Clinical trial NCT03602066
Conditions: Head and Neck Carcinoma
Sponsor: University of Arizona
Start date: 2019-02-14; Completion date: 2022-11-07
Enrollment: 14 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This pilot randomized phase II trial studies who well chlorine dioxide sterilization works in reducing oral mucositis in patients with stage I-IV head and neck cancer who are undergoing radiotherapy. Chlorine dioxide sterilization may help to treat oral mucositis.

Detailed description:
PRIMARY OBJECTIVES:

I. To evaluate and compare CloSYS Unflavored Oral Rinse effect on the incidence of severe oral oral mucositis (OM) (\>= World Health Organization \[WHO\] grade 3).

SECONDARY OBJECTIVES:

I. To evaluate and compare CloSYS Unflavored Oral Rinse effect on the time to onset of severe OM (\>= WHO grade 3).

II. To evaluate and compare CloSYS Unflavored Oral Rinse effect on the duration of severe OM (\>= WHO grade 3).

III. To evaluate and compare CloSYS Unflavored Oral Rinse effect in patient reported outcomes as measured by the Oral Mucositis Weekly Questionnaire (OMWQ).

IV. To evaluate and compare CloSYS Unflavored Oral Rinse effect on rates of radiation therapy (RT) interruption.

V. To evaluate and compare CloSYS Unflavored Oral Rinse effect on the oral microbiome.

VI. To evaluate and compare CloSYS Unflavored Oral Rinse effect on salivary TNFalpha, IL-1beta, and IL-6 levels.

OUTLINE: Patients are randomized into 1 of 2 arms.

ARM I: Patients receive chlorine dioxide sterilization oral rinse over 30 seconds twice daily (BID) from the start of radiation therapy (RT) to the evening prior to the 1 month RT follow-up appointment.

ARM II: Patients receive placebo oral rinse over 30 seconds BID from the start of RT to the evening prior to the 1 month RT follow-up appointment.

After completion of study, patients are followed up on day 1.

Arms: arm 1: Patients receive chlorine dioxide sterilization oral rinse over 30 seconds BID from the start of RT to the evening prior to the 1 month RT follow-up appointment. arm 2: Patients receive placebo oral rinse over 30 seconds BID from the start of RT to the evening prior to the 1 month RT follow-up appointment.
Interventions: intervention 1: Chlorine Dioxide Sterilization intervention 2: Laboratory Biomarker Analysis intervention 3: Placebo
Intervention descriptions: intervention 1: Given via oral rinse intervention 2: Correlative studies intervention 3: Given via oral rinse

Protocol documents:
Prot_SAP_000.pdf: 
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Protocol version date:  August 30, 2022 
 
 
 
Study Title: A Pilot Phase II Study Evaluating the Role of Stabilized Chlorine 
Dioxide on Mucositis for Patients Undergoing Head and Neck Radiotherapy 
 
 
Principal Investigator:  
Jared R. Robbins, MD 
Associate Professor  
Radiation Oncology 
University of Arizona  
Tucson, AZ  85719 
(520) 694-0861 
 
Study Site:   
 
The University of Arizona  
Department of Radiation Oncology 
 
 
 
 
3838 North Campbell Avenue, Bldg #2 
 
 
 
 
Tucson, AZ 85724  
 
NCT #:  
 
 
NCT03602066 
 
 
 
 
 
 
 
 
 
 
 
Protocol Version  
August 30, 2022 
 
 


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Protocol version date:  August 30, 2022 
 
Investigator Agreement 
 
I have read, understand and will adhere to the protocol as written, that any changes to 
the protocol will be approved by the sponsor or sponsor-investigator and the IRB, 
except changes to eliminate an immediate hazard to study subjects.  
 
I agree to conduct this study in accordance with the current International Conference on 
Harmonization (ICH) guidance, the Good Clinical Practice (GCP) guidance, the 
Declaration of Helsinki, FDA regulations, local IRB and legal requirements.  
 
 
 
 
 
 
__________________________   
 
 
_______________________ 
Signature 
 
 
 
 
 
 
Date (MM/DD/YY) 
 
 
 
 
___Jared R. Robbins, MD____ 
Name of Principal Investigator 
 
 
 


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Protocol version date:  August 30, 2022 
Table of Contents 
1. 
INTRODUCTION / SYNOPSIS .................................................................................................................. 6 
1.1 
Phase ............................................................................................................................................. 6 
1.2 
Indication ...................................................................................................................................... 6 
1.3 
Endpoints ...................................................................................................................................... 6 
1.4 
Patient Population ........................................................................................................................ 7 
2. 
STUDY DESIGN....................................................................................................................................... 7 
2.1 
Phase ............................................................................................................................................. 7 
2.2 
Number of centers ........................................................................................................................ 7 
2.3 
Number of subjects ....................................................................................................................... 8 
2.4 
The subject participation time period .......................................................................................... 8 
3. 
OBJECTIVES ........................................................................................................................................... 9 
3.1 
Primary Objective .......................................................................................................................... 9 
3.2 
Secondary Objectives .................................................................................................................... 9 
4. 
BACKGROUND AND RATIONALE ........................................................................................................... 9 
4.1 
Disease/Condition ......................................................................................................................... 9 
4.2 
Investigational Product, Intervention, Device ............................................................................ 10 
4.3 Preclinical Experience ....................................................................................................................... 10 
4.4 
Clinical Experience ...................................................................................................................... 10 
5. INVESTIGATIONAL PRODUCT, INTERVENTION, DEVICE .......................................................................... 11 
5.1 
Investigational Product, Intervention or Device ......................................................................... 11 
5.2 
Investigational product, intervention or device supply .............................................................. 11 
5.3 
Investigational product, Intervention or Device Accountability ................................................. 11 
5.4 
Storage ........................................................................................................................................ 12 
5.5 
Preparation ................................................................................................................................. 12 
5.6 
Handling ...................................................................................................................................... 12 
6.SUBJECT ELIGIBILITY ................................................................................................................................. 12 
6.1 
Inclusion Criteria ......................................................................................................................... 12 
6.2 
Exclusion Criteria ......................................................................................................................... 13 
6.3 
Enrollment ................................................................................................................................... 13 
7.STUDY PLAN ............................................................................................................................................. 14 
7.1 
IP/treatment/intervention/device regimen ............................................................................... 14 
7.2 
Pre-medications .......................................................................................................................... 16 
7.3 
Rescue Medications .................................................................................................................... 16 
7.4 
Excluded medications/treatments .............................................................................................. 16 
8.REQUIREMENTS FOR TREATMENT ........................................................................................................... 16 


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8.1 
Standard dose/treatment/device use ......................................................................................... 16 
8.2 
Dose IP/intervention/device modification ................................................................................. 16 
8.3 
IP treatment/intervention/devise delays ................................................................................... 16 
8.4  
Definition of a Dose Limiting Toxicity (DLT) ................................................................................ 17 
9.STUDY PROCEDURES ................................................................................................................................ 17 
9.1 
Screening and Baseline ............................................................................................................... 17 
9.2 
Registration/Randomization ....................................................................................................... 17 
9.3 
On IP treatment/Intervention/Device ........................................................................................ 17 
9.4 
End of IP treatment/Intervention/Device ................................................................................... 18 
9.5 
Follow up ..................................................................................................................................... 18 
9.6 
Early Treatment Termination ...................................................................................................... 18 
9.7 
Off Study ..................................................................................................................................... 18 
10.PHARMACOKINETIC STUDIES ................................................................................................................. 18 
11.DATA AND SAFETY MONITORING PLAN ................................................................................................. 19 
11.1. Identification of the DSMB obligated for oversight responsibilities ........................................... 19 
11.2. Identification of the entity obligated for routine monitoring duties .......................................... 19 
11.3. Monitoring progress and data review process ........................................................................... 19 
11.4 
Process to implement study closure when significant risks or benefits are  identified ............. 21 
11.5 
Description of adverse events and reporting procedures .......................................................... 21 
11.6 
Plan for assuring data accuracy and protocol compliance ......................................................... 22 
11.7 
Identification of the sponsor or funding agency, as applicable .................................................. 23 
12.   ADDITIONAL SAFETY REPORTING REQUIREMENTS ............................................................................ 23 
13. QUALITY ASSURANCE MEASURES ....................................................................................................... 23 
14. RECIST CRITERIA .................................................................................................................................. 23 
15. REMOVAL OF SUBJECTS ...................................................................................................................... 23 
16. STATISTICAL CONSIDERATIONS ........................................................................................................... 24 
17. ANALYSIS ............................................................................................................................................. 24 
17.1 
Safety Analysis ............................................................................................................................ 24 
17.2  Efficacy Analysis .......................................................................................................................... 24 
17.3 
Interim Analysis ........................................................................................................................... 25 
18. REGULATORY OBLIGATIONS ............................................................................................................... 25 
18.1 
Informed Consent ....................................................................................................................... 25 
18.2 
Institutional Review Board .......................................................................................................... 25 
19. ADMINISTRATIVE PROCEDURES.......................................................................................................... 25 
19.1  Investigator Responsibilities ....................................................................................................... 25 
19.2 
Data and Safety Monitoring Board Protocol Review .................................................................. 25 


