Clinical trial NCT04409873
Conditions: COVID-19 SARS-CoV 2 Severe Acute Respiratory Syndrome Coronavirus 2 Virus Disease Coronavirus Infections Pharyngeal Diseases
Keywords: Anti-Infective Agents, Local Mouthwashes Gargle Saliva
Sponsor: University of California, San Francisco
Start date: 2021-03-31; Completion date: 2022-09-10
Enrollment: 54 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
In this pilot trial, 150 confirmed COVID-19 individuals will be randomly assigned to 1 of 5 groups: distilled water, CloSYS Ultra Sensitive Rinse (Rowpar Pharmaceutical Inc., USA), Oral-B Mouth Sore (Oral-B, USA), Crest Pro-Health Multi-Protection (Crest, USA), or Listerine Zero (Johnson and Johnson, USA).

Study participants will be asked to rinse/gargle with 10-20ml (according to the rinse instructions) of the assigned solutions 4 times per day, for 30-60 seconds, for 4 weeks.

Detailed description:
SARS-CoV-2, the virus causing COVID-19, has affected vulnerable individuals, especially those with comorbidities, and high exposure health care workers (HCWs). Typically, the virus first colonizes in the upper respiratory tract (URT) causing clinical symptoms such as coughing, sore throat, and then is transferred to the lower respiratory tract (LRT) which can lead to severe pneumonia, acute respiratory distress syndrome (ARDS), sepsis, and death, if it is not managed.

The World Health Organization (WHO) has presented comprehensive guidelines underscoring personal hygiene measures including respiratory hygiene against SARS, MERS, influenza, and now SARS-CoV-2 / COVID-19. While personal protection equipment (PPE), personal hygiene measures, environmental infection control, and physical distancing are crucial in mitigating disease transmission, respiratory hygiene measures do not prevent SARS-CoV-2 colonization in URTs and LRTs of infected individuals (symptomatic and asymptomatic).

Experimental and clinical research studies on infections similar to COVID-19 such as SARS, MERS, and H5N1 have shown that using antiseptic mouthwash/gargling solutions, such as products containing chlorhexidine gluconate (CHG), polyvinylpyrrolidone iodine (PVP-I), chlorine dioxide (ClO2), cetylpyridinium chloride (CPC), and hydrogen peroxide (H2O2) can reduce viral load. A randomized controlled trial (N=387) showed efficacy and cost-effectiveness of gargling with water or a product containing PVP-I (3X/day, 20 seconds) on URTIs in healthy volunteers (18-65 years) over 60 days from a societal perspective; in vitro studies have shown that CloSYS, an over the counter mouthwash containing ClO2, was effective on inactivating SARS-CoV as well as disinfecting dental unit waterlines, and biofilm control in ultrasonic dental scaling units. A recent study has shown that CloSYS Ultra Sensitive Oral Rinse reduced the viral load of SARS CoV 2, SARS CoV, and Influenza A H3N2 to a varying extent. The data show that the viral load reduction of SARS CoV 2 by Ultra Sensitive rinse was 10 fold more than the reduction of SARS CoV in 30s. Recently, the US Centers for Disease Control (CDC) and the American Dental Association (ADA) have recommended using a mouthwash containing 1.0-1.5% H2O2 as a pre-procedural rinse before dental treatment to potentially reduce SARS-CoV-2 load; however, no in vivo clinical studies have been conducted to support this claim.

The aim of this pilot trial is to evaluate the effect of four over-the-counter antiseptic mouthwash/gargling solutions compared to a control (distilled water) to reduce SARS-CoV-2 load. In addition, study participants will be assessed for the severity of their clinical symptoms during the study period. The 4-week protocol was selected as studies have shown that patients can continue to shed the virus and potentially transmit it to the others for a 2 to 4 week period. An interim analysis is planned when 10 participants per arm (50 total) complete the study using the alpha-spending function with O'Brien-Fleming boundary rule.

Arms: arm 1: Over the counter: Distilled water arm 2: Over the counter: Oral-B Mouth Sore (Oral-B, USA) contains hydrogen peroxide (H2O2) arm 3: Over the counter: Crest Pro-Health Multi-Protection (Crest, USA) contains cetylpyridinium chloride (C21H38ClN) arm 4: Over the counter: CloSYS Ultra Sensitive Rinse (Rowpar Pharmaceutical Inc., USA) contains stabilized chlorine dioxide (ClO2) arm 5: Over the counter: Listerine Zero (Alcohol-Free)(Johnson and Johnson, USA) (C30H52O3)
Interventions: intervention 1: Oral-B Mouth Sore mouthwash intervention 2: Crest Pro-Health Multi-Protection mouthwash intervention 3: CloSYS Ultra Sensitive Rinse mouthwash intervention 4: Distilled water intervention 5: Listerine Zero Mouthwash Product
Intervention descriptions: intervention 1: Rinse and gargle 4 times daily, for 60 seconds, for 4 weeks. intervention 2: Rinse and gargle 4 times daily, for 60 seconds, for 4 weeks. intervention 3: Rinse and gargle 4 times daily, for 45 seconds, for 4 weeks. intervention 4: Rinse and gargle 4 times daily, for 60 seconds, for 4 weeks. intervention 5: Rinse and gargle 4 times daily, for 30 seconds, for 4 weeks.

