mouthrinses have promising in vitro data with SARS-CoV-2 and in vivo data with other 
enveloped viruses, with a proposed mechanism of membrane disruption.3,11 Ethanol 
solutions (21% EtOH) have been shown to reduce SARS-CoV-2 viral infectivity levels in 
vitro, and in patients suffering Herpes Simplex Virus-1 flare-ups, a 30 second oral rinse 
significantly reduced salivary infectivity for over 30 minutes.12 Povidone-iodine (PVP), is 
a fast-acting, broad-spectrum antiseptic with antiviral activity in vitro and is commonly 
encountered in dental settings outside the US.13 PVP is effective in reducing SARS-CoV-
2 viral titers by 99.99%, lasting up to 20 minutes in vitro, suggesting it could be effective 

in reducing transmission during dental procedures.14,15 Chlorhexidine Gluconate (0.12%) 
has been widely adopted by practices despite discouraging in vitro data.3,11 Finally, 
hydrogen peroxide mouthrinse (1.5% w/v H2O2) is a widely available oral antiseptic 
capable of hydroxylating membranes, and is the only preprocedural mouthrinse currently 
recommended by the ADA to prevent transmission, despite no studies evaluating its 
efficacy against SARS-CoV-2.16 No in vivo clinical data exists to-date for PVP or H2O2 
mouthrinses.  
Despite promising in vitro data, there is a dearth of in vivo clinical trials interrogating the 
efficacy of mouthrinses on reducing salivary SARS-CoV-2 viral infectivity. Given the 
rapid transmission and widespread distribution of the COVID-19 pandemic, the 
development of protocols and treatments mitigating transmission represent a major and 
urgent unmet public health need; this study will rigorously address the in vivo utility of 
widely available oral rinses in limiting SARS-CoV-2 viral infectivity and their 
acceptability in the dental healthcare setting. 5–8 
Like other enveloped viruses, coronaviruses are surrounded by a lipid bilayer which allows 
for spike glycoproteins required for infection to be inserted.3 The mouth rinses proposed 
in this study have shown success in previous studies in disrupting this lipid envelope in 
other viruses, and there is promise that they will do the same to the SARS-CoV-2 virus. 
Effect size: Based on similar viral infectivity studies evaluating mouth rinse efficacy 
against enveloped viruses, we used power calculations to estimate effect size and determine 
sample size as a function of length of mouthrinse time course. With ten simultaneous 
comparisons (T1-T0, T2-T1, T2-T0, T3-T0, T3-T2, T3-T1, T4-T3, T4-T2, T4-T1, T4-T0) 
= 60 minute time course, the sample size n=78 can detect the effect size in Cohen’s d =0.7, 
which is between medium and large, under 80% power and 0.016 type-I error rate by 
Bonferroni correction.12 
Subjects will be required to rinse their mouths with an unlabeled/blinded mouthrinse for 
60 seconds. Per the instructions on the product label, it is recommended to rinse for 30-60 
seconds. The longer rinse-time was chosen for this study in order to maximize the 
antiviral activity of the proposed mouthrinses. The mouthrinses chosen for this study are 
all commercially available products with known antiviral effects. 
The intervention will be administered orally because an oral mouth rinse will be utilized, 
and the dosage will range from 10 mL to 20 mL, dependent on the dosage instructions on 
the product label. The duration of the intervention will be 60 minutes, with collection of 
pooled saliva samples pre-rinse and every 15 minutes (0 min, 15 min, 30 min, 45 min, 
and 60 min) post-rinse. This time period was chosen to be similar to that of a typical 
dental appointment. Multiple saliva samples will be collected to show how long the 
proposed mouth rinses are successful in deactivating the virus. 

Known and potential risks include breach of confidentiality, slight discomfort, and a low 
chance of a previously undiagnosed allergy. To minimize risk of breach of 
confidentiality, all patients will be assigned a unique study ID. There will be no other 
subject identifiers on samples or data. The linkage file that relates patient's names to 
study ID numbers will be stored on a UNC secure server. All desktops computers and 
servers are kept in locked facilities and in accordance with UNC IT/security safeguards 
and policies.  All providers and study researchers have been trained in and abide by 
HIPAA procedures, and confidential patient information will be stored on secured, 
encrypted UNC servers. Patients will already have been provided with their COVID 
status through their healthcare providers, and this confidential medical information will 
not be released by any study personnel. Some patients may perceive the mouth rinse as 
causing some discomfort if they dislike the flavor or the bubbling sensation. A small 
fraction of patients (rare <1%) may be allergic to the mouth rinse. We will ask detailed 
questions in our screening protocol if patients are allergic to any component of the 
mouthrinses, but if a patient answers no to all screening questions regarding allergy and 
then they are allergic, it may lead to an adverse allergic reaction.  
The following table lists adverse effects associated with each mouthrinse proposed in this 
study: 
mouthrinse* 
Adverse effects 
26.9% ethanol 
plus essential 
oils 
● 
If more than used for rinsing is accidentally swallowed, get medical 
help or contact a Poison Control Center right away 
0.5% w/v 
povidone-iodide 
(PVI)  
● 
Local irritation, skin burns and sensitivity reactions have been 
reported rarely. 
● 
Anaphylactic reactions, anaphylactoid reactions and anaphylactic 
shock have been reported uncommonly with products containing 
povidone-iodine or povidone. 
● 
Excess iodine can produce goitre and hypothyroidism or 
hyperthyroidism. Such effects have occasionally been seen with 
extensive or prolonged use of povidone iodine.  
● 
Other effects that have been reported are metabolic acidosis and acute 
renal failure. 
● 
Possible unwanted effects: Like all medicines, Betadine Gargle and 
Mouthwash can cause unwanted effects, although these are 
uncommon. Allergic-type reactions, including a rash or difficulty in 
breathing, have been reported uncommonly with products containing 
povidone iodine (the active ingredient in Betadine Gargle and 
Mouthwash). Although rare, Betadine Gargle and Mouthwash could 
also cause itching and burns on your skin. Applying very large 

amounts of medicines containing povidone iodine, or using such 
products over a long period of time, can occasionally cause thyroid 
disorders (symptoms of which include weight loss, increased appetite 
and sweating or lacking energy and weight gain). Other effects that 
have been reported are increased amounts of acid in your blood (the 
main symptoms are shortness of breath, confusion and lethargy) and 
kidney failure (symptoms include difficulty in urinating, thirst and a 
dry mouth and pain in one side of your back). Should you suffer from 
any of these unwanted effects, or if you notice any unwanted effects 
not listed in this leaflet, stop using Betadine Gargle and Mouthwash 
straight away and tell your doctor or pharmacist. 
1.5% w/v  
hydrogen 
peroxide rinse 
● 
If irritation persists for 7 days, is severe, is due to orthodontic 
appliances and/or dentures, or swelling or fever develops, the patient's 
condition needs to be re-evaluated by a doctor or dentist. 
● 
Overdose can injure the gums and continued use of hydrogen peroxide 
may cause reversible hypertrophy of the papillae of the tongue known 
as ‘black hairy tongue’ therefore using this product at high doses or 
for long periods of time is not recommended. 
● 
Some cases of mucosal irritation and swelling of the oral tissues have 
been reported specially with high doses or in continued use. 
0.12% 
Chlorhexidine 
Gluconate 
● 
The most common side effects associated with chlorhexidine 
gluconate oral rinses are: (1) an increase in staining of teeth and other 
oral surfaces, (2) an increase in calculus formation, and (3) an 
alteration in taste perception; see WARNINGS and PRECAUTIONS. 
Oral irritation and local allergy-type symptoms have been 
spontaneously reported as side effects associated with use of 
chlorhexidine gluconate rinse. The following oral mucosal side effects 
were reported during placebo-controlled adult clinical trials: aphthous 
ulcer, grossly obvious gingivitis, trauma, ulceration, erythema, 
desquamation, coated tongue, keratinization, geographic tongue, 
mucocele, and short frenum. Each occurred at a frequency of less than 
1.0%. Among postmarketing reports, the most frequently reported oral 
mucosal symptoms associated with chlorhexidine gluconate oral rinse 
are stomatitis, gingivitis, glossitis, ulcer, dry mouth, hypesthesia, 
glossal edema, and paresthesia. Minor irritation and superficial 
desquamation of the oral mucosa have been noted in patients using 
chlorhexidine gluconate oral rinses. There have been cases of parotid 
gland swelling and inflammation of the salivary glands (sialadentitis) 
reported in patients using chlorhexidine gluconate oral rinse. 
 
