Conditions: Caries
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 15
Healthy volunteers: yes

Brief summary:
The purpose of this study is to evaluate the fluoride dose response of different dentifrice fluoride concentrations - 0, 250, 1100 and 2800 ppm fluoride as sodium monofluorophosphate (MFP) and a fifth leg of 1100 ppm Stannous Fluoride (SnF2) using an in situ caries model.

Detailed description:

Arms:
arm 1: 0 ppm F (placebo, negative control), 250 ppm F as MFP (dose-response control), 1100 ppm F as MFP (reference), 2800 ppm F as MFP (dose-response control) arm 2: 0 ppm F (placebo, negative control), 250 ppm F as MFP (dose-response control), 1100 ppm F as MFP (reference), 2800 ppm F as MFP (dose-response control) arm 3: 0 ppm F (placebo, negative control), 250 ppm F as MFP (dose-response control), 1100 ppm F as MFP (reference), 2800 ppm F as MFP (dose-response control) arm 4: 0 ppm F (placebo, negative control), 250 ppm F as MFP (dose-response control), 1100 ppm F as MFP (reference), 2800 ppm F as MFP (dose-response control) arm 5: 1100 ppm SnF2 toothpaste only

Interventions: intervention 1: 0 ppm F intervention 2: 250 ppm F as MFP intervention 3: 1100 ppm as MFP intervention 4: 2800 ppm F as MFP intervention 5: 1100 ppm SnF2
Intervention descriptions: intervention 1: Each subject will be randomly assigned to this treatment during one of the four periods for this crossover study. intervention 2: Each subject will be randomly assigned to this treatment during one of the four periods for this crossover study. intervention 3: Each subject will be randomly assigned to this treatment during one of the four periods for this crossover study. intervention 4: Each subject will be randomly assigned to this treatment during one of the four periods for this crossover study. intervention 5: Each subject will be assigned to this treatment during the fifth period for this crossover study.

Protocol document:
Prot_SAP_000.pdf: 
Clinical Study Protocol 2020154 
Amendment 1  
CONFIDENTIAL 
Page 1 of 29 
 
The Procter & Gamble Company 
Mason Business Center 
8700 Mason-Montgomery Road 
Mason, OH USA 45040 
 
 
EVALUATION OF THE FLUORIDE DOSE RESPONSE OF MFP DENTIFRICE USING IN 
SITU CARIES MODEL 
 
Date of Protocol: January 21, 2021 
Date of Amendment 1:  May 19, 2021 
 
Study Number 2020154 
 
 
Sponsor: 
The Procter & Gamble Company 
Worldwide Clinical Investigations—Oral 
8700 Mason-Montgomery Road 
Mason, OH 45040 
Principal Investigator 
Location, Phone 
Domenick T Zero, DDS, MS,  
Oral Health Research Institute 
415 Lansing Street 
Indianapolis, IN 46202-2876 
 
Phone: 317-274-8822 
FAX: 317-274-5425 
Email: dzero@iu.edu 
 
 
Investigator’s Agreement Statement: 
I have read, I understand, and I will conduct the 
study according to this Protocol and Good Clinical 
Practices. 
 
 
 
Domenick Zero, DDS, MS 
(Date) 
 
 Signatures below indicate approval of the Protocol Amendment 1. 
Clinical Scientist – Malgorzata Klukowska, DDS, PhD 
 
 
Statistician – Yuanshu Zou, PhD 
 
 
Clinical Data Manager – Marsha Gabbard, AA 
 
 
 
Clinical Trial Manager – Molly Seeck BSN, JD 
 
 
Svetlana Farrell, DDS, PhD, Senior Director, Global 
Oral Care-Clinical Operations and Performance 
Testing 
 
Julie Grender, PhD, Research Fellow, Health Care 
Statistics and Data Management 
 
 
 
 
 
 
PII 
PII 
PII 


Clinical Study Protocol 2020154 
Amendment 1  
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Page 2 of 29 
 
TABLE OF CONTENTS 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS..........................................................3 
1. 
Clinical Study Information ......................................................................................................4 
1.1 
Background................................................................................................................4 
1.2 
Investigational Products ...............................................................................................5 
1.3 
Summary of Findings ...................................................................................................5 
1.4 
Known Potential Risks and Benefits ...............................................................................5 
1.5 
Investigational Product Dosage .....................................................................................5 
1.6 
Ethical Compliance......................................................................................................6 
1.7 
Target Population........................................................................................................6 
1.8 
Literature and Data......................................................................................................7 
2. 
Investigational Plan ..............................................................................................................7 
2.1 
Overall Study Design and Plan ......................................................................................7 
2.2 
SELECTION AND WITHDRAWAL OF STUDY POPULATION ......................................... 12 
3. 
TREATMENTS .................................................................................................................. 13 
3.2 
Assessment of Efficacy .............................................................................................. 14 
3.3 
Assessment of Safety ................................................................................................ 15 
4. 
Laboratory Methods............................................................................................................ 18 
4.1 
Specimen Preparation ............................................................................................... 18 
4.2 
Lesion Creation ........................................................................................................ 19 
4.3 
6.3 Lesion Quality ..................................................................................................... 19 
4.4 
Efficacy Measurements and Evaluations ....................................................................... 19 
4.5 
Laboratory Data ........................................................................................................ 20 
4.6 
Specimen Retention .................................................................................................. 20 
5. 
Statistical Plan ................................................................................................................... 20 
5.1 
Statistical Methods .................................................................................................... 20 
5.2 
Hypotheses .............................................................................................................. 20 
5.3 
Number of Subjects ................................................................................................... 21 
5.4 
Selection of Subjects ................................................................................................. 21 
6. 
Data Management Plan....................................................................................................... 21 
6.1 
Access to Source Data/Documents .............................................................................. 22 
6.2 
Data Handling and Recordkeeping............................................................................... 22 
7. 
Ethics ............................................................................................................................... 22 
8. 
Financing and Insurance ..................................................................................................... 23 
9. 
Publication Policy ............................................................................................................... 23 
 
 
 
 


Clinical Study Protocol 2020154 
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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS  
 
Abbreviation 
Definition 
AE(s) 
Adverse Event(s) 
ANCOVA 
Analysis of Covariance 
ANOVA  
Analysis of Variance 
B&A 
Balance & Assignment 
CFR 
Code of Federal Regulations 
CRF(s) 
Case Report Form(s) 
ECRF 
Electronic Case Report Forms (system) 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
IRB/IEC 
Institutional Review Board/Independent Ethics Committee 
ICH 
International Council for Harmonization 
ITT 
Intent-to-Treat 
MFP 
Sodium Monofluorophosphate 
PP 
Per Protocol 
SAE 
Serious Adverse Events  
SMH 
 
SnF2 
Surface Microhardness 
 
Stannous Fluoride 
OSHT 
Oral Soft/Hard Tissue Exam  
OST 
Oral Soft Tissue  
EFU 
Enamel Fluoride Uptake  
 
 
 
 