Protocol documents:
Prot_SAP_000.pdf: 
Title 
Effect of Antiseptic Mouthwash/Gargling Solutions and Pre-procedural Rinse on SARS-CoV-2 
Load (COVID-19): A Structured Summary of a Study Protocol for a Randomized Controlled 
Trial 
 
ClinicalTrials.gov Identifier:  
NCT04409873 
 
Version: 1.6  
Date: Mar-12-2021 
 
 


Title 
Effect of Antiseptic Mouthwash/Gargling Solutions and Pre-procedural Rinse on SARS-CoV-2 
Load (COVID-19): A Structured Summary of a Study Protocol for a Randomized Controlled 
Trial 
Protocol contributors 
Sepideh Banava, DDS, MSc, MPH  
Stuart A. Gansky, DrPH 
 
Abstract 
Background: SARS-CoV-2, the virus causing the COVID-19 pandemic, is a health threatening 
disease especially for people with comorbidities, and for people with high exposure such as 
health care workers (HCWs) and frontline workers. The virus typically first colonizes in the 
upper respiratory tract (URT) and can cause clinical symptoms such as cough and sore throat; the 
virus can move to the lower respiratory tract (LRT) potentially leading to severe pneumonia, 
acute respiratory distress syndrome (ARDS), sepsis, and death. The aim of this pilot clinical trial 
is to investigate the ability of four over-the-counter (OTC), alcohol-free and fluoride-free 
antiseptic mouthwashes to inactivate SARS-CoV-2 in vivo versus distilled water as a control. 
Another objective of the study is to assess changes in self-reported clinical symptom onset or 
worsening (e.g. healthcare utilization and hospitalization). In addition, this study will also 
examine tobacco use, marijuana smoking, or electronic cigarette vaping as possible effect 
modifiers of the effect of OTC antiseptic mouthwash on SARS-CoV-2. 
Methods: In this trial, 150 confirmed COVID-19 positive volunteers will be randomly assigned 
to 1 of 5 groups: distilled water (H2O); Chlorine Dioxide (ClO2) (CloSYS Ultra Sensitive, 


Rowpar Pharmaceutical Inc., USA); Hydrogen Peroxide (H2O2) (Oral-B Mouth Sore, Proctor & 
Gamble, USA); Cetylprydinium Chloride (CPC) (Crest Pro-Health Multi-Protection, Proctor & 
Gamble, USA); or Essential Oils (EO) (Listerine Zero, Johnson & Johnson, USA). Study 
participants will be masked to the extent possible to the rinse assigned. After collecting a 
baseline unstimulated saliva, throat wash (gargle lavage), and oropharyngeal swab samples, they 
will be asked to rinse and gargle with 10-20ml (2-4 teaspoons) of the assigned mouthwash 4 
times per day, for 30-60 seconds, for 4 weeks. Post-baseline samples will be collected on days 7, 
and 28 to compare the viral load.  
Discussion: This pilot trial will build on in vitro studies and small, limited clinical studies of 
antiseptic mouth rinses to inactivate the SARS-CoV-2 virus. Considering the many benefits of 
prevention over treatment, reducing incident cases and decreasing disease severity will be crucial 
in providing a safe and healthy environment for communities, including HCWs during a pandemic 
of a novel respiratory coronavirus.  
Trial registration: This clinical trial was registered on clinicaltrials.gov on June 1, 2020. The 
Clinical Trial Identifier is NCT04409873. 
Objectives: The objectives of the study are as follows: 
- 
To determine the ability of four over-the-counter (OTC) antiseptic mouthwashes on 
inactivating SARS-CoV-2 versus distilled water as control.  
- 
To assess changes in self-reported clinical symptom onset or worsening (e.g. healthcare 
utilization and hospitalization). 
- 
To examine tobacco use, marijuana smoking, or electronic cigarette vaping as possible 
modifiers of the effect of OTC antiseptic mouthwash on SARS-CoV-2.  


