Clinical trial NCT04748783
Conditions: COVID19 Coronavirus Infection SARS CoV-2 Infection
Keywords: Inactivation of SARS CoV-2 Virus Antiseptic Mouth Rinses
Sponsor: University of North Carolina, Chapel Hill
Start date: 2021-03-26; Completion date: 2021-04-07
Enrollment: 2 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
Subjects (125) will be randomized to one of five mouthrinses and will be asked to give a saliva sample immediately before and after a 30-60 second mouthwash.

Saliva samples will be collected from subjects at 15-minute intervals thereafter up to one hour (15, 30, 45 and 60 min). The saliva will be used for RT-PCR detection of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) and viral infectivity assays, along with quantitative cytokine and chemokine concentration (pg/mL, Luminex).

Subjects will complete a short survey on the taste and experience of using the mouthwash.

Peripheral blood will be collected at the end of salivary collection. Subjects, except controls, will be provided materials and oral hygiene instruction related to daily use of oral hygiene products. In the seven-day period between study visit one and study visit two, subjects will be directed to brush with Colgate toothpaste (at least twice per day) and rinse with the Colgate mouthrinse (according to on-label procedures). Controls are asked to carry out their typical oral hygiene regimen with the products they typically use.

All subjects keep a daily diary of oral hygiene performance, product usage, COVID-19 symptoms and exposures. Subjects complete study visit two one week after the baseline visit during which additional salivary (1 time point, 2 mL of saliva over 5 min, no rinse) will occur and blood samples collected. each subject will undergo a periodontal exam.

Detailed description:
Randomized, double-blind prospective trial to test the efficacy and acceptability of therapeutic, antiseptic mouth rinses to inactivate SARS-CoV-2 virus in saliva of SARS-CoV-2+ patients 18-65 years of age. The study will enroll 30 outpatient SARS-CoV-2+ subjects per mouth rinse, (to achieve a total of 25 subjects with full data sets per mouth rinse,) with 5 mouth rinses, requiring us to enroll 125 SARS-CoV-2+ patients. Patients will be randomized to their mouthrinse. These patients will have already had a confirmed SARS-CoV-2+ test prior to enrollment. There will be no stratification to our randomization. All mouthrinses are commercially available Colgate products and will be used according to on-label instructions.

Subjects will be randomized to a mouthrinse and will be asked to give a saliva sample immediately before and after a 30-60 second mouthwash.

Saliva samples will be collected from subjects at 15-minute intervals thereafter up to one hour (15, 30, 45 and 60 min). The saliva samples will be stored and used for RT-PCR detection of SARS-CoV-2 virus and viral infectivity assays, along with quantitative cytokine and chemokine concentration (pg/mL, Luminex).

Subjects will also complete a short survey on the taste and experience of using the mouthwash.

Peripheral blood will be collected at the end of salivary collection. Subjects, except controls, will be provided materials and oral hygiene instruction related to daily use of oral hygiene products provided by the study. In the seven-day period between study visits, subjects will be directed to brush with Colgate toothpaste (at least twice per day) and rinse with the Colgate mouthrinse (according to on-label procedures). Controls are not given Colgate products and will be asked to carry out their typical oral hygiene regimen with the products they typically use. All subjects will be asked to keep a daily diary of oral hygiene performance, product usage, COVID-19 symptoms and exposures.

Subjects will be scheduled to return to the research clinic at one week after the baseline assessment, during which additional salivary (1 time point, 2 mL of saliva over 5 min, no rinse will occur) and blood samples will be collected. At the conclusion of sample collection, the subject will undergo a periodontal exam. Study participation concludes following the periodontal exam at the end of the second on site study visit. Patients with any periodontitis or gingivitis diagnosis will be informed of their diagnosis and will be referred to an oral healthcare provider for further treatment. This study involves two 90-minute visits.

Arms: arm 1: Subject participants will rinse mouth one time for 60 seconds with 10 mL of sterile water arm 2: Subject participants will rinse mouth one time for 60 seconds with 10 mL Peroxyl (1.5% w/v hydrogen peroxide) rinse arm 3: Subject participants will rinse mouth one time for 60 seconds with 10 mL Periogard (0.12% Chlorhexidine Gluconate) rinse. arm 4: Subject participants will complete an on-label sequential rinse starting with Peroxyl (1st) 10ml for 60 seconds and then Periogard (2nd) 15ml for 30 seconds. arm 5: Subject participants will rinse mouth one time with Colgate Total Zero Fresh Breath (0.075% Cetylpyridinium Chloride) 20ml for 30 seconds
Interventions: intervention 1: Water intervention 2: Peroxyl intervention 3: Periogard intervention 4: Colgate Total Zero
Intervention descriptions: intervention 1: Subject participants will rinse mouth one time for 60 seconds with 10 mL of sterile water. intervention 2: Subject participants will rinse mouth one time for 60 seconds with 10 mL Peroxyl (1.5% w/v hydrogen peroxide) rinse intervention 3: Subject participants will rinse mouth one time for 60 seconds with 10 mL Periogard (0.12% Chlorhexidine Gluconate) rinse. intervention 4: Subject participants will rinse mouth one time with Colgate Total Zero Fresh Breath (0.075% Cetylpyridinium Chloride) 20ml for 30 seconds

Protocol documents:
Prot_SAP_000.pdf: 
 
Antiviral Efficacy and Acceptability of Therapeutic Antiseptic Mouth 
Rinses for Inactivation of 
COVID SARS-2 Virus 
 
 
 
 
NCT number NCT04748783 
Document Date 01/28/2021 
 


1 
 
 
 
 
 
 
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, UNC Adams School of Dentistry 
Jennifer Webster, DDS, PhD, Professor, UNC Adams School of Dentistry 
 
Version: 2.0 (January 28, 2021)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


2 
 
Contents 
2.2 Study Rationale ....................................................................................................................................... 9 
4. SELECTION AND ENROLLMENT OF PARTICIPANTS .................................................................. 15 
4.1 Inclusion Criteria .................................................................................................................................. 15 
4.2 
Exclusion Criteria ........................................................................................................................... 16 
5. STUDY INTERVENTIONS ...................................................................................................................... 19 
5.1 Interventions, Administration, and Duration .................................................................................... 19 
5.2 Handling of Study Interventions ......................................................................................................... 20 
5.3 Concomitant Interventions .................................................................................................................. 21 
5.3.1 Allowed Interventions ................................................................................................................... 21 
5.3.2 Required Interventions ................................................................................................................. 21 
5.3.3 Prohibited Interventions ............................................................................................................... 21 
6.2 Description of Evaluations ................................................................................................................... 22 
6.2.1 Screening Evaluation ..................................................................................................................... 23 
6.2.2 Enrollment, Baseline, and/or Randomization ............................................................................. 23 
6.2.3 Blinding .......................................................................................................................................... 24 
6.2.5  Completion/Final Evaluation ....................................................................................................... 25 
8.1  Specification of Safety Parameters..................................................................................................... 29 
8.2  Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters ..................... 29 
8.3  Adverse Events and Serious Adverse Events .................................................................................... 29 
8.4  Reporting Procedures .......................................................................................................................... 29 
8.5  Follow-up for Adverse Events ............................................................................................................ 30 
8.6   Safety Monitoring ............................................................................................................................... 30 
Tertiary Outcomes ...................................................................................................................................... 32 
10.2   Sample Size and Randomization ..................................................................................................... 33 
10.3   Definition of Populations .................................................................................................................. 33 
10.4   Study Stopping Rules ....................................................................................................................... 34 
10.5   Outcomes ........................................................................................................................................... 34 
10.5.1   Primary Outcome ...................................................................................................................... 35 
10.5.3   Tertiary Outcomes ..................................................................................................................... 35 
10.6   Data Analyses .................................................................................................................................... 36 
11.2 
Data Management........................................................................................................................... 37 
10.3 
Quality Assurance........................................................................................................................... 38 
11.3.1  Training ....................................................................................................................................... 38 


3 
 
11.3.2  Quality Control Committee ....................................................................................................... 39 
11.3.3  Metrics ......................................................................................................................................... 39 
11.3.4  Protocol Deviations ..................................................................................................................... 40 
11.3.5  Monitoring ................................................................................................................................... 40 
12. PARTICIPANT RIGHTS AND CONFIDENTIALITY ....................................................................... 40 
12.1 
Institutional Review Board (IRB) Review .................................................................................... 40 
12.2 
Informed Consent Forms ............................................................................................................... 40 
12.3   Participant Confidentiality .............................................................................................................. 41 
12.4 
Study Discontinuation .................................................................................................................... 41 
13. COMMITTEES ........................................................................................................................................ 41 
14. PUBLICATION OF RESEARCH FINDINGS ...................................................................................... 42 
15. REFERENCES ......................................................................................................................................... 42 
16. SUPPLEMENTS/APPENDICES ............................................................................................................ 43 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