Clinical Study Protocol 2020154 
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1. 
CLINICAL STUDY INFORMATION 
1.1 
Background 
Fluoride toothpaste is the most widely used form of fluoride delivery worldwide. Fluoride dentifrices have 
shown in numerous clinical trials to be effective anticaries agents and have been the subject of several 
systematic quantitative evaluations (Marinho et al., 2003; Twetman et al., 2003), which provide the 
highest standard of evidence for the effectiveness of fluoride dentifrice. Marinho et al. (2003) based their 
conclusions on a meta-analysis of 70 trials of the effectiveness of fluoride dentifrice for the prevention 
of dental caries in children compared to placebo. They found evidence that the use of fluoride dentifrices 
has a caries-inhibiting effect (average reduction in Decay, Missing, and Filled Surfaces (DMFS) of 24%) 
on the permanent dentition. In addition, they concluded that the effectiveness of fluoride dentifrice may 
be relatively greater in individuals with a higher caries experience, with increased fluoride concentration, 
increased frequency of use, and with supervised brushing. There was no evidence that the effect was 
dependent on background exposure to fluoridated water. Twetman et al. (2003) reached similar overall 
conclusions from their systematic review.  
The current levels of fluoride dentifrice products that are marketed worldwide generally fall in the range 
between 1000 – 1500 ppm F, although dentifrices with lower fluoride concentrations are marketed in 
some countries. All fluoride toothpaste sold in the US is in the 1000-1100 ppm F range. Walsh et al. 
(2010) reported based on a network meta-analysis that the relative anticaries effects of fluoride 
toothpastes increased with higher fluoride concentration. Based on 74 trials involving the caries scores 
(DMFS) in the mixed or permanent dentition, the anticaries effect of fluoride toothpaste was 23% for 
1000/1055/1100/1250 ppm F and 36% 2400/2500/2800 ppm F; however, toothpastes with 440/500/550 
ppm F and below did not show a statistically significant effect compared to placebo.  
Clinical caries trials remain the standard for evaluating the safety and efficacy of fluoride dentifrice 
products. However, due to the high cost involved in conducting clinical caries trials, several surrogate 
measures of fluoride efficacy have been introduced. These include in situ caries models, rat caries 
models, in vitro demineralization and remineralization studies, and fluoride uptake studies. These model 
systems have been extensively reviewed at a Conference held in Rochester, NY in 1994 (Adv Dent Res 
9:169-340, 1995). While intra-oral models have been in use for the past forty years, a "Consensus 
Conference of Intraoral Models" (ADA, Chicago, Sept. 1990) clearly identified the need for validation of 
intra-oral caries models for their potential use as methods of evaluating the efficacy of fluoride dentifrices 
and other fluoride-containing dental products. Based on this conference and the Rochester Models 
conference, there is general agreement among researchers in the field that appropriately validated in 
situ models represent an acceptable approach for testing the anticaries potential of fluoride products. 
The use of intraoral appliance models overcome the ethical and practical problems associated with 
human studies by permitting the evaluation of subtle changes in the mineral status of enamel specimens 
occurring at a well-controlled test site mounted in the subject's own partial denture. Intraoral models 
serve as bridges between the clinical situation and the more controlled laboratory environment. 
Philosophically, these models should, as far as possible, be representative of what occurs in the clinical 
situation, yet keep variables under control so that meaningful outcome differences can be detected in a 
reasonable number of subjects (Zero, 1995). 
Previous work by our group has shown that a modification of the Koulourides intra-oral model 
(Koulourides et al., 1974) has sufficient sensitivity and reproducibility to respond in dose-response 
manner to meet the requirements for model validation (Proskin et al., 1992). The model, which uses 
partially demineralized enamel as the starting hard tissue substrate, permits the evaluation of the ability 
of the test dentifrice to enhance net remineralization and measure fluoride bioavailability under clinically 
relevant conditions. Our current model has been validated based on its response to different dentifrice 
fluoride concentrations - 0, 250, 500 and 1100 ppm fluoride (Zero et al., 1994; Zero, 1995; Zero et al., 
2005) as well as in several more commercially funded in situ studies testing fluoride dentifrice products 
that also included fluoride dose response controls (Zero et al., 2018). 
Our intraoral model system uses the surface microhardness (SMH) test to assess changes in the mineral 
content of enamel. A very precise instrument (Wilson 2100 Hardness Tester) with a Knoop diamond 
under a fixed load is used to place indentations into the enamel surface. The length of the indentations 
is accurately measured using an image analysis system and correspond directly to the depth of 


Clinical Study Protocol 2020154 
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penetration of the diamond into the enamel surface (30:1 ratio). This method can be considered 
analogous to the dentist’s use of a sharp dental probe to detect clinical dental caries; however, the SMH 
test is hundreds-fold more sensitive and accurate. Using the SMH test, the hardness of sound enamel 
can be measured and compared with the hardness of enamel after exposure to a caries 
(demineralization) challenge and after exposure to conditions that favor remineralization. An increase in 
the indentation length compared to the baseline indentation length indicates softening of the enamel 
and demineralization. A decrease in the indentation length represents rehardening of the enamel surface 
and remineralization. The SMH test is one of the few methods that can be used to accurately determine 
the very early changes occurring at the enamel surface during the caries process (Zero, 1995). For early 
enamel lesions, a decrease in SMH has been shown to be linearly related to mineral loss 
(demineralization) (Zero et al., 1990). White (1988) reported that for shallow enamel lesions, an increase 
in SMH was highly correlated with remineralization as measured by microradiography. 
Another advantage of our in-situ model system is that fluoride uptake can be measured on the same 
enamel 
specimens 
that 
are 
used 
to 
evaluate 
changes 
in 
mineral 
status 
(demineralization/remineralization) of enamel. Fluoride uptake testing using in vitro models is accepted 
by the American Dental Association as one of the profile methods for generic efficacy testing of 
dentifrices in their Seal Acceptance Program and by the Food and Drug Administration as one of the 
testing procedures for fluoride dentifrice drug products in the Final Monograph - Anticaries Drug 
Products for Over-the-Counter Human Use. An important advantage of determining F uptake using an 
intraoral appliance model is that studies are conducted under clinically relevant conditions, which very 
closely approximate the in vivo situation. Human subjects apply the test dentifrice by brushing their teeth 
during the intraoral study. The fluoride is delivered in the presence of physiologically secreted saliva and 
there are intermittent cycles of demineralization and remineralization during the experimental period as 
with the natural caries process. Furthermore, when the active fluoride agent in a dentifrice is sodium 
monofluorophosphate (MFP), plaque and saliva present in the oral environment provide the necessary 
phosphatases to hydrolyze MFP to release free fluoride ion (Pearce and Dibdin, 1995). This will take 
place only to a limited extent when using non-biologic in vitro testing methods. Thus, the determination 
of intraoral fluoride uptake should provide a better estimation of the true fluoride bioavailability of fluoride 
dentifrice products than determination of fluoride uptake in vitro. 
 
1.2 
Investigational Products 
• 
0 ppm F toothpaste and Oral-B® Indicator soft toothbrush.  
• 
250 ppm F sodium monofluorophosphate toothpaste and Oral-B® Indicator soft toothbrush. 
• 
1100 ppm F sodium monofluorophosphate toothpaste and Oral-B® Indicator soft toothbrush.  
• 
2800 ppm F sodium monofluorophosphate toothpaste and Oral-B® Indicator soft toothbrush. 
• 
1100 ppm gluconate-stabilized SnF2 toothpaste and Oral-B® Indicator soft toothbrush. 
 
1.3 
Summary of Findings 
Previous in-situ caries model dose response studies have evaluated the dose response of different 
dentifrice fluoride concentrations through the use of this in situ caries model.  
 
1.4 
Known Potential Risks and Benefits 
This study is for research purposes only. There may be no direct benefit to the subjects from participation 
in the study. Others may benefit from the knowledge gained. There may be other risks of study 
participation that are unknown. There is no guarantee that the subjects will receive any medical or dental 
benefits from participating in this research study. 
 