- 
To follow-up with participants after the 1 month of assigned study mouthwash for the 
next 11 months to evaluate acceptability of the assigned mouthwash after one month, 
clinical symptoms, and continued mouthwash use.  
Keywords: Mouthwashes; SARS CoV-2; Randomized Clinical trial; Saliva; Real-Time 
Polymerase Chain Reaction 
{Note: Numbering in curly brackets refers to items in the SPIRIT guidelines for clinical trial 
protocols. https://www.spirit-statement.org/} 
Title {1} 
Effect of Antiseptic Mouthwash/Gargling Solutions and Pre-
procedural Rinse on SARS-CoV-2 Load (COVID-19): A structured 
summary of a study protocol for a randomized controlled trial 
Trial registration {2a 
and 2b}. 
ClinicalTrials.gov Identifier: NCT04409873 
Protocol version {3} 
Version 1.6: Mar-12-2021 
Funding {4} 
- 
University of California Office of the President Emergency 
COVID-19 Research funded by the California Breast Cancer 
Research Program, Grant #R00RG2901 
- 
NIH/NIDCR T32DE007306 for Dr. Banava’s time 
- 
NIH/NIDCR U01DE025507-S1 for pilot trial coordination 
- 
Proctor & Gamble for in-kind products 
- 
Rowpar Pharmaceutical Inc. for funding and in-kind product 
Author details {5a} 
Sepideh Banava 


Division of Oral Epidemiology & Dental Public Health, Department 
of Preventive and Restorative Dental Sciences, University of 
California, San Francisco (UCSF), San Francisco, USA 
- Center to Address Disparities in Children's Oral Health (known as 
CAN DO), UCSF, San Francisco, USA 
Stuart A. Gansky 
- Division of Oral Epidemiology & Dental Public Health, Department 
of Preventive and Restorative Dental Sciences, UCSF, San Francisco, 
USA 
- Center to Address Disparities in Children's Oral Health (known as 
CAN DO), UCSF, San Francisco, USA 
- Philip R. Lee Institute for Health Policy Studies, UCSF, San 
Francisco, USA 
- Bakar Computational Health Sciences Institute, UCSF, San 
Francisco, USA 
- Institute for Global Health Sciences, UCSF, San Francisco, USA 
Name and contact 
information for the 
trial sponsor {5b} 
Stuart A. Gansky 
Division of Oral Epidemiology & Dental Public Health, Department 
of Preventive and Restorative Dental Sciences, UCSF, Box #1361 
San Francisco, CA, USA 94143. Email: stuart.gansky@ucsf.edu 
Role of sponsor {5c} 
Principal Investigator 
 
 


Introduction 
Background and rationale {6a} 
As of July 20, 2021, about a year and half after the onset of coronavirus disease 2019 (COVID-
19) pandemic in 2020, about 34 million COVID positive cases and 607,000 deaths have been 
reported in the United States (US)1. Practicing public health safety measures during this period, 
along with the initiation of COVID-19 vaccination in January 2020 in the US has decreased daily 
cases to about 8,000 cases1, however, there are pre-symptomatic, asymptomatic, and 
symptomatic cases that could transmit virus through respirable and inspirable particles that can 
be inhaled into the oronasopharynx and colonized in the upper respiratory tract (URT) causing 
clinical symptoms such as fever, dry cough, sore throat, and nasal congestion. Colonizing in the 
lower respiratory tract (LRT) causes serious conditions such as severe pneumonia, acute 
respiratory distress syndrome (ARDS), sepsis, and death if the disease is not managed properly3–
5. Therefore, practicing preventive measures seems to help mitigating the risk of transmission 
and clinical manifestations.  
The COVID-19 risk, hospitalization, and death have been reported to be higher among 
American Indian or Alaska Native (AI/AN), Black or African American, and Hispanic or 
Latinos6 with higher percentage among AI/AN than White2. Additionally, people with 
comorbidities such as diabetes, cardiovascular diseases, and chronic renal diseases suffer 
drastically by COVID-19. Frontline health care workers (HCWs) are at higher risk for exposure 
to infectious particles’ spread when in contact with COVID-19 infected individuals. According 
to the United States (US) Centers for Disease Control and Prevention, the trend of new cases 
among HCP shows a decrease from about 13,000 in April 2020 to 190 cases in July 2021. 
Additionally, as of July 20, 2021, of 517,341 cases of COVID-19 among HCP, about 1700 