4 
 
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 
Jennifer Webster, Principal Investigator, 4506 KOHS, 385 S. Columbia St., Chapel Hill, NC 
27599, (919) 537-3581, jennifer_cyriaque@unc.edu 
Christina Graves, Co-Investigator, 4352 Genome Sciences Building, Chapel Hill, NC 27514, 
(919) 962-7140, 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, Dental Research Project Manager , GO Health Clinic, 2040 Old Dental, 385 S. 
Columbia St., Chapel Hill, NC 27599, (919) 537-3424, sherrill_phillips@unc.edu 
Clare Bocklage, Study Coordinator, 385 S. Columbia St., Chapel Hill, NC 27599, (919) 
843-2596, eclare@live.unc.edu 
Samantha Jhingree, Research Assistant, 385 S. Columbia St., Chapel Hill, NC 27599, (336) 
207-8569, samjhing@live.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  
 
 
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)  
 
 
 
 
 
 
 


5 
 
PRÉCIS 
Study Title 
Antiviral Efficacy and Acceptability of Therapeutic Antiseptic Colgate Mouth Rinses for 
Inactivation of COVID SARS-2 Virus 
Objectives 
The primary objective is to quantify the impact of Colgate antiseptic mouthwashes on salivary 
infectivity in SARS-CoV-2+ patients. The secondary objectives are to quantitatively determine 
inflammatory cytokine and chemokine levels (pg/mL) in SARS-CoV-2+ patient blood and saliva 
and to evaluate the incidence of periodontal disease among study participants.  
Design and Outcomes  
Randomized, double-blind prospective trial to test the efficacy and acceptability of therapeutic, 
antiseptic mouth rinses to inactivate SARS-CoV-2 virus in saliva of SARS-CoV-2+ patients 18-65 
years of age. All mouthrinses are commercially available Colgate products 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 30-60 second mouthwash. Saliva samples will 
be collected from patients at 15-minute intervals thereafter up to one hour (15, 30, 45 and 60 min). 
The samples will be stored and used for RT-PCR detection of SARS-CoV-2 virus and viral 
infectivity assays, along with quantitative cytokine and chemokine concentration (pg/mL, 
Luminex). Patients will also complete a short survey on the taste and experience of using the 
mouthwash. Peripheral blood will be collected at the end of salivary collection. Participants, except 
controls, will be provided materials and oral hygiene instruction related to daily use of oral hygiene 
products provided by the study. In the seven-day period between study visits, participants will be 
directed to brush with Colgate toothpaste (at least twice per day) and rinse with the Colgate 
mouthrinse (according to on-label procedures). Controls are not given Colgate products and will be 
asked to carry out their typical oral hygiene regimen with the products they typically use. All 
participants will be asked to keep a daily diary of oral hygiene performance, product usage, 
COVID-19 symptoms and exposures. Daily diary data confirms compliance with oral hygiene 
protocol, and is needed for data inclusion and analysis, as it relates to secondary and tertiary 
outcomes for visit 2. Participants will be scheduled to return to the GoHealth Clinical Research Unit 
at one week after the baseline assessment, during which additional salivary (1 time point, 2 mL of 
saliva over 5 min, no rinse will occur) and blood samples will be collected.  At the conclusion of 
sample collection, the participant will undergo a periodontal exam. Study participation concludes 
following the periodontal exam at the end of the second on site study visit. Patients with any 
periodontitis or gingivitis diagnosis will be informed of their diagnosis and will be referred to an 
oral healthcare provider for further treatment. Patients’ final periodontal status will be a tertiary 
outcome of the periodontal exam and will be reported as the percentage of participants categorized 
into each diagnostic group; patients with a periodontal diagnosis will be informed of the findings 
and will be referred to an oral healthcare provider for further treatment. Participants who provide 
salivary and blood samples and follow study protocols will receive a $30 gift card as compensation 
at the end of visit 1 and $50 at the end of visit 2. This study involves two 90-minute visits. 
 
Interventions and Duration 
Subjects will have a brief oral exam and will be asked to rinse with an unlabeled/blinded antiviral 
mouth rinse for 30-60 sec and provide 2 mL of saliva prior to the rinse and 2 mL of saliva 


6 
 
immediately post-rinse, 15 min post-rinse, 30 min post-rinse, 45 min post-rinse, and 60 min post-
rinse. Subjects will also be asked to complete a short survey about the rinsing experience. A blood 
sample will be taken. Subjects will be onsite for a 90 min initial visit appointment. Patients will be 
sent home with Colgate toothpaste and mouthrinse for a daily oral health regimen, where 
participants will brush and rinse twice daily and maintain a take-home diary on their oral care 
regimen and COVID-19 symptoms. Patients will return for a second visit one week later for a 
salivary sample, blood draw and periodontal exam.  
Sample Size and Population 
We will enroll 30 outpatient SARS-CoV-2+ subjects per mouth rinse, (to achieve a total of 25 
subjects with full data sets per mouth rinse,) with 5 mouth rinses, requiring us to enroll 125 SARS-
CoV-2+ patients. Patients will be randomized to their mouthrinse. These patients will have already 
had a confirmed SARS-CoV-2+ test prior to enrollment. There will be no stratification to our 
randomization. In addition to having a SARS-CoV-2+ test, patients will need to be at least 18 y.o.a. 
and at most 65 y.o.a. and in fair oral health without any known allergies or reactions to commercial 
dental products or mouthrinses. Patients who are pregnant, nursing/lactating, have a cognitive or 
developmental disability, have kidney dysfunction, have an active oral herpes flare up, have any 
oral viral infection and/or flare up, have significant mucosal tears, growths or damage to their 
mouth, have uncontrolled severe periodontal disease with bleeding gums, or those with severe 
xerostomia will be excluded from the study. There are no exclusion criteria based on gender or 
race.  
 
Table of Abbreviations and Definition of Terms 
Abbreviation / Term 
Definition 
Additional details or range of 
value 
baseline 
Refers to the blood and saliva sample 
collection time point prior to mouth rinse use 
at study visit 1. 
 
BOP 
Bleeding on probing- sites where the 
gingiva/gum bleeds after probing 
0 not bleeding; 1 bleeding 
CDART 
Carolina Data Acquisition and Reporting Tool 
0mm to 20mm, recession plus 
probing depth 
CAL 
Clinical attachment level 
Probing depth plus recession 
(mm) 
Co-I 
Co-investigator 
CT 
Cycle threshold 
refers to the number of cycles 
needed to amplify viral RNA to 
reach a detectable level 
Cytokine / chemokines assessed in this 
study 
Interleukin-6 (IL-6), Interleukin-10 (IL-10), 
Interleukin-8 (IL-8), C-X-C Motif Chemokine 
Ligand 10 (CXCL-10) 
EDTA 
Ethylenediaminetetraacetic acid 


7 
 
FBS 
Fetal bovine serum 
GI 
Modified Gingival index 
0-3 
hr 
hours 
infectivity 
Reduction factor in infectivity 
MEM 
Minimum essential media 
mL 
milliliters 
mRNA 
Messenger ribonucleic acid 
OD 
Optical density 
Measured in nm on a 
spectrophotometer retrofitted with 
a plate reader 
PBS 
Phosphate buffered saline 
PFU 
Plaque forming units 
pg 
picogram 
PHI 
Protected health information 
PI 
Principle investigator or modified Plaque 
Index (depends on context) 
0-3 
PD, PPD 
Periodontal Probing depth (PPD, PD); pocket 
depth- Refers to pocket depth (measured in 
mm) upon periodontal probing 
0 to 15mm; measured with a 
periodontal probe 
RDC 
Respiratory Diagnostic Center 
 
RF 
Reduction factor 
RF values [ = log10 ( loadpre / loadpost )] 
RF will be calculated as the 
difference in the quotient of 
the infection titre before 
(‘control titration’) and after 
the mouthrinse  (‘remaining 
virus’). The log10 titre and its 
(double) standard deviation 
(SD) will be calculated as well 
as the variance of the RF. 
RNA 
Ribonucleic acid 
RQ 
Relative quantification 
for RQ the −ΔΔCt values [ =log2 (2−ΔΔCt )] 
RQ will be calculated to 
determine viral transcript changes 
across the time course for each 
individual participant. RQ is 
calculated using the  2(-delta 
delta Ct) method.  
RT-PCR 
Reverse transcription - polymerase chain 
reaction 
SD 
Standard deviation 


8 
 
sec 
seconds 
y.o.a. 
Years of age 
°C 
Degrees celsius 
µL 
microliters 
 
1.1 Primary Objective: 
The primary objective of this study is to determine the efficacy of antiseptic mouthwashes on 
reducing SARS-CoV-2 cellular infectivity in COVID+ patient saliva. We hypothesize that SARS-
CoV-2+ patients will have a >20% 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 primary objective will focus on quantifying SARS-CoV-2 viral infectivity (Reduction Factor 
(RF) calculated from plaque forming units [PFU] / mL) and viral titer (Relative Quantification (RQ) 
ratio) using an in vitro infectivity assay and RT-PCR. 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 SARS-CoV-2+ 
transmission. 
1.2 Secondary Objective:  
The secondary objectives are to measure cytokine and chemokine levels (pg/mL) in samples 
taken from SARS-CoV-2+ patients and also to determine the frequency of periodontal disease 
among SARS-CoV-2+ patients. We hypothesize that SARS-CoV-2+ patients using Colgate oral 
health products will have lower inflammatory marker levels than controls. Furthermore, we 
hypothesize that SARS-Cov-2+ patients will have a greater incidence of periodontal disease than 
the general population. 
 