1.5 
Investigational Product Dosage 
Each subject will be randomized to each of the treatment groups below: 
• 
0 ppm F toothpaste  
• 
250 ppm F sodium monofluorophosphate toothpaste  


Clinical Study Protocol 2020154 
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• 
1100 ppm F sodium monofluorophosphate toothpaste  
• 
2800 ppm F sodium monofluorophosphate toothpaste  
Subjects willing to participate in a fifth leg of the study will receive 1100 ppm gluconate-stabilized SnF2 
toothpaste as the investigational product.  
 
Subjects will be instructed to brush twice daily (morning and at bedtime) with their assigned dentifrice 
for 21 days following the brushing instructions found in the Appendix. Subjects will be instructed to brush 
the biting surfaces of their back teeth (but not enamel specimens) with a full ribbon of study dentifrice 
for one timed minute while wearing their partial dentures, followed by rinsing with the 15 mL of measured 
tap water for 10 seconds and then expectorating. Subjects who are regular floss users will be allowed 
to continue flossing; use of any non-study related products will be disallowed. Compliance with brushing 
procedures will be recorded in subject diaries (see Appendix), as well as any de novo or changes in 
medical conditions, medications or treatments.  
 
1.6 
Ethical Compliance 
Prior to study initiation, the Investigator must obtain institutional review and approval of the Protocol, the 
consent form, and other necessary study-related documents in compliance with the US Code of Federal 
Regulations, Title 21, Part 56 or the ICH-GCP Consolidated Guidelines, Chapter 3 and in compliance 
with Procter & Gamble SOP CTN-CL-504 (“Institutional Review Board/Independent Ethics Committee 
Review and Approval”). The Investigator will maintain any original authorization letter(s) and will be 
available for review by the Sponsor. IRB approval letters should include the study title, Sponsor study 
number, the address of the IRB, date of request, and the signature of the IRB chairperson or designate. 
Additionally, the letter must acknowledge that both the Protocol and consent form have been approved 
by the IRB. The study will not begin until the Sponsor has received confirmation of IRB approval. The 
IRB shall also review the investigation at least once a year during study execution. The Investigator will 
notify the IRB when the study is terminated and provide confirmation that the study has been closed 
with the IRB to the Sponsor. 
 
The Investigator will obtain written informed consent for each subject prior to participation in the study, 
per the US Code of Federal Regulations, Title 21, Parts 50.25 and 50.27 and ICH-GCP, Chapter 4, 
subpart 4.8 and in compliance with Procter & Gamble SOP CTN-CL-503 (“Informed Consent Form, 
Ethics Approval and Investigator Use”). Subjects, or their legal guardian, are required to read, sign and 
date an IRB approved consent form with the Investigator also maintaining a signed and dated copy. The 
subject or legal guardian will be given a copy of the consent form. All study procedures must be 
explained in non-technical terms. 
 
Changes to the Protocol following IRB approval affecting the safety of subjects, scope or objectives of 
the investigation, or the scientific quality of the study will be documented as amendments. Such changes 
will require the Sponsor, Investigator, and IRB approval prior to implementation, unless immediate action 
is required to safeguard subject safety. Administrative or minor changes (e.g., typographical errors, 
changes in Sponsor personnel, etc.) will be documented as revisions but may not need to be submitted 
as amendments unless required by the IRB. Any change in the Sponsor’s monitoring staff, Clinical Trial 
Manager or Medical Monitor during the conduct of the study, will be reported to the Investigator.  
 
During the course of the trial, the clinical site will allow monitoring by the Sponsor (Clinical Trial Manager 
or designee) to check compliance with the Protocol, regulations and guidelines, adequacy of the 
equipment and facilities, and satisfactory data collection. 
 
1.7 
Target Population 
The target population of this study is generally healthy subjects ranging from 18-85 years with a normal 
salivary flow rate.  
 


Clinical Study Protocol 2020154 
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1.8 
Literature and Data 
Clinical aspects of de/remineralization of teeth: proceedings of Models Conference 1994. Rochester, 
New York, June 11–14, 1994. Adv Dent Res. 1995;9:169–340. 
"Consensus Conference of Intraoral Models" (ADA, Chicago, Sept. 1990) 
Gelhard TBFM, ten Cate JM, Arends J: Rehardening of artificial enamel lesions in vivo. Caries Res 
1979;13:80-83. 
Koulourides, T, Phantumvanit, P, Munksgaard, EC, Housch, T. An intraoral model used for studies of 
fluoride incorporation in enamel. J Oral Pathol 1974;3:185–196. 
Marinho VC, Higgins JP, Sheiham A, Logan S (2003c). Fluoride toothpastes for preventing dental caries 
in children and adolescents. Cochrane Database Syst Rev 1:CD002278. 
Pearce EI, Dibdin GH. The diffusion and enzymic hydrolysis of monofluorophosphate in dental plaque. 
J Dent Res 1995;74:691-697. 
Proskin HM, Chilton NW, Kingman A. Interim report of the ad hoc committee for the consideration of 
statistical concerns related to the use of intra-oral models in submissions for product claims approval 
to the American Dental Association. J Dent Res 71 1992;(Spec Iss):949-952. 
Sakkab NY, Cilley WA, Haberman JP. Fluoride in deciduous teeth from an anti-caries clinical study. J 
Dent Res 1984;63:1201-1205. 
Twetman S, Axelsson S, Dahlgren H, Holm AK, Kallestal C, Lagerlöf F, et al. Caries-preventive effect 
of fluoride toothpaste: a systematic review. Acta Odontol Scand 2003;61:347-355. 
Walsh T, Worthington HV, Glenny AM, Appelbe P, Marinho VC, Shi X. Fluoride toothpastes of different 
concentrations for preventing dental caries in children and adolescents. Cochrane Database Syst Rev 
2010;1:CD007868. 
White DJ. Use of synthetic polymer gels for artificial carious lesion preparation. Caries Res  
1987;21:228-242. 
White DJ. Reactivity of fluoride dentifrices with artificial caries. II Effect of subsurface lesions: F uptake, 
F distribution, surface hardening and remineralization. Caries Res 1988;22:27-36. 
Zero DT: In situ caries models. Adv Dent Res 1995;9:214–230. 
Zero D, Chin JR, Kelly S, Eckert G, Venell JA, Engelman EE. Efficacy of natural toothpastes in an in 
situ caries model. J Dent Res 84(Spec Iss A): Abst. 3259, 2005, (www.dentalresearch.org). 
Zero, DT, Lippert F, Hara AT, Creeth J, Newby E, Butler A, Karwal R, Constantin P, Bosma ML. In situ 
anticaries efficacy of dentifrices with different formulations - a pooled analysis of results from three 
randomized clinical trials. J Dent 2018;77:93–105. doi: 10.1016/j.jdent.2018.07.014  
Zero D, Featherstone JDB, Fu J, Hayes AL, Vogel GL. Response of a de/remineralization in situ model 
to fluoride dentifrice (abstract). Caries Res 1994;28:208. 
Zero DT, Rahbek I, Fu J, Proskin HM, Featherstone JDB. Comparison of the iodide permeability test, 
the surface microhardness test and mineral dissolution of bovine enamel following acid challenge. 
Caries Res 1990:24:181-188. 
 
2. 
INVESTIGATIONAL PLAN  
2.1 
Overall Study Design and Plan  
The purpose of this study is to evaluate the fluoride dose response of different dentifrice fluoride 
concentrations - 0, 250, 1100 and 2800 ppm fluoride as sodium monofluorophosphate (MFP) using an 
in-situ caries model. 
 
The purpose of Period 5 is to evaluate the performance and sensitivity of the in-situ caries model to a 
SnF2 toothpaste. 