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19.3 
Multicenter Trials ........................................................................................................................ 26 
20. SUBJECT CONFIDENTIALITY ................................................................................................................. 26 
21. STUDY DOCUMENTATION AND ARCHIVE ........................................................................................... 26 
22. DATA ................................................................................................................................................... 26 
23. PROTOCOL DEVIATIONS ...................................................................................................................... 27 
24. KARNOFSKY PERFORMANCE STATUS SCALE DEFINITIONS ................................................................. 28 
25. COMMON TOXICITY CRITERIA (CTCAE) ............................................................................................... 29 
26. STUDY SCHEDULE ................................................................................................................................ 30 
27. Tobacco Assessment Questionnaire ................................................................................................... 32 
28. OMWQ-HN and PRO-CTCAE.................................................................................................................. 33 
29. REFERENCES ........................................................................................................................................ 37 
30. APPENDICES ........................................................................................................................................ 39 
30.1 
Appendix A: Whole Unstimulated Saliva (WUS) Collection and Analyses Standard Operating 
Procedures .............................................................................................................................................. 39 
30.2 
Appendix B: Microbiome Collection and Analyses Standard Operating Procedures ................. 40 
 
 
 


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1. INTRODUCTION / SYNOPSIS 
Oral mucositis (OM) is an extremely common and often therapy limiting toxicity in 
patients receiving radiation therapy (RT) for head and neck cancer (HNC) (1).  The 
pathobiology of OM is complex and some evidence suggests that changes within 
the oral microbiome during RT may play a contributing role (2).  Chlorine dioxide 
is a known broad spectrum antiseptic against bacterial, fungal, and viral 
pathogens, and has been shown effective in the treatment of drinking water (3).  
Stabilized chlorine dioxide based rinsing products are now commercially available 
(CloSYS®) for the treatment of halitosis and oral hygiene use (4). It is believed that 
regulating the oral microbiome during RT could perhaps mitigate the incidence, 
onset, and duration of OM (5). No studies to date have evaluated the role of 
chlorine dioxide based oral rinses in preventing and treating OM associated with 
RT for HNC patients. 
1.1 
Phase  
This is a Phase II, single center, prospective, randomized, double-blind, placebo-
controlled study assessing the effects of stabilized chlorine dioxide oral rinse 
versus placebo on incidence, time to onset, duration, and patient reported 
outcomes on radiation therapy related OM, RT interruption, and changes in oral 
microbiome and cytokine composition. 
 
1.2 
Indication 
Stabilized chlorine dioxide oral rinse for the treatment of RT related OM. 
1.3 
Endpoints  
Primary: 
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect on the 
incidence of severe OM as measured by the World Health Organization 
(WHO) scale (≥WHO Grade 3) 
 
Secondary: 


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• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect on the 
time to onset of severe OM (≥WHO Grade 3) 
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect on the 
duration of severe OM (≥WHO Grade 3) 
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect in 
patient reported outcomes as measured by the Oral Mucositis Weekly 
Questionnaire (OMWQ) (6) and Patient Reported Outcomes version of the 
Common Terminology Criteria for Adverse Events (PRO-CTCAE)  
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect on rates 
of RT interruption 
 
 
            Exploratory: 
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect on the 
oral microbiome  
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect on 
salivary TNFα, IL-1β, and IL-6 levels 
1.4 
Patient Population 
 
Eligible subjects must be able to understand and sign the study specific subject 
consent form.  They must be >18 years of age, have a Karnofsky Performance 
Status Scale (KPS)of >60 with confirmed histologic or cytologic diagnosis of HNC 
(stage I-IV) for which they are receiving definitive or post-operative treatment with 
either RT alone or in combination with systemic therapy.  Subjects may not have 
previous allergies to chlorine dioxide products and must fulfill the inclusion and 
exclusion criteria found in Section 6.  Approximately 24 subjects will be 
randomized in a 1:1 ratio to stabilized chlorine dioxide rinse and placebo rinse. 
Twenty evaluable subjects (10 in each group) are needed in this pilot study to 
meet 81% statistical power (assuming a one-sided alpha of 0.05). We plan to 
recruit an additional 4 subjects to account for a 20% drop out/study non-
compliance rate to ensure we have 20 evaluable subjects.  
2. STUDY DESIGN 
2.1 
Phase 
This is a Phase II, single center, prospective, randomized, double-blind, placebo-
controlled study. Approximately 24 subjects will be randomized in a 1:1 ratio to 
stabilized chlorine dioxide oral rinse and placebo oral rinse. 
2.2 
Number of centers 
 
One 
 
This study will be performed by The University of Arizona Cancer Center 
Radiation Oncology Department at both the Banner University North Medical 
Campus Tucson (BUMC-N) and Orange Grove Clinic (OGC). 
 


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2.3 
Number of subjects 
Approximately 24 will be recruited for this pilot study. Twenty evaluable subjects 
are needed to meet 81% statistical power (assuming a one-sided alpha of 0.05). 
We plan to recruit 4 additional subjects to account for a 20% drop out/study non-
compliance rate.  
2.4 
The subject participation time period 
Subjects will be enrolled in the study for approximately 3 months, including 
screening time, treatment time, and follow-up at 30 days post RT.  
 
Subjects enrolled in this study are undergoing treatment at BUMC-N or OGC as 
part of standard of care for the treatment of HNC.  Study subjects will be treated 
with either stabilized chlorine dioxide oral rinse or placebo oral rinse twice daily 
during RT.  Typical RT schedule is 6 to 7 weeks in length with treatments 
administered Monday through Friday.  Subjects will begin the oral rinse 
(stabilized chlorine dioxide or placebo) on the morning of RT day 1.  Whole 
unstimulated salivary collections, OM scoring, and validated OMWQ and PRO-
CTCAE patient surveys will be completed at baseline (pre-RT), during weeks 2, 
4, and 6 of RT during their planned on-treatment visits (OTVs), and at their 1-
month post-RT follow-up appointment.  The number of days and cause of RT 
treatment interruption will also be tracked.   
 
SCHEMA 
 
REGISTER 
Head and 
Neck 
Cancer  
 
 
Radiation 
Alone 
(Group 1) 
 
STRATIFY 
RANDOMIZE 
ARM 1  
Placebo Oral Rinse 
BID 
Radiation 
plus 
Systemic 
Therapy 
(Group 2) 
ARM 2  
Stabilized Chlorine 
Dioxide Oral Rinse 
BID 
 


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Protocol version date:  August 30, 2022 
3. 
OBJECTIVES 
3.1 
Primary Objective 
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect on the 
incidence of severe OM as measured by the World Health Organization 
(WHO) scale (≥WHO Grade 3) 
 
3.2 
Secondary Objectives 
• To evaluate and compare ClōSYS® UnflavoredOral Rinse effect on the time to 
onset of severe OM (≥WHO Grade 3) 
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect on the duration 
of severe OM (≥WHO Grade 3) 
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect in patient 
reported outcomes as measured by the Oral Mucositis Weekly Questionnaire 
(OMWQ) (6) and PRO-CTCAE 
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect on rates of RT 
interruption 
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect on the oral 
microbiome  
• To evaluate and compare ClōSYS® Unflavored Oral Rinse effect on salivary 
TNFα, IL-1β, and IL-6 levels 
 
4. 
BACKGROUND AND RATIONALE 
4.1 Disease/Condition 
It is estimated that 61,760 new cases of head and neck cancer (HNC) will be 
diagnosed in the United States in 2016 (7).  The majority of HNC patients receive 
radiation therapy (RT) as part of their care and nearly all are expected to develop 
treatment related oral mucositis (OM) (1).  This often therapy limiting toxicity 
presents a challenge to practitioners and patients as it is known to reduce both 
quality of life and prognosis (8)(9).  The pathobiology of OM is complex and involves 
alterations in mucosal barrier integrity, epithelial cell kinetics, extracellular matrix 
composition, vascular integrity, inflammatory mediators, and the oral microbiome 
(10)(11).  In cases of high grade OM, infection and ulcerations may lead to a breach 
in the mucosal barrier, sepsis, and even death. 
 
Few effective agents are available today for the prevention, delay, or reduction of RT 
related OM. Palifermin, a recombinant human keratinocyte growth factor, was 
demonstrated to improve the incidence, onset, and duration of high grade (WHO 3-
4) OM in two independent phase III randomized clinical trials for patients receiving 
definitive and post-operative HNC RT, however both studies showed no impact on 
patient reported pain scores, RT compliance, or disease related outcomes (12)(13).  
Though FDA approved, the results of these studies have called into question the role 
of palifermin for OM, as it is a difficult drug to administer with weekly IV infusions 
during RT.   
 


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4.2 Investigational Product, Intervention, Device 
Chlorine dioxide is a known antimicrobial disinfectant which has been shown to be 
effective in the treatment of drinking water and is FDA approved for this indication 
among others (3). Clinically, chlorine dioxide has also been proven efficacious in the 
treatment of halitosis, which is thought to be related to its effect on decreasing the 
amount of volatile sulfur compounds and their associated bacterial organisms within 
the oral cavity (4).  Commercially available products including ClōSYS® Unflavored 
Oral Rinse containing 0.1% (w/v) stabilized chlorine dioxide, are widely available for 
personal oral hygiene use. 
 
Evidence exists that RT related OM is associated with a shift in the oral microbiome 
toward opportunistic species as resultant from alterations in salivary production and 
composition (2).  It is believed that regulating the microbiome could eliminate, delay, 
or reduce the effects of OM as demonstrated by a meta-analysis of clinical trials 
evaluating various antimicrobials, none which included chlorine dioxide based 
products (5).  In addition to its broad spectrum antiseptic properties (bacterial, 
fungal, and viral), chlorine dioxide products are alcohol-free, sulfate-free, well-
tolerated, available in rinse form, and possess anti-inflammatory properties which 
are critical to the prevention of OM (14). 
 