1.3 Tertiary Objective (Exploratory):  
The tertiary objective of this study is to use post-rinse surveys to evaluate patient perceptions of 
the mouthrinse and feedback on their willingness to use the product (units: percentage of 
respondents). Additionally, a periodontal exam will be performed to assess periodontal health. 
Patients’ final periodontal status will be the tertiary outcome of the periodontal exam and will be 
reported as the percentage of participants categorized into each diagnosis. This tertiary outcome is 
exploratory. 
 
 
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 


9 
 
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 and Povidone-Iodine (PVP) solutions have been 
shown to reduce SARS-CoV-2 viral infectivity levels and titers in vitro.12-15 Cetylpyridinium chloride 
has promising in vitro data for inactivating SARS-CoV-2 infectivity.16 Chlorhexidine Gluconate 
(0.12%) has been widely adopted by practices with variable antiviral 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 a preprocedural mouthrinse recommended by the ADA to 
prevent transmission.17 No in vivo clinical data regarding SARS-COV-2 exists to-date for most oral 
rinses including 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 
 
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.3,11-17 No in vivo clinical data regarding SARS-COV-2 exists to-
date for most oral rinses including 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. 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 


10 
 
promise that they will do the same to the SARS-CoV-2 virus. Effect size: Using data from Meiller et 
al. we calculated power at four time points.18 Time points included: Post-0 (immediately after rinse), 
Post-30 (30 min after rinse), and Post-60 (60 min after rinse). In addition, we calculated a Post-45, 
which was the midpoint between 30 and 60 min in the Meiller et al. data. Based on this viral study 
evaluating mouth rinse efficacy against infectivity of Herpes Simplex Virus, an enveloped virus, 
power calculations were determined to estimate effect size and determine sample size as a function 
of length of mouth rinse time course.18 The sample size was justified by a paired T-test on outcomes 
between time points. With three simultaneous comparisons (t1-t0, t2-t1, t2-t0) over 30 min, an n=16 
sample size can detect the effect size in Cohen’s d=0.7, which is between medium and large, with 
80% power and 0.016 type-I error rate by Bonferroni correction. Therefore, with four simultaneous 
comparisons (t0-t1, t0-t2, t0-t3, t0-t4 ) the sample size required for each arm is ~25 participants per group to 
discern oral rinse efficacy at any given time point.  
 
Subjects will be required to rinse their mouths with an unlabeled/blinded mouthrinse for 30-
60 sec. Per the instructions on the product label, it is recommended to rinse for 30-60 sec, 
depending on the rinse (on-label directions will be followed). 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 min, 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. 
 
Peripheral blood will be collected at the end of salivary collection.Participants will be 
provided materials and oral hygiene instruction related to daily use of oral hygiene products 
provided by the study. Participants will be asked to keep a daily diary of oral hygiene performance, 
product usage, COVID-19 symptoms and exposures. During the week between study visits, 
participants will be directed to brush with Colgate toothpaste (at least twice per day) and rinse with 
the Colgate mouthrinse (according to on label instructions). Participants will be scheduled to return 
to the GoHealth Clinical Research Unit at one week after the first visit, during which additional 
salivary (1 time point, 2 mL of saliva over 5 min, no antiseptic mouth rinse will occur) and blood 
samples will be collected  (2 - 5 mL in EDTA-containing blood collection tubes).  At the conclusion 
of sample collection, the participant will undergo a periodontal exam. Study participation concludes 
following the periodontal exam.  
Samples will be evaluated at both timepoints for inflammation-related cytokines and 
chemokines using pre validated quantitative multiplex assays (Luminex technologies), diaries will 
be reviewed and periodontal charting will be evaluated for differences between patient groups. A 
periodontal exam will consist of periodontal probing depths (PD), clinical attachment level (CAL) 
and bleeding on probing (BOP) at six sites for each tooth excluding third molars. Once collected, 
the periodontal exam data will be evaluated and the periodontal status for each participant will be 
assigned via the case definitions for periodontitis, gingivitis, and clinical periodontal health as 
described in the 2017 World Workshop on the Classification of Periodontal and Peri-Implant 
Diseases and Conditions and the 2012 Centers for Disease Control and Prevention and American 
Academy of Periodontology case definitions.19, 20 Patients will be informed of their periodontal status. 
Patients with any periodontitis or gingivitis diagnosis will be informed of their diagnosis and will be 


11 
 
referred to an oral healthcare provider for further treatment. Patients’ final periodontal status will be 
the tertiary outcome of the periodontal exam and will be reported as the percentage of participants 
categorized into each diagnostic group. This study involves two on-site 90-minute visits. 
 
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 SARS-CoV-2 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 
1.5% w/v  hydrogen 
peroxide rinse 
(Peroxyl) 
• 
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 (Periogard) 
• 
The most common (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. 


12 
 
1:1.5% w/v  hydrogen 
peroxide rinse 
(Peroxyl) 
2nd: 0.12% 
Chlorhexidine 
Gluconate (Periogard) 
• 
No additional adverse effects documented from sequential use. 
• 
Please see above for adverse effects of each rinse individually. 
• 
For the sequential Peroxyl (1st) and Periogard (2nd) rinses, both oral rinses will 
be conducted in an on label fashion. The patient will begin by rinsing with 10 
mL of Peroxyl for 60 seconds and then expectorate the mouthrinse into an 
empty tube. Then the patient will rinse with 15 mL of Periogard for 30 seconds, 
and spit this mouthrinse into a separate tube. 
0.075% 
Cetylpyridinium 
Chloride (Colgate 
Total Zero) 
• 
If more than used for rinsing is accidentally swallowed, get medical help or 
contact a Poison Control Center right away 
Sterile Water 
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 
One of the aims of this placebo-controlled, randomized, double-blinded study is to measure 
the efficacy of antiseptic mouthwashes on reducing SARS-CoV-2 cellular 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 7 days of symptom onset. Subjects’ samples are 
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 give a baseline salivary sample (2 mL over 5 min) and then asked to rinse with an mouth 
rinse for 30-60 sec (on-label instructions) according to the table below: 
 mouthrinse* 
Number of final participants 
(sample size)  
Group 
1.5% w/v hydrogen peroxide rinse 
(Peroxyl) 
25**  
Experimental 
0.12% Chlorhexidine Gluconate (Periogard) 25 
Experimental 


13 
 
1st Peroxyl; 2nd Periogard^ 
25 
Experimental 
0.075% Cetylpyridinium Chloride (Colgate 
Total Zero) 
25 
Experimental 
Sterile Water 
25 
Control 
*All mouthrinses are commercially available in the USA, routinely used in clinical settings, and will 
be used in an on-label manner. 
**30 participants will be enrolled per regimen to ensure that complete data will be obtained from at 
least 25 participants per regimen. We assume that ≤ 5 enrollees will drop out or have incomplete 
data.30 participants will be enrolled per arm to collect full data sets from 25 participants per arm, 
assuming some patients will need to be replaced due to inability to complete a visit (missing data).  
^For the sequential Peroxyl (1st) and Periogard (2nd) rinses, both oral rinses will be conducted in 
an on-label fashion. The patient will begin by rinsing with 10 mL of Peroxyl for 60 sec and then 
expectorate the mouthrinse into an empty tube. Then the patient will rinse with 15 mL of Periogard 
for 30 seconds and spit this mouthrinse into another empty tube.  
 
After the participant expectorates the mouth rinse, research staff will collect another salivary 
sample (T0, 2 mL over 5 min). During a 10 min 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. At 15 minutes 
post-rinse, the participant will be asked to provide an additional saliva sample (2 mL over 5 min), 
followed by another 10-minute recuperation period. This sequence will be repeated for 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).  
The samples will be stored and used for determining SARS-CoV-2 viral load by RT-PCR 
and inflammation-associated cytokine and chemokine concentration (pg/mL).   The impact of 
mouthrinses on viral load in samples will be using a Relative quantification (RQ) ratio. A cycle 
threshold cut off (see sections 9.4-9.5) of 22 will be used for determining which samples will be 
used in downstream viral infectivity assays. Viral infectivity will be calculated (see sections 9.4-
9.5) and yield a Reduction Factor as quantified output. Peripheral blood will be collected at the end 
of salivary collection (2 - 5 mL in EDTA-containing blood collection tubes).  
Participants will be provided materials and oral hygiene instruction related to daily use of 
oral hygiene products provided by the study. During the week between study visits, participants will 
be directed to brush with Colgate toothpaste (at least twice per day) and rinse with the Colgate 
mouthrinse according to on-label instructions (10-20 mL, 30-60 seconds, 2-3 times per day 
depending on mouthrinse).  
Participants will be asked to keep a daily diary of oral hygiene performance, product usage, 
COVID-19 symptoms and exposures. Participants will be given a $30 gift card for their 
participation in visit 1.  Participants will be scheduled to return to the GoHealth Clinical Research 
Unit at one week after the baseline assessment, during which additional salivary (1 time point, 2 mL 