0.1% 
Cetylpyridinium 
Chloride 
● 
If more than used for rinsing is accidentally swallowed, get medical 
help or contact a Poison Control Center right away 

0.9% NaCl 
No contraindications 
 
*All mouthrinses are commercially available in the USA, routinely used in clinical settings, and 
will be used in an on-label manner. 
3. STUDY DESIGN 
Aims of this placebo-controlled, randomized, double-blinded study are to determine the 
efficacy of antiseptic mouthwashes on reducing SARS-CoV-2 viral RNA, protein antigen 
levels, and infectivity in COVID+ patient saliva (described above).  Asymptomatic and 
symptomatic SARS-CoV-2+ subjects will be enrolled in the study (according to Inclusion 
and Exclusion Criteria - Section A.3). Symptomatic SARS-CoV-2+ subjects will be 
enrolled within 10 days of symptom onset. Subjects will be seen and samples collected in 
the GoHealth Clinic (Adams School of Dentistry, UNC Chapel Hill, North Carolina, USA).  
Asymptomatic SARS-CoV-2+ subjects will be enrolled within one week of likely 
exposure. Study participants who meet all inclusion criteria without any exclusion criteria 
will be randomized to either an experimental or control group using block randomization.  
Participants will be asked to give a baseline salivary sample (5ml over 10 minutes) and then asked 
to rinse with an unlabeled (blinded) antiseptic mouth rinse for 60 seconds according to the table 
below: 
mouthrinse* 
Number of participants (sample 
size)  
Group 
26.9% ethanol plus essential oils 
80 
Experimental 
0.5% w/v povidone-iodide (PVI)  
80 
Experimental 
1.5% w/v  hydrogen peroxide rinse 80 
Experimental 
0.12% Chlorhexidine Gluconate 
80 
Experimental 
0.1% Cetylpyridinium Chloride 
80 
Experimental 

0.9% NaCl 
80 
Control 
*All mouthrinses are commercially available in the USA, routinely used in clinical settings, and 
will be used in an on-label manner. 
After the participant expectorates the mouth rinse, research staff will collect another 
salivary sample (T0, 2 mL over 5 minutes). During a 10 minute recuperation period, the 
study participants will answer demographics survey questions and questions regarding the 
taste, color, flavor and general acceptability of using the mouthrinse as part of routine 
dental and medical visits. Patients will be asked to smell an ammonia ampule (“smelling 
salts”) to evaluate sense of smell, as loss of olfaction is indicative of the acute infection 
phase. At 15 minutes post-rinse, the participant will be asked to provide an additional saliva 
sample (2 mL over 5 minutes), followed by another 10 minute recuperation period. This 
sequence will be repeated for at the 30, 45 and 60 minute time points post rinse. At 60 
minutes, the study participant will give their final salivary sample (2 mL over 5 min), and 
study participation will conclude.   
All salivary samples will be collected on ice in sterile 15 mL conical vials labelled 
“COVID Core” and using pre-printed labels including patient’s random alphanumeric 
study ID. Saliva samples will be cataloged and stored at the UNC Delta COVID 
Translational Initiative (also known as the Covid-19 Core) for future qRT-PCR, viral 
infectivity, and rapid antigen or sandwich ELISA assays.  Samples will be transported on 
ice from the collection site to 4403A Koury Hall in leak proof biohazard bags. Samples 
will be processed according to the sample processing and outcomes procedure (below).  
Following study participation completion, participants will receive $50 compensation in 
the form of a Visa gift card. 
During the period that the COVID+ study participants are present in the clinic and giving 
saliva samples, we will monitor oxygen saturation and inquire about their comfort. If 
participants exhibit signs of distress or if oxygen saturation drops, research study staff will 
contact emergency medical responders (present in the building) to visit the patient and if 
deemed appropriate, the participants will be escorted to UNC hospital (adjacent building). 
This time course analysis design will allow us to determine if any of the antiseptic mouthwashes 
are effective at reducing SARS-CoV-2 in salivary secretions of COVID-19+ study participants as 
well as to determine the therapeutic window if effective. We have designed this study to 
recapitulate a standard oral exam and procedure time (~1 hr in length).  Further, the design will 
allow for a kinetic analysis of viral shedding in salivary secretions and to determine the most 
effective mouth rinse. 
 

Sample processing and outcomes 
Sample processing: Following collection, saliva samples will be processed according to existing 
BSL2+ SOP protocols (COVID Core, UNC (PI: Wallet and Bush) by pre-authorized personnel. 
Samples will be assessed for 1) molecular presence of SARS-CoV-2, 2) salivary antibody and 
cytokine responses to SARS-CoV-2 and 3) in vitro infectibility. 
1. Molecular analysis: For molecular analysis, including viral RNA and protein antigen 
analysis, samples will be inactivated by adding 2 mL of extraction buffer to each 500 uL 
saliva aliquot (4:1 ratio) and mixed by inversion. These samples will be further aliquoted 
at 250uL in 1.5 mL Eppendorf tubes and stored in an “Inactivated Samples -80 °C Freezer” 
until further analysis. 
2. Antibody and cytokine responses: an aliquot of saliva will be inactivated in a 56 °C 
water bath for 30 minutes. 250 uL aliquots will be transferred into 1.5 mL cryovials and 
stored in the “Inactivated Samples -80 °C Freezer” in 4403A Koury Hall until further 
analysis.  
3. In vitro infectivity: Samples will be stored in the “Activated Samples -80 °C Freezer” 
in 4403A Koury Hall until further analysis.  
At analysis, all samples will be serially diluted in sterile PBS (10-10 fold dilutions). Plaque 
assays will be performed using Vero E6 cells at confluency in 96-well cell culture plates. 
Briefly, cell cultures will be washed with sterile PBS and samples containing virus will 
then be plated in duplicate (100 µL per well). Plates will be incubated at 37 °C for 45 
minutes with occasional rocking. Then 2 mL of 0.5% agarose in minimal essential media 
(MEM) containing 2% FBS and antibiotics will be added per well. Plates will be incubated 
at 37 °C for 72 hours, fixed with 10% buffered formalin, followed by the removal of the 
overlay, and then stained with 0.2% crystal violet to visualize plaque forming units (PFU). 
Average PFUs will be evaluated by OD. All assays will be performed in BSL-3 laboratory 
setting. (PMID: 32475066)  
Unprocessed saliva exceeding immediate experimental capacity will be stored in aliquots 
of 250 uL volume in 1.5 mL Eppendorf tubes and stored in “Activated Samples -80C 
Freezer.”  
Survey data: The survey data will be analyzed and used to determine clinical and other 
factors impacting applicability of each of the mouth rinses.  
 
 
 

 
4. SELECTION AND ENROLLMENT OF PARTICIPANTS 
4.1   
Inclusion Criteria 
Subjects must meet all of the following inclusion criteria to be eligible for enrollment in 
the study: 
● Diagnosed COVID+ status by physician. Either became symptomatic in the prior 
10 days, or if not symptomatic, likely infected/exposed within the prior 10 days. 
● Individuals (all sex, all gender) at least 18 years of age and at most 65 years of 
age and in good oral health without any known allergies to commercial dental 
products or cosmetics.  
● Evidence of a personally signed and dated informed consent document indicating 
the subject (or legally acceptable representative) has been informed of all 
pertinent aspects of the trial and all of their questions have been answered. 
● Able to comprehend and follow the requirements of the study (including 
availability on scheduled visit dates) based on research site personnel’s 
assessment.  
● Females of childbearing potential will have a negative urine pregnancy test (on 
site) or be physically incapable of pregnancy (implants or injections, Intrauterine 
device, Bilateral tubal ligation, Hysterectomy, Ovariectomy, Women post-
menopausal)  
4.2   
Exclusion Criteria 
Subjects presenting with any of the following will not be included in the study: 
● Patients who have been eating or drinking within an hour of the study 
● Patients under 18 years old and older than 65 years old 
● Subjects presenting with and/or self-reporting any of the following will not be 
included in the study: 
○ history of significant adverse effects following use of oral hygiene 
products such as toothpastes and mouthrinses. (self-reported) 
○ Self-reported allergy to iodine, ethanol, essential oils (Eucalyptol, 
Menthol, Methyl salicylate, Thymol), hydrogen peroxide, chlorhexidine 
gluconate, peroxyl, listerine, betadine, peridex, cetylpyridinium chloride, 
and other components in the mouth rinses (methyl salicylate, ethanol, 
saccharin sodium, glycerin, propylene glycol, sorbitol, FD&C blue no. 1, 
Poloxamer 407, Benzoic acid, Zinc chloride, Sodium benzoate, Sucralose, 
PEG-40 sorbitan diisostearate, potassium sorbate, citric acid). 
○ History of serious medical conditions that, at the discretion of the 
Investigator, will disqualify the subject. (Self-reported) 

○ A history of severe dry mouth (xerostomia), drug-induced xerostomia 
(antidepressants, anticonvulsants, antihypertensives), or Sjogren’s 
syndrome  
○ A history of recent (within the last 30 days) or current recent oral herpes 
flare up, candida (thrush) infection, apthous ulcer flare up, current/active 
severe periodontal disease, or other recent oral viral infection or flare up 
within the past 30 days (self-reported) 
○ Current history of alcohol or drug abuse (self-reported).  
○ History of drinking water or eating food within an hour of the study visit.  
○ History of drinking alcohol within 12 hours of the study visit.  
○ History of using a commercial mouthrinse within 24 hours of the study 
visit. 
○ Participation in any study involving oral care products, concurrently or 
within the previous 30 days. (self-reported) 
○ Positive pregnancy test, reported pregnancy or lactation (this criterion is 
due to oral tissue changes related to pregnancy and nursing which can 
affect interpretation of study results. Additionally, women are advised to 
check with their physician before using Povidone-iodine during pregnancy 
and lactation, which cannot occur in a blinded, randomized trial.) 
○ Other severe acute or chronic medical or psychiatric condition or 
laboratory abnormality that may increase the risk associated with trial 
participation or investigational product administration or may interfere 
with the interpretation of trial results and, in the judgment of the 
Investigator, would make the subject inappropriate for entry into this trial.  
○ Patient with developmental/cognitive disability that cannot self-consent, 
comprehend and follow the requirements of the study based on research 
site personnel’s assessment.  
○ Patient who has or ever had a thyroid problem, including swelling 
(nodular colloid goitre, endemic goitre or Hashimoto’s thyroiditis) 
○ Patients currently having lithium therapy for depression 
○ Patients with sizable mucosal tears, abrasions, growths or burns in the 
mouth  
○ Patients with kidney dysfunction 
● The following table lists exclusion criteria specific to each mouth rinse proposed 
in the study: 
mouthrinse* 
Exclusions 
26.9% ethanol plus 
essential oils 
● 
Do not use in children under 12 