 
We propose that the addition of twice daily usage of ClōSYS® Unflavored Oral 
Rinse to standard oral hygiene care during RT for HNC will decrease OM incidence, 
increase time to onset, decrease duration, and improve patient reported outcomes 
related to OM.  Additionally, we hypothesize that ClōSYS® Unflavored Oral Rinse 
will reduce OM in patients receiving RT for HNC by mechanisms via changes in 
microbiome and inflammatory cytokine compositions.  Lastly, we believe that 
decreased OM will potentially lead to less RT interruption.  We hope that the results 
of this study will provide preliminary data for future phase III clinical trial testing. 
4.3 Preclinical Experience 
The safety of chlorine dioxide and its metabolites (chlorite and chlorate) have been 
studied in animals extensively.  Initial evaluations of high dose chlorine dioxide 
based products have shown adverse hematological, thyroid, and reproductive 
effects. In a review by Lyman Condie, these animal studies are highlighted in 
monkey, rat, and mouse experiments, which have shown that at high doses of 
exposure (>10mg/L or >10mg/kg/d) changes in several key physiologic endpoints 
have been clearly documented (15).  Some studies have shown that chlorine dioxide 
and its metabolites (chlorite and chlorate) can cause oxidation of hemoglobin into 
methemoglobin and a subsequent reduction in red cell count by virtue of hemolytic 
anemia.  Other studies have shown decreases in rat T4 levels leading to functional 
hypothyroidism.  Further studies have shown sperm head abnormalities in mice 
exposed to high levels of chlorine dioxide for 5 days.  To our knowledge, no animal 
studies have specifically evaluated chlorine dioxide in its potential protective effects 
on OM. 
4.4 Clinical Experience 
In humans, the over ingestion of sodium chlorate (a similar compound to chlorine 
dioxide) based herbicides revealed a minimal lethal dose of 220 mg/kg of sodium 
chlorate in adult humans according to one study (16).  Another study in humans 
showed that chlorine dioxide and its metabolites (chlorite and chlorate) are well 
tolerated at doses of 2.5 mg or less (<0.04 mg/kg assuming 70 kg average man) 


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Protocol version date:  August 30, 2022 
when ingested daily for 12 weeks, except potentially in patients with glucose-6-
phosphate dehydrogenase (G6PD) deficiency (17).  A prospective epidemiological 
study in humans exposed to chlorine dioxide (0.25 – 1.1 mg/L) after ingesting 
drinking water for 12 consecutive weeks showed no adverse effects when compared 
to a control group ingesting water without chlorine dioxide (18). 
 
There are no data we are aware of evaluating the safety and efficacy of ClōSYS® 
Unflavored Oral Rinse for the prevention, delay, or reduction in RT related OM in 
patients being treated for HNC.  As mentioned above, a meta-analysis of other 
similar orally available, topically delivered antimicrobials and anti-inflammatory 
agents have shown equipoise for mitigating RT induced OM (5). ClōSYS® 
Unflavored Oral Rinse is an FDA approved, commercially available product which is 
approved for 15 ml twice daily (30 second) rinse applications (19)(20). Two clinical 
trials of 15 patients each have demonstrated that chlorine dioxide rinsing is safe and 
effective in the treatment of halitosis and reduction of plaque build-up, while none 
reporting toxicities associated with the rinse (4)(21).  
5. INVESTIGATIONAL PRODUCT, INTERVENTION, DEVICE 
 
5.1 
Investigational Product, Intervention or Device  
ClōSYS® Unflavored Oral Rinse containing 0.1% (w/v) stabilized chlorine dioxide 
used twice daily with 15 mL per rinse for 30 seconds 
5.2 
Investigational product, intervention or device supply 
The IP (ClōSYS® Unflavored Oral Rinse) is a commercially available, over-the-
counter, product which is currently FDA approved for the treatment of halitosis 
and general oral hygiene care.  The following products will be supplied by 
Rowpar Pharmaceuticals Inc. specifically for the purpose of this study in 16 oz. 
white, opaque bottles, along with measuring cups, requiring no additional 
preparation.  ClōSYS® Unflavored Oral Rinse or Placebo Oral Rinse will be 
labelled in such a way that they are not identifiable by subjects or investigators. 
 
a. ClōSYS® Unflavored Oral Rinse (also referred as ClōSYS® Unflavored 
Alcohol-Free Oral Rinse). 
b. Unflavored Placebo Oral Rinse. The study placebo will be identical in 
appearance to the IP and without stabilized chlorine dioxide. 
 
5.3 
Investigational product, Intervention or Device Accountability 
Because the IP (ClōSYS® Unflavored Oral Rinse) and Placebo Oral Rinse will be 
supplied directly to the Banner UMC Research (BUMC-N) pharmacy for use in 
this study, the receipt of shipment, dispensing and return or destruction records 
will be tracked using the Investigational Agent Accountability Record or other 
applicable and similar record.  Once a signed order for IP is received by 
pharmacy, the IP will be given to the research staff to dispense to the study 
participant.  Participants will be asked to keep a rinse diary, which will include 
date, time, and length of rinsing.  All containers will be returned by the patient 


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and then taken to pharmacy by the research staff.  The amount of rinse solution 
taken will be documented by taking the initial volume of rinse given to the patient 
and subtracting any oral rinse not used by measuring the returned residual 
volume of solution.  The unused remaining rinse solution will then be destroyed 
in accordance with the BUMC Pharmacy policy.  We plan to distribute and collect 
rinse solution bottles at weekly intervals. 
 
5.4 
Storage 
IP (ClōSYS® Unflavored Oral Rinse) and placebo will be stored at room 
temperature per manufacturer recommendations in the BUMC-N pharmacy with 
limited access to personnel.  
5.5 
Preparation 
Once an order is written by the treating doctor and received by pharmacy, the 
pharmacist or technician will dispense the randomized, blinded study drug.  A 
member of the research staff will pick up the prescription bottle and will give the 
bottle directly to the subject. 
5.6 
Handling 
There is no special handling required for the IP. 
6.SUBJECT ELIGIBILITY 
Investigators will maintain an electronic subject log (in the UACC OnCore system and/or 
Redcap) of all potential (i.e. consented) study subjects, which will include as applicable 
(demographics, informed consent, eligibility, treatment assignment, on treatment, off 
treatment, follow up and off study dates).  
 
6.1 
Inclusion Criteria  
a. Able to provide properly obtained written informed consent 
b. Age 18 years or older 
c. Pathologically-confirmed diagnosis of head and neck malignancy (stage I-IV) 
d. Planned to receive high dose RT ≥50 Gy to visualizable oral cavity and/or 
oropharyngeal mucosa, with or without administration of concurrent systemic 
therapy. 
e. Karnofsky Performance Status of ≥ 60, within 90 days of registration 
f. Baseline hematocrit > 20 within 90 days of registration to the study 
g. Willingness to complete OMWQ and PRO-CTCAE  forms at each designated 
time point along with oral rinse diary 
h. Life expectancy ≥ 3 months 
i. Willing to tolerate oral rinsing for 30 second intervals 
j. Negative serum pregnancy test in females of childbearing age 
k. Must be willing to use an effective form of birth control if female and of 
childbearing potential 


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6.2 
Exclusion Criteria 
a) Known hypersensitivity to chlorine dioxide products 
b) Chlorine dioxide product usage within the past 7 days prior to registration for 
this study 
c) Utilization of any antibiotic medications (orally topical or systemic) within past 
7 days prior to registration for this study 
d) Utilization of supraphysiological corticosteroid medication (>10 mg oral 
prednisone or equivalent dose) for chronic indication  
e) Sjogrens disease 
f) Medically documented glucose-6-phosphate dehydrogenase (G6PD) 
deficiency  
g) Planned daily RT of less than 5 weeks duration  
h) Known history of HIV or AIDS 
i) Current pregnancy 
 
 
6.3 
Enrollment 
 
The source of subjects is the patient population at the Banner University Medical 
Group/University of Arizona Cancer Center. Patients who are scheduled as part 
of their routine care to consult with the radiation oncologist for definitive treatment 
of HNC (stage I-IV) will be provided with information regarding this study.  
 
 
 
Before subjects may be entered into the study, a copy of the written institutional 
review board (IRB) approval of the protocol, informed consent form (ICF), and all 
other applicable subject information and/or recruitment material will be on file at 
the University of Arizona.  All efforts will be made to consent the subject at an 
already scheduled appointment with the radiation oncologist.  It is possible they 
would need to come in for an additional appointment.  This could be either at 
their initial consult visit or during their CT simulation appointment.  Subjects will 
have the opportunity to take their time in making a decision to join the study and 
will be encouraged to take the Informed Consent Form home and discuss it with 
whomever they would like.  The treating physician will explain to the subjects the 
risks and benefits. They will be informed that their participation is voluntary, and 
lack of participation will not affect the subject’s relationship with the treating staff 
or our facility.  Subjects will be consented by either the PI, or the Research Staff 
of the Radiation Oncology department. The written informed consent document 
will be signed and personally dated by the subject and completed to a fully 
executed informed consent document and processed per the University of 
Arizona standard operating procedures. 
 
Subjects will also be made aware that should they consent, they can withdraw 
their consent at any time.   When new information becomes available, it will be 
given to the patient as soon as possible, either at their next visit, or if that is more 
than 30 days away, by phone call.  Once an approved revised consent form is 
submitted and approved by the IRB, the subject will be asked to sign the new 
consent and the subject will be notified of the change to the consent form.  The 
AZCC Verification of Consent form will be completed at the time the subject is 
consented.  This form includes information pertaining to who is present at 


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consenting, that the consent form was reviewed with the subject, the subject 
understood the consent form, and the date and the time of the consent.  Also 
documented will be the version of the Subject Consent form and the HIPAA form.   
 