14 
 
of saliva over 5 min, no rinse will occur) and blood samples  (2 - 5 mL in EDTA-containing blood 
collection tubes) will be collected.  
At the conclusion of sample collection, the participant will undergo a periodontal exam and 
will receive a $50 gift card for their participation in visit 2. Study participation concludes following 
the periodontal exam after visit 2.  
Missing data may occur due to participants being unable to complete a study visit due to 
health concerns or insufficient salivary production. Any missing data will be excluded from the 
study analysis. We do not anticipate that this will bias the data in any way. To accommodate this 
eventuality, we plan to enroll 30 participants per arm, to be able to have 25 participants per arm 
with full datasets.  
All salivary samples will be collected on ice in sterile 50 mL conical vials and blood will be 
collected in EDTA-containing blood collection tubes. All collection tubes will be labelled “COVID 
Core” using pre-printed labels including the participants random alphanumeric study ID. Saliva 
samples will be cataloged and stored at the UNC Delta COVID Translational Initiative (also 
referred to as the COVID Core) for future RT-PCR, cytokine and chemokine concentration 
(Luminex) and viral infectivity assays.  Samples will be transported on ice in a secondary container 
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).  
Participants who successfully provide salivary and blood samples and follow study protocols will 
receive a $30 gift card as compensation at the end of visit 1 and $50 at the end of visit 2. This study 
involves two, 90 minute on site visits. 
During the period that the study participants are present in the clinic and giving saliva samples, 
we will monitor patient wellbeing and inquire about their comfort. If participants exhibit signs of 
distress or if oxygen saturation drops, research study staff will contact emergency medical responders 
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 investigate if any of the antiseptic 
mouthwashes are effective at reducing SARS-CoV-2 in salivary secretions of 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 (~1.5 hrs in length).  Further, the design will allow for a 
kinetic analysis of viral shedding in salivary secretions and to identify 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: Webster, Wallet and Maile) by pre-authorized 
personnel. Samples will be assessed for 1) molecular presence of SARS-CoV-2, 2) salivary antibody 
responses to SARS-CoV-2, 3) in vitro infectibility and 4) inflammatory biomarkers. 
1. Molecular analysis: For molecular analysis, samples will be inactivated by adding 2 mL 
of TriZol LS reagent to each 500 µL saliva aliquot (4:1 ratio) and mixed by inversion. These 
samples will be further aliquoted at 250 µL in 1.5 mL Eppendorf tubes and stored in an 
“Inactivated Samples -80 °C Freezer” until further analysis. 


15 
 
2. Antibody responses: an aliquot of saliva will be inactivated in a 56 °C water bath for 30 
min. 250 µL 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 min 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 PFU 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 µL 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 identify clinical and other factors 
impacting applicability of each of the mouth rinses.  
4. Detection of Inflammatory biomarkers: Inflammation-associated cytokine and 
chemokine concentrations (IL-6, CXCL-10, IL-8, and IL-10 [pg/mL]) will be quantified from 
saliva and blood samples using multiplex analyte analysis (Luminex technology) and using 
internal, manufacturer supplied standards of known concentration (pg/mL).  These analyses 
will be performed by the DELTA Translational Core according to COVID Core SOP (PI: 
Wallet and Maile). The COVID Core staff routinely performs these analyses on samples from 
SARS-CoV-2+ research study participant samples.  
 
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 SARS-CoV-2+ status. Either became symptomatic in the prior 7 days, or if not 
symptomatic, likely infected/exposed within the prior 7 days. All patients listed from the 
UNC Respiratory Diagnostic Center (RDC) have a confirmed SARS-CoV-2 infection and 
have consented to be contacted for research purposes. For patients contacting study 
coordinators for enrollment, who were not tested in the RDC, they must provide written 
proof of positive SARS-CoV-2 status in the prior 7 days.  
• Individuals (all sex, all gender) at least 18 y.o.a. and at most 65 y.o.a. and in good oral 
health without any known allergies to commercial dental products or cosmetics. ASA class I 
or II prior to SARS-CoV-2 infection. 
• 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.  


16 
 
• 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 y.o.a. and older than 65 y.o.a. 
• Subjects presenting with and/or self-reporting any of the following will not be included in 
the study: 
o history of significant adverse effects following use of oral hygiene products such as 
toothpastes and mouthrinses. (self-reported) 
o Self-reported allergy to hydrogen peroxide, peroxyl, chlorhexidine gluconate, periogard, 
peridex, colgate total zero, colgate total, cetylpyridinium chloride, essential oils 
(Eucalyptol, Menthol, Methyl salicylate, Thymol), 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). 
o History of serious medical conditions that, at the discretion of the Investigator, will 
disqualify the subject. (Self-reported) 
o A history of severe dry mouth (xerostomia), severe drug-induced xerostomia 
(antidepressants, anticonvulsants, antihypertensives), or Sjogren’s syndrome  
o 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) 
o Current history of alcohol or drug abuse (self-reported).  
o History of drinking water or eating food within an hour of the study visit.  
o History of drinking alcohol within 12 hours of the study visit.  
o History of using a commercial mouthrinse within 24 hours of the study visit. 
o Participation in any study involving oral care products, concurrently or within the 
previous 30 days. (self-reported) 
o 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 
Chlorhexidine Gluconate during pregnancy and lactation, which cannot occur in a 
blinded, randomized trial.) 
o Other severe acute or chronic medical or psychiatric conditions 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.  
o Patient with developmental/cognitive disability that cannot self-consent, comprehend 
and follow the requirements of the study based on research site personnel’s assessment.  
o Patients with sizable mucosal tears, abrasions, growths or burns in the mouth  
o Patients with kidney dysfunction 


17 
 
 
 
The following table lists exclusion criteria specific to each mouth rinse proposed in the study: 
  Mouthrinse* 
Exclusions 
1.5% 
w/v  hydrogen 
peroxide rinse 
(Peroxyl) 
• 
Pregnancy and lactation 
o 
Although there is not sufficient specific clinical data on the use of 1.5 
o 
% 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 
(Periogard) 
• 
Carcinogenesis, Mutagenesis, Impairment of Fertility 
o 
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 
o 
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 
o 
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 
o 
Clinical effectiveness and safety of 0.12% Chlorhexidine Gluconate have not 
been established in children under the age of 18. 
1st: Peroxyl 
2nd: Periogard 
• 
No additional exclusions from sequential use are documented. 
• 
Please see above for adverse effects of each rinse individually. 
0.075% 
Cetylpyridinium 
Chloride 
(Colgate Total 
Zero) 
• 
Do not use in children under 6 
Sterile Water 
No contraindications 


18 
 
*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 (ASA 
Class I or II prior to SARS-CoV-2 
infection), decided at hte discretion of the study 
coordinator.  
Known allergies or significant adverse reactions following the use of oral 
hygiene products (toothpastes, mouthrinses), commercial cosmetics, and any 
ingredient in the mouthrinses (list will be provided).  
Diagnosed SARS-CoV-2+ status (within 
the past 48 hours) who became symptomatic in 
the prior 7 days, or if not symptomatic, was 
likely infected/exposed within the prior 7 days. 
Established history of severe Xerostomia (drug-induced or  autoimmune dry 
mouth), renal disease, Hepatitis C Virus (HCV), severe periodontal disease with 
actively bleeding gums, significant oral abrasions/ulcers or growths, current 
alcohol abuse, and/or current recreational drug abuse. Other severe acute or 
chronic medical or psychiatric conditions 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 the participant 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. 
  
 


19 
 
 
 
4.3  Study Enrollment Procedures 
Outpatient subjects will be recruited from patients seen in the UNC Respiratory Diagnostic 
Center (RDC) who have tested positive for SARS-CoV-2+ and consented to be contacted for 
participation in COVID-related research studies. The potential subjects will have signed a prior 
facility consent at the RDC confirming their willingness to share their name and contact information 
to be contacted for study participation in COVID-19 related research. We will not receive 
information on subjects that decline this internal consent. A brief review of the patient's Epic chart 
will take place for medical screening and to identify any missing contact information, as needed. 
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, both 
appointments 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 SARS-CoV-2+ 
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.  
All of the consenting eligible patients will be enrolled in the study and will then be allocated 
to the treatment regimens by randomization A statistician 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 2 mL over 5 min. The patient will then be asked to rinse with an 
unlabeled/blinded antiseptic mouth rinse for 60 sec. Once the participant expectorates the mouth 
rinse, a time 0, 2 mL salivary sample will be collected over 5 min. During the 10 min recuperation 
period post rinse, the participant will answer 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. At 15 min post-rinse, the patient will be asked to provide an additional 2 mL 
saliva sample over the course of 5 min, followed by another 10 min break. This sequence repeats for 