1% w/v povidone-
iodide (PVI)  
● 
Hypersensitivity to iodine, polyvinyl pyrrolidine or to any excipient.  
● 
History of abnormal thyroid function or goitre (in particular nodular 
colloid goitre, endemic goitre and Hashimoto’s thyroiditis).  
○ 
Use of this preparation may interfere with tests of thyroid 
function. Iodine is absorbed through burns and broken skin 
and to a lesser extent through intact skin and may lead to toxic 
levels of iodine in the blood, particularly in patients with renal 
insufficiency. If symptoms occur suggesting changes in 
thyroid function, these should be investigated. In patients with 
impaired renal function, blood levels of iodine should be 
monitored 
● 
Regular use should be avoided in patients on concurrent lithium 
therapy.  
○ 
Use with concurrent lithium therapy has been shown to exhibit 
additive hypothyroidic effects. Absorption of iodine from 
povidone iodine through either intact skin or broken skin may 
interfere with thyroid function tests. Contamination with 
povidone iodine of several types of tests for the detection of 
occult blood in faeces or blood in urine may produce false-
positive results. 
● 
Do not use in children of 6 years and under. 
● 
Pregnant or nursing mothers 
○ 
Iodine freely crosses the placenta and is secreted in breast 
milk. Thyroid function disorders have been reported in the 
offspring of mothers exposed to pharmacological doses of 
iodine. Povidone iodine should not be used regularly during 
pregnancy unless there is no alternative treatment available. 
● 
Do not use Betadine Gargle and Mouthwash 10mg/ml Oral Solution: - 
if you are allergic (hypersensitive) to povidone iodine or any of the 
other ingredients listed - if you currently have or have ever had a 
thyroid problem, including swelling (nodular colloid goitre, endemic 
goitre or Hashimoto’s thyroiditis), as using Betadine Gargle and 
Mouthwash in these circumstances can further affect the function of 
your thyroid - if you are currently having lithium therapy for 
depression as this type of medicine can interact with Betadine Gargle 
and Mouthwash to affect the function of your thyroid - on children of 6 
years and under. Talk to your doctor or pharmacist before using 
Betadine Gargle and Mouthwash 10mg/ml Oral Solution - if your skin 
is broken (for example, due to a burn) as using Betadine Gargle and 
Mouthwash in these circumstances may cause toxic levels of iodine to 
be absorbed into your blood - if you currently have or have ever had a 
kidney problem as using Betadine Gargle and Mouthwash in these 
circumstances may cause toxic levels of iodine to be absorbed into your 
blood - when you know you are due to have a thyroid, faeces (stool) or 
urine test as Betadine Gargle and Mouthwash may affect the results. 

1.5% w/v  
hydrogen peroxide 
rinse 
● 
Pregnancy and lactation 
○ 
Although there is not sufficient specific clinical data on the 
use of 1.5 
○ 
% w/v  hydrogen peroxide rinse in this patient group, self-
administration without medical advice is not recommended. 
● 
Do not use in children under 12 
0.12% 
Chlorhexidine 
Gluconate 
● 
Carcinogenesis, Mutagenesis, Impairment of Fertility 
○ 
In a drinking water study in rats, carcinogenic effects were not 
observed at doses up to 38 mg/kg/day. Mutagenic effects were 
not observed in two mammalian in vivo mutagensis studies 
with chlorhexidine gluconate. The highest doses of 
chlorhexidine used in a mouse dominant-lethal assay and a 
hamster cytogenetics test were 1000 mg/kg/day and 250 
mg/kg/day, respectively. No evidence of impaired fertility was 
observed in rats at doses up to 100 mg/kg/day. 
● 
Pregnancy: Teratogenic Effects 
○ 
Reproduction studies have been performed in rats and rabbits 
at chlorhexidine gluconate doses up to 300 mg/kg/day and 40 
mg/kg/day, respectively, and have not revealed evidence of 
harm to the fetus. However, adequate and well-controlled 
studies in pregnant women have not been done. Because 
animal reproduction studies are not always predictive of 
human response, this drug should be used during pregnancy 
only if clearly needed. 
● 
Nursing Mothers 
○ 
It is not known whether this drug is excreted in human milk. 
Because many drugs are excreted in human milk, caution 
should be exercised when 0.12% Chlorhexidine Gluconate is 
administered to a nursing woman. In parturition and lactation 
studies with rats, no evidence of impaired parturition or of 
toxic effects to suckling pups was observed when 
chlorhexidine gluconate was administered to dams at doses up 
to 100 mg/kg/day. 
● 
Pediatric Use 
○ 
Clinical effectiveness and safety of 0.12% Chlorhexidine 
Gluconate have not been established in children under the age 
of 18. 

0.1% 
Cetylpyridinium 
Chloride 
● 
Do not use in children under 6 
0.9% NaCl   
 No contraindications 
*All mouthrinses are commercially available in the USA, routinely used in clinical settings, and will be used in an on-
label manner. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Summary of Inclusion and Exclusion Criteria for Mouthrinse RCT 
Inclusion Criteria 
Exclusion Criteria 
Individuals (all sex, all gender) at least 18 years of age and at 
most 65 years of age in good oral health and with stable 
physical health, decided at the discretion of the study 
coordinator.  
Known allergies or significant adverse reactions following the 
use of oral hygiene products (toothpastes, mouthrinses), 
commercial cosmetics, iodine, and any ingredient in the 
mouthrinses (list will be provided).  
Diagnosed COVID+ status (within the past 48 hours) who 
became symptomatic in the prior 10 days, or if not 
symptomatic, was likely infected/exposed within the prior 10 
days. 
Established 
history 
of 
Xerostomia 
(drug-induced 
or  
autoimmune dry mouth), renal disease, thyroid problems 
(including 
swelling, 
Hoshimoto’s 
thyroiditis, 
Goiter), 
Hepatitis C Virus (HCV), severe periodontal disease with 
bleeding gums, significant oral abrasions/ulcers or growths, 
alcohol abuse, recreational drug abuse, and/or current 
treatment with lithium. Other severe acute or chronic medical 
or psychiatric condition or laboratory abnormality that may 
increase the risk associated with trial participation or 
investigational product administration or may interfere with 
the interpretation of trial results and, in the judgment of the 
Investigator, would make the subject inappropriate for entry 
into this trial.  
Evidence of a personally signed and dated informed consent 
document indicating the subject (or legally acceptable 
representative) has been informed of all pertinent aspects of the 
trial and all of their questions have been answered. 
Drinking or eating within an hour of the study visit.  
Consuming alcohol within 12 hours of the study visit.  
Rinsing with a commercial mouthrinse within 24 hours of the 
study visit.  
Able to comprehend and follow the requirements of the study 
(including availability on scheduled visit dates) based on 
research site personnel’s assessment.  
Developmental/cognitive disability such that patient cannot 
self-consent, comprehend and follow the requirements of the 
study based on research site personnel’s assessment.  
Females of childbearing potential will have a negative urine 
pregnancy test (on site) or be physically incapable of pregnancy 
(implants or injections, Intrauterine device, Bilateral tubal 
ligation, 
Hysterectomy, 
Ovariectomy, 
Women 
post-
menopausal)  
Positive pregnancy test, reported pregnancy or lactation. 

  
A history of recent (within the last 30 days) or current recent 
oral herpes flare up, candida (thrush) infection, apthous ulcer 
flare up, current/active severe periodontal disease with 
bleeding gums, or other recent oral viral infection or flare up 
within the past 30 days. 
  
4.3   
Study Enrollment Procedures 
Outpatient subjects will be recruited from patients seen in the UNC Respiratory Distress 
Clinic (RDC) and other UNC testing facilities who have tested positive for COVID+ and 
consented to be contacted for participation in COVID-related research studies. The subjects are 
being approached by phone or email have signed a prior facility consent at the Respiratory 
Distress Clinic confirming their willingness to share their name and contact information to be 
contacted for study participation in COVID related research. We will not receive information on 
subjects that decline this internal consent.  Subjects will be contacted for recruitment by phone 
and (if unreachable by phone) by secure email by study personnel; patients will have the study 
rationale and risks explained and will be provided time to ask questions and consider 
participation. If patients are interested in participating, a single appointment will be scheduled 
for them at the Adams School of Dentistry Go Health Clinical research core. Consent 
documentation will be signed in person and in private in the Go Health Clinical Research Core.  
For Spanish speaking patients and/or parents, Spanish forms will be provided and 
communication will occur through a Spanish translator. All patients are adults and over normal 
cognitive capacity, and therefore will be able to consent for themselves. A list of COVID+ 
subjects that have consented to be contacted for research, will be provided by the RDC clinic, 
sent via secure UNC servers or secure UNC email to our research team's clinical coordinator, and 
this list will include names and contact details (PHI). This list is updated daily and provided to 
approved research sites, including ours. 
At the visit, a consented patient will answer screening questions regarding inclusion and 
exclusion criteria. Answers will be entered into the CDART research database managed by 
UNC. Prior to finalizing these screening questions, women participants of childbearing potential 
will be asked to provide a urine sample in the restroom, to undergo a rapid pregnancy test by  
study  personnel. Any pregnant or lactating patients will be excluded.  
Our study statistician, Dr. Kevin Moss, will create a block randomization schedule and 
perform these computations. All patients will be consented and then fill out our screening 
questions for inclusion and exclusion to confirm eligibility prior to being assigned to the 
randomization schedule. 
 