deaths (0.33%) have been reported (CDC) which has increased compared to April 2020 death 
cases 27 of 9282 (0.29%)3, however, it has decreased since vaccination started. Considering the 
staffing shortages, losing one HCP in this period could increase pressure on the health care 
system and reduce the workforce, hence, in addition to the vaccination as a primary prevention 
method, finding methods to decrease COVID-19 transmission via infectious particles can save 
lives among HCWs and in the community.  
The World Health Organization (WHO) has presented comprehensive guidelines 
underscoring personal hygiene measures including respiratory hygiene against SARS, MERS, 
influenza, and now COVID-198. While personal protection equipment (PPE), personal hygiene 
measures, and environmental infection control are crucial in mitigating disease transmission, the 
current recommended respiratory hygiene measures has not prevented SARS-CoV-2 
colonization in URTs and LRTs of infected individuals, or reducing the severity of clinical 
symptoms. Experimental and clinical research studies on diseases similar to COVID-19 such as 
SARS, MERS, and H5N1 have shown that using antiseptic mouthwash or gargling solutions, 
such as products containing chlorhexidine gluconate (CHG), polyvinylpyrrolidone iodine (PVP-
I), chlorine dioxide (ClO2), cetylpyridinium chloride (CPC), and hydrogen peroxide (H2O2) 
could reduce viral load9–11. A randomized controlled trial (N=387) showed efficacy and cost-
effectiveness of gargling with water or a product containing PVP-I (3X/day, 20 seconds) on URT 
infections in healthy volunteers (18-65 years) over 60 days from a societal perspective12; An in 
vitro study has shown that CloSYS Ultra Sensitive oral rinse could reduce the viral load of SARS 
CoV 2, SARS CoV and Influenza A H3N2 to varying extent; the viral load reduction of SARS 
CoV 2 by CloSYS Ultra Sensitive oral rinse was 10-fold more than reduction of SARS CoV in 
30s13. In another in vitro study, three of eight OTC antiseptic mouth rinses were able to 


inactivate three strains of SARS-CoV2 in 30s, however, the other five rinses partially inactivated 
the virus. The active ingredients of those three effective rinses were benzalkonium chloride, 
chlorhexidine, and povidone iodine14. A recent clinical trial has shown that using 1% PVP-I as 
mouthwash, gargle, and eye or nose drop could significantly reduce COVID-19 related 
morbidity, mortality, and hospitalization15. 
The CDC's initial interim guidelines for dental settings during COVID-19 suggested that 
using a pre-procedural rinse with an antimicrobial product (such as chlorhexidine gluconate, 
essential oils, hydrogen peroxide, povidone-iodine, or cetylpyridinium chloride) could have a 
potential effect in reducing the level of oral microorganisms in aerosols and spatter generated 
during dental procedures7. In April 2020, the CDC and the American Dental Association (ADA) 
recommended using a mouthwash containing 1.0-1.5% H2O2 as a pre-procedural rinse before 
dental treatment to reduce SARS-CoV-2 load potentially; however, no US clinical studies have 
been conducted to support this claim. 
This study aims to assess the effect of four over-the-counter (OTC) antiseptic 
mouthwash/gargling solutions compared to a distilled water control on the SARS-CoV-2 load in 
unstimulated saliva, throat wash (gargle lavage), and oropharyngeal swab samples. Also, study 
participants will be assessed for the severity of their clinical symptoms during the study period. 
The 4-week protocol was selected as studies show people with COVID can continue to shed the 
virus and potentially transmit to the others for a 2- to 4-week period. This study will also 
examine tobacco use, marijuana smoking, or vaping as possible effect modifiers of antiseptic 
mouthwash on SARS-CoV-2. Study participants will be followed up daily via a very short daily 
questionnaire and every 3 months for the next 11 months after the study is finished for longer-
term follow-up via text messaging.  


The null hypothesis of the study is that there is no difference between any OTC antiseptic 
mouthwash solution (Chlorine Dioxide (ClO2), Hydrogen Peroxide (H2O2), Cetylprydinium 
Chloride (CPC), Essential Oils (EO), and distilled water (H2O) in reducing SARS-CoV-2 load in 
unstimulated saliva, throat wash (gargle lavage), and oropharyngeal swab samples (baseline to 4 
week follow-up). 
Objectives {7} 
1a. To assess and compare the SARS-CoV-2 viral load in unstimulated saliva and throat wash 
(gargle lavage) samples before and after rinse at baseline in Chlorine Dioxide (ClO2), Hydrogen 
Peroxide (H2O2), Cetylprydinium Chloride (CPC), and Essential Oils (EO) groups versus 
distilled water (H2O) group (pre-procedural rinse primary outcome measure).1b. To assess the 
SARS-CoV-2 viral load reduction of throat wash (gargle lavage) samples from baseline to 4 
weeks in Chlorine Dioxide (ClO2), Hydrogen Peroxide (H2O2), Cetylprydinium Chloride (CPC), 
and Essential Oils (EO) groups versus distilled water (H2O) group (homecare reduction primary 
outcome measure). 
2. To assess the clinical symptom onset progression and health care utilization (e.g. 
hospitalization) from baseline to 4 weeks comparing Chlorine Dioxide (ClO2), Hydrogen 
Peroxide (H2O2), Cetylprydinium Chloride (CPC), Essential Oils (EO) to distilled water 
(secondary outcome measures). 
3. To assess if tobacco use, marijuana smoking, or vaping are effect modifiers of primary 
outcome measures. 
4. To evaluate study participants’ in a 12-month follow up.  
Trial design {8} 
This is a single center, 5-arm, parallel groups, double-blinded (participant, outcomes assessor), 