20 
 
the 30, 45 and 60-min time points. At 60 min, the patient will give a final 2 mL salivary 
sample.  Peripheral blood will be collected at the end of salivary collection (2 - 5 mL in EDTA-
containing blood collection tubes).  
Participants will be provided materials and oral hygiene instruction related to daily use of 
oral hygiene products provided by study sponsors. In the one week between study visits, 
participants will be directed to brush with Colgate toothpaste (at least twice per day) and rinse with 
the Colgate mouthrinse (2-3 times per day based on-label directions). Participants will be asked to 
keep a daily diary of oral hygiene performance, product usage, COVID symptoms and exposures. 
Study personnel will monitor daily diary adherence and will reach out to participants between visits 
1 and 2 to ensure home oral hygiene protocol adherence by phone or email. Participants will be 
given a $30 gift card for their participation in visit 1.   
Participants will be scheduled to return to the GoHealth Clinical Research Unit at one week 
after the baseline assessment, during which additional salivary (1 time point, 2 mL of saliva over 5 
min, no rinse will occur) and blood samples will be collected.  At the conclusion of sample 
collection, the participant will undergo a periodontal exam and will receive a $50 gift card for their 
participation in visit 2. Once collected, the periodontal exam data will be evaluated and the 
periodontal status for each participant will be assigned via the case definitions for periodontitis, 
gingivitis, and clinical periodontal health as described in the 2017 World Workshop on the 
Classification of Periodontal and Peri-Implant Diseases and Conditions and the 2012 Centers for 
Disease Control and Prevention and American Academy of Periodontology case definitions.19, 20 
Patients will be informed of their periodontal status. Patients with any periodontitis or gingivitis 
diagnosis will be informed of their diagnosis at the time of the visit and will be referred to an oral 
healthcare provider for further treatment. This study involves two on-site 90 minute visits. Study 
participation concludes at the end of the second on site visit with no additional follow-up, as results 
are not relevant to the patient's clinical course and treatment management.  
 
5.2 Handling of Study Interventions 
Mouthrinses will be acquired from the following sources: 
mouthrinse* 
Source: Colgate company providing their commercially available, 
commercially packaged and ready-for-sale products 
1.5% w/v Hydrogen 
Peroxide rinse 
Peroxyl- Colgate 
0.12% Chlorhexidine 
Gluconate 
Periogard- Colgate 
0.075% Cetylpyridinium 
Chloride 
Colgate Total Zero- Colgate  


21 
 
Sterile water 
CVS 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 in a 50 mL sterile conical tube.  The 
conical tube will be unlabeled so both the participant and study personnel will be blinded to 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 mouthrinse options 
and using commercially available Colgate toothpaste and mouthrinse at home, as 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 30-60 seconds (as specified on the label) and 
then will spit it out, followed by donating saliva and blood samples. 
5.3.3 Prohibited Interventions 
Patients who have brushed their teeth, drank or eaten within an hour of the study visit will be 
directed to wait until an hour has elapsed; if they cannot wait, the patient 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 currently 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 30-60 sec (as directed on the label) 
before expectorating, and then providing saliva samples at 15 min time points and a blood sample.  
 
 
 
 
 


22 
 
 
 
 
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) 
Visit 2 
Informed Consent Form  
X 
X  
 
Demographics 
X 
 X 
Medical History 
X 
X  
X 
Current Medications 
X 
X 
X 
Blood Chemistries 
X 
X 
Urine Analysis- pregnancy 
X 
Vital Signs 
X 
X 
Brief Oral Exam 
X 
X 
Periodontal Exam 
X 
Inclusion/Exclusion 
Criteria 
X  
X 
Enrollment/Randomization 
 X 
X 
Treatment Admin.Form 
  
X  
Concomitant Medications 
X  
X 
X 
Adverse Events 
X  
X 
X 
 
6.2 Description of Evaluations 
Subjects are required to be onsite for two 90-minute appointments. Subjects will be asked to 
rinse with an unlabeled/blinded antiviral mouth rinse for 30-60 sec and provide 2 mL of saliva prior 
to the rinse and 2 mL of saliva immediately post-rinse, 15 min post-rinse, 30 min post-rinse, 45 min 
post-rinse, and 60 min post-rinse. Subjects will also be asked to complete a short survey about the 
rinsing experience. A blood sample will also be collected, and a brief oral exam will occur.  At the 
end visit, a single saliva sample and blood sample will be taken, along with a periodontal exam.   


23 
 
 
 
 
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 trained study coordinators. 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. 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. A brief oral exam will take place to evaluate for existing severe oral trauma, severe 
periodontal disease, tumors or infection. 
• 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 RDC who have tested 
positive for SARS-CoV-2+ and consented to be contacted for participation in COVID-19-related 
research studies. The subjects being approached by phone or email have signed a prior facility 
consent at the RDC confirming their willingness to share their name and contact information to be 
contacted for study participation in COVID-19-related research. We will not receive information on 
subjects that decline this internal consent. A brief review of the patient's Epic chart will take place 
for medical screening and to identify any missing contact information, as needed. 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, appointments will be scheduled for them at the 
Adams School of Dentistry Go Health Clinical research core. Subjects must be able to attend both 
visits, to be enrolled. Consent documentation will be signed in person and in private in the Go 
Health Clinical Research Core. A list of SARS-CoV-2+ subjects that have consented to be 
contacted for research, will be provided by the RDC clinic, sent via secure UNC servers or secure 


24 
 
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 
In this application, "baseline" refers to the initial saliva sample taken before the participant uses a 
mouthwash." For this baseline saliva sample, we will measure the salivary viral load and infectivity. 
The initial brief oral exam will also be a baseline visual inspection. 
 
Randomization 
A statistician will create a block randomization schedule and perform these computations; 
assignments will be placed in organized sequentially numbered opaque envelopes (SNOEs), which 
are opened as participants are enrolled. Block randomization will provide for group equality. Our 
sample is limited to adults aged 18-65years, to limit age variability and exclude people at highest 
risk. We will not preemptively stratify our sample based on our demographic variables (age, gender, 
race, BMI). However, after we have collected our data, we will check if there is an effect of these 
demographic features and then include an adjustment if appropriate.  
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. A secondary 
study coordinator will use a block randomization schedule, organized into SNOEs, to determine the 
mouthrinse for the next patient, and load it into an unmarked tube. This secondary study coordinator 
will have no interaction with the patients and the primary study coordinators and lab technicians. 
The primary study coordinator 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 mouthrinse. If a subject 
identifies the mouthrinse due to sensation or taste, this will not affect the primary and secondary 
outcomes as viral load and infectivity are quantitative laboratory assays. The primary study 
coordinator 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 conical to provide 
to the patient for the rinse. The laboratory technician who will conduct the RT-PCR, viral infectivity 
and cytokine/chemokine analyte analyses will be blinded to the mouthrinse used prior to the 
salivary sample collection.  
Prior to enrollment of participants, the trial statistician, Kevin Moss, will prepare the 
randomization schedule which will be concealed from all other study personnel. To conceal the 
treatment assignments, Wendy Lamm will prepare a set of sequentially numbered, opaque 
envelopes (SNOEs). The SNOEs will be used during recruitment to allocate enrollees to treatment 
regimens; i.e., for the next patient enrolled, the next envelope will be opened by an approved 
research staff who has been delegated responsibility for preparing the mouthrinse tubes for the 
enrollees. 


25 
 
Kevin Moss will set up a block randomization schedule that the team will abide by for 
assignment of enrolled subjects using sequentially numbered, opaque envelopes (SNOEs). 
 
 
 
6.2.4  Follow-up Visits 
This study requires two 90 minute visits. 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 two 90 minute visits. Participation is completed after the 2nd visit. For the 
second visit, participants will be scheduled to return to the GoHealth Clinical Research Unit at one 
week after the baseline assessment, during which additional salivary (1 time point, 2 mL of saliva 
over 5 min, no rinse will occur) and blood samples will be collected.  At the conclusion of sample 
collection, the participant will undergo a periodontal exam and will receive a $50 gift card for their 
participation in visit 2. Study participation concludes after visit 2 with no additional follow-up, as 
results are not relevant to the patient’s clinical course and treatment management.  
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, insufficient saliva production, 
missed appointment, or subject no longer meets the eligibility criteria. The reason for 
discontinuation will be documented in the CDART record.  
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 visit 1 $30 gift card and will 
be replaced. This would lead to missing data at the uncompleted time points. If a patient suffers 
from dry mouth, such that s/he cannot produce salivary sample volumes needed for later time points 
(45 min or 60 min post-rinse) but s/he produced sufficient baseline, immediate, 15 min and 30 min 
post-rinse samples, the participant will still be compensated with the visit 1 $30 gift card and 
parking vouchers, and will remain in the study for visit 2 participation, if willing. At the conclusion 
of the visit 2 on site appointment (after saliva and blood samples have been collected and the 
periodontal exam is completed), participants will receive a $50 gift card and parking vouchers.  If a 
participant fails to carry out the at-home oral hygiene regimen with diary, cannot provide salivary or 
blood samples at visit 2, or cannot undergo the oral exam, they will not be provided the $50 gift 
card for visit 2.  Missing or deficient samples will be noted in CDART. 
  


26 
 
7.0  Clinical Dental Measures 
Clinical dental measures include a comprehensive periodontal examination including the following 
measures and indices: UNC Modified Plaque Index (Greene & Vermillion); Modified Gingival 
Index, Löe and Silness ; Periodontal Probing Depth (PPD), Clinical attachment level (CAL) and 
Bleeding on Probing (BOP). 
  