 

 
5. STUDY INTERVENTIONS 
5.1   
Interventions, Administration, and Duration 
Patients who meet all inclusion criteria without any exclusions will be asked to give a 
baseline salivary sample of 5ml over 10 minutes. The patient will then be asked to rinse 
with an unlabeled/blinded antiseptic mouth rinse for 60 seconds. Once s/he expectorates 
the mouth rinse, a time 0, 2ml salivary sample will be collected over 5 minutes. Then the 
patient will have 10 minutes to recuperate, while answering survey questions about their 
demographics and regarding the taste, color, flavor and acceptability of using the mouth 
rinse as part of routine dental and medical visits. Patients will be asked to smell an 
ammonia ampule ('smelling salt') to evaluate sense of smell, as loss of olfaction is 
indicative of the acute infection phase. At 15 minutes post-rinse, the patient will be asked 
to provide an additional 2ml saliva samples over 5 minutes, followed by another 10 
minute break. This sequence repeats for the 30, 45 and 60 minute time points. At 60 
minutes, the patient will give their last 2ml salivary sample, and study participation will 
conclude.  Participants will be provided with a gift card for their participation.  
5.2   
Handling of Study Interventions 
Mouthrinses will be acquired from the following sources: 
mouthrinse* 
Source: Online pharmacies and reputable 
marketplaces with over the counter mouthrinses 
26.9% ethanol plus essential oils 
Listerine’s Original Mouthrinse- Amazon or CVS 
online store 
1% w/v Povidone-iodide (PVI)  
Betadine Mouthrinse- Amazon or CVS online 
store 
1.5% w/v  Hydrogen Peroxide rinse 
Oral B Mouth Sore Mouthrinse- Amazon or CVS 
online store 
0.12% Chlorhexidine Gluconate 
Peridex- CVS Pharmacy and Adams School of 
Dentistry Dispensary 
0.1% Cetylpyridinium Chloride 
Crest Pro-Health Mouthrinse- Amazon or CVS 
online store 
0.9% NaCl  
Amazon or CVS online store 

*All mouthrinses are commercially available in the USA, routinely used in clinical settings, and 
will be used in an on-label manner. 
Mouthrinses will be prepared by research study staff under sterile conditions in a 10mL sterile 
syringe.  The syringe will be unlabeled and opaque so both the participant and study personnel 
will be blinded to the color of the solution.   
Any adverse events or protocol deviations will be reported to the UNC IRB.  Weekly meetings of 
the internal quality control committee will allow us to closely monitor such events.  
5.3   
Concomitant Interventions 
There are no concomitant interventions other than the mouthrinse and a short survey.  
5.3.1 Allowed Interventions 
There are no additional interventions, other than mouth rinsing with one of the 6 
options previously described. If patients have an allergic reaction, EMS will be 
contacted for management, and therefore no rescue medications are indicated.  
5.3.2 Required Interventions 
The patient will rinse with a blinded mouthrinse for 60 seconds and then will spit it 
out, followed by donating saliva samples. 
5.3.3 Prohibited Interventions 
Patients who have drank or eaten within an hour of the study visit will be excluded. 
Patients who have used a mouthrinse within 48 hours of the study will be excluded.  
Patients who have participated in a study of other oral products (toothpaste, 
mouthrinses) within the prior 30 days will be excluded.  
Patients who are abusers of alcohol or recreational drugs, and have used such 
products within 48 hours will be excluded.  
5.4   
Adherence Assessment 
Adherence would be defined as rinsing with the mouthrinse for a full 60 seconds 
before expectorating, and then providing saliva samples at 15 minute time points.  
 
 
 
 

 
6. STUDY PROCEDURES 
6.1   
Schedule of Evaluations 
Assessment 
Screening: Visit-1 (Day-14 to Day -1) 
Baseline, Enrollment, Randomization, 
Intervention: Visit 1 (Day 0) 
Informed Consent 
Form  
X 
X  
Demographics 
X 
 X 
DXA 
X 
X  
Medical History 
X 
X  
General Physical 
Examination 
 
 
Current Medications 
X 
X 
Blood Chemistries 
 
 
Hematology 
 
 
Urine Analysis- 
pregnancy 
 
X 
Vital Signs 
 
X 
Inclusion/Exclusion 
Criteria 
X  
X 
Enrollment/Randomiz
ation 
 X 
X 

Treatment 
Administration Form 
  
X  
Concomitant 
Medications 
X  
X 
Adverse Events 
X  
X 
6.2   
Description of Evaluations 
Subjects are required to be onsite for a 70-90 minute, single visit appointment. Subjects 
will be asked to rinse with an unlabeled/blinded antiviral mouth rinse for 60 seconds and 
provide 5 mL of saliva prior to the rinse and 2 mL of saliva immediately post-rinse, 15 
minutes post-rinse, 30 minutes post-rinse, 45 minutes post-rinse, and 60 minutes post-
rinse. Subjects will also be asked to complete a short survey about the rinsing experience. 
6.2.1 Screening Evaluation 
Consenting Procedure 
Patients will be contacted first by phone and, if not available, then by email. The consent 
process will be conducted by Samantha Jhingree. The study's purpose and requirements 
will be explained and the patient will have the opportunity to ask questions and will have 
time to consider participation. Interested patients will make an appointment to visit the Go 
Health Clinical Research Unit at Adams School of Dentistry for this study.  Study 
participation includes only one visit. The consent forms will be reviewed and signed by 
interested patients in person in the Go Health Clinical Research Unit at Adams School of 
Dentistry prior to study participation. For Spanish speaking patients and/or parents, 
Spanish forms will be provided and communication will occur through a Spanish translator. 
All patients are adults and over normal cognitive capacity, and therefore will be able to 
consent for themselves. 
Screening 
● At the visit, a consented patient will answer screening questions regarding 
inclusion and exclusion criteria. Answers will be entered into the CDART 
research database managed by UNC.  
● Prior to finalizing these screening questions, women participants of childbearing 
potential will be asked to provide a urine sample in the restroom, to undergo a 
rapid pregnancy test by study personnel. Any pregnant or lactating patients will 
be excluded.  
 

6.2.2 Enrollment, Baseline, and/or Randomization 
Enrollment 
Outpatient subjects will be recruited from patients seen in the UNC Respiratory Distress 
Clinic (RDC) who have tested positive for COVID+ and consented to be contacted for 
participation in COVID-related research studies. The subjects being approached by phone 
or email have signed a prior facility consent at the Respiratory Distress Clinic confirming 
their willingness to share their name and contact information to be contacted for study 
participation in COVID related research. We will not receive information on subjects that 
decline this internal consent.  Subjects will be contacted for recruitment by phone and (if 
unreachable by phone) by secure email by study personnel; patients will have the study 
rationale and risks explained and will be provided time to ask questions and consider 
participation. If patients are interested in participating, a single appointment will be 
scheduled for them at the Adams School of Dentistry Go Health Clinical research core. 
Consent documentation will be signed in person and in private in the Go Health Clinical 
Research Core. A list of COVID+ subjects that have consented to be contacted for 
research, will be provided by the RDC clinic, sent via secure UNC servers or secure UNC 
email to our research team's clinical coordinator, and this list will include names and 
contact details (PHI). This list is updated daily and provided to approved research sites, 
including ours. 
Baseline Assessments 
There are no baseline assessments required as this study only requires a 70-90 minute 
single visit appointment. 
Randomization 
Our statistician Dr. Kevin Moss will create a block randomization schedule and perform 
these computations. All patients will be consented and then fill out our screening 
questions for inclusion and exclusion to confirm eligibility prior to being assigned to the 
randomization schedule. 
The researcher in charge of collecting the salivary samples will be blinded to the 
mouthwash solution, as they will be given a pre-aliquoted mouthrinse in an unlabeled 
storage tube to provide to the patient for the rinse.  
6.2.3 Blinding 
At their onsite visit, subjects will be provided with an unlabeled/blinded mouthrinse. The 
researcher in charge of collecting the salivary samples will be blinded to the mouthwash 
solution as well, as they will be given a pre-aliquoted mouthrinse in an unlabeled storage 
syringe to provide to the patient for the rinse. Our statistical consultant, Dr. Kevin Moss, 

will set up a block randomization schedule that the team will abide by for assignment of 
enrolled subjects.  
6.2.4 Follow-up Visits 
This study requires only one 70-90 minute, single visit appointment. No diagnostic tests 
will be run as part of this clinical trial. All participants will already have known COVID+ 
status, and therefore no follow-up reporting is needed.   
6.2.5 Completion/Final Evaluation 
This study requires only one 70-90 minute, single visit appointment. Subjects will be 
asked to rinse with an unlabeled/blinded antiviral mouth rinse for 60 seconds and provide 
5 mL of saliva prior to the rinse and 2 mL of saliva immediately post-rinse, 15 minutes 
post-rinse, 30 minutes post-rinse, 45 minutes post-rinse, and 60 minutes post-rinse. 
Subjects will also be asked to complete a short survey about the rinsing experience. 
Subjects may withdraw from the trial at any time at their own request, or they may be 
withdrawn at any time at the discretion of the Investigator or Sponsor for safety, 
behavioral or administrative reasons.  
The Investigator may discontinue a subject if, in the opinion of the Investigator, the 
subject is no longer a suitable candidate for the study. Possible reasons for the 
discontinuation of a subject are including but not limited to: adverse event, protocol 
deviation, missed appointment, subject no longer meets the eligibility criteria. 
Patients may be removed from the study due to an allergic reaction to a mouth rinse or 
due to failure or inability to comply with study procedures.  
If a patient suffers from xerostomia/dry mouth, such that s/he cannot produce the baseline 
salivary sample volume or the immediate, 15 minute and 30 minute post-rinse saliva 
volumes, s/he will be removed from the study with a parking voucher, but without a gift 
card and will be replaced. If a patient suffers from dry mouth, such that s/he cannot 
produce salivary sample volumes needed for later time points (45 minutes or 60 minutes 
post-rinse) but s/he produced sufficient baseline, immediate, 15 minute and 30 minute 
post-rinse samples, s/he will still be compensated with a gift card and parking vouchers, 
and will remain in the study. Missing or deficient salivary samples will be noted in 
CDART. 
 