randomized controlled trial of adults who are COVID-19 positive using over-the-counter (OTC) 
mouthwash/gargling solutions: Chlorine Dioxide (ClO2) (CloSYS Ultra Sensitive, Rowpar 
Pharmaceutical Inc., USA); Hydrogen Peroxide (H2O2) (Oral-B Mouth Sore, Proctor & Gamble, 
USA); Cetylprydinium Chloride (CPC) (Crest Pro-Health Multi-Protection, Proctor & Gamble, 
USA); Essential Oils (EO) (Listerine Zero, Johnson & Johnson, USA) versus distilled water as a 
control.  
Methods: Participants, interventions and outcomes 
Study setting {9} 
Consenting community-dwelling San Francisco Bay Area (California, USA) participants positive 
for COVID-19.  
Eligibility criteria {10} 
Inclusion criteria 
• Adults 18 years and older 
• Tested positive for COVID-19 with a sample collected in the prior 7 days 
• 
Ability to read and speak in English or Spanish 
• 
Ability to participate in the study for 4 weeks 
• 
Ability to gargle 
• 
Not having any oral condition that might worsen with gargling solutions 
• 
Not having an allergy to a mouthwash ingredient that has been used before 
• 
Not using another mouthwash/gargling solution 
• 
Not taking antimicrobial medications (antibacterial, antiviral (e.g., Truvada), antibiotics 
including off-label FDA-approved medications such as Remdesivir) 


• 
Have a cellphone and agree to receive text messages for reminders to use mouthwash during 
the day and for follow-up visits 
• 
Ability to meet with study staff via videoconference (e.g., Zoom, FaceTime, WhatsApp) 
Exclusion criteria 
• Those who experience severe clinical symptoms which require hospitalization  
• Individuals who receive antiviral medications that could potentially affect viral load in their 
saliva samples 
• Pregnancy or lactation for women due to potential aversions to mouthwash solution 
taste/smell 
Who will take informed consent? {26a} 
Study Co-PI or a trained bilingual study staff member will confirm the study participants’ 
eligibility based on inclusion/exclusion criteria before obtaining informed consent and the 
required waiver/authorization form included in supplemental IRB documents. 
Additional consent provisions for collection and use of participant data and biological 
specimens {26b} 
The informed consent form (ICF) explains the participant sample collection procedure, lab test, 
and potential future studies on samples. The ICF explains test results being kept confidential and 
participants’ ability to discontinue the study at any time, without any interference in their 
COVID-19 related or other health care.  
Interventions 
Explanation for the choice of comparators {6b} 
The comparator control group is distilled water (H2O) which may help clear microbes from the 
oral cavity and throat without deactivating SARS-CoV-2 virus. A published randomized clinical 


trial and cost-benefit analysis has shown gargling with plain water to be cost effective in 
reducing URT infections (Sakai et al. 2008). Participants in the control group will be asked to 
rinse and gargle for 60 seconds with 20ml (4 teaspoons) 4 times per day (including after each 
meal and before bedtime), for 4 weeks. 
Intervention description {11a} 
The four OTC alcohol-free, fluoride-free, gluten-free mouthwash intervention groups are as 
follows: Chlorine Dioxide (ClO2) (CloSYS Ultra Sensitive, Rowpar Pharmaceutical Inc., USA); 
Hydrogen Peroxide (H2O2) (Oral-B Mouth Sore, Proctor & Gamble, USA); Cetylprydinium 
Chloride (CPC) (Crest Pro-Health Multi-Protection, Proctor & Gamble, USA); Essential Oils 
(EO) (Listerine Zero, Johnson & Johnson, USA), which were selected based on being OTC 
alcohol-free and fluoride-free products legally marketed in the US, each with a different main 
active ingredient. Participants will be asked to rinse and gargle for 30-60 seconds with 10-20 ml 
(2-4 teaspoons) of the assigned mouthwash 4 times per day (including after each meal and before 
bedtime), for 4 weeks.  
Criteria for discontinuing or modifying allocated interventions {11b} 
In the rare case that a study participant has an adverse experience possibly, probably, or 
definitely related to the assigned oral rinse, he or she will be instructed to discontinue using the 
rinse to see if the problem resolves. At the discretion of the participant and investigators, after a 
problem resolves, the participant may resume using the rinse. If the problem does not resolve, the 
participant will be instructed to not resume using the rinse; if possible, the participant will be 
encouraged to still provide the follow-up samples and report symptoms. If a participant’s clinical 
symptoms worsen, he or she will be instructed to contact his or her primary care physician to get 