 
The examination for all measurements will follow the sequence below (FDI numbering system): 
Quadrant 1 Buccal:  teeth 8 - 1 
Quadrant 2 Buccal:  teeth 1 – 8 
Quadrant 2 Lingual:  teeth 8 – 1 
Quadrant 1 Lingual:  teeth 1 - 8 
Quadrant 3 Buccal:  teeth 8 – 1 
Quadrant 4 Buccal:  teeth 1 – 8 
Quadrant 4 Lingual:  teeth 8 – 1 
Quadrant 3 Lingual:  teeth 1 – 8 
  
 
  
The comprehensive periodontal exam will yield a diagnosis of periodontal disease or health. 
Once collected, the periodontal exam data will be evaluated and the periodontal status for each 
participant will be assigned via the case definitions for periodontitis, gingivitis, and clinical 
periodontal health as described in the 2017 World Workshop on the Classification of Periodontal 
and Peri-Implant Diseases and Conditions and the 2012 Centers for Disease Control and Prevention 
and American Academy of Periodontology case definitions.19, 20 Patients will be informed of their 
periodontal status. Patients with any periodontitis or gingivitis diagnosis will be informed of their 
diagnosis and will be referred to an oral healthcare provider for further treatment. Patients’ final 
periodontal status will be the tertiary outcome of the periodontal exam and will be reported as the 
percentage of participants categorized into each diagnostic group. 
 
Study procedures and Periodontal evaluations  
Prior to the first subject study visit, study examiner(s) will be trained and calibrated for 
accuracy and repeatability in using the UNC Modified Gingival Index (GI), Loe and Silness, UNC 
Modified Plaque Index (Green & Vermillion), periodontal pocket depths, and clinical attachment 
levels using a UNC 15 periodontal probe. All examiners have a kappa > 0.90 for all calibration 
sessions. Calibration training will be conducted on an annual basis according to the guidelines set 
forth in this document including: Periodontal determination of plaque (PI), gingival inflammation 
(GI), gingival bleeding (BOP), pocket probing depths (PD) and clinical attachment levels (CAL). 
These parameters are customarily used in clinical dental studies to measure the disease status of 


27 
 
gingivitis and periodontitis. Probe penetration and depth may vary with the degree of inflammation, 
probing force, angulation, position, and instrument tip diameter. 
  
Other confounding factors include patient discomfort, accuracy of probe markings, 
anatomical differences in tooth crown and roots, and technique variability within and between 
examiners. Thus, all of these measurements are important to consider in periodontal clinical studies 
and this training session is designed to minimize the inter-examiner variance. 
 
7.1  UNC Modified Gingival Index (GI), Loe and Silness 
Full mouth gingival scores shall be visually assessed by segmenting marginal and papillary units, 6 
sites per tooth: distobuccal, buccal, mesiobuccal and distolingual, lingual, mesiolingual surfaces. 
0 = Normal gingiva 
1 = Mild inflammation; slight change in color, slight edema. No bleeding on probing. 
2 = Moderate inflammation: redness, edema, and glazing. Bleeding on probing 
3= Severe inflammation; marked redness and edema. Ulceration. Tendency for spontaneous 
bleeding. 
  
7.2  UNC Modified Plaque Index (Greene & Vermillion) 
Full mouth plaque assessment shall be assessed using the UNC Modified Plaque Index (Greene and 
Vermillion). Plaque scores shall be visually assessed at 6 sites per tooth (distobuccal, buccal, 
mesiobuccal and distolingual, lingual, mesiolingual surfaces) on a scale of 0-3. 
0 =No debris or stain present on the clinical crown. 
1 =Soft debris covering not more than 1/3 of the clinical crown (cervical 3rd), or presence 
of extrinsic stains without other debris regardless of surface area covered. 
2 =Soft debris covering more than 1/3, but not more than 2/3 (middle 3rd) of the clinical 
crown. 
3 =Soft debris covering more than 2/3 of the clinical crown 
  
7.3  Pocket depth (PD): Linear distance from the gingival margin (GM) to base of the pocket. If a 
PD reading falls between two millimeter readings, the rule shall be to round down and the lower of 
the two readings will be recorded. 
  
7.4  Bleeding on Probing (BOP): Presence or absence of bleeding to manual probing recorded as a 
dichotomous variable. 
0 =No bleeding within 10 seconds after probing. 
1 =Bleeding within 10 seconds after probing. 
  
7.5  Clinical Attachment Level (CAL): Linear distance from the cemento-enamel junction (CEJ) 
to base of the pocket. If a CAL reading falls between two millimeter readings, the rule shall be to 
round down and the lower of the two readings will be recorded. 
 
8. SAFETY ASSESSMENTS 


28 
 
The following table lists possible adverse effects associated with each mouthrinse proposed in this 
study: 
mouthrinse* 
Adverse effects 
1.5% w/v  Hydrogen 
Peroxide rinse 
(Peroxyl) 
• 
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 (Periogard) 
• 
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. 
1st: 1.5% w/v Hydrogen 
Peroxide rinse 
(Peroxyl) 
2nd: 0.12% Chlorhexidine 
Gluconate (Periogard) 
• 
No additional adverse effects documented from sequential use. 
• 
Please see above for adverse effects of each rinse individually. 
0.075% Cetylpyridinium 
Chloride (Colgate Total 
Zero) 
• 
If more than used for rinsing is accidentally swallowed, get medical help or 
contact a Poison Control Center right away 
Sterile Water 
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 and collection of saliva carry minimal risk for 
participants, apart from the very rare, undiagnosed allergy, and the unlikely event that the sample is 


29 
 
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.  
 
 
8.1  Specification of Safety Parameters 
Mouthrinse and saliva collection carries low risk and minimal to no safety concerns to the 
participants. Blood draws and oral exams are routine healthcare screening procedures that carry 
minimal safety concern to 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.  
 
8.2  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, tooth pastes, and collection of saliva and blood carry minimal risk for participants, 
apart from the very rare, undiagnosed allergy, which would be managed through proper referral to 
the neighboring UNC hospital. 
 
8.3  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 and toothpastes and collection of saliva and 
blood 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.  
8.4  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.  
 


30 
 
8.5  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. 
8.6   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. 
 
9. 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, insufficient saliva production, 
missed appointment, failure to comply with oral hygiene regimen, subject no longer meets the 
eligibility criteria. The reason for discontinuation will be documented in the patient’s CDART 
record.  
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 visit 1 $30 gift card and will be replaced. If a participant suffers from dry 
mouth, such that they cannot produce salivary sample volumes needed for later time points (45 min 
or 60 min post-rinse) but produced sufficient baseline, immediate, 15 min and 30 min post-rinse 
samples, s/he will still be compensated with the visit 1 $30 gift card and parking vouchers, and will 
remain in the study for visit 2 participation, if willing.  
At the conclusion of the visit 2 appointment (after saliva and blood samples have been 
collected and the periodontal exam is completed), participants will receive a $50 gift card and 
parking vouchers.  If a participant fails to carry out the at-home oral hygiene regimen with diary, 
cannot provide salivary or blood samples at visit 2, or cannot undergo the oral exam, s/he will not 
be provided the $50 gift card for visit 2.  Missing or deficient samples will be noted in CDART. 
 
 
10. STATISTICAL CONSIDERATIONS 
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.  
 


31 
 
 
10.2   Research Hypotheses 
The central hypothesis of this study is that COVID-19+ patients will have a >20% reduction in SARS-
CoV-2 viral infectivity following oral rinse with an antiseptic mouthrinse and that this reduction will 
persist for at least 30 minutes.  
The secondary hypothesis is that COVID-19+ patients using Colgate oral health products will have 
lower inflammatory marker levels than controls. Furthermore, we hypothesize that COVID-19+ 
patients will have a greater incidence of periodontal disease than the general population. 
 
Sample Size Considerations 
Using data from Meiller et al. we calculated power at four time points.18 Time points included: Post-
0 immediately after rinse, Post-30 (30 min post rinse), and Post-60 (60 min post rinse). In addition, 
we calculated a Post-45, the midpoint between 30 and 60 min, in the Meiller et al. data. Based on this 
viral study evaluating mouth rinse efficacy against viral infectivity of Herpes Simplex Virus, an 
enveloped virus, power calculations were determined to estimate effect size and determine sample 
size as a function of length of mouth rinse time course.18 The sample size was justified by a paired T-
test on outcomes between time points. With three simultaneous comparisons (t1-t0, t2-t1, t2-t0) over 30 
min, an n=16 sample size can detect the effect size in Cohen’s d=0.7, which is between medium and 
large, with 80% power and 0.016 type-I error rate by Bonferroni correction. Therefore, with four 
simultaneous comparisons (t0-t1, t0-t2, t0-t3, t0-t4 ) the sample size required for each arm is ~25 participants 
per group to discern oral rinse efficacy at any given time point (minutes post rinse). 
Statistical analysis of our data will be performed by Kevin Moss, the statistician on this 
project.Statistical analysis of our data will be performed by Kevin Moss, the statistician on this 
project. Kevin Moss will be involved in all stages of this project to help ensure statistical rigor. 
 