 
 

 
7. SAFETY ASSESSMENTS 
The following table lists possible adverse effects associated with each mouthrinse proposed in 
this study: 
mouthrinse* 
Adverse effects 
26.9% ethanol plus 
essential oils 
● 
If more than used for rinsing is accidentally swallowed, get medical 
help or contact a Poison Control Center right away 
1% w/v povidone-
iodide (PVI)  
● 
Local irritation, skin burns and sensitivity reactions have been 
reported rarely. 
● 
Anaphylactic reactions, anaphylactoid reactions and anaphylactic 
shock have been reported uncommonly with products containing 
povidone-iodine or povidone. 
● 
Excess iodine can produce goitre and hypothyroidism or 
hyperthyroidism. Such effects have occasionally been seen with 
extensive or prolonged use of povidone iodine.  
● 
Other effects that have been reported are metabolic acidosis and 
acute renal failure. 
● 
Possible unwanted effects: Like all medicines, Betadine Gargle and 
Mouthwash can cause unwanted effects, although these are 
uncommon. Allergic-type reactions, including a rash or difficulty in 
breathing, have been reported uncommonly with products containing 
povidone iodine (the active ingredient in Betadine Gargle and 
Mouthwash). Although rare, Betadine Gargle and Mouthwash could 
also cause itching and burns on your skin. Applying very large 
amounts of medicines containing povidone iodine, or using such 
products over a long period of time, can occasionally cause thyroid 
disorders (symptoms of which include weight loss, increased appetite 
and sweating or lacking energy and weight gain). Other effects that 
have been reported are increased amounts of acid in your blood (the 
main symptoms are shortness of breath, confusion and lethargy) and 
kidney failure (symptoms include difficulty in urinating, thirst and a 
dry mouth and pain in one side of your back). Should you suffer 
from any of these unwanted effects, or if you notice any unwanted 
effects not listed in this leaflet, stop using Betadine Gargle and 
Mouthwash straight away and tell your doctor or pharmacist. 
1.5% w/v  hydrogen 
peroxide rinse 
● 
If irritation persists for 7 days, is severe, is due to orthodontic 
appliances and/or dentures, or swelling or fever develops, the 
patient's condition needs to be re-evaluated by a doctor or dentist. 
● 
Overdose can injure the gums and continued use of hydrogen 
peroxide may cause reversible hypertrophy of the papillae of the 

tongue known as ‘black hairy tongue’ therefore using this product at 
high doses or for long periods of time is not recommended. 
● 
Some cases of mucosal irritation and swelling of the oral tissues have 
been reported specially with high doses or in continued use. 
0.12% Chlorhexidine 
Gluconate 
● 
The most common side effects associated with chlorhexidine 
gluconate oral rinses are: (1) an increase in staining of teeth and 
other oral surfaces, (2) an increase in calculus formation, and (3) an 
alteration in taste perception; see WARNINGS and 
PRECAUTIONS. Oral irritation and local allergy-type symptoms 
have been spontaneously reported as side effects associated with use 
of chlorhexidine gluconate rinse. The following oral mucosal side 
effects were reported during placebo-controlled adult clinical trials: 
aphthous ulcer, grossly obvious gingivitis, trauma, ulceration, 
erythema, desquamation, coated tongue, keratinization, geographic 
tongue, mucocele, and short frenum. Each occurred at a frequency of 
less than 1.0%. Among postmarketing reports, the most frequently 
reported oral mucosal symptoms associated with chlorhexidine 
gluconate oral rinse are stomatitis, gingivitis, glossitis, ulcer, dry 
mouth, hypesthesia, glossal edema, and paresthesia. Minor irritation 
and superficial desquamation of the oral mucosa have been noted in 
patients using chlorhexidine gluconate oral rinses. There have been 
cases of parotid gland swelling and inflammation of the salivary 
glands (sialadentitis) reported in patients using chlorhexidine 
gluconate oral rinse. 
 
0.1% 
Cetylpyridinium 
Chloride 
● 
If more than used for rinsing is accidentally swallowed, get medical 
help or contact a Poison Control Center right away 
0.9% NaCl  
No contraindications 
 
*All mouthrinses are commercially available in the USA, routinely used in clinical settings, and 
will be used in an on-label manner. 
The use of on-label, widely used mouthrinses (like Listerine) and collection of saliva 
carry minimal risk for participants, apart from the very rare, undiagnosed allergy, and 
the unlikely event that the sample is swallowed. If the case of allergy, UNC Hospital 
emergency services will be contacted. In the case of swallowing, a Poison Control 
Center will be contacted.  
 
 

7.1   
Specification of Safety Parameters 
Mouthrinse and saliva collection carries low risk and minimal to no safety concerns 
to the participants. Results of analysis from known COVID+ patients will not require 
any medical follow-up or safety concerns. As a result, the investigator will monitor 
subject data, without a safety monitoring board. The investigator and team will meet 
weekly to review safety concerns, unanticipated problems or adverse events.  
7.2   
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters 
The investigators and study coordinators will meet weekly to review unanticipated 
problems with recruitment or adverse events. There is only one site for this study. The 
use of on-label, widely used mouthrinses (like Listerine) and collection of saliva carry 
minimal risk for participants, apart from the very rare, undiagnosed allergy, which 
would be managed through proper referral to the neighboring UNC hospital. 
7.3   
Adverse Events and Serious Adverse Events 
An adverse event (AE) is generally defined as any unfavorable and unintended 
diagnosis, symptom, sign (including an abnormal laboratory finding), syndrome or 
disease which either occurs during the study, having been absent at baseline, or if 
present at baseline, appears to worsen. Adverse events are to be recorded regardless 
of their relationship to the study intervention.  
A serious adverse event (SAE) is generally defined as any untoward medical 
occurrence that results in death, is life threatening, requires inpatient hospitalization 
or prolongation of existing hospitalization, results in persistent or significant 
disability/incapacity, or is a congenital anomaly. 
The use of on-label, widely used mouthrinses (like Listerine) and collection of saliva 
carry minimal risk for participants, apart from the very rare, undiagnosed allergy, or 
the unlikely event that a participant slips or falls during the study, which would be 
managed through proper referral to the neighboring UNC hospital. 
The investigator and team will meet weekly to review safety concerns, unanticipated 
problems or adverse events. All adverse events will be reported to the IRB and the 
committee overseeing the study within 30 days.  
7.4   
Reporting Procedures 
The PI will be responsible to monitor the overall study, including both research data 
and clinical procedures. The PI will be responsible for reporting all adverse events to 
the necessary parties. The investigator and team will meet weekly to review safety 
concerns, unanticipated problems or adverse events. All adverse events will be 
reported to the IRB and the committee overseeing the study within 30 days.  
 

7.5   
Follow-up for Adverse Events 
In the case of an adverse event, participants will be referred to the neighboring UNC 
hospital. All follow-up visits and procedures regarding adverse events will be handled 
through the UNC Hospital system. A representative from our team will call the 
participant one week after the adverse event occurs to check-in. 
7.6   
Safety Monitoring 
The investigator will monitor subject data, without a safety monitoring board. The 
investigator and team will meet weekly to review safety concerns, unanticipated 
problems or adverse events. 
8. INTERVENTION DISCONTINUATION 
Subjects may withdraw from the trial at any time at their own request, or they may be 
withdrawn at any time at the discretion of the Investigator or Sponsor for safety, 
behavioral or administrative reasons.  
The Investigator may discontinue a subject if, in the opinion of the Investigator, the 
subject is no longer a suitable candidate for the study. Possible reasons for the 
discontinuation of a subject are including but not limited to: adverse event, protocol 
deviation, missed appointment, subject no longer meets the eligibility criteria. 
Patients may be removed from the study due to an allergic reaction to a mouth rinse or 
due to failure or inability to comply with study procedures.  
If a patient suffers from xerostomia/dry mouth, such that s/he cannot produce the baseline 
salivary sample volume or the immediate, 15 minute and 30 minute post-rinse saliva 
volumes, s/he will be removed from the study with a parking voucher, but without a gift 
card and will be replaced. If a patient suffers from dry mouth, such that s/he cannot 
produce salivary sample volumes needed for later time points (45 minutes or 60 minutes 
post-rinse) but s/he produced sufficient baseline, immediate, 15 minute and 30 minute 
post-rinse samples, s/he will still be compensated with a gift card and parking vouchers, 
and will remain in the study. Missing or deficient salivary samples will be noted in 
CDART. 
 