the required medical care for his or her health. A participant can withdraw him- or herself at any 
time without concern for repercussions about their health care. 
Strategies to improve adherence to interventions {11c} 
During the study, adherence data will be collected through a daily REDCap questionnaire 
distributed via SMS text message, asking about the frequency and duration of mouthwash use, as 
well as clinical symptoms and healthcare utilization.  
Relevant concomitant care permitted or prohibited during the trial {11d} 
The study participants should not use any other mouthwashes or take any antimicrobial 
medication (antibacterial, antiviral, antibiotics including off-label usage such as Remdesivir) 
during the study. They can continue practicing their usual home oral hygiene care (tooth 
brushing with a toothpaste, and flossing). If they have a dental emergency (e.g. dental infection 
such as abscess or pulp infection) they should contact their regular dentist for dental treatments 
according to their dentist’s COVID protocols.  
Provisions for post-trial care {30} 
The antiseptic mouthwash solutions to be used by study participants are safe over-the-counter 
solutions that are available without requiring dentist's prescriptions, to improve oral health 
conditions. Unless individuals might be allergic to the ingredients of the study mouthwashes, 
they will not face any harm and risks when using the solutions. However, research team will 
provide their phone numbers and emails if participants confront any problems during the study 
period. If study participants feel any stress and discomfort due to their COVID-19 status, they 
will be advised to contact their physician and COVID-19 hotlines as soon as possible to receive 
required health services. After the study is finished, participants will be asked a few questions by 
texting a link to an online questionnaire designed in REDCap in about every 3 months over the 


next 11 months to see how they are doing, their clinical symptoms, acceptability of using the 
assigned mouthwash during the trial, and their potential use after the trial.  
Outcomes {12} 
Primary outcome Measure:  
- 
Change in SARS-Cov-2 viral load: Change in RT-PCR SARS-Cov-2 viral load in 
unstimulated saliva and throat wash (gargle lavage) samples  (Time Frame: Baseline to 4 
weeks)  
Secondary Outcomes Measures:  
- 
Change in self-reported (questionnaire) clinical symptom onset. A symptom checklist will 
include: cough, runny nose, scratchy/sore throat, fever, chills, fatigue, muscle ache, 
shortness of breath, diarrhea/nausea/vomiting, loss of taste/smell, and red /painful eye 
(Time Frame: Baseline to 4 weeks)  
- 
Change in healthcare utilization and hospitalization (Time Frame: Baseline to 4 weeks)  
Other Pre-specified Outcome Measures:  
- 
Change in SARS-Cov-2 viral load in tobacco users, marijuana smokers, or vapers (Time 
Frame: Baseline to 4 weeks)  
- 
Change in self-reported (questionnaire) clinical symptom onset in tobacco users, marijuana 
smokers, or vapers. A symptom checklist will include: cough, runny nose, scratchy/sore 
throat, fever, chills, fatigue, muscle ache, shortness of breath, diarrhea/nausea/vomiting, 
loss of taste/smell, and red /painful eye (Time Frame: Baseline to 4 weeks)  
- 
Change in healthcare utilization and hospitalization in tobacco users, marijuana smokers, 
or vapers (Time Frame: Baseline to 4 weeks) 
Participant timeline {13} 


Study participants will remain in the trial for 4 weeks. After completing the intervention, they 
will be followed up for the next 11 months via text messaging to ask about clinical symptoms 
and healthcare utilization, experience using mouthwash, and their interest in using mouthwash 
after the trial.  
Sample size {14} 
A total of 150 eligible individuals will be enrolled in the trial after providing informed consent 
and randomly assigned to 1 of 5 groups. This trial is sized to provide precise estimates of effect 
size for a future definitive trial with at-risk individuals. However, this trial would be able to 
detect a rather large effect: a sample size of 24 for a mouthwash group and 24 for the distilled 
water control group is estimated to have 80% power to detect a 2-fold difference in mean values 
between the 2 groups with a 2-sided alpha=0.05. Assuming 80% retention through 28 days, 30 
participants per group at baseline (150 total) will be enrolled.  
Recruitment {15} 
Two recruitment routes have been planned for this trial:  
1- Upon referring to testing centers at UCSF individuals will receive the trial details via a letter 
or flyer, and if tested positive for COVID-19, they will contact study staff and enroll into the 
trial.  
2- The UCSF Clinical and Translational Science Institute (CTSI) Participant Recruitment 
Program (PRP) provides new COVID+ patient lists enabling us to contact COVID+ patient who 
are interested in participating in research studies.  
After study staff confirm their eligibility, participants will receive the sample collection package 
and written instructions at their home along with scheduling a videoconference to collect saliva 
samples and use the assigned mouthwash during the 4-week period.  