Subjects will be registered by the University of Arizona Research Pharmacy.  
Once subjects complete the screening process for this study and are deemed 
eligible, required information will be given to the pharmacy staff to register the 
subject in a stratification/randomization system designed by biostatistician 
Denise Roe, DrPH.  Each subject will be identified by a unique sequential subject 
identification number.  This number will be used to identify the subject throughout 
the clinical study and will be used on all applicable study documentation related 
to that subject. The subject identification number will remain constant throughout 
the study.  The sponsor will be providing study IP with blinded labeling and only 
they will know which label corresponds with active agent or placebo. 
 
7.STUDY PLAN 
7.1 
IP/treatment/intervention/device regimen 
Subjects will be stratified by whether or not they will be receiving concurrent 
systemic therapy and then randomly assigned to receive either ClōSYS® 
Unflavored Oral Rinse twice daily or placebo oral rinse twice daily from the start 
of RT to their 1-month RT follow-up appointment.  Assignment of IP will be done 
in a double-blinded fashion.  Both active rinse and placebo rinse will be 
formulated to look identical in appearance. 
 
Subjects will receive verbal and written oral hygiene instructions and 11 bottles of 
either (16 oz. each) of ClōSYS® Unflavored Oral Rinse or placebo oral rinse. 
Subjects will also receive measuring cups for dispensing the rinse (15 ml per 
dose) and a diary for recording usage in order to track their rinsing adherence, as 
well as notating any subjective effects possibly attributable to the rinse.  
 
Treatment with IP will start on the morning of day 1 of RT and subjects will be 
advised to use the IP (preferably after meals) at approximately 8 a.m. ± 3 hours 
and 8 p.m. ± 3 hours, with 15 mL per rinse for 30 seconds during each session.  
They will be instructed to take nothing by mouth for 20 minutes following their 
rinse to allow it to take effect.  They will note in their patient’s log the date, time, 
and duration of rinsing.  Subjects will be instructed to continue with other 
standard oral hygiene practices we offer to our RT patients, including alkaline 
saline rinses, honey, miracle mouthwash, etc.  
 
Baseline tobacco history including type of tobacco, number of pack-years, 
duration of smoking, and quit date if applicable will be recorded. This will be 
captured using the Tobacco Assessment Questionnaire (see section 27).  
Baseline dental evaluation counting number of teeth and generally graded 
condition (excellent, good, fair, poor) will be recorded.  Baseline weight, BMI, and 
history of recent (3 months) weight loss will be recorded.  Nutritional intake status 
(solids and liquids, liquids only, no oral intake) and utilization of enteral versus 


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parenteral feeding will be noted.  HPV status of malignancy will be recorded.  
One week up until registration, concomitant medications will be collected. 
 
Subjects will be seen once per week per standard of care for on-treatment visits 
(OTVs).  Radiation treatments may be 6 or 7 weeks at the discretion of the 
treating physician.  Vital signs and weight measurement along with grading of 
common head and neck side effects per common terminology criteria for adverse 
events (CTCAE v5.0) will be performed.  CTCAE items will include generalized 
weakness, skin reaction, weight loss, dry mouth, sore throat, dysphagia, 
anorexia, dysgeusia, voice alteration, and neck edema. Physical examination will 
include focused evaluation of the head and neck region, with particular attention 
paid to documenting skin reaction and oral mucositis (OM).  
 
At baseline (for example during CT-simulation), during weekly OTVs, and at their 
1-month post-RT follow-up appointment, physician grading of OM will occur via 
intraoral examination utilizing the World Health Organization (WHO) Scale, as 
described in the table below: 
 
At baseline (for example during CT-simulation), during weekly OTVs, and at their 
1-month post-RT follow-up appointment, intraoral photo-documentation will be 
taken and uploaded into patient specific Cerner electronic medical record charts 
utilizing the Cerner Camera Capture application via handheld mobile devices. 
These will be filed with study identifier only and not the subject name.   
 
Patient reported assessment of OM will be evaluated at baseline (for example 
during CT-simulation), at OTVs during weeks 2, 4, and 6 of RT, and at their 1-
month post-RT follow-up appointment, with the OMWQ (6) and PRO-CTCAE.  
The OMWQ questionnaire contains 9 items with the total ranging from 0 to 54. 
 
Whole Unstimulated Saliva (WUS) will be collected at baseline (prior to first 
treatment), at OTVs during weeks 2, 4, and 6 of RT, and at the 1-month post-RT 
follow-up appointment. Further information on WUS collection and analyses is 
provided in Appendix A. 
 


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Microbiome samples will also be collected at baseline (prior to first treatment), at 
OTVs during weeks 2, 4, and 6 of RT, and at the 1-month post-RT follow-up 
appointment, immediately following collection of patient WUS.  Further 
information on microbiome collection and analyses is provided in Appendix B.   
7.2 
Pre-medications 
Not applicable 
7.3 
Rescue Medications  
Not applicable 
7.4 
Excluded medications/treatments  
Orally topical or systemic antibiotic agents (such as chlorhexidine, nystatin, 
poviodine-iodine, clotrimazole, clindamycin, benzydamine, etc) are not allowed 
and are not to be used within 7 days prior to registration.  Directed antibiotics 
prescribed during or after RT for suspected infection related to treatment (ie oral 
thrush, neck abscess, cellulitis, etc) are allowed but will be documented 
appropriately. 
8.REQUIREMENTS FOR TREATMENT  
8.1 
Standard dose/treatment/device use  
The standard dose of ClōSYS® Unflavored Oral Rinse for the prevention and 
treatment of plaque build-up and halitosis is 15 ml (containing 0.1% [w/v] 
stabilized chlorine dioxide) for 30 seconds twice daily, which equates to a total of 
30 mg of chlorine dioxide exposure over the oral mucosa.  This dosage is the 
FDA approved and manufacture recommended amount (19)(20). Two 
randomized trials for the treatment of halitosis also utilized a similar dosage and 
found no significant adverse effects when compared against a placebo rinse 
(4)(21).  
8.2 
Dose IP/intervention/device modification  
Dose modifications will be considered at weekly OTVs during patient evaluations, 
should the patient report bothersome effects thought to be associated with use of 
the rinse.  Dose reduction will occur by eliminating the PM rinse for one week 
followed by further assessment at the following OTV (approximately 7 days later).  
Should the patient continue to describe bothersome effects thought likely due to 
the rinse, the patient will be taken off the rinse at the discretion of the treating 
physician following discussion with the patient. 
8.3 
IP treatment/intervention/devise delays 
Subjects will begin oral rinsing the morning of day 1 of RT.  The IP will be used 
twice per day continuously to the evening prior to their 1-month post-RT follow-up 
visit. If RT interruptions occur while taking the IP, subjects should continue IP per 
protocol, and additional IP may need to be given to the patient to complete the 


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schedule of treatment. All subjects will take a full continuous course of IP unless 
instructed by the PI to discontinue because of unacceptable toxicity as deemed 
appropriate by the PI.  Subjects will be advised to rinse with the IP (preferably 
following meals) at 8 am ± 3 hours and 8 pm ± 3 hours.   
 
If a subject misses a scheduled dose of IP within the recommended window, they 
should skip the missed dose and resume treatment at the next scheduled time.  
Treatment course will not be extended beyond the 1-month post-RT follow-up to 
compensate for missed doses.  Subjects will stop treatment on the evening prior 
to their 1-month post-RT follow-up appointment with their Radiation Oncologist 
and return any remaining unused IP as described in Section 5.   
    
If more than 7 consecutive days are missed of rinsing with the IP, the patient will 
be taken off study. 
8.4  
Definition of a Dose Limiting Toxicity (DLT) 
Subjects will continue on IP twice daily from day 1 of RT to the evening prior to 
their 1-month post-RT follow-up appointment with their Radiation Oncologist or 
unless instructed to stop IP as described elsewhere.  If the subject meets the 
criteria listed above to be taken off the study, they will discontinue IP, but all 
assessments will continue to be collected.  There are no known pre-specified 
DLTs associated with the IP. 
 
9.STUDY PROCEDURES  
9.1 
Screening and Baseline 
Potential subjects will enter the screening period of the study after a completely 
executed informed consent has been obtained.  See Section 26 (Study 
Schedule) for full list of baseline evaluations. 
 
9.2 
Registration/Randomization 
All regulatory requirements must be in place prior to subject registrations.  
  
Single center study subject identification be assigned sequentially. Screen failure 
and subjects who withdraw prior to registration will have –“SF” or “WD” added to 
the end of the subject ID. 
 
The stratification/randomization procedure utilized will be created and will employ 
a system of prepared envelopes and blocks of random numbers.  
 
 
9.3 
On IP treatment/Intervention/Device  
Registered subjects will be treated with either ClōSYS® Unflavored Oral Rinse 
(0.1% w/v) at 15 ml per rinse for 30 seconds each twice daily or placebo oral 


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rinse at 15 ml per rinse for 30 seconds each twice daily, from the morning of day 
1 of RT to the evening prior to their 1-month post-RT follow-up appointment with 
their Radiation Oncologist.  Subjects will be instructed to place the rinse in their 
mouth, continuously swish and gargle the solution, and spit the rinse out after 30 
seconds, without swallowing the rinse. It will be requested that they do the rinse 
at 8am (+/- 3 hours) and 8pm (+/- 3 hours) each day that they are receiving 
radiation treatments.  In addition, they will be requested to do the rinse twice per 
day on weekends even though they do not receive radiation treatment on the 
weekends.  Once starting the IP subjects will be asked to complete a rinse diary 
with the time, date, and duration of each rinse taken.  They will also be asked to 
complete a patient observation form listing any side effects and/or problems with 
using the rinse that they may encounter.  
 