Clinical Study Protocol 2020154 
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2.1.1 
Primary Endpoints 
The primary endpoint is the enamel fluoride uptake on Day 21 and the primary comparison between 
pastes with 0 and 2800ppm fluoride concentrations, and all other treatment comparisons and time points 
being of secondary interest. The secondary endpoints include % SMH recovery and SMH change (see 
4.4), 
2.1.2 
Design Type 
This is a randomized, four-treatment, 4-period crossover, double blind clinical trial. The target population 
is approximately 16 adult volunteers. During the course of the study, all subjects will be asked to 
voluntarily participate in a fifth period, not included as part of the crossover design.  Those subjects 
willing to participate in Period 5 will sign an amended informed consent form and will all receive the 
same product during that period. Two to three days before the start of each treatment period the subjects 
will have their teeth cleaned to remove all accessible plaque and calculus and will be provided with a 
non-fluoride dentifrice to use until their next visit. At the beginning of each testing period, two gauze-
covered 4 mm round partially demineralized human enamel specimens will be placed in the buccal 
flange area of the subject’s mandibular partial denture. In addition, two gauze-covered 4 mm round 
partially demineralized human enamel specimens will be placed in the buccal surface of two posterior 
denture teeth of the same side of the partial denture. Once specimens are placed, subjects will wear 
their partial dentures twenty-four hours a day and use their assigned toothpaste twice daily, as 
instructed, until their next visit. Subjects will return at 7 and 14 days to have one specimen removed per 
visit, and again at 21 days to have the remaining two specimens removed. The specimen removal 
sequence will occur in a predetermined order starting with the anterior denture tooth specimen, posterior 
denture tooth specimen, and then the two buccal flange specimens. Following the 21-day specimen 
removal visit, the subjects will undergo a four- to five-day washout period followed by another cleaning 
and two- to three-day lead in period. This process will be repeated until all subjects have used all four 
test products. Changes in the mineral content of the enamel specimens will be assessed using the SMH. 
Enamel fluoride uptake (EFU) will be determined using the microdrill enamel biopsy technique.  
 
          Table 1. Study Schedule by Procedure Type and Visit 
PROCEDURE 
VISIT 1 
VISIT 2, 7, 12, 
17, 22  
VISIT 3, 8, 13, 
18, 23 
VISIT 4, 9, 
14, 19, 24 
VISIT 5, 10, 
15, 20, 25 
VISIT 6, 11, 16, 
21, 26 
 
SCREENING 
PROPHY 
BEGIN 
TREATMENT  
7 DAY 
REMOVAL 
14 DAY 
REMOVAL 
END1 
TREATMENT 
Informed Consent 
X 
 
 
 
 
 
Medical History Review 
X 
X 
X 
X 
X 
X 
Demographics 
X 
 
 
 
 
 
Oral Soft Tissue Exam  
X 
X 
X 
 
 
X 
Oral Hard Tissue Exam  
X 
X (v2 only) 
 
 
 
X (v21 and 
v26) 
Salivary Flow 
X 
 
 
 
 
 
Inclusion/Exclusion 
Criteria 
X 
 
 
 
 
 
Continuance Criteria 
 
X 
X 
X 
X 
X 
Randomization 
 
 
X (v3 only) 
 
 
 
Dental Prophylaxis 
 
X 
 
 
 
 
Placement of specimens 
 
 
X 
 
 
 
Issue washout toothpaste 
 
X 
 
 
 
 
Return washout toothpaste 
 
 
X 
 
 
 


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Issue study products and 
diary 
 
 
X 
 
 
 
Return study products and 
diary 
 
 
 
 
 
X 
General Comments 
X 
X 
X 
X 
X 
X 
Adverse Events 
 
X 
X 
X 
X 
X 
Subject Accountability 
 
 
 
 
 
X1 (v21 or 
26) 
 
1 For those subjects continuing on Period 5, end of study activities will occur at Visit 26 
 
2.1.3 
Mitigating Bias 
Subjects will be randomly assigned to a treatment sequence (see section 3.1.2). Identities of the study 
investigational products will be disguised with a study label. Similar sized kit boxes will be used to ensure 
product blinding. All treatment products will be dispensed in a secluded area, away from the study 
examiner, to maintain the study blind.  All subjects will receive the same product for Period 5. 
 
The treatment product(s) each subject will receive will not be disclosed to the Principal Investigator, 
examiners, study center personnel, subjects, contract monitors, contract vendors, or the Sponsor, 
except for select site personnel responsible for usage instruction and supervised use.  Period 5 product 
will be blinded to analytical personnel. 
 
2.1.4 
 Labeling and Shipping 
The identity of the toothpaste will be disguised. Subjects will receive a kit box containing 1 tube of 
toothpaste, a timer, a toothbrush and product use instructions to be used throughout the duration of their 
study period. Subjects will receive a new kit box at the beginning of each study period.   
 
The kit boxes will be labeled by letter code (except Period 5). Kit box labels will also contain the study 
number, emergency phone number, distributor name/address, appropriate caution statements, content 
statement and other information as required by internal regulations and clinical SOPs. The shipping 
containers will be labeled with the clinical site address and a content statement listing study number and 
kit box numbers contained within. Supplemental products will be provided to site.  
 
The site will be provided with a code breaker report in a sealed envelope. The sealed code breaker 
report contains documents that list the kit box number or treatment code while the identity of the 
treatment products is hidden by an opaque scratch-off seal (except Period 5). If the study blind needs 
to be broken, an individual subject’s investigational product may be ascertained by opening the sealed 
code breaker report, locating the subject’s kit box number or treatment code and scratching off the 
opaque seal to reveal the treatment identity. The sealed code breaker report will be opened if a clinically 
serious AE occurs or management of the subject requires knowledge of the identity of the investigational 
product. The Investigator should immediately inform the Sponsor that the code will be broken and record 
the date, time and reason for breaking the code in writing. After the study is complete and the study 
database has been finalized and locked, the site will return the code breaker report to the Sponsor using 
the self-addressed, stamped envelope provided by the Sponsor.  
 
2.1.5 
Duration of Subject Participation  
Visit 1 (Screening) 
Subjects who have been recruited for the study via a telephone interview will sign in at the study site for 
this and all subsequent visits. At this time, subjects will be asked to read and sign duplicate copies of 
an informed consent statement, authorization for the release of health information for research form and 
demographic form. Demographic information and study entry criteria will be obtained and documented 
on the appropriate case report form (CRF). Upon review of these documents, an update of their medical 


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history/medications and the inclusion/exclusion criteria, each subject will be given an oral soft/hard 
tissue exam (OSHT) prior to their acceptance into the study. Their mandibular partial denture will be 
examined to determine if specimens can be held in one buccal flange area and the posterior denture 
teeth of the same side, as previously described. They will then provide a stimulated and unstimulated 
saliva sample to determine salivary flow rate. If no contraindications to their participation are discovered 
and the subject meets the study requirements, the subject will be accepted into the study.  
 