Assignment of interventions: allocation 
Sequence generation {16a} 
The permuted block allocation sequence stratified on language (English/Spanish) will be 
generated in SAS and uploaded to REDCap to randomly assign participants to one of the five 
mouthwash groups.   
Concealment mechanism {16b} 
The randomization will be concealed in REDCap; once participant’s eligibility is confirmed, 
consent form is signed, and the baseline questionnaire completed, then the assignment is 
revealed. No study staff involved in recruitment or procedures with participants or participants 
know the allocation sequence since it is revealed via REDCap after qualifying steps.  
Implementation {16c} 
Following release of the REDCap allocation sequence for an individual participant, study staff 
will obtain the relevant kit and deliver to participants.   
Assignment of interventions: Blinding 
Who will be blinded {17a} 
Participants will be blinded/masked to the extent possible by covering product labels, but the 
solutions have different colors, flavors, and smells so they may guess which group they have 
been assigned. Lab staff performing assays (outcome assessors) and data analysts will be 
blinded/masked to intervention group.  
Lab staff will receive participants’ samples with barcode stickers with only the study ID number.  
The data analyst will receive data set with coded group identifier.   
Procedure for unblinding if needed {17b} 


We will not unblind the study groups for outcome assessors unless subsequent actions to assure 
participants’ safety would be affected.   
Data collection and management 
Data will be collected via various questionnaires (eligibility, baseline, daily and long-term follow 
ups) designed in REDCap. The data collection will be supervised and assessed regularly for 
completeness and inconsistencies.  
Plans for assessment and collection of outcomes {18a} 
We have designed questionnaires (eligibility, baseline, daily and long-term follow ups, sample 
collection, and RT-PCR results) in REDCap to collect data from participants and assess 
outcomes. We will train research and lab staff regarding each related steps and data collection. 
We have pilot tested assessments. The data collection will be supervised and assessed regularly 
for discrepancies.  
Plans to promote participant retention and complete follow-up {18b} 
We have planned to send daily reminders to participants to use their assigned mouthwashes and 
follow-up the next day to see if they have used it and if there were problems.  
At any time during the trial, if a participant discontinues from the study or deviates from 
intervention protocols, we will record the information.  
Data management {19} 
We have planned to use unique barcodes on sample collection kits and participants’ packages to 
be scanned during the trial and automatically recorded in an Excel file on a UCSF secured laptop 
and the Cloud. Only PIs know about the codes. Sepideh Banava (SB) will supervise data entry 
and its quality on a regular basis. Data will be linked to a REDCap form.  
Confidentiality {27} 


Email address and telephone number are the only personal identifying information which will be 
kept confidential in REDCap on UCSF secured servers. Participant information will be kept 
confidential. After the study and its follow-up in 12 months is finished, email address and phone 
number will be deleted.  
Plans for collection, laboratory evaluation and storage of biological specimens for genetic 
or molecular analysis in this trial/future use {33} 
The study procedures include collecting saliva and oropharyngeal samples from participants on 3 
study days (day 1, 7, 28), assaying them, and storing them for future potential research use.   
Statistical methods 
Statistical methods for primary and secondary outcomes {20a} 
Primary outcome measure: To assess and compare the SARS-CoV-2 viral load in unstimulated 
saliva and throat wash (gargle lavage) samples at baseline, days 7 and 28, we will compare the 
viral load (cycle threshold, Ct) among active groups versus the control (water) and within groups 
on day 28 using proportional hazards regression models with right censoring for undetermined 
Ct, stratifying on replicate for a marginal repeated measures survival model.  
We follow the intention-to-treat (ITT) approach for efficacy analyzing participant data as-
randomized; a safety analysis will tabulate any adverse experiences. The ITT group will include 
participants who are randomized after meeting the eligibility criteria and receive at least one 
study mouthwash dose and have PCR data from collected samples. A safety analysis group will 
include participants who received study mouthwashes and have safety evaluation data after using 
the mouthwash at least once. 
Interim analyses {21b} 


We plan to conduct an interim analysis after 10 participants in each group (N=50) have finished 
the 28-day period (using mouthwash). The alpha-spending approach with an O’Brien-Fleming 
stopping rule will be used. 
Methods for additional analyses (e.g. subgroup analyses) {20b} 
Similar proportional hazards regression analyses will be conducted for baseline (after rinse) and 
day 7.  We will assess the relationship between study results and baseline variables 
(demographics, clinical symptoms, tobacco use, etc.). 
Methods in analysis to handle protocol non-adherence and any statistical methods to 
handle missing data {20c} 
Since PCR Ct data (inversely proportional to viral load) are expected to include right censored 
data when the assay is stopped before sufficient amplified viral genetic material is detected, we 
plan to use statistical analyses which can accommodate right censoring.  Alternatively, we may 
use multiple imputation for Ct values above the detection level (e.g. 32, 35, or 40) and combine 
the estimates and variability across multiple imputations. 
Plans to give access to the full protocol, participant level-data and statistical code {31c} 
We will make the protocol available as per ClinicalTrials.Gov requirements. We do not plan to 
provide access to participant-level data. We may make statistical code available with 
publications. 
Oversight and monitoring 
Composition of the coordinating center and trial steering committee {5d} 
SG, study PI and supervisor, and SB, co-PI form the steering committee. They will have regular 
weekly meetings to review and supervise various study steps and train/lead study staff. They will 
work with different study staff groups (e.g. recruitment, data management, and lab staff) on a 