See Section 26 (Study Schedule) for weekly evaluations while on IP. 
9.4 
End of IP treatment/Intervention/Device 
Subjects will complete IP the evening prior to their 1-month post-RT follow-up 
appointment with their Radiation Oncologist. 
9.5 
Follow up 
Subjects will be seen for one month [+21 days] follow-up visit after the end of RT.  
During these visits, routine vitals, review of systems, physical examination, and 
adverse event documentation will be performed.  See treatment plan calendar.  
Additionally, any remaining unused IP, rinse diary, and observation forms will be 
collected.  Patients will complete their final OMWQ and PRO-CTCAE, have OM 
scored by their treating physician, and have their final WUS and microbiome 
samples collected. 
 
See Section 26 (Study Schedule) for weekly evaluations while on IP. 
9.6 
Early Treatment Termination 
Subjects that terminate IP treatment prior to completing the planned treatment 
portion of the protocol will still be seen as per the study schedule and have an 
early termination visit at their 1-month post-RT follow-up appointment with their 
Radiation Oncologist. However, it the subject decides to withdraw from the study, 
no further information will be collected.  
9.7 
Off Study 
Subjects will be considered off study after their 1-month post-RT follow-up 
appointment with their Radiation Oncologist.  
10.PHARMACOKINETIC STUDIES 
Not applicable. 


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11.DATA AND SAFETY MONITORING PLAN 
11.1. Identification of the DSMB obligated for oversight responsibilities: 
The University of Arizona Cancer Center Data (UACC) and Data Safety 
Monitoring Board (DSMB) will provide ongoing oversight for this trial.  UACC will 
act as the DSMB of record. The Clinical Trials Office (CTO) QA/QC team, the 
UACC DSMB’s monitoring arm, will ensure operational integrity to study 
operations through regular monitoring visits. This study has been assigned 
institutionally as a Low Risk level by the UACC DSMB. 
 
11.2. Identification of the entity obligated for routine monitoring duties: 
Routine monitoring will be provided by the University of Arizona Cancer Center 
Quality Assurance/Quality Control (QA/QC) Program to ensure that the 
investigation is conducted according to protocol design and regulatory 
requirements.  
 
The investigator will perform real time monitoring by reviewing UACC QA/QC 
periodic reports within 2 weeks of receipt.  The PI and the study team will review 
data monthly to ensure patient safety and integrity of data. 
 
All visits will be conducted according to the FDA principles described in the Code 
of Federal Regulations and following pertinent Guidance. 
 
11.3. Monitoring progress and data review process: 
Routine monitoring of subject data will be conducted at least quarterly by the 
UACC QA/QC Program.   
The first routine monitoring visit will include at a minimum: 
• Informed consent – 50% of cases enrolled;  
• Subject eligibility – 10% of cases, up to two subjects; 
• Data review – 10% of cases, up to two subjects. 
 
All subsequent monitoring visits will consist of randomly selected subject cases 
based on current enrollment and include continuing review of previously selected 
cases, as applicable. 
 
A monitoring visit report and follow-up letter will be completed approximately two 
weeks after the routine monitoring visit; a copy will be maintained in the study 
file. The monitor will request additional source documentation, clarification, 
information, or corrections to the CRF and/or regulatory records from the Clinical 
Research Coordinator (CRC) or other applicable staff responsible for the study 
and resolution of queries/findings.  Documentation of such a request will be 
maintained with a copy of the monitor’s visit report for follow-up at the next 
monitoring visit. Electronic records will be available in the institutional database 
or provided by the QA/QC Program staff.  
 
The Principal Investigator will ensure the accuracy, completeness, legibility, and 
timeliness of the data reported in the Case Report Form (CRF), or other 
acceptable data formats. Source documentation supporting the study data should 
indicate the subject’s participation in the trial and should document the dates and 
details of study procedures, adverse events, and patient status.  


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Case report forms, including but not limited to the inclusion/exclusion criteria 
form, adverse event and serious adverse event forms, and rinse compliance 
forms will be completed via the institution database,  Redcap. Trials using paper 
CRFs will have the data entered with a black ball-point pen or typed. Corrections 
to the forms should not obscure the original entry and should be made by striking 
the incorrect information with a single line. Each strike should be accompanied 
by the initials of the corrector and the correction date. All subject forms and study 
files will be stored in a secure area limited to authorized staff. 
 
Note: Routine monitoring of regulatory documents will be conducted at least 
annually by the UACC QA/QC Program. 
 
 
 
 
 
The Radiation Oncology Staff will generate Case Report Forms (CRFs) for this 
study. Entries made in the CRF must be either verifiable against source 
documents, or have been directly entered into the CRF, in which case the entry 
in the CRF will be considered as the source data. The source data parameter to 
be verified and the identification of the source document must be documented. 
Corrections to CRFs and source data will be made only by authorized members 
of the study staff, clearly entered, initialed, and dated. The Investigator will sign a 
final CRF to indicate that, to his/her knowledge, they are complete and accurate. 
If further changes are made after this, the Investigator will be made aware of the 
corrections and his/her approval will be documented by re-signing.  
 
As well, the study may be audited by regulatory agencies at any time. applicable 
regulatory agencies, and/or applicable ethical review boards with direct access to 
the original source documents. 
 
The following topics are planned to be reviewed during each visit. Additional 
subject matter as appropriate will also be reviewed.   
 
Initiation Visit: The initiation visit will be conducted soon after initiation of the 
Study. 
1. Protocol 
2. Credentials of key personnel 
3. Facility readiness  
4. Study products storage 
5. Approval by the appropriate IRB 
6. All study forms, including the informed-consent form and case report forms 
7. Eligibility criteria 
8. Randomization of subjects 
 
Mid-study Visit: The mid-study visit will be scheduled after enrollment of about 
60% of the total number of subjects.  
1. Any change in key personnel 
2. Workflow of the study 


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3. Informed consent form for subjects enrolled after the initiation visit 
4. Eligibility criteria for subjects enrolled after the initiation visit 
5. Protocol violations and deviations, if any 
6. Any changes in the facility 
7. Budget and payments 
8. Subject dropouts 
9. Adverse events reporting, if any 
 
 
Closeout Visit: The closeout visit will be conducted after completion the Study.  
1. Any change in key personnel 
2. Informed consent form for subjects enrolled after the mid-study visit 
3. Eligibility criteria for subjects enrolled after the mid-study visit 
4. Protocol violations and deviations, if any 
5. Any changes in the facility 
6. Subject dropouts 
7. Case report forms for all subjects 
8. Corrections of data by authorized personnel 
9. Adverse events reporting, if any 
10. Completeness of all site source documents 
11. Final accountability of all IP 
 
 
11.4 Process to implement study closure when significant risks or benefits are 
 
identified: 
 
If a Common Terminology Criteria for Adverse Events (CTCAE Version 5.0) 
grade 5 toxicity occurs that is more likely than not, directly related to the IP or 
placebo rinse, the study will be discontinued. 
 
 
 
 
 
11.5 Description of adverse events and reporting procedures: 
 
 
ADVERSE EVENTS  
 
An adverse event (AE) is any untoward medical occurrence in a patient or clinical 
investigation subject administered a pharmaceutical product and that does not 
necessarily have a causal relationship with this treatment. An AE can therefore 
be any unfavorable and unintended sign, symptom, or disease temporally 
associated with the use of a medicinal (investigational) product, whether or not 
related to the medicinal (investigational) product.  
 
Adverse events will be recorded on the UACC adverse events record form and 
reviewed by the Principal Investigator. For this study we will focus on collecting 
the AEs that may be related to the investigational product and/or radiation 
therapy/chemotherapy with specific focus on generalized weakness, skin 
reaction, weight loss, dry mouth, sore throat, dysphagia, anorexia, dysgeusia, 
voice alteration, and neck edema. 
 
All adverse events will be classified using NCI Common Terminology Criteria for 
Adverse Events (CTCAE) version 5.0 and will address: 
• Grade 


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• Relationship to study drug (not related, unlikely, possible, probable, definitely)  
• Relationship to radiation and/or chemotherapy (not related, unlikely, possible, 
probable, definitely) 
• Causality other than study drug (disease related, concomitant medication 
related, intercurrent illness, other)   
• Date of onset, date of resolution 
• Frequency of event (single, intermittent, continuous) 
• Event outcome (resolved, ongoing, death) 
• Action taken (none, held, dose reduced, discontinued, medication given) 
 
 
SERIOUS ADVERSE EVENTS 
 
A serious adverse event (SAE) is any untoward medical occurrence that at any 
dose:  
 
1) Results in death;  
 
2) Is life-threatening; 
 
3) Requires in-patient hospitalization or prolongation of an existing hospital stay;  
 
4) Results in disability persistent or significant disability/incapacity, or: 
 
5) Is a congenital anomaly/birth defect.  
 
Note: A SAE may also be an important medical event, in the view of the 
investigator that requires medical or surgical intervention to prevent one of the 
outcomes listed above.  
 
All serious adverse events, regardless of attribution, and any deaths will be 
reported within 24 hours of notification of the event to the sponsor and, if 
applicable, any collaborating entity. All serious adverse events and any deaths 
will be reported to the DSMB and to the University of Arizona Human Subjects 
Protection Program per the guidelines set forth in University of Arizona Cancer 
Center Data and Safety Monitoring Board Charter, Table 5: Adverse Event 
Reporting.  
 
 
All submitted serious adverse events will be processed by the DSMB Coordinator 
monthly for initial trend analysis and then reviewed by the DSMB Chair. The 
assigned QA/QC Monitor will review the SAE reporting process to confirm 
reporting requirements are met. 
 