 
 
 

 
9. STATISTICAL CONSIDERATIONS  
9.1   
General Design Issues 
A double-blinded, randomized controlled trial was chosen as the study design. It allows us to 
collect human based data on the effects of commercially available mouthrinses on salivary 
infectivity and viral load. A crossover study was not pursued because the repeated use of different 
mouthrinses is likely to confound the results.  A 1 hour time-course was chosen to correspond with 
the average length of most dental procedures.  Fifteen minute intervals were chosen to evaluate 
viral kinetics over time, and to see when infectivity rebounds post- mouthrinse.  
Statistical Hypothesis 
The central hypotheses of this study are that a 60 second antiseptic mouth rinse will effectively 
reduce detectable viral RNA and protein antigen levels of SARS-CoV-2 for a minimum of 30 
minutes duration.  
The tertiary hypotheses are that a 60 second antiseptic mouth rinse will effectively reduce viral 
infectivity (>50%) of SARS-CoV-2 for a minimum of 30 minutes duration, and that mouth rinses 
will be well tolerated in a clinical care setting by a majority of patients and providers.  
Sample Size Considerations 
Based on similar viral infectivity studies evaluating antiviral mouth rinse efficacy, we were able 
to run power calculations to estimate effect size and determine sample size as a function of length 
of mouthrinse time course. The sample size is justified by a paired t-test on outcomes between 
time points. With three simultaneous comparisons (T1-T0, T2-T1, and T2-T0 = 30 minute time 
course), the sample size n=6 can detect the effect size in Cohen’s d =0.7, which is between medium 
and large, over 80% power and 0.016 type-I error rate by Bonferroni correction.  With six 
simultaneous comparisons (T1-T0, T2-T1, T2-T0, T3-T0, T3-T2, T3-T1) = 45 minute time course, 
the sample size n=53 can detect the effect size in Cohen’s d =0.7, which is between medium and 
large, over 80% power and 0.016 type-I error rate by Bonferroni correction. With ten simultaneous 
comparisons (T1-T0, T2-T1, T2-T0, T3-T0, T3-T2, T3-T1, T4-T3, T4-T2, T4-T1, T4-T0) = 60 
minute time course, the sample size n=78 can detect the effect size in Cohen’s d =0.7, which is 
between medium and large, over 80% power and 0.016 type-I error rate by Bonferroni correction. 
Statistical analysis of our data will be performed by Dr. Kevin Moss, the statistician on this project. 
Primary Outcomes 
The analyses will compare viral RNA level (qRT-PCR) and protein antigen level (sandwich 
ELISA or rapid antigen) of SARS-CoV-2 between mouth rinse types and across the time course. 
Data generated from the qRT-PCR and sandwich ELISA or rapid antigen assays will be presented 
as mean standard deviation for normally distributed data and as median [Interquartile range] for 

non-normally distributed data. P values will be reported as two tailed. Between group comparisons 
will be performed using a Student’s t test or Mann-Whitney U test. Normality will be assessed 
using the Kolmorgorov-Smirnov test, and logistic regression will be performed with robust 
standard errors.  
 Tertiary Outcomes 
The viral infectivity analysis will compare infectivity levels between mouth rinse types and across 
the time course for calculation of the reduction factor (RF). The RF will be calculated as the 
difference in the quotient of the infection titre before (‘control titration’) and after the mouthrinse  
(‘remaining virus’). Therefore, the log10 titre and its (double) standard deviation (SD) were 
calculated as well as the variance of the RF. 
Survey- Statistical analysis will be conducted by Odum Institute’s statistical consultant, Chris 
Wiesen, PhD. We will use descriptive statistics, frequency counts and means to evaluate feasibility 
and acceptability of using point-of-care mouthrinses in clinical workflow. 
 
9.2   
Sample Size and Randomization 
Based on similar viral infectivity studies evaluating antiviral mouth rinse efficacy, we were able 
to run power calculations to estimate effect size and determine sample size as a function of 
length of mouthrinse time course. The sample size is justified by a paired t-test on outcomes 
between time points. With three simultaneous comparisons (T1-T0, T2-T1, and T2-T0 = 30 
minute time course), the sample size n=6 can detect the effect size in Cohen’s d =0.7, which is 
between medium and large, under 80% power and 0.016 type-I error rate by Bonferroni 
correction.  With six simultaneous comparisons (T1-T0, T2-T1, T2-T0, T3-T0, T3-T2, T3-T1) = 
45 minute time course, the sample size n=53 can detect the effect size in Cohen’s d =0.7, which 
is between medium and large, under 80% power and 0.016 type-I error rate by Bonferroni 
correction. With ten simultaneous comparisons (T1-T0, T2-T1, T2-T0, T3-T0, T3-T2, T3-T1, 
T4-T3, T4-T2, T4-T1, T4-T0) = 60 minute time course, the sample size n=78 can detect the 
effect size in Cohen’s d =0.7, which is between medium and large, under 80% power and 0.016 
type-I error rate by Bonferroni correction. 
Dr. Kevin Moss, who is the statistical consultant on this project, will create a block randomization 
schedule and perform these computations. All patients will be consented and then fill out our 
screening questions for inclusion and exclusion to confirm eligibility prior to being assigned to the 
randomization schedule. 
The researcher responsible for collecting the salivary samples will be blinded to the mouthwash 
solution, as they will be given a pre-aliquoted mouthrinse in an unlabeled storage syringe to 
provide to the patient for the rinse. The syringe will be opaque to mask any coloring of the solution.  

The PI, Co-I, study coordinators and study personnel who work for GoHealth are authorized to 
break the blinding if a patient suffers an allergic or adverse reaction to one of the mouthrinses.  
Though highly unlikely as all mouthrinses are sold commercially at Walgreens and CVS, adverse 
or allergic reactions are possible. Using the numerical code associated with the participant and 
mouthrinse syringe, the study personnel can reference the block randomization schedule to 
determine the mouthrinse type, to inform the emergency medical workers, poison control or the 
emergency room what type of mouthrinse product led to the reaction.  
9.3   
Definition of Populations 
Our population includes patients 18-65 years old who have tested positive with 
COVID-19 and are either asymptomatic but exposed in the prior 10 days, or are 
symptomatic with symptom development in the prior 10 days. Inclusion and 
exclusion criteria (described elsewhere) will be met.  We are not treating patients for 
their COVID-19 infection. 
9.4   
Interim Analyses and Stopping Rules 
An interim analysis will be conducted after 120 patients are enrolled and samples 
collected; block randomization will be planned to randomize in batches of 120 
participants, with 20 randomized to each mouthrinse. Therefore, we can do an interim 
analysis with an n=120.  This sample size is sufficient to determine antiviral efficacy at 
30 minutes post-mouthrinse.  If mouthrinses are ineffective at reducing viral infectivity at 
timepoints 15 and 30 minutes, then that mouth rinse arm will be discontinued for further 
enrollment and will be removed from the block randomization calendar, with initiation of 
a subsequent trial with fewer arms. This will allow us to enroll sufficient numbers of 
patients to statistically evaluate longer time point efficacy for the mouthrinses with 
antiviral efficacy (the longer time points require larger samples sizes for sufficient 
statistical power). 
 
If no mouthrinse has antiviral effects at this interim analysis point, the study will be 
stopped due to lack of efficacy. Because all oral rinse products are commercially 
available, low risk products that have previously undergone extensive safety testing and 
approval procedures, we do not anticipate a situation where safety concerns to 
participants necessitates halting the study. Adverse events will be reported and reviewed 
by our internal quality control committee.  Medical problems that occur in our facility 
will be reported as needed to the IRB board and emergency medical personnel will be 
contacted to care for the patient and potentially bring them to the adjacent UNC medical 
hospital. If a significant number of adverse events or reactions are witnessed (defined as 
more than 1 per every 20 patients), a safety review will be triggered and study personnel 
will look at the records in an unblinded fashion to see if one mouthrinse is causing 
reactions. If that is the case, the mouthrinse study arm associated with adverse events will 

be discontinued, but the mouthrinse arms associated with minimal adverse events will 
continue.   
9.5   
Outcomes 
All oral rinse products are commercially available, low risk products. Therefore, 
adverse outcomes are anticipated to be extremely rare, and will be reviewed weekly 
by the study team/committee.  Any adverse event will be documented in our CDART 
database system. 
 The qRT-PCR assay for our primary outcome will be documented in an excel 
spreadsheet generated by the qRT-PCR assay quantification device, available weeks 
to months after sample collection when the experiments are conducted. If qRT-PCR 
proves unsuccessful, antigen-based assays will be used instead to quantify viral 
protein antigen level (Spike or N protein) using a sandwich ELISA or rapid antigen 
assays. These outcomes will be documented in excel spreadsheets generated by study 
personnel, available weeks to months after sample collection when the experiments 
are conducted. Samples will be run in large batches to limit batch effects. 
The viral infectivity assay for our tertiary outcome will be documented in an excel 
spreadsheet generated by the viral infectivity assay quantification device, available 
weeks to months after sample collection when the experiments are conducted. Survey 
results will also yield excel spreadsheets of data, available shortly after data 
collection completion.  
The outcomes do not include patient treatment response, and therefore the outcomes 
will not be reviewed and adjudicated by an outside committee.  The study personnel 
will constitute their own committee that meets weekly, and they will review and 
evaluate the data once these experiments are conducted. The committee will not be 
masked to the participant’s group assignment; the statistician will conduct analyses 
and then the committee will evaluate the data to reach scientific conclusions on which 
mouthrinse is more effective. 
9.5.1 Primary Outcomes  
The primary objectives of this study are to determine the efficacy of antiseptic 
mouthwashes on reducing SARS-CoV-2 viral RNA and protein antigen levels in 
COVID+ patient saliva. We hypothesize that COVID-19+ patients will have a >95% 
reduction in SARS-CoV-2 viral RNA and protein antigen levels following a 60 second 
oral rinse with an antiseptic mouthrinse and that this reduction will persist for at least 
30 minutes. These primary objectives will assess SARS-CoV-2 viral load based on viral 
RNA measured by quantitative real time PCR and viral protein antigen levels measured 
by rapid antigen or sandwich ELISA assays. These objectives provides urgently needed 
data to guide healthcare workers on the proper use of specific commercially available 