Primary Outcomes 
The analyses will compare viral titre (RT-PCR) and viral infectivity levels between mouth rinse types 
and across the time course. 
Table: Variable Types for Primary Outcomes 
 
Viral Infectivity (Continuous) 
Viral Load (Continuous) 
Age, Years (Continuous) 
 
 
Race (Categorical) 
 
 
Sex (Categorical) 
 
 
BMI (Continuous) 
 
 
Time (Categorical) 
 
 
StudyGroup (Categorical) 
 
 
 


32 
 
  
Data generated from the RT-PCR assays (relative quantification (RQ) ratio, numerical value) 
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 continuous 
numerical scales, with multiple testing adjustments. After performing the main analysis using a linear 
model specified a priori, various sensitivity analyses will be performed. These will include an analysis 
of residuals which will rely on graphical methods, examination of skewness, kurtosis, and other 
descriptive statistics. The sensitivity analyses may also include a Shapiro-Wilk test of the null 
hypothesis Ho ‘the underlying distribution of the residuals is Gaussian’. The sensitivity analyses may 
also include assessment of the impact of using a modified version of the linear model analysis in 
which the scale of the dependent variable is transformed (e.g., log10 or some other Box-Cox 
transformation). The sensitivity analyses will be used only to guide our level of trust in the main 
results.  
For the primary analysis, viral infectivity and viral load, we will first determine if these 
variables are normally distributed.  However, we anticipate these variables to be right skewed. They 
will be log transformed if they are not normally distributed.  We will then compare the demographic 
variables (age, race, sex, age and BMI) and groups (study arms).  We will use Chi-square and 
ANOVA statistics to determine if the demographic variables are evenly distributed amongst the 
groups.  If not we will adjust for these variables in subsequent analyses.  In the rare event there are 
multiple demographic variables that are found to be significantly different by group we will create a 
propensity score to adjust for these variables in subsequent analyses.  
The primary analysis will be ANCOVA models with viral infectivity at time Post 0 minute, 
Post 1 minute, post 15 minute, post 30 minute, post 45 minute and post 60 minute adjusting for time 
Pre 0 minute.  We will also perform a repeated measures analysis looking at viral infectivity over 
time, with the advantage of having one model with more power, while the ANCOVA model may give 
us a clearer answer if the rebound effect of viral infectivity is exponential.  We will adjust for any 
demographic variables that are found to be significantly different between groups. We will adjust for 
any demographic variables that are found to be significantly different between groups. 
The viral infectivity analysis will compare infectivity levels between mouth rinse types and 
across the time course for calculation of the reduction factor (RF calculated from PFU/mL, numerical 
value). 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. 
 
Secondary Outcomes 
Inflammation-associated cytokine and chemokine concentration (pg/mL) will be quantified using a 
Luminex bioanalyzer containing pre-validated internal standards of known concentration. Saliva and 
serum analyte concentrations will be compared between groups using descriptive statistics. 
Periodontal exam results will be reviewed to categorize patients’ periodontal status which will be 
reported as frequencies.    
Tertiary Outcomes 
The tertiary outcomes of this study are a periodontal exam and post-rinse surveys to evaluate patient 
perceptions of the mouthrinse, their symptoms and feedback on their willingness to use the product. 
Percentage of respondents will be gathered and reported for surveys. Periodontal exam data will be 
evaluated and the periodontal status for each participant will be assigned via the case definitions for 
periodontitis, gingivitis, and clinical periodontal health as described in the 2017 World Workshop 


33 
 
on the Classification of Periodontal and Peri-Implant Diseases and Conditions and the 2012 Centers 
for Disease Control and Prevention and American Academy of Periodontology case definitions.19, 20 
Patients’ final periodontal status will be the tertiary outcome of the periodontal exam and will be 
reported as the percentage of participants categorized into each diagnostic group. 
 
 
10.2   Sample Size and Randomization 
Using data from Meiller et al. we calculated power at four time points.18 Time points included: 
Post-0 immediately after rinse, Post-30 (30 min post rinse), and Post-60 (60 min post rinse). In 
addition, we calculated a Post-45, which was the mid-point between 30 and 60 min in the Meiller et 
al. data. Based on this viral study evaluating mouth rinse efficacy against infectivity of Herpes 
Simplex Virus, an enveloped virus, power calculations were determined to estimate effect size and 
determine sample size as a function of length of mouth rinse time course.18 The sample size was 
justified by a paired t-test on outcomes between time points. With three simultaneous comparisons 
(t1-t0, t2-t1, t2-t0) over 30 min, an n=16 sample size can detect the effect size in Cohen’s d=0.7, which is 
between medium and large, with 80% power and 0.016 type-I error rate by Bonferroni correction. 
Therefore, with four simultaneous comparisons (t0-t1, t0-t2, t0-t3, t0-t4 ) the sample size required for each 
arm is ~25 participants per group to discern oral rinse efficacy at any given time point (minutes post 
rinse).  
Kevin Moss, who is the statistical consultant on this project, will create a block randomization 
schedule and perform these computations. SAS Institute (SAS Cary, NC) Proc PLAN will be used to 
create the randomization schedule.  Block randomization will be used to ensure equal and random 
assignment into each study arm.  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. We will closely collaborate on Kevin Moss throughout all stages of this 
study to ensure statistical rigor.  
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 conical tube to 
provide to the patient for the rinse.  
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 proposed mouth rinses are commercially available over the counter, 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.  
 
 
10.3   Definition of Populations 
Our population includes patients 18-65 y.o.a. who have tested positive with COVID-19 and are 
either asymptomatic but exposed in the prior 7 days or are symptomatic with symptom development 
in the prior 7 days. Inclusion and exclusion criteria (described elsewhere) will be met.  We are not 
treating patients for SARS-CoV-2. 
 
 


34 
 
 
10.4   Study Stopping Rules 
Analysis will occur at several points, while enrolling and collecting samples from 125 patients, in 
batches of 25 randomized participants. Analysis will occur after enrollment of 25, 50, 100 and then 
125 participants and will include viral load and replication (as measured by changes in genomic, 
strand, and subgenomic expression and will be evaluated and compared between treatment groups). 
No interim analyses of data will be performed. mRNA obtained from salivary samples will be 
analyzed and will be assessed by RT-PCR at time points 0 and 30 min. Infectivity assays will be 
subsequently performed on salivary samples collected at time points 0 min (‘baseline’) and 30 min 
post rinse with cycle threshold of 22 or lower (CT22) as determined by RT-PCR using primers 
specific for SARS-CoV-2 viral products.  If the cycle threshold is above 22 (CT22), samples are 
considered to contain insufficient virus for use in viral infectivity assays. This cycle threshold has 
been empirically determined by the Baric laboratory (personal communication, Dr. Ralph Baric). 
 
If saliva samples from patients 6-7 days post-diagnosis around found to routinely have insufficient 
viral load at interim analysis (at n=25 or 50), the study will be modified for more stringent 
enrollment criteria (with inclusion up to 5 days post-diagnosis rather than 7, as salivary viral load 
diminishes with time after infection). 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 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.   
10.5   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 primary and secondary outcomes of RT-PCR (RQ), viral infectivity (RF), and 
cytokine/chemokine analyte concentration (pg/mL) for each sample type and each participant will 
be stored as a comma-separated values file analyzed using Excel (or similar) software. This data 
will be available to research study personnel weeks to months after sample collection when the 
experiments are conducted. Samples will be run in batches resulting in large spreadsheets of data. 
Periodontal exam data will be entered into CDART and can be exported as a comma-separated 
values file.  
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. 


35 
 
10.5.1   Primary Outcome  
The primary objective of this study is to determine the efficacy of antiseptic mouthwashes on reducing 
SARS-CoV-2 cellular infectivity in participant saliva. We hypothesize that COVID-19+ patients will 
have a >20% 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 primary 
objective will focus on quantifying SARS-CoV-2 viral infectivity and mRNA level using an in vitro 
infectivity assay (Reduction factor (RF) calculated from PFU/mL) and RT-PCR (relative 
quantification [RQ] ratio). 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.  
 
Outcome assessments will be conducted weeks to months after the study visit, when RT-PCR and 
viral infectivity assays are routinely conducted in the COVID Core and the laboratory of Dr. Ralph 
Baric, respectively. 
10.5.2   Secondary Outcomes 
The secondary objectives are to measure inflammation-associated cytokine and chemokine 
concentration (pg/mL) in saliva and blood samples taken from SARS-CoV-2+ participants and also 
to determine the frequency of periodontal disease among participants. We hypothesize that SARS-
CoV-2+ participants using Colgate oral health products will have lower blood and salivary 
inflammation-associated cytokine and chemokine concentrations (pg/mL) than non-intervention 
controls. Furthermore, we hypothesize that SARS-CoV-2+ participants will have a greater 
incidence of periodontal disease than the general population. Outcome assessments will be 
conducted weeks to months after the study visit, when statistical analyses are conducted. 
10.5.3   Tertiary Outcomes  
The tertiary outcomes of this study are a periodontal exam and post-rinse surveys to evaluate patient 
perceptions of the mouthrinse, their symptoms and feedback on their willingness to use the product. 
Percentage of respondents will be gathered and reported for surveys. Periodontal exam data will be 
evaluated and the periodontal status for each participant will be assigned via the case definitions for 
periodontitis, gingivitis, and clinical periodontal health as described in the 2017 World Workshop 
on the Classification of Periodontal and Peri-Implant Diseases and Conditions and the 2012 Centers 
for Disease Control and Prevention and American Academy of Periodontology case definitions.19, 20 
Patients’ final periodontal status will be the tertiary outcome of the periodontal exam and will be 
reported as the percentage of participants categorized into each diagnostic group. These tertiary 
outcomes are exploratory and therefore have no hypotheses assigned to them. 
 