11.6 Plan for assuring data accuracy and protocol compliance: 
Routine study activity and safety information will be reported to the DSMB on a 
quarterly basis, or more frequently if requested. These reports will include: 
• Study activity, cumulative and for the period under review; 
• Safety (narrative description on non-serious and serious adverse events); 
• Predetermined protocol early stopping rules for efficacy/futility;    
• Monitoring and protocol compliance; 
• Comments;  
• Attachments (AE data reviewed by the PI to compile the report, SAE letters 
and reports, results of any review(s), applicable correspondence with the IRB 
or other regulatory agencies.  
 
 
Data, safety and study progress will be reported to: 


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• Human Subjects Protection Program (IRB) at least annually; 
• Sponsor (if applicable) at least every six months. 
 
11.7 Identification of the sponsor or funding agency, as applicable: 
The PI will immediately notify, in writing, the funding agency, if applicable, any 
action resulting in a temporary or permanent suspension of the study. A copy of 
this correspondence will also be forwarded to the DSMB and the Scientific 
Review Committee (SRC).  
12.   ADDITIONAL SAFETY REPORTING REQUIREMENTS  
Serious adverse events will be reported to the Data Safety Monitoring Board as 
well as the institutional IRB within 24 hours of notification of the event to the PI.  
Serious adverse events will be reported using the FDA MedWatch form to inform 
the DSMB, and Report information that is Potentially Problematic to the 
institutional IRB. 
13. 
QUALITY ASSURANCE MEASURES 
Per the UACC DSMB Charter, Internal Ad Hoc audits may be performed on any 
UACC clinical trial if identified for audit, the audit will be conducted by an 
identified audit team per the UACC DSMB Charter. A QA/QC representative will 
coordinate the audit team functions and a written audit report will be provided to 
the principal investigator and the DSMB. 
14. 
RECIST CRITERIA 
Not applicable 
15. 
REMOVAL OF SUBJECTS 
Subjects have the right to withdraw from the study at any time and for any reason 
without prejudice to their future medical care by the physician or at the institution. 
If this occurs, the investigator, or designee, is to discuss with the subject the safe 
and appropriate processes for discontinuation from the investigational product, 
intervention or device. 
 
Subjects that wish to discontinue active IP treatment/intervention/device may 
elect to continue with the other protocol required assessments. The investigator 
should discuss with the subject the options for continuation of the study schedule 
of assessments (i.e. blood work, scans, physical exams, diaries) and collection of 
data, including endpoints and adverse events. 
 
The investigator or designee must document the change in status of the subject’s 
participation in the study and as applicable, the level of follow up that is agreed to 


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by the subject (i.e. agrees to follow up exams, adverse event review, phone 
contact, but not to further treatment and/or procedures).  
 
Subject withdrawal of consent for a study indicates that the subject does not wish 
to receive further protocol required therapies or procedures, and the subject does 
not wish to, or is unable to continue further study participation. Subject data only 
up to the time when consent is withdrawn will be included in the analysis of the 
study.  
16. 
STATISTICAL CONSIDERATIONS 
The primary statistical analysis will compare the proportion of patients who 
develop severe OM (>WHO Grade 3) in patients randomized to ClōSYS Oral 
Rinse versus placebo.  Analysis will be performed by estimating the proportion 
with exact 95% binomial confidence interval for each group separately.  
Statistical testing will use a chi-square test.  As an alternative, Fisher’s Exact 
Test will be used if the minimum expected value is < 5.  
 
The secondary statistical endpoint of time to onset of severe OM will be tabulated 
and the median week to onset will be computed for each group.  Comparison will 
be performed using a Wilcoxon Rank Sum Test (as the values are unlikely to be 
normally distributed).  Duration of severe OM (number of weeks) will be analyzed 
similarly. The proportion of patients in each group with RT interruption will be 
tabulated and compared using a chi-square test. 
 
The longitudinal measurements of the OMWQ and PRO-CTCAE, microbiome 
components and pro-inflammatory cytokines will be compared between groups 
using a linear mixed-effects model to account for the correlation among the 
measurements within an individual. 
 
To justify the proposed sample size, we assume that 9/10 (90%) of the placebo 
control patients will experience severe OM within the 6-week period.  Ten 
patients per group will allow us to detect a decrease to 40% (4/10) in the patients 
randomized to ClōSYS® Unflavored Oral Rinse with 81% statistical power 
(assuming a one-sided alpha of 0.05).    
17. 
ANALYSIS 
17.1 Safety Analysis  
Given the pilot nature of this study and limited number of participants, there will 
be no formal planned interim safety analysis unless an SAE is documented and 
thought more likely than not directly related to the IP.  We will conduct a safety 
analysis if deemed necessary upon review of the SAE by the DSMB.  Otherwise 
safety analysis will be performed upon the conclusion of the study. 
17.2  Efficacy Analysis  
Efficacy analysis will be conducted at the completion of the study.  


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17.3 Interim Analysis 
None planned. 
18. 
REGULATORY OBLIGATIONS 
18.1 Informed Consent 
Before a subject’s participation in the clinical study, the PI or identified designee 
is responsible for obtaining written informed consent from the subject after 
adequate explanation of the aims, methods, anticipated benefits, and potential 
hazards of the study and before any protocol specified procedures, 
investigational product, intervention or device are administered or initiated.  
18.2 Institutional Review Board   
A copy of the protocol, proposed ICF, and all other applicable subject information 
will be submitted to the IRB for written approval. A copy of the written approval of 
the protocol and ICF must be on file at the institution before recruitment of 
subjects into the study.   
 
The investigator is responsible for obtaining IRB approval/renewal at least 
annually throughout the duration of the study. Copies of the investigator’s reports 
and the IRB continuance of approval must be on file at the institution.  
 
The investigator must submit study information to the IRB as required by all 
applicable guidelines and requirements. The investigator will obtain IRB approval 
for subsequent protocol amendments; except changes to eliminate an immediate 
hazard to study subjects, and changes to the informed consent document from 
the IRB prior to implementation.  
 
The investigator will notify the IRB of deviations from the protocol or serious 
adverse events occurring at the site and other serious adverse event reports 
occurring at or received from participating centers as applicable for multi-center 
trials following the IRB policies and procedures. 
19. 
ADMINISTRATIVE PROCEDURES 
19.1  Investigator Responsibilities 
The PI will conduct this study in accordance with the current International 
Conference on Harmonization (ICH) guidance, the Good Clinical Practice (GCP) 
guidance, the Declaration of Helsinki, FDA regulations, local IRB and legal 
requirements.  
  
19.2 Data and Safety Monitoring Board Protocol Review 
Initial DSMB protocol review will be conducted prior to SRC and IRB 
submissions.  
 


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Any protocol revision or amendment that includes a potential change to any 
section of data and safety monitoring plan must be reviewed and approved by 
the DSMB prior to the protocol amendment submission to the IRB.  
19.3 Multicenter Trials 
Not applicable. This is a single center trial. 
20. 
SUBJECT CONFIDENTIALITY  
The principal investigator will ensure that the subject’s confidentiality is 
maintained in compliance with Federal regulations, the International Conference 
on Harmonization (ICH), and Good Clinical Practice (GCP) Guidelines.  
 
Oversight entities and/or regulatory authorities will be permitted direct access to 
review the subject’s original medical records, electronic medical records or 
certified copies for verification of study-related procedures and data. Direct 
access includes examining, analyzing, verifying, and reproducing any records 
and reports that are important to the evaluation of the study.  
21. 
STUDY DOCUMENTATION AND ARCHIVE  
The investigator will maintain a list of appropriately qualified persons to whom 
he/she has delegated study duties. All persons authorized to make entries and/or 
corrections on CRFs will be included on the Delegation of Responsibilities Form.  
 
Source documents, data, and records from which the subject’s CRF data are 
obtained include, but are not limited to, hospital records, clinical/office/research 
charts, laboratory and pharmacy records, diaries, microfiches, radiographs, and 
correspondence. Source data will include information necessary for the 
reconstruction and evaluation of the trial.  
 
The principal investigator or sponsor-investigator is responsible for maintaining a 
comprehensive and centralized filing system of all study-related (essential) 
documentation as required per ICH Guidelines. This can be accomplished by the 
PI, through the site’s standard operating procedures and/or the institutions 
infrastructure. 
 
The investigator will follow ICH Good Clinical Practice Guidelines and the Code 
of Federal Regulations for records and record retention.  
22. 
DATA 
Applicable data as specified as required in the protocol will be reported/submitted 
in the case report form (CRF). Data reported in the case report forms that are 
derived from source documents must be consistent with the source documents or 
the discrepancies must be explained.  In addition, data will be entered into 


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RedCap which is currently being used by many BUMC-T departments for their 
study data bases.   
 
Additional procedures and assessments may be performed as the institution’s 
standard of care; however, these data should remain in the medical records and 
should not be provided as part of the clinical study data unless it pertains to a 
serious adverse event.  
 
The investigator is required to prepare and maintain adequate and accurate case 
histories that record all observations and other data pertinent to the investigation 
on each individual administered the investigational product/intervention/device, or 
employed as a control in the investigation.  
 
At the end of the study, the original samples will be destroyed. Remaining 
extracted DNA samples will be preserved for further sequencing. All remaining 
biospecimens will be destroyed after 6 years.  
 
23. 
PROTOCOL DEVIATIONS  
The investigator will conduct the study in conformance with this protocol, 
generally accepted standards of Good Clinical Practice and all applicable federal, 
state and local laws, rules, and regulations.  
 