mouthwashes capable of reducing SARS-CoV-2 in saliva and potentially limiting 
COVID-19+ transmission.  
Outcome assessments will be conducted weeks to months after the study visit, when 
 
qRT-PCR and sandwich ELISA or rapid antigen assays are conducted in the COVID core 
 
and the Baric lab.   
9.5.2  Tertiary Outcomes 
The first tertiary objective will focus on quantifying SARS-CoV-2 viral infectivity 
using an in vitro infectivity assay. We hypothesize that COVID-19+ patients will have 
a >95% reduction in SARS-CoV-2 viral infectivity following a 60 second oral rinse 
with an antiseptic mouthrinse and that this reduction will persist for at least 30 minutes. 
This objective provides urgently needed data to guide healthcare workers on the proper 
use of specific commercially available mouthwashes capable of reducing SARS-CoV-
2 viral infectivity and potentially limiting COVID-19+ transmission. Our second 
tertiary objective is to determine the feasibility and acceptability of using antiseptic 
mouthwashes by patients in healthcare settings. We hypothesize that patients will 
welcome the use of commercially available antiseptic mouthrinses into clinical 
practice.  Patients enrolled in the randomized mouthrinse study will be surveyed on the 
taste, appearance, sensation, and acceptability of using the mouthrinse in healthcare 
settings. Understanding acceptability of a mouthrinse protocol is critical to 
implementation and therefore the ability to reduce COVID-19 transmission risk in 
clinical settings. 
Outcome assessments will be conducted weeks to months after the study visit, when 
viral infectivity assays are conducted in the COVID core and the Baric lab.   
Survey results will be evaluated using bivariate, descriptive statistics and non-
parametric methods as appropriate for the question type. Outcome assessments will be 
conducted weeks to months after the study visit, when statistical analyses are 
conducted. 
9.6   
Data Analyses 
qRT-PCR and sandwich ELISA or rapid antigen: Data will be presented as mean standard 
deviation for normally distributed data and as median [Interquartile range] for non-normally 
distributed data. P values will be reported as two tailed. Between group comparisons will be 
performed using a Students t test or Mann-Whitney test. Normality will be assessed using the 
Kolmorgorov-Smirnov test, and logistic regression will be performed with robust standard errors. 
Methods adapted from Bullard et al 2020. 
Viral infectivity: The viral infectivity analysis will compare infectivity levels between mouth rinse 
types and across the time course for calculation of the reduction factor (RF). The RF will be 
calculated as the difference in the quotient of the infection titre before (‘control titration’) and after 

the mouthrinse  (‘remaining virus’). Therefore, the log10 titre and its (double) standard deviation 
(SD) were calculated as well as the variance of the RF. 
Survey results will be evaluated using bivariate, descriptive statistics and non-parametric methods 
as appropriate for the question type.  
Power Calculation: Based on similar viral infectivity studies evaluating mouth rinse efficacy 
against other enveloped viruses, we used power calculations to estimate effect size and determine 
sample size as a function of length of mouthrinse time course.12 The sample size is justified by a 
paired t-test on outcomes between time points.  With ten simultaneous comparisons (T1-T0, T2-
T1, T2-T0, T3-T0, T3-T2, T3-T1, T4-T3, T4-T2, T4-T1, T4-T0) = 60 minute time course, the 
sample size n=78 can detect the effect size in Cohen’s d =0.7, which is between medium and large, 
under 80% power and 0.016 type-I error rate by Bonferroni correction. Analyses will compare 
viral RNA level (qRT-PCR), protein level (sandwich ELISA and rapid antigen) or viral infectivity 
levels between mouth rinse types and across the time course. ANCOVA models will be utilized to 
look at changes in viral load and infectivity logarithmically before and after treatment.  
 
 
 
 
 
 
 
 
 
 
 
 
 

 
10. DATA COLLECTION AND QUALITY ASSURANCE  
10.1 
Data Collection Forms 
Consented patients' data (including screening questions, demographics, and the survey on 
mouth rinse acceptability) will be collected using the CDART dental toolkit program, 
which stores data on a secure UNC maintained server and meets federal guidelines for 
clinical study data acquisition. The CDART electronic forms and database will serve as 
our CRFs and source data. IRB approved study personnel will ask participants the 
questions and will enter responses into this encrypted, password protected database. Data 
will be collected in this program using participant's study ID and no other personal health 
identifiers (PHI). A separate linkage file, securely stored on UNC servers, will be utilized 
to associate the random alphanumeric study ID with patient identifier, so samples and 
data will not be associated with patient identifiers. Data will only be transmitted among 
the research team using the school of dentistry secure research servers. Demographic data 
will be transmitted among the research team through access to the secure CDART 
research database system, stored on secure UNC servers, which study personnel will 
access only through encrypted computers via secure user accounts. 
 
10.2 
Data Management 
An Excel key will be created with study ID (random alphanumeric code) to identify 
patients, saved in a secure folder on a UNC SOD server. This linkage file will be stored 
separately from data and destroyed at the close of the study. All data collected from 
participants and results from sample analysis will be coded with unique numerical 
identifiers and stored in the UNC CDART clinical research database and on a secure 
UNC-CH server. Only trained, IRB-approved study personnel will enter and access data 
in the secure CDART research database. 
Consented patients' data (including screening questions, demographics, and the survey on 
mouth rinse acceptability) will be collected using the CDART dental toolkit program, 
which stores data on a secure UNC maintained server and meets federal guidelines for 
clinical study data acquisition. Only HIPAA and human subjects trained, IRB-approved 
study personnel will collect, enter and manage patient data; study coordinators will serve 
in this capacity.  
Data will be collected in the CDART research database using participant's study ID and 
no other PHI identifiers, on approved, carefully designed digital forms within CDART. 
CDART forms will include questions with drop downs, radio button multiple choice and 
text field entry, depending on the question type. There will be separate digital forms for 
inclusion/exclusion screening questions (before sample collection begins), demographic 

questions, and questions regarding mouthrinse experience (taste, color, sensation)  and 
willingness to use a mouthrinse in various clinical settings (e.g. dentist or doctor 
appointments). A separate linkage file, securely stored on UNC servers, will be utilized to 
associate the random alphanumeric study ID with patient identifier, so samples and 
CDART data will not be associated with patient identifiers.  
Data will only be transmitted among the research team using the SOD secure research 
server. Demographic data will be transmitted among the research team through access to 
the secure CDART research database system, stored on secure UNC servers, which study 
personnel will access only through encrypted computers via secure user account. 
Linkage files containing identifiers will be deleted and permanently removed from all 
servers and study computers at the conclusion of the study with acceptance of associated 
manuscripts. Saliva specimens will be labeled with a study ID (random alphanumeric 
code) that is not identifiable without the linkage file, and therefore will not be identifiable 
after linkage file destruction.  
The quality control committee (described below) will be in charge of monitoring these 
forms and the CDART database.  
10.3 
Quality Assurance 
10.3.1  Training 
All study personnel have been trained in health privacy (HIPAA) and human subjects 
research through the CITI program and are certified to work with patient data on clinical 
trials. Study coordinators and healthcare providers are trained in and approved for the use 
of the CDART electronic research database and EPIC medical record system. Technical 
research personnel performing viral PCR and infectivity assays are approved to operate in 
their BL3 laboratory facilities and have specialized, lab-based training in these molecular 
techniques. They have also undergone training in the use of universal precautions and 
management of  human biological samples along with bloodborne pathogen training. 
FInally, the GoHealth Clinical Research core has highly experienced research 
coordinators who will monitor all study staff to ensure protocol compliance and quality 
assurance.    
10.3.2  Quality Control Committee 
The investigators and study coordinators will meet weekly as an internal quality control 
committee to review consent documents, CDART data forms, and unanticipated 
problems with recruitment or adverse events. There is only one site for this study. The 
use of on-label, widely used mouthrinses (like Listerine or Crest) and collection of saliva 
carry minimal risk for participants, apart from the very rare, undiagnosed allergy, which 

would be managed through proper referral to the neighboring UNC hospital. Therefore, 
there is no study quality control committee.  
10.3.3  Metrics 
Every salivary sample will be collected in a conical tube with millimeter markings to 
monitor salivary volume. If insufficient saliva is collected, the study personnel collecting 
the samples from participants will note insufficient volume in the CDART database for 
each applicable time point and will indicate in CDART if the subject must be withdrawn 
and replaced. If a patient is unable to generate sufficient saliva for the 0 min, 15 minutes 
or 30 minutes post-rinse time points, the subject will be removed from the study and this 
will be noted in CDART along with their insufficient samples. If a subject is unable to 
generate sufficient saliva for time points 45 or 60 minutes post-rinse, the patient will 
remain in the study but their insufficient samples will be noted in CDART.   
For qRT-PCR, viral infectivity, and sandwich ELISA or rapid antigen assays, all salivary 
samples will be run in triplicate with standard experimental controls (negative and 
positive) and protocol quality checks performed by research technical personnel in the 
COVID core, Baric, and Wallet laboratories.  
10.3.4  Protocol Deviations 
Protocol deviations will be recorded within the source documentation for each participant 
within the CDART study database by study personnel during the study visits.  The 
investigators and study coordinators will meet weekly to review unanticipated problems, 
adverse events and protocol deviations recorded in the CDART study database. All such 
events will be appropriately reported to the IRB board of UNC.  
10.3.5  Monitoring 
The quality control committee will meet weekly to review adverse events, protocol 
deviations, data quality in CDART, and collection of consent forms. The GoHealth 
Clinical Research core has highly experienced research coordinators who will monitor all 
study staff to ensure protocol compliance. A representative from the GoHealth core will  
participate in the weekly quality control committee meetings to review CDART records 
and consent forms. 
 