General comments, if any, will be recorded on the appropriate CRF. 
Visit 2 (Prophy Period 1)  
Two to three days prior to the beginning of Treatment Period 1, subjects will return for a professional 
dental prophylaxis. Subjects will answer have their medical history/medication information updated and 
answer continuation questions. Subjects will then receive an oral soft tissue exam (OST) and oral hard 
tissue exam (at the first period only).  The subject’s mandibular partial denture will be prepared to hold 
the round enamel specimens by drilling out hollows in the partial dentures at the specimen sites in the 
buccal flange area and the buccal surface of two denture teeth on the same side of the partial denture. 
A temporary dental material (DentuSil™) will be placed in the drilled-out areas of the partial denture. 
Subjects will be given fluoride-free toothpaste to use at home for the next two to three days until they 
return for their next visit. They will be encouraged to remove their partial denture at night until their next 
visit. 
Visit 3 (Begin Treatment Period 1)  
Each subject will receive an OST examination, answer continuation questions and have their medical 
history/medication information updated. Subjects will be asked to return the washout product they 
received at Visit 2 and will be given their randomized study product and diary for product use. Subjects 
will be instructed on the brushing method (see Appendix). The four enamel partially demineralized 
specimens will be placed in the buccal flange/posterior teeth areas on one side of the subject's 
mandibular partial denture. All subjects will be instructed to wear their partial denture containing the 
enamel specimens 24 hours a day except during the cleaning of their natural teeth (twice per day) and 
for short periods to rinse their mouth out with tap water after meals and snacks.  
Visit 4 (7 Day Removal)  
Seven (7) days following Visit 3, the subjects will return to the Institute and answer continuance criteria 
questions and update their medical history/medication information. One enamel specimen will be 
removed from anterior denture tooth site of their partial denture and the hollow will be filled with a 
temporary dental material (DentuSil™). The subjects will be told to continue using their assigned product 
as instructed. 
Visit 5 (14 Day Removal)  
Fourteen (14) days following Visit 3, the subjects will return to the Institute and answer continuance 
criteria questions and update their medical history/medication information. One enamel specimen will 
be removed from posterior denture tooth site of their partial denture and the hollow will be filled with a 
temporary dental material (DentuSil™). The subjects will be told to continue using their assigned product 
as instructed. 
Visit 6 (End Treatment Period 1)  
Twenty-one (21) days following Visit 3, the subjects will return to the Institute and receive an OST 
examination, answer continuance criteria questions and update their medical history/medication 
information. Subjects will be asked to return the study product and diary they received at Visit 3. The 
remaining two enamel specimens in the buccal flange site will be removed from their partial denture and 
the hollows will be filled with a temporary dental material (DentuSil) 
 
Period 2, 3, 4 & 5 
The procedures outlined above (Visit 2-6) will be repeated until each subject has used each of the four 
test products, followed by a 5th test product in Period 5. At Visit 21 (and also at Visit 26 for those subjects 


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participating in Period 5), subjects will receive an OSHT examination, have their mandibular partial 
denture cleaned through sonication, if applicable, have their partial denture repaired (if not going on to 
another in situ study) and their participation in the study will end. 
 
Subject Accountability 
If, for any reason, a subject does not complete the study, an explanation will be entered on the Subject 
Accountability CRF. All data gathered on the subject prior to discontinuation will be made available to 
the Sponsor. 
 
General Comments and Adverse Events 
General comments can be recorded at any time during the study. Adverse Events (AEs) will be 
documented on the AE CRF. Any self-reported AE that remains unresolved by the end of the study 
should be followed up until resolution by the investigator/designee, and the resolution should be 
documented only as source documentation. If a subject is unreachable to determine whether the AE 
has been resolved, the attempts to contact the subject should be documented as source documentation. 
Examiner observed AEs that are unresolved at the end of the study are followed to resolution at the 
discretion of the Medical Monitor. 
 
 
2.1.5.1 
Lifestyle Requirements 
1. For 2-3 days following each cleaning visit subjects must discontinue all regular oral hygiene 
practices (products and procedures) and use only the study fluoride-free toothpaste and toothbrush 
provided, twice daily, with the exception of interdental cleaners, e.g., dental floss, if this is their 
normal practice.  
2. For the 21 days of each treatment period, subjects may use only the study product assigned to 
them and the provided toothbrush twice daily, after breakfast and just before going to bed. Subjects 
may floss if this is their normal practice. 
3. Subjects must wear their lower partial denture 24 hours a day, except when cleaning it, during each 
21-day treatment period. Subjects will be encouraged to remove their mandibular partial denture at 
night during the washout and lead in periods. 
4. Subjects may use a non-zinc fixative like Poligrip® on their upper denture but no adhesive is 
permitted in the lower partial denture. 
5. Subjects must refrain from eating canned sardines during the course of the study and may not eat 
hard candy or nuts when the specimens are in place. 
 
2.1.6 
Discontinuation Criteria 
Discontinued subjects are those who do not complete final evaluations and/or procedures outlined in 
the protocol because of one of the following:  
a) an AE;  
b) significant protocol deviation that, in the opinion of the Investigator, may compromise the study 
results;  
c) voluntary withdrawal; or  
d) withdrawal at the Investigator’s discretion.  
The Investigator/Sponsor may recommend dropping a subject from the study at any time. 
Recommendations to drop a subject may include but are not limited to: misuse of study product, not 
following study procedures, or a serious protocol violation. Any subject who is discontinued from the 
study that has a continuing AE will have follow-up communications about the AE until the AE is resolved 
to the satisfaction of the Investigator/Medical Monitor. 
 
2.1.7 
Product Accountability 
Study products will be stored in a secure area, under environmental condition as required by label 
instructions or as described in this protocol and dispensed only under the authorization of the 
Investigator. The storage condition shall be properly documented. The study site will record and log both 
the receipt and dispensation of all test products (used and unused) by using forms provided by the 


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Sponsor or suitable forms provided by the site. Study products will be returned to the Sponsor following 
the trial, or alternatively, they will be destroyed at the clinical site provided the site has an existing SOP 
for the destruction of clinical materials and prior written approval from the Sponsor. 
 
2.1.8 
Treatment Codes 
The site will be provided with a code breaker report in a sealed envelope (except for Period 5). The 
sealed code breaker report contains documents that list the kit box number or treatment code while the 
identity of the treatment products is hidden by an opaque scratch-off seal. If the study blind needs to be 
broken, an individual subject’s investigational product may be ascertained by opening the sealed code 
breaker report, locating the subject’s kit box number or treatment code and scratching off the opaque 
seal to reveal the treatment identity. The sealed code breaker report will be opened if a clinically Serious 
Adverse Event (SAE) occurs or management of the subject requires knowledge of the identity of the 
investigational product. The Investigator should immediately inform the Sponsor that the code will be 
broken and record the date, time and reason for breaking the code in writing. After the study is complete 
and the study database has been finalized and locked, treatment codes will be unblinded.  
 
2.1.9 
Source Data 
The Investigator has the responsibility for ensuring that all source documents (i.e., study and/or medical 
records) and CRFs are completed and maintained according to the study protocol and is available at 
the site. Any CRF used as a source document must be identified as such in the Investigator Notebook. 
 
2.2 
SELECTION AND WITHDRAWAL OF STUDY POPULATION 
 
2.2.1 
Inclusion Criteria  
In order to be included in the study, each subject must: 
i. 
Be between 18 and 85 years of age; 
ii. 
Provide written informed consent prior to participation and be given a signed copy of the 
informed consent form; 
iii. 
Be in general good health as determined by the Investigator based on a review of the health 
history/update for participation in the trial; 
iv.      Be wearing a removable mandibular partial denture with sufficient room in one posterior buccal 
flange area to accommodate two 4 mm round enamel specimens and room on the same side 
to accommodate two 4 mm round specimens in the buccal surface of two posterior denture 
teeth;  
v.      Be willing and capable of wearing their removable partial denture 24 hours a day for four (4), 
three-week treatment periods; 
vi.      Be willing to allow study personnel to drill specimen sites (as described in #iv) in their 
mandibular partial denture; 
vii.      Be in good medical and dental health with no active caries or periodontal disease (NOTE: 
subjects presenting at screening with caries may continue in the study if their carious lesions 
are restored prior to beginning treatment 1); 
viii.      Have a salivary flow rate in the range of normal values (unstimulated whole saliva flow rate ≥ 
0.2 mL/min; gum base stimulated whole saliva flow rate ≥ 0.8 mL/min). 
 