Approvals or waivers for protocol deviations will be obtained from the IRB prior 
to occurring, except changes to eliminate an immediate hazard to study subjects. 
If immediate verbal approval is obtained, it will be documented by the research 
staff obtaining the approval and followed by a written protocol deviation form per 
the site standard operating procedures. The sponsor or the sponsor-investigator 
will sign the Protocol Deviation (Waiver) Approval Form or other similar 
document. The original will be filed in the regulatory binder and a copy will be 
placed in the subject’s research file. 
 
 


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24. 
KARNOFSKY PERFORMANCE STATUS SCALE DEFINITIONS 
 
 
Able to carry on normal activity and to 
work; no special care needed. 
  100   Normal no complaints; no 
evidence of disease. 
90 
Able to carry on normal activity; 
minor signs or symptoms of 
disease. 
80 
Normal activity with effort; some 
signs or symptoms of disease.  
Unable to work; able to live at home and 
care for most personal needs; varying 
amount of assistance needed. 
70 
Cares for self; unable to carry on 
normal activity or to do active 
work. 
60 
Requires occasional assistance, 
but is able to care for most of his 
personal needs. 
50 
Requires considerable assistance 
and frequent medical care.  
Unable to care for self; requires equivalent 
of institutional or hospital care; disease 
may be progressing rapidly. 
40 
Disabled; requires special care 
and assistance. 
30 
Severely disabled; hospital 
admission is indicated although 
death not imminent. 
20 
Very sick; hospital admission 
necessary; active supportive 
treatment necessary. 
10 
Moribund; fatal processes 
progressing rapidly. 
0 
Dead 
 
 
 


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25. 
COMMON TOXICITY CRITERIA (CTCAE) 
CTCAE version 5.0 
 
 
Expedited Reporting Requirements for Adverse Events 
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR 
Part 312) 
 
NOTE: Investigators MUST immediately report to the sponsor ANY Serious Adverse 
Events, whether or not they are considered related to the investigational agents(s) 
intervention (21 CFR 312.64) 
 
An adverse event is considered serious if it results in ANY of that following outcomes: 
1. Death 
2. A life-threatening adverse event 
3. An adverse event that results in inpatient hospitalization or prolongation of 
existing hospitalization for ≥ 24 hours 
4. A persistent or significant incapacity or substantial disruptive of the ability to 
conduct normal life functions 
5. A congenital anomaly birth defect 
6. Important Medical Events (IME) that may not result in death, be life 
threatening, or require hospitalization may be considered serious when, based 
upon medical judgement, they may jeopardize the patient or subject and may 
require medical or surgical intervention to prevent one of the outcomes listed in 
the definition (FDA 21 CFR 312.32, ICH E2A and ICH E6). 
Toxicity will be scored using CTCAE Version 5.0 for toxicity and adverse event 
reporting.  A copy of the CTCAE Version 5.0 can be downloaded from the CTEP 
homepage (http://ctep.cancer.gov).  All appropriate treatment areas should have 
access to a copy of the CTCAE Version 5.0.  The investigator must evaluate each 
adverse experience for its relationship to the test drug/treatment and for its 
seriousness. 
 
 
 


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Protocol version date:  August 30, 2022 
26. 
STUDY SCHEDULE 
 
 
 
Test or 
Procedure 
Baseline 
(Prior to 
first 
treatme
nt) 
 
Week 
1 
(±1 
day) 
Week 
2 
 (±1 
day) 
Week 
3 
 (±1 
day) 
 
Week 
4 
(±1 
day) 
Week 
5  
(±1 
day) 
Week 
6 
 (±1 
day) 
Week 7 
(if 
applica
ble) 
(±1 
day) 
1-month 
post-RT 
follow-up 
visit (+21 
days) 
 
Informed 
consent 
 
X 
 
 
 
 
 
 
 
 
 
Medical 
history, 
BMI and 
tobacco 
history 1 
X 
 
 
 
 
 
 
 
 
 
Concurren
t meds 2 
X 
     
X 
 
X 
 
X 
 
X 
 
Physical 
Exam 
including 
dental 
eval and 
photos 3 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Hct 4 
X 
 
 
 
 
 
 
 
X 
 
Malignanc
y info 5  
 
X 
 
 
 
 
 
 
 
 
 
Registrati
on & 
Randomiz
ation 
 
X 
 
 
 
 
 
 
 
 
 
OMWQ 
and PRO 
CTCAE 6 
X 
 
X 
 
X 
 
X 
 
X 
 
WUS and 
Microbiom
e 
Collection 
7 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
X 
 
Dispense 
IP/placeb
o  
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
Adverse 
Event 
Check 8 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
Rinse 
Diary and 
Observati
onal Form 
9 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X  
 


31 | P a g e  
Protocol version date:  August 30, 2022 
 
 
 
Test or 
Procedure 
Baseline 
(Prior to 
first 
treatme
nt) 
 
Week 
1 
(±1 
day) 
Week 
2 
 (±1 
day) 
Week 
3 
 (±1 
day) 
 
Week 
4 
(±1 
day) 
Week 
5  
(±1 
day) 
Week 
6 
 (±1 
day) 
Week 7 
(if 
applica
ble) 
(±1 
day) 
1-month 
post-RT 
follow-up 
visit (+21 
days) 
 
Collect 
Unused 
IP/placeb
o 10 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
Document
ation of 
RT 
treatment 
interruptio
ns  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
1= Complete medical history will include demographics and will be performed within 90 
days of registration. Tobacco history will be collected using the Tobacco Assessment 
Questionnaire.  
2= Review medications including usage of any chlorine dioxide, antimicrobials, or 
corticosteroids within last 7 days prior to registration. Additionally, will review usage of 
honey, baking soda and salt rinses, magic mouthwash, viscous lidocaine, doxepin 
solution, chlorhexidine, and other antimicrobials, corticosteroids, anti-inflammatories, etc 
during study period.  Only medications related to either cancer therapy or treatment of a 
related adverse events will be collected. No other concomitant medications will be 
tracked.  
3= All PEs to include vitals (BP, pulse, temperature, weight), KPS scoring, and OM 
grading per WHO scoring criteria (see section 7.1)  
4= Hematocrit within 90 days of study registration 
5= Confirmation of cytologic or histologic evidence of HNC and documentation of stage 
6= Oral Mucositis Weekly Questionnaire and Patient Reported Outcomes Common 
Terminology Criteria for Adverse Events 
7= Whole Unstimulated Salivary and Microbiome Collection (see section 7.1) 
8= Common Terminology Criteria for Adverse Events (CTCAE) v.5 will be used for 
grading of generalized weakness, skin reaction, weight loss, dry mouth, sore throat, 
dysphagia, anorexia, dysgeusia, voice alteration, and neck edema   
9= Instruct patients on rinse diary to include time, date, and duration of rinses and to 
note any potential effects thought related to rinse on observation form.  Will dispense 
rinse diary and observation form at baseline visit (prior to the start of week 1 of RT).  
Will collect and replace both the rinse diary and observation form each week during 
OTVs 
10= Collect and record by volume amount of unused IP/product 
 
 


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27. 
Tobacco Assessment Questionnaire 
 


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28. OMWQ-HN and PRO-CTCAE 
 


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Protocol version date:  August 30, 2022 
 
 
 


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Protocol version date:  August 30, 2022 
29. 
REFERENCES 
 
1. Trotti A, Bellm LA, Epstein JB, et al. Mucositis incidence, severity and associated 
outcomes in patients with head and neck cancer receiving radiotherapy with or without 
chemotherapy: a systematic literature review. Radiotherapy and oncology. 
2003;66:253–262. 
2. De Ryck T, Duprez F, Bracke M, et al. Dynamic shifts in the oral microbiome during 
radiotherapy. CLINICAL RESEARCH IN INFECTIOUS DISEASES. 2015;2. 
3. Aieta EM, Berg JD. A review of chlorine dioxide in drinking water treatment. Journal 
(American Water Works Association). 1986:62–72. 
4. Shinada K, Ueno M, Konishi C, et al. Effects of a mouthwash with chlorine dioxide on 
oral malodor and salivary bacteria: a randomized placebo-controlled 7-day trial. Trials. 
2010;11:14. 
5. Donnelly JP, Bellm LA, Epstein JB, et al. Antimicrobial therapy to prevent or treat oral 
mucositis. The Lancet Infectious Diseases. 2003;3:405–412. 
6. Epstein JB, Beaumont JL, Gwede CK, et al. Longitudinal evaluation of the oral 
mucositis weekly questionnaire‐head and neck cancer, a patient‐reported outcomes 
questionnaire. Cancer. 2007;109:1914–1922. 
7. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA: a cancer journal for 
clinicians. 2016;66:7–30. 
8. Bese NS, Hendry J, Jeremic B. Effects of prolongation of overall treatment time due 
to unplanned interruptions during radiotherapy of different tumor sites and practical 
methods for compensation. International Journal of Radiation Oncology* Biology* 
Physics. 2007;68:654–661. 
9. Russo G, Haddad R, Posner M, et al. Radiation treatment breaks and ulcerative 
mucositis in head and neck cancer. The Oncologist. 2008;13:886–898. 
10. Sonis ST. The pathobiology of mucositis. Nature Reviews Cancer. 2004;4:277–284. 
11. Al-Dasooqi N, Sonis ST, Bowen JM, et al. Emerging evidence on the pathobiology 
of mucositis. Supportive care in cancer. 2013;21:3233–3241. 
12. Le Q-T, Kim HE, Schneider CJ, et al. Palifermin reduces severe mucositis in 
definitive chemoradiotherapy of locally advanced head and neck cancer: a randomized, 
placebo-controlled study. Journal of Clinical Oncology. 2011;29:2808–2814. 
13. Henke M, Alfonsi M, Foa P, et al. Palifermin decreases severe oral mucositis of 
patients undergoing postoperative radiochemotherapy for head and neck cancer: a 
randomized, placebo-controlled trial. Journal of Clinical Oncology. 2011;29:2815–2820. 
14. Mohammad AR, Giannini PJ, Preshaw PM, et al. Clinical and microbiological 
efficacy of chlorine dioxide in the management of chronic atrophic candidiasis: an open 
study. International dental journal. 2004;54:154–158. 