 
 

 
11. PARTICIPANT RIGHTS AND CONFIDENTIALITY 
11.1 
Institutional Review Board (IRB) Review 
This protocol and the informed consent document and any subsequent modifications 
will be reviewed and approved by the IRB or ethics committee responsible for 
oversight of the study. Consent forms are separate from this protocol document and 
were submitted as part of the IRB application. 
11.2 
Informed Consent Forms 
Outpatients, who have tested positive for COVID-19 and consented to be contacted for 
study enrollment in the UNC Respiratory Distress Clinic (RDC), will be contacted to 
participate in this study by research personnel. The subjects being approached by phone 
or email have signed a prior facility consent at the Respiratory Distress Clinic confirming 
their willingness to share their name and contact information to be contacted for study 
participation in COVID related research. We will not receive information on subjects that 
decline this internal consent.  Patients will be contacted first by phone and, if not 
available, then by email. The study's purpose and requirements will be explained and the 
patient will have the opportunity to ask questions and will have time to consider 
participation. Interested patients will make an appointment to visit the Go Health Clinical 
Research Unit at Adams School of Dentistry for this study.  Study participation includes 
only one visit. The consent forms will be reviewed and signed by interested patients in 
person in the Go Health Clinical Research Unit at Adams School of Dentistry.  
A signed consent form will be obtained from each participant. For participants who 
cannot consent for themselves, such as those with a legal guardian (e.g., person with 
power of attorney), this individual must sign the consent form, though minors and 
patients who are intellectually delayed and unable to consent for themselves will be 
excluded. The consent form will describe the purpose of the study, the procedures to be 
followed, and the risks and benefits of participation. A copy will be given to each 
participant or legal guardian and this fact will be documented in the participant’s record. 
For Spanish speaking patients and/or parents, Spanish forms will be provided and 
communication will occur through a Spanish translator. All patients are adults and over 
normal cognitive capacity, and therefore will be able to consent for themselves. 
At the visit, a consented patient will answer screening questions regarding inclusion and 
exclusion criteria. Answers will be entered into the CDART research database managed 
by UNC. Prior to finalizing these screening questions, women participants of 
childbearing potential will be asked to provide a urine sample in the restroom, to undergo 
a rapid pregnancy test by study personnel. Any pregnant or lactating patients will be 
excluded.  

Our study statistician, Dr. Kevin Moss, will create a block randomization schedule and 
perform these computations. All patients will be consented and then fill out our screening 
questions for inclusion and exclusion to confirm eligibility prior to being assigned to the 
randomization schedule. 
11.3 
Participant Confidentiality 
An Excel Linkage File key will be created with study ID (random alphanumeric 
code) to identify patients, saved in a secure folder in a UNC School of Dentistry 
(SOD) server. This linkage file will be stored separately from data and destroyed at 
the close of the study. All data collected from participants and results from sample 
analysis will be coded with unique numerical identifiers and stored in the UNC 
CDART clinical research database and on a secure UNC-CH server. Data will only be 
transmitted among the research team using the SOD secure research server. 
Demographic data will be transmitted among the research team through access to the 
secure CDART research database system, stored on secure UNC servers, which study 
personnel will access only through encrypted computers via secure user account. All 
research personnel will be trained in maintaining patient confidentiality and HIPAA. 
Any data, specimens, forms, reports, and other records that leave the site will be 
identified only by a participant identification number (Participant ID, PID) to 
maintain confidentiality. We do not anticipate any data, specimens, forms reports and 
other records leaving the site as sample storage and analysis will occur on site. All 
paper records will be kept in a locked file cabinet, and all digital records will be 
stored in the encrypted, secure, password protected CDART research database. All 
computer entry and networking programs will be done using PIDs only. Information 
will not be released without written permission of the participant, except as necessary 
for monitoring by IRB, the FDA, the NCCIH, and the OHRP. 
There is little to no potential for deductive disclosures from the survey portion of 
this study. Patients will be asked about the the experience rinsing with a 
commercially available mouth rinse (such as Listerine).  They will asked to rate the 
taste, appearance/color, sensation, and their willingness to use the mouth rinse in the 
future. General demographics will be asked such as gender, race, ethnicity and age 
bracket. Their identity cannot be deduced from this information.  
11.4 
Study Discontinuation 
The study may be discontinued at any time by the IRB, the NCCIH, the OHRP, the FDA, 
or other government agencies as part of their duties to ensure that research participants 
are protected. 
 

 
12. COMMITTEES 
The IRB committee will review and approve all study documents, and adverse events and 
protocol deviations will be reported to them for review as well.  
13. PUBLICATION OF RESEARCH FINDINGS 
Any presentation, abstract or manuscript will be made available for review and approval 
by the sponsor and all authors prior to submission.  
14. REFERENCES 
1.        State Officials Announce Latest COVID-19 Facts Including New Data on Racial 
Demographics and Expanded Health Care Worker Data. California Department of Public 
Health(2020). 
2.        Sorveglianza Integrata COVID-19 in Italia. Instituto Superiore di 
Sanitahttps://portale.fnomceo.it/wp-content/uploads/2020/04/Infografica_9aprile-ITA.pdf 
(2020). 
3.        O’Donnell, V. B. et al.Potential Role of Oral Rinses Targeting the Viral Lipid 
Envelope in SARS-CoV-2 Infection. Function 1, (2020). 
4.        Harrel, S. K. & Molinari, J. Aerosols and splatter in dentistry: A brief review of 
the literature and infection control implications. J. Am. Dent. Assoc.(2004) 
doi:10.14219/jada.archive.2004.0207. 
5.        Izzetti, R., Nisi, M., Gabriele, M. & Graziani, F. COVID-19 Transmission in 
Dental Practice: Brief Review of Preventive Measures in Italy. Journal of Dental 
Research(2020) doi:10.1177/0022034520920580. 
6.        Ather, A., Patel, B. & Ruparel, N. Coronavirus Disease 19 (COVID-19): 
Implications for Clinical Dental Care. J. Endod.(2020). 
7.        Baghizadeh Fini, M. What dentists need to know about COVID-19. Oral 
Oncology(2020) doi:10.1016/j.oraloncology.2020.104741. 
8.        Cleveland, J. L. et al.Transmission of blood-borne pathogens in US dental health 
care settings: 2016 update. J. Am. Dent. Assoc.(2016) doi:10.1016/j.adaj.2016.03.020. 
9.        Pfefferle, S. et al.Low and high infection dose transmission of SARS-CoV-2 in the 
first COVID-19 clusters in Northern Germany. medRxiv(2020) 
doi:10.1101/2020.06.11.20127332. 

10.      Ge, Z. yu, Yang, L. ming, Xia, J. jia, Fu, X. hui & Zhang, Y. zhen. Possible 
aerosol transmission of COVID-19 and special precautions in dentistry. Journal of 
Zhejiang University: Science B(2020) doi:10.1631/jzus.B2010010. 
11.      Carrouel, F. et al.COVID-19: A Recommendation to Examine the Effect of 
Mouthrinses with ß-Cyclodextrin Combined with Citrox in Preventing Infection and 
Progression. J. Clin. Med.(2020) doi:10.3390/jcm9041126. 
12.      Dennison, D. K., Meredith, G. M., Shillitoe, E. J. & Caffesse, R. G. The antiviral 
spectrum of Listerine antiseptic. Oral Surgery, Oral Med. Oral Pathol. Oral 
Radiol.(1995) doi:10.1016/S1079-2104(05)80124-6. 
13.      Hasheminia, D. et al.The efficacy of 1% betadine mouthwash on the incidence of 
dry socket after mandibular third molar surgery. J. Clin. Exp. Dent.(2018) 
doi:10.4317/jced.54444. 
14.      Challacombe, S. J., Kirk-Bayley, J., Sunkaraneni, V. S. & Combes, J. Povidone 
iodine. British Dental Journal(2020) doi:10.1038/s41415-020-1589-4. 
15.      Kirk-Bayley, J., Challacombe, S., Sunkaraneni, V. & Combes, J. The Use of 
Povidone Iodine Nasal Spray and Mouthwash During the Current COVID-19 Pandemic 
May Protect Healthcare Workers and Reduce Cross Infection. SSRN Electron. J.(2020) 
doi:10.2139/ssrn.3563092. 
16.      COVID-19 Frequently Asked Questions. American Dental Assocation: Center for 
Professional Sucess(2020). 
17.      Sethuraman, N., Jeremiah, S. S. & Ryo, A. Interpreting Diagnostic Tests for 
SARS-CoV-2. JAMA - Journal of the American Medical Association(2020) 
doi:10.1001/jama.2020.8259. 
15. SUPPLEMENTS/APPENDICES 
No supplements or appendices.  All forms are submitted as part of the IRB application.