2.2.2 
Exclusion Criteria  
Subjects may be excluded from study participation due to: 
i. 
Currently being pregnant, intending to become pregnant during the study period, or breast 
feeding; 
ii. 
Currently having any medical condition that could be expected to interfere with the subject’s 
safety during the study period;  


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iii. 
Currently taking antibiotics or having taken antibiotics in the two weeks prior to beginning 
treatment 1; 
iv. 
Having participated in another clinical study or receipt of an investigational drug within 30 days 
of beginning treatment 1; or 
v. 
Taking fluoride supplements, required to use a fluoride mouthrinse, or have received a 
professional fluoride treatment in the two weeks preceding specimen placement; 
vi. 
Currently taking or have ever taken bisphosphonate drugs (e.g., Fosamax, Actonel and Boniva) 
for the treatment of osteoporosis. 
 
2.2.3 
Continuance Criteria 
Subjects may be excluded from study participation and/or the analysis due to: 
i. 
Use of any non-study oral hygiene products since the previous study visit; 
ii. 
Having any diseases or condition that might interfere with the safe participation in the study;  
iii. 
Having an inability to undergo study procedures; 
iv. 
Significant protocol deviation that, in the opinion of the Investigator, may compromise the 
study results;  
v. 
Use of antibiotics since the previous study visit; 
vi. 
Being unwilling or incapable of wearing their removable partial denture 24 hours a day for a 
fifth, three-week treatment period (asked at Period 5, Visit 22 only).  
 
2.2.4 
Subject Withdrawal Criteria 
Subject participation is strictly voluntary, and a subject may withdraw from the research study at any 
time. Subjects may withdraw or take away permission to use and disclose health information at any time 
by sending written notice to the study doctor. If a subject withdraws permission, the subject will not be 
able to continue being in the research study. When permission has been withdrawn, no new health 
information will be gathered after that date. Information that has already been gathered may still be used 
and given to Sponsor. 
 
The Investigator/Sponsor may recommend dropping a subject from the study at any time. 
Recommendations to drop a subject may include but are not limited to: misuse of study product, not 
following study procedures, or a serious protocol violation. 
 
Subjects that withdraw or drop from the research study will not be replaced.  
 
If, for any reason, a subject does not complete the study, an explanation will be entered on the Subject 
Accountability CRF. All data gathered on the subject prior to discontinuation will be made available to 
the Sponsor. 
 
3. 
TREATMENTS 
 
3.1.1 
Product Usage 
 
Washout Product:  
Subjects will be instructed to brush twice daily (morning and at bedtime) with their washout toothpaste 
and toothbrush following the brushing instructions found in the Appendix. 
 
Treatment Product: 
Subjects will be instructed to brush twice daily (morning and at bedtime) with their assigned dentifrice 
for 21 days following the brushing instructions found in the Appendix. Subjects will be instructed to brush 
the biting surfaces of their back teeth (but not enamel specimens) with a full ribbon of study dentifrice 
for one timed minute while wearing their partial dentures, followed by rinsing with the 15 mL of measured 
tap water for 10 seconds and then expectorating. Subjects who are regular floss users will be allowed 


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to continue flossing; use of any non-study related products will be disallowed. Compliance with brushing 
procedures will be recorded in subject diaries (see Appendix), as well as any de novo or changes in 
medical conditions, medications or treatments.  
 
 
3.1.2 
Method of Assigning Subjects to Treatment Groups  
 
 
Study Design 
n 
Number of Treatment Periods 
Treatment Sequences 
Crossover 
16 
4 
ABCD, BDAC. CADB, DCBA 
 
 
Treatment Sequence Schedule 
Eligible subjects will be randomly assigned to one of the 4 pre-specified treatment sequences. Subjects 
will be assigned to a treatment sequence in the order they come to the site for their first treatment period 
using a computer-generated treatment sequence schedule. The site will keep the treatment sequence 
schedule while the study is on-going. Subjects will complete the treatment sequence in the order 
assigned to them. Should a subject miss a treatment period, that treatment in the sequence will be 
skipped. 
 
Period 5:  The crossover design does not apply to Period 5.  All subjects will receive the same product 
in Period 5. 
 
3.1.3 
Prior and Concomitant Therapy  
Medical history and concomitant medication will be recorded as source documentation.  
 
3.1.4 
Treatment Compliance  
Product use compliance will be assessed by determining the average weight of each type of test product 
prior to dispensing (five tubes per product will be weighed and average weight determined). At the end 
of each treatment period, subjects will be required to return all product containers (including empty ones) 
to the study site. The tubes will be individually weighed upon return and that number subtracted from 
the average weight will be recorded on the treatment dispensing log. 
 
The amount of test product used for each study treatment will be compared to daily product usage 
recorded on the subject’s diary. Significant discrepancies will be discussed with the study subject, as 
needed. Subjects who miss 15% or more treatments within the three-week treatment period will be 
considered non-compliant. The reason for non- compliance will be noted in the subjects’ study records. 
All subjects will be instructed to use only the assigned products in place of normal oral hygiene for the 
duration of the study. 
 
3.2 
Assessment of Efficacy 
3.2.1   Sample Collection 
Salivary Flow Assessment  
Salivary flow will be assessed during the screening visit. For the unstimulated collection, subjects will sit 
quietly for five minutes before beginning the test. During the five-minute test time, they will be told to 
allow their saliva to pool, emptying into a collection cup whenever they feel the need to swallow. 
For the stimulated collections, subjects will chew unflavored gum base for one minute and then swallow 
any pooled saliva. They will then chew the gum base for two minutes, during which time they will empty 
any pooled saliva into a collection cup.  
The samples will be weighed and the salivary flow rates determined. The unstimulated saliva flow rate 
must be ≥ 0.2 mL/min and the stimulated saliva flow rate must be ≥ 0.8 mL/min for study qualification. 


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Diary for Home Use 
At the start of each treatment period, subjects will be provided with a diary (see Appendix) to record the 
date, and the time of the morning (a.m.) and evening (p.m.) of each brushing and any deviation from the 
brushing regimen. In addition, subjects will also record any new or changes in pre-existing medical 
conditions, medications or non-drug treatments or any change in signs or symptoms that may occur.  
Subjects will be required to bring the completed diary to the end of treatment visit. Study staff will review 
the Diary with the subject to confirm treatment compliance and clarify listed medical conditions, 
medications and non-drug treatments. 
 
Intra-oral Appliance  
The in-situ model involves the placement of gauze-covered enamel specimens in the subject’s 
mandibular partial denture (see Figure 1). The subject’s denture will be prepared for the study by 
creating two hollows in the buccal flange area on one side of the partial denture and in two denture teeth 
on the same side large enough to accommodate the enamel specimens. Four gauze-covered 4 mm 
round enamel specimens will be mounted on the partial denture as described previously. The enamel 
specimens will be mounted in place with DentuSil™ - Silicone Soft Reline Material (The Harry 
J.Bosworth® Company, Skokie, IL) or an equivalent material. The DentuSil™ material will be placed in 
the drilled sites and the enamel specimens carefully inserted so that they be mounted flush with the 
surface of the buccal flange/denture teeth when fully seated. Great care will be taken to avoid 
contaminating the enamel surface of the specimens with the luting material. Upon completion of the 
study the subject’s partial denture will be repaired. The location where the enamel specimens will be 
placed on the subject’s partial denture are not functional parts of the denture, and the experimental 
procedures will not cause any permanent damage to the denture.  
 