38 | P a g e  
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15. Condie, Lyman W. "Toxicological problems associated with chlorine dioxide." 
Journal (American Water Works Association) (1986): 73-78. 
 
16. Helliwell, M., and J. Nunn. "Mortality in sodium chlorate poisoning." British medical 
journal 1.6171 (1979): 1119. 
 
17. Lubbers, Judith R., Sudha Chauan, and Joseph R. Bianchine. "Controlled clinical 
evaluations of chlorine dioxide, chlorite and chlorate in man." Environmental health 
perspectives 46 (1982): 57. 
 
18. MICHAEL, GEORGE E., et al. "Chlorine Dioxide Water Disinfection: A Prospective 
Epidemiology Study." Archives of Environmental Health: An International Journal 36.1 
(1981): 20-27. 
 
19. Ratcliff, James L., Elizabeth A. Renken, and Jessica K. Ward. "Method for 
determining the effectiveness of stabilized chlorine dioxide in a mouth rinse." U.S. 
Patent No. 7,875,460. 25 Jan. 2011. 
 
20. U.S. Food and Drug Administration (FDA) 21 CFR, Parts 210 and 211, the current GMP 
regulations. 
21. Shinada, Kayoko, et al. "A randomized double blind crossover placebo-controlled 
clinical trial to assess the effects of a mouthwash containing chlorine dioxide on oral 
malodor." Trials 9.1 (2008): 71. 
 
 


39 | P a g e  
Protocol version date:  August 30, 2022 
30. 
APPENDICES 
 
30.1 Appendix A: Whole Unstimulated Saliva (WUS) Collection and Analyses 
Standard Operating Procedures 
 
To collect WUS, each patient will be asked to refrain from eating, drinking, smoking, and 
oral hygiene procedures for at least 1 h before saliva collection. Patient will be asked to 
rinse with plain water prior to resting for 5 minutes with head tilted forward and no 
swallowing.  Pooled saliva in the floor of mouth will then be expectorated every 60 
seconds into a dedicated collection device (SpeciMAX™ Saliva Collection; Thermo 
Fisher) for a total of 5 minutes, as previously described(1-3).  Protease inhibitor cocktail 
has to be added immediately after sample collection to minimize protein degradation. 
WUS samples will be individually placed in labeled containers with the patient’s unique 
sequential subject identification number and week of collection.  These samples will be 
transferred to the Microbiome Core on the same day, aliquoted and stored at -80°C in a 
secure location until utilized for salivary cytokine analysis.  The samples will be 
destroyed after analyses are complete. Remaining extracted DNA samples will be 
preserved for further sequencing. Six years after the samples are collected all remaining 
biospecimens will be destroyed. 
 
WUS samples will be centrifuged at at 2600x g for 15 min to remove cell pellets and 
debris, and supernatant divided into 100 µL aliquots to reduce freeze thaw cycles.  
Multiplexed quantification of pro-inflammatory cytokines IL-1β, IL-6, IL-8/CXCL8, and 
TNFα will be conducted with customized xMAP Human High Sensitivity T Cell 
MAGNETIC Bead Panel HSTCMAG-28SK-04 (Sigma-Millipore) using MagPix instrument 
(Luminex) in the Microbiome Core laboratory. Three serial dilutions of each sample will 
be made in duplicate and xMAP assays will be performed per the manufacturer’s protocol. 
In cases where severe contamination of WUS samples with blood is observed, samples 
will be cleared with HemogloBindTM (Biotech Support Group catalog H0145-05), 
according to the manufacturer’s protocol. 
 
Reference: 
1. Navazesh M. Methods for collecting saliva. Annals of the New York Academy of 
Sciences. 1993;694:72–77. 
2. Ondrey FG, Dong G, Sunwoo J, et al. Constitutive activation of transcription 
factors NF‐κB, AP‐1, and NF‐IL6 in human head and neck squamous cell 
carcinoma cell lines that express pro‐inflammatory and pro‐angiogenic cytokines. 
Molecular carcinogenesis. 1999;26:119–129. 
3. Rhodus NL, Cheng B, Myers S, et al. The feasibility of monitoring NF‐κB 
associated cytokines: TNF‐α, IL‐1α, IL‐6, and IL‐8 in whole saliva for the 
malignant transformation of oral lichen planus. Molecular carcinogenesis. 
2005;44:77–82. 
 
 
 


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Protocol version date:  August 30, 2022 
30.2 Appendix B: Microbiome Collection and Analyses Standard Operating 
Procedures 
 
Microbiome collection will be performed by gently swabbing over areas of visible 
mucositis, or areas of high dose radiation where mucositis would be most likely to 
present, utilizing disposable medical sterile swabs (OMNIgen ORAL OMR-110, DNA 
Genotek).  Microbiome samples will be individually placed in labeled containers with the 
patient’s unique sequential subject identification number and week of collection.  These 
samples can be stored at room temperature for at least one month and then will be 
stored in a secure location at the Microbiome Core, at -80°C until utilized for microbiome 
analyses. An aliquot of WUS collected as described in Appendix A will be used for 
microbiome analysis. The samples will be destroyed after analyses are complete.  
Remaining extracted DNA samples will be preserved for further sequencing. Six years 
after the samples are collected all remaining biospecimens will be destroyed. 
 
The analyses will be performed as per institutional technique: 
 
DNA isolation and the V4 of the 16S rRNA gene amplicon generation: 
 
DNA is isolated from oral swabs using the DNeasy PowerSoli Pro Kit (Qiagen, USA) 
with the bead beating step following manufacturer’s protocol to ensure isolation of DNA 
from gram positive and gram negative organisms including difficult to isolate species 
such as those in the genus Bifidobacterium. The hypervariable V4 region of the 16S 
rRNA gene will be amplified from each sample using barcoded reverse primers (806R) 
and common forward primer (515F). Both reverse and forward primes are extended with 
the sequencing primer pads, linkers, and Illumina adapters (Caporaso JG, Lauber CL, 
Walters WA, Berg-Lyons D, Huntley J, Fierer N, et al. Ultra-high-throughput microbial 
community analysis on the Illumina HiSeq and MiSeq platforms. The ISME Journal. 
2012;6(8):1621-4), and with MyFi™ Mix (Bioline Meridian, Cat No. BIO-25050). The 
PCR will be performed on LightCycler 96 (Roche) in the final volume of 40µL. 
Amplicons will be quantified using Quant-It PicoGreen dsDNA Assay kit (ThermoFisher 
Scientific, Cat No. P7589), according to the manufacturer’s protocol. Equal amount of 
amplified DNA (240 ng) from each sample will be pooled and cleaned using UltraClean 
PCR Clean-Up Kit (MoBio, Cat No. 12500).  
 
 
16S rRNA gene sequencing: 
 
Pooled amplicons were diluted and denatured with 0.2N NaOH to the final DNA 
concentration in the library of 7.5-8.0pM. The library was sequenced at the Microbiome 
Core at the Steele Children’s Research Center, University of Arizona, using MiSeq 
platform (Illumina) and custom primers (Caporaso JG, Lauber CL, Walters WA, Berg-
Lyons D, Huntley J, Fierer N, et al. Ultra-high-throughput microbial community analysis 
on the Illumina HiSeq and MiSeq platforms. The ISME Journal. 2012;6(8):1621-4). Due 
to the limited sequence diversity among 16S rRNA amplicons, 5% of the PhiX 
Sequencing Control V3 (Illumina, Cat No. FC-110-3001) made from phiX174, will be 
used to spike the library to increase diversity. Paired-end sequencing, 150 cycles each 
(PE150) will be used to sequence the amplified fragments.  
 
Sequence analysis: 


41 | P a g e  
Protocol version date:  August 30, 2022 
 
The raw sequencing data will be demultiplexed using the idemp script 
(https://github.com/yhwu/idemp). Filtering, dereplication, chimera identification, and 
merging of paired-end reads will be performed with dada2 R package (Callahan BJ, 
McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: High-resolution 
sample inference from Illumina amplicon data. Nature Methods. 2016;13(7):581-3). The 
Amplicon Sequence Variants (ASVs) taxonomy will be assigned using the Ribosomal 
Database Project (RDP) classifier (Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive 
Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial 
taxonomy. Appl Environ Microbiol. 2007;73(16):5261-7) against SILVA database 
release 138 (or newer if available, Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, 
Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data 
processing and web-based tools. Nucleic Acids Res. 2013;41:D590-6). 
Taxonomic richness and evenness (Shannon and Simpson indices) will be calculated 
and statistical significance within in each experiment was calculated using Kruskal-
Wallis rank sum test followed by Dunn’s multiple comparison test with Bonferroni 
correction (dunn.test R package). Differences in microbial communities will evaluated 
using non-metric multidimensional scaling (NMDS) ordination analysis on Bray-Curtis 
distances followed by permutational multivariate analysis of variance (PERMANOVA) to 
analyze the contribution of different metadata variables to microbial communities 
composition dissimilarities. Also, to investigate and visualize the association between 
metadata variables and their effect on the species distribution pattern, redundancy 
analysis will used in vegan R package. The obtained results will be visualized with a 
ggplot2 (ver 3.3.2) package and with heatplus (ver. 3.11) R package. Changes in 
microbiome will be correlated with collected bio-medical variables using stats R 
package and visualized using cor package.