 
 
 
 
 
 


36 
 
Table: Study Variables and Covariates 
X Independent variables 
Y Dependent variables for 
primary and secondary outcomes 
Covariates 
Mouthrinse Group 
Viral infectivity (continuous 
positive variable) 
Age (years) 
Time (in minutes, 0, 15, 
30, 45 and 60 minutes, 
visit 1 and 2) 
Viral load (continuous positive 
variable) 
Gender (m/f) 
Cytokine level (continuous 
positive variable) 
Race (White, Native Hawaiian or 
other pacific islander, Asian, 
African American, Hispanic or 
Latino, American Indian or Alaska 
Native) 
BMI Body mass index (continuous 
range: 0-56) 
 
 
10.6   Data Analyses 
qRT-PCR: 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 Student’s 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. 
 
Inflammatory Markers: Inflammation-associated cytokine and chemokine concentrations (IL-6, CXCL-10, IL-
8, and IL-10 [pg/mL]) will be quantified from saliva and blood samples collected at each visit and across the 
salivary collection time course using multiplex analyte analysis (Luminex technology) and using internal, 
manufacturer supplied standards of known concentration (pg/mL).  
The analysis plan will  include use of sensitivity analyses to evaluate the robustness/fragility of the study’s 
main results to reasonable perturbations of the statistical methods and assumptions used in the main analysis.  
We will first describe the demographics (age, race, sex and BMI) for each study arm.  If significant 
differences are found between groups, we will adjust the remainder of the analysis.  The main analysis will 
be a repeated measures linear regression model(s).  If multiple demographics are found to be significantly 
different the propensity scores will be created and final models will be adjusted by propensity scores.  In the 
primary analysis, viral load and replication as measured by changes in genomic, strand, and subgenomic 
expression, will be evaluated and compared between treatment groups.  Repeated measures linear regression 
models will be utilized to look at changes in viral load logarithmically before and after treatment. Similarly, 


37 
 
in the secondary analyses, the repeated measures linear regression will be used to compare the changes in 
other continuous outcomes (such as immunological parameters) from baseline to specific time points 
outlined. Statistical significance will be tested using a two-sided 0.05 level with adjustments for multiple 
testing.  Multiple testing correction will be made by Tukey adjustments (for multiple study arms) and False 
Discovery Rate (FDR) where appropriate (i.e. multiple inflammatory markers).   
 
All statistical estimates of population parameters will be tabulated along with corresponding confidence 
intervals (CIs) and/or standard errors (SEs) to convey level of precision. All P- values will be reported as 
continuous numerical outputs (and not as dichotomous results). All hypothesis tests yielding large p-value 
(e.g., p > α) will be reported as being inconclusive. 
Power Calculation: Using data from Meiller et al. we calculated power at four time points.18 Time 
points included: Post-0 (immediately post rinse), Post-30 (30 min post rinse), and Post-60, (60 min 
post rinse). In addition, we calculated a Post-45, which was the midpoint between 30 and 60 min in 
the Meiller et al. data. Based on this viral study evaluating mouth rinse efficacy against infectivity of 
Herpes Simplex Virus, an enveloped virus, power calculations were determined to estimate effect 
size and determine sample size as a function of length of mouth rinse time course.18 The sample size 
was justified by a paired t-test on outcomes between time points. With three simultaneous 
comparisons (t1-t0, t2-t1, t2-t0) over 30 min, an n=16 sample size can detect the effect size in Cohen’s 
d=0.7, which is between medium and large, with 80% power and 0.016 type-I error rate by Bonferroni 
correction. Therefore, with four simultaneous comparisons (t0-t1, t0-t2, t0-t3, t0-t4 ) the sample size required 
for each arm is ~25 participants per group to discern oral rinse efficacy at any given time point 
(minutes post rinse). 
 
 
11. DATA COLLECTION AND QUALITY ASSURANCE 
11.1 
Data Collection Forms 
Consented patients' data (including screening questions, demographics (age in years, race, sex (m/f) 
and BMI (continuous)), 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. 
 
11.2 
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 


38 
 
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), medical history, substance use history, 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. 
Home diary entries will be made on paper or using a secure UNC Qualtrix account link, depending 
on patients’ technological capabilities. 
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 accounts. 
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.  
 
10.3 
Quality Assurance 
11.3.1  Training 
All study personnel have been trained in health privacy (HIPAA), human subjects research and 
good clinical practices 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.    
 
 


39 
 
 
11.3.2  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 
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.  
11.3.3  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 min or 30 min 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 min post-rinse, the participant 
will remain in the study but their insufficient samples will be noted in CDART. If a patient is unable 
to tolerate the blood draws or periodontal exam, this will be noted in CDART and the patient will be 
withdrawn from the study.   
For RT-PCR, viral infectivity and multiplex Luminex 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, Webster and Wallet 
laboratories. SARS-CoV-2 viral infectivity and RNA level will be measured using an in vitro 
infectivity assay (Reduction factor [RF] calculated from PFU / mL) and RT-PCR (relative 
quantification [RQ] ratio). Cytokine and chemokine concentration (pg/mL) will be measured using 
a prevalidated multiplexed quantitative analyte assay on a bioanalyzer (Luminex technologies). 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.  
Outcome assessments will be conducted weeks to months after the study visit, when RT-PCR, viral 
infectivity and cytokine/chemokine assays are conducted in the COVID Core and the laboratory of 
Dr. Ralph Baric. 
The secondary objectives are to measure inflammation-associated cytokine and chemokine 
concentration (pg/mL) in saliva and blood samples taken from SARS-CoV-2+ participants and also 
to quantify the frequency of periodontal disease among study participants. The tertiary objectives of 
this study are a periodontal exam and to use post-rinse surveys to evaluate patient perceptions of the 
mouthrinse and feedback on their willingness to use the product (units: percentage of respondents). 
These tertiary outcomes are exploratory. Periodontal exams will be scored and reported according 
to the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and 
Conditions, co-presented by the American Academy of Periodontology (AAP) and the European 
Federation of Periodontology (EFP).19 
 
 
 


40 
 
 
11.3.4  Protocol Deviations 
Protocol deviations will be recorded within source documentation (CDART) for each participant by 
study personnel during the study visits. The investigator and study coordinators will meet weekly to 
review unanticipated problems, adverse events and protocol deviations. All such events will be 
appropriately reported to the IRB board of UNC.  
11.3.5  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. 
 
12. PARTICIPANT RIGHTS AND CONFIDENTIALITY 
12.1 
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. 
12.2 
Informed Consent Forms 
Outpatients, who have tested positive for SARS-CoV-2 and consented to be contacted for study 
enrollment in the UNC Respiratory Diagnostic Center (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 RDC confirming their willingness to share their name and contact information 
to be contacted for study participation in COVID-19 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 two visits, one week apart. 
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. 
 


41 
 
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,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. 
 
12.3   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 accounts. 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 experience rinsing with a commercially available mouth rinse, about their 
COVID-19 related symptoms and about their oral healthcare. General demographics will be asked 
such as gender, race, ethnicity and age bracket. Their identity cannot be deduced from this 
information.  
12.4 
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. 
13. 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.  
 
 


42 
 
 
14. 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. 
 
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(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). 
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Practice: Brief Review of Preventive Measures in Italy. Journal of Dental Research(2020) 
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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. 
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doi:10.1101/2020.06.11.20127332. 
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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 


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May Protect Healthcare Workers and Reduce Cross Infection. SSRN Electron. J.(2020) 
doi:10.2139/ssrn.3563092. 
16.      Statkute, E., Rubina, A., O’Donnell, V., Thomas, D., & Stanton, R. The Virucidal 
Efficacy of Oral Rinse Components Against SARS-CoV-2 In Vitro. BiorXiv. 
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17.      COVID-19 Frequently Asked Questions. American Dental Association: Center for 
Professional Success (2020). 
18. 
Meiller TF, Silva A, Ferreira SM, Jabra-Rizk MA, Kelley JI, DePaola LG. Efficacy 
of Listerine Antiseptic in reducing viral contamination of saliva. J Clin Periodontol. 
2005;32(4):341-346. doi:10.1111/j.1600-051X.2005.00673.x 
19. 
Tonetti et al. J Periodontol 89 (Suppl 1): S159-S172 [2018]; Trombelli et al. J 
Periodontol 89 (Suppl 1): S46-S73 [2018]; Chapple et al. J Periodontol 89 (Suppl 1) S74-
S84 [2018] 
20.  
Eke et al. J Periodontol 83: 1449-1454 [2012] 
 
16. SUPPLEMENTS/APPENDICES 
No supplements or appendices.  All forms are submitted as part of the IRB application.