regular basis to assess trial steps and solve any problems/errors that might rise.   
Composition of the data monitoring committee, its role and reporting structure {21a} 
SG, the PI, is a biostatistician and clinical trialist who will instruct and supervise study staff on 
designing questionnaires, data collection, and data analyses. SB, the co-PI, is a dental clinician 
who will work closely with and report to SG.  
Adverse event reporting and harms {22} 
REDCap will be configured with Twilio to send a daily text message to participants with a link 
to a very short REDCap questionnaire asking about previous day mouthwash use and possible 
adverse experiences.  
Frequency and plans for auditing trial conduct {23} 
Study PIs will audit trial conduct and trial procedures weekly to resolve possible errors and make 
decisions about required modifications, staff re-training, etc.  
Plans for communicating important protocol amendments to relevant parties (e.g. trial 
participants, ethical committees) {25} 
Study PIs will communicate with the UCSF ethics committee (institutional review board) before 
making any modifications during the study.  
Dissemination plans {31a} 
We plan to disseminate study results via publications. (If the interim analysis shows important 
early findings, we will share that information with the health research community via preprint 
such as MedRxiv.) We will report aggregate study results to ClinicalTrials.gov for the public.  
Discussion 
This clinical trial was originally designed to be conducted in-person at the UCSF School of 
Dentistry to collect saliva samples, but since specific settings and supplies are required to 


examine COVID positive patients in-person, we were instructed to modify the study protocol so 
participants could receive sample collection kits in their homes and staff instruct them via 
videoconference software, telephone, and written instructions to guide them through the process.  
Abbreviations 
SARS-CoV-2: Severe Acute Respiratory Syndrome- CoronaVirus-2 
COVID-19: Corona Virus Disease-2019 
Declarations 
• 
Acknowledgments 
The authors are grateful for the support from Danielle Stephens, Rocio Cisneros, and 
Nancy Cheng for preparing for this trial. 
• 
Authors' contributions 
SB conceived and conceptualized the idea, designed the study, prepared the UCSF IRB 
and ClinicalTrials.Gov application and developed study documents, contributed in 
preparing the budget, applied for funding, developed study logistics, prepared 
presentations, and drafted the protocol. SG contributed to conceptualization and study 
design, the UCSF IRB and ClinicalTrials.Gov application and developing study 
documents, prepared the budget, applied for funding and prepared contracts to receive the 
funding, contributed to study logistics, and edited this protocol. They both read and 
approved the final protocol. 
• 
Funding 
- 
T32DE007306 (National Institute of Health (NIH)/National Institute of Dental and 
Craniofacial Research (NIDCR) supports SB during the study) 


- 
U01DE025507-S1 (National Institute of Health (NIH)/National Institute of Dental and 
Craniofacial Research (NIDCR) supports some clinical trials coordinating center 
assistance) 
- 
R00RG2901 (University of California Office of President (UCOP) Emergency 
COVID19 Research Funding: California Breast Cancer Research Program 
- 
Rowpar Pharmaceutical Inc.  
• 
Availability of data and material 
Study PIs will review requests for study data on a case by case basis. 
• 
Ethics approval and consent to participate 
This clinical trial has been approved by the Institutional Review Board at the University 
of California San Francisco (UCSF) (IRB number: 20-30874) on March 17, 2021.  The 
reference number is 284660.  
The informed consent forms for study participants have been approved by the UCSF IRB. 
Study staff will obtain consent from participants after confirming their eligibility and 
before sending the testing package. The participants or their legal guardian will sign an 
electronic consent after their eligibility is confirmed prior to inclusion into the study. 
Information from all participants will be kept confidential, and those who do not wish to 
continue being in the trial can withdraw from the study at any time. 
• 
Consent for publication 
Not applicable. 
• 
Competing interests 
The authors declare that they have no competing interests. 
• 
Authors’ information (optional) 


1. Division of Oral Epidemiology & Dental Public Health, Department of Preventive and 
Restorative Dental Sciences, University of California San Francisco (UCSF), San 
Francisco, USA 
2. Professor and Lee Hysan Chair of Oral Epidemiology, Associate Dean for Research, 
School of Dentistry, Director of Center to Address Disparities in Children's Oral Health 
(known as CAN DO), University of California San Francisco (UCSF), San Francisco, 
USA 
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