Prior to placement in the subjects’ partial dentures, all enamel specimens will be sterilized by exposure 
to ethylene oxide. 
 
 
FIGURE 1  
Lower Partial Denture Appliance with specimens placed in the buccal flange and buccal surface of 
posterior denture teeth 
 
 
  
 
3.3 
Assessment of Safety 
 
3.3.1 
Safety Variables 
Oral Examination 
The study dentist will complete an oral soft and hard tissue (OSHT) examination at screening, oral hard 
tissue (OHT) at Visit 2 and 21(and Visit 26 for those subjects participating in Period 5) and an oral soft 
tissue (OST) examination at each study visit with the exception of the 7-day and 14-day removal visits. 


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The exams will be conducted via a visual examination of the oral cavity and perioral area utilizing a 
standard dental light, dental mirror, gauze, and periodontal probe and tongue blade, as needed. The 
soft tissue structures examined will involve the labial mucosa including lips, buccal mucosa, 
mucogingival folds, gingival mucosa, hard and soft palate, tonsillar and pharyngeal areas, Edentulous 
ridge/Retromolar area, tongue, sublingual area/floor of mouth, submandibular area, major salivary 
glands, head and neck and TMJ. Observations will be listed as “Normal” and “Abnormal” and 
abnormalities will be described.  
 
The hard tissue structures examined will include assessing for enamel irregularities, tooth fracture, 
pathologic tooth wear, cavitated lesions, active non-cavitated lesions, residual roots, faulty restorations 
and implants. Observations will be listed as “Absent” or “Present” and conditions noted as present will 
be described. 
 
All abnormal findings are recorded and categorized by their location with hard tissue findings categorized 
as “other-oral.” An AE is recorded if a new abnormal finding is noted after treatment application or any 
abnormal finding noted prior to treatment application increases in severity after treatment is applied.  
 
 
3.3.2 
Adverse Event Management  
An adverse event (AE) is any untoward medical occurrence in a subject administered a product and 
which does not necessarily have a causal relationship with this treatment. An AE can therefore be any 
unfavorable and unintended sign (including an abnormal laboratory finding), symptom or disease 
temporally associated with the use of a medicinal (investigational) product, whether or not related to the 
medicinal (investigational) product. 
 
A serious adverse event (SAE) is defined as an event which suggests a definite hazard or handicap to 
the subjects. SAEs are any events resulting in death, life threatening situation, disability or permanent 
damage, hospitalization or prolongation of existing hospitalization, or congenital anomaly/birth defects; 
events requiring intervention to prevent permanent impairment/damage; or other serious (important) 
medical events. 
 
All other AEs that do not fit within these criteria will be considered “non-serious.” Unexpected adverse 
event/reaction is any occurrence the nature or severity of which is not consistent with the AE provided 
in the current summary of product characteristics. All other AEs are considered “expected.”  
 
When an AE is suspected, all relevant evaluations will be carried out and appropriate treatment will be 
recommended. Additional follow-up will be performed as necessary and recorded as source 
documentation, with the results provided to the Sponsor. Subjects that experience any clinically 
significant AE will remain under medical supervision until the Principal Investigator/Medical Monitor 
recommends appropriate follow-up treatment or deems the AE to be resolved, stabilized, or no longer 
serious enough to warrant follow-up. 
 
When an Investigator is notified of a serious AE, the Investigator must promptly (within 24 hours) notify 
the Sponsor (Clinical Trial Manager or the Medical Monitor) of the serious event, regardless of causality. 
Within 5 working days, a written and/or electronic report describing the circumstances of the event must 
be submitted to the Sponsor. The Investigator will be responsible for SAE reporting to the IRB. 
 
3.3.3 
Adverse Event Reporting 
AE Recording 
When an AE occurs after written informed consent has been obtained but before the first dose of study 
drug, the AE will be considered a nontreatment-emergent AE. Only serious nontreatment-emergent AEs 
related to study procedures will be recorded on the CRF. All nontreatment-emergent AEs that are non-
serious or unrelated to study procedures should be documented by updating the medical history and 
general comments as applicable.  
 


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An AE that occurs from the time the subject receives the first dose of study drug until dismissed from 
the study will be considered a treatment-emergent AE. All treatment-emergent AEs will be collected. 
The severity, action taken, causality, outcome, and category of all recorded AEs will be recorded. 
 
Severity 
Severity refers to the extent to which an AE affects daily activities. Severity will be categorized according 
to the following criteria: 
• Mild: Normal activities unaltered; 
• Moderate: Normal activities altered; 
• Severe: Unable to undertake normal activities. 
 
The term “severity” is not the same as “serious.” Seriousness, not severity, serves as the guide for 
defining regulatory reporting obligations. 
 
Action Taken 
If any action was taken due to the AE, it should be recorded. 
• None: No action was taken by the subject. 
• Discontinued: Investigator recommended, or subject stopped using the study products. 
• Reduced/Interrupted: Investigator recommended or subject reduced or interrupted the instructed 
product usage. 
 
Causality 
Causality refers to the relationship of the AE to study drug. Assessment of causality is the responsibility 
of the Principal Investigator at each site. If this responsibility is delegated to a sub-investigator, this 
should be appropriately documented in the delegation sheet. Causality will be categorized according to 
the following criteria: 
• Doubtful: There is no medical evidence to suggest that the AE may be related to study drug usage, 
or there is another more probable medical explanation. 
• Possible: There is medical evidence to suggest that the AE may be related to study drug usage. 
However, other medical explanations cannot be excluded as a possible cause. 
• Probable: There is strong medical evidence to suggest that the AE is related to study drug usage. 
• Not Related: There is no evidence to suggest the adverse event is related to study drug usage. 
 
Category 
Category refers to the region or area where the AE occurred. Categories are listed 0-9.  
• 0 = Non-oral related 
• 1 = Perioral area/lips 
• 2 = Labial mucosa/buccal mucosa 
• 3 = Mucolabial fold/mucobuccal fold 
• 4 = Gingiva/free and attached 
• 5 = Palate/hard and soft 
• 6 = Oropharynx/uvula 
• 7 = Tongue 
• 8 = Sublingual 
• 9 = Other oral 
 
3.3.4 
Adverse Event Follow-up 
Any self-reported AE that remains unresolved by the end of the study should be followed up until 
resolution by the investigator/designee, and the resolution should be documented only as source 
documentation. If a subject is unreachable to determine whether the AE has been resolved, the attempts 
to contact the subject should be documented as source documentation. Examiner observed AEs that 
are unresolved at the end of the study are followed to resolution at the discretion of the Medical Monitor. 
 


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4. 
LABORATORY METHODS 
4.1 
Specimen Preparation 
Extracted human teeth will be used as the hard tissue test substrate for the preparation of the 4 x 4 mm 
and the 4 mm round specimens. The teeth will be collected and transported to OHRI in a saturated 
thymol solution. Upon receipt, the teeth will be sorted and cleaned. The teeth will then be stored in 
saturated thymol solution during sample preparation procedures. 
 
Teeth will be selected based on the following criteria: 
• 
Free of caries and major restorations; 
• 
No discoloration and no markings, such as cracks, when viewed under a microscope at 20× 
magnification; 
• 
Sufficient tooth surface to provide a large size specimen to meet study requirements. 
 
A core of enamel 4 mm in diameter will be prepared from each human tooth by cutting perpendicularly 
to the buccal surface with a hollow-core diamond drill bit. Each block will be ground and polished to 
create flat surfaces [Zero et al., 1990]. 
 
The round specimens will be ground and polished to create planar parallel dentin and enamel surfaces. 
All grinding/polishing will be done on a polishing surface moistened with deionized water (dw). The 
dentin side will be ground flat using 500 grit silicon carbide paper, followed by grinding and polishing of 
the enamel side. A small orientation cut will be placed on each block (see Figure 2). The enamel surface 
of each specimen will be ground using 1200 grit silicon carbide paper followed by 2400 and then 4000 
grit silicon carbide paper. After the grinding procedures are completed, the enamel specimens will be 
placed under running dw for three minutes, sonicated in deionized water (dw) for three minutes and then 
placed under running dw for three minutes. The polishing step will involve the use of a 1 micrometer 
(μm) diamond suspension on a polishing cloth. The enamel specimens will then be rinsed under a steady 
stream of dw for three minutes, sonicated for three minutes in 2% microliquid soap and then rinsed again 
with dw for three minutes. Resulting specimens will have a thickness range of 1.8 to 2.2 mm. The 
specimen will have a minimum polished surface of 3 mm × 3 mm in the center of the enamel surface. 
  
Figure 2 
4 mm Round Human Enamel Specimen 
  
 
 


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4.2 
Lesion Creation 
The round enamel specimens will be partially demineralized using a modification of the method 
described by White [1987]. The human enamel specimens will be immersed for 24 hours at 37º C under 
static conditions in 40 ml of an acid buffer (0.05 mol/L lactate), 50% saturated with respect to 
hydroxyapatite and with 0.2% (wt/vol) carbopol 907 (BF Goodrich Co., USA) added (pH adjusted to 5.0 
using KOH or NaOH), and then rinsed thoroughly with deionized water. The demineralized enamel 
specimens will then be stored in a moist environment to prevent dehydration. 
 
4.3 
6.3 Lesion Quality 
Lesions will be inclined towards an overhead light until a reflection is obtained. Acceptable lesions with 
an intact surface-zone will have a continuous, uniform shiny surface, with no matt areas. Lesions with 
matt area(s) will be rejected. The following criteria will be used to select specimens for inclusion in the 
study: 
•     The lesioned areas of each specimen should be of equal and uniform opacity; and 
•     The lesioned areas of each specimen should possess a surface shine when exposed to light, 
thereby indicating an intact surface. 
 
4.4 
Efficacy Measurements and Evaluations 
4.4.1 
Surface Microhardness 
The SMH test will be used to assess changes in the mineral status of partially demineralized enamel 
specimens. SMH will be measured using a Wilson 2100 Hardness Tester. Each enamel specimen will 
be secured on a 1-inch square acrylic block with sticky wax and then placed on the microhardness 
tester. Five baseline indentations spaced 100 µm apart will be placed with a Knoop diamond under a 50 
gram load in the center of a flattened, polished sound enamel specimen. SMH will be determined by 
measuring the length of the indentations using Wilson 2100 - Clemex CMT Software (version 6.0.011). 
For enamel specimens to be acceptable for use in the study, the mean of the five baseline indentation 
length must be 43 µm with a standard deviation of ≤ 3µm. 
 
After in vitro demineralization, the enamel specimens will be again SMH tested by placing five 
indentations 100 µm to the left of the baseline indentations (see Figure 2). To qualify for the study, the 
mean (n = 5) indentation lengths of the partially demineralized specimens must fall within a range of 
100-140 µm with a standard deviation of ≤ 10 µm. After 7, 14 and 21 days of intra-oral exposure the 
enamel specimens will be again SMH-tested by placing five indentations 100 µm to the right of the 
baseline indentations (see Figure 2). The extent of remineralization will be calculated based on the 
method of [Gelhard et al., 1979]. 
 
 
%SMH recovery = (D1-R)/(D1-B) ×100 
      
B = indentation length (µm) of sound enamel specimen at baseline  
D1 = indentation length (µm) after in vitro demineralization 
R = indentation length (µm) after intra-oral exposure. 
 
Additionally, the absolute remineralization change will be computed as: SMH change= D1-R 
 
4.4.2 
Enamel Fluoride Uptake 
The microdrill enamel biopsy technique as described by [Sakkab et al., 1984] will be used to analyze 
the fluoride content of the partially demineralized enamel specimens. Each enamel specimen will be 
mounted perpendicular to the long axis of a drill bit attached to a specially designed microdrill, and drilled 
to a depth of ~100 µm through the entire lesion (four cores per specimen; see Figure 2). 
The drilling and sample collection will be performed in a static-controlled atmosphere to prevent loss of 
enamel powder due to charging effects. The enamel powder sample, pooled from the four drilling 
samples, will be transferred to an analyzer cup cap. 20 microliters (μl) of 0.5 Molar (M) Perchloric Acid 
(HClO4) will be added to the enamel powder and the cap gently swirled to dissolve the powder. To the 
analyzer cap containing the 20 μl of HClO4/enamel powder, 40 μl of citrate/ ethylene-diamine-tetraacetic 
acid (EDTA) buffer and 40 μl of de-ionized water will be added and immediately analyzed for fluoride 


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content using a fluoride specific electrode and pH/Ion meter. The diameter of the drill hole will be 
determined using a calibrated microscope interfaced with an image analysis system. The amount of 
fluoride-uptake by enamel will be calculated based on the amount of fluoride divided by the area of the 
enamel cores and expressed as µg F/cm2.  
 
4.5 
Laboratory Data 
As each specimen is analyzed, individual Excel files will be generated and imported into a validated 
relational database management system. After the analyses are completed, all enamel specimen 
surface microhardness, and enamel fluoride uptake data will be reviewed by the principal investigator 
before secure transfer to the sponsor. The investigator will document this review and documentation will 
be filed with the laboratory study files. 
 
4.6 
Specimen Retention 
Laboratory specimens will be retained by the study site for twelve months following database lock. 
 
5. 
STATISTICAL PLAN 
5.1 
Statistical Methods 
Efficacy Analyses  
The mean % SMH recovery and SMH change will be calculated using the five sets of indentations within 
each enamel specimen. The mean fluoride uptake will be calculated using the four samples within each 
enamel specimen. The mean % SMH recovery, SMH change and mean fluoride uptake will be computed 
for each enamel specimen at the different time points (1 specimen for 7 and 14 days and 2 specimens 
for 21 days) within a subject; if a subject is missing an enamel specimen for the 21-day reading, the 
single available enamel specimen will be used. The treatments will be compared using analysis of 
variance models (ANOVA) or analysis of covariance (ANCOVA) suitable for a crossover study.  
 
To assess for potential carryover effects on the primary endpoint, the models will include random effects 
for subject and fixed effects for study period, product and carryover term. If the carryover term is not 
significant at the 0.1 level (p>0.1) then the final crossover model will not include the carryover term.  
 
A repeated measures crossover model will also be carried out to explore the treatment time effect. This 
model will include a random effect for subject and fixed effects for study period, product, treatment times 
(7, 14 and 21 days) and treatment time by product interaction . For SMH change, B (indentation length 
(µm) of sound enamel specimen at baseline) or D1 (indention length (µm) after challenge) will be added 
as a covariate in the model. Additionally, the product by covariate interaction will also be assessed in 
this model. A 10% significance level will be used for all product comparison tests. No alpha-level 
adjustments for multiple comparisons adjustments will be applied.   
 
If the data does not satisfy the normality criterion, log transformation or analogous nonparametric 
methods will be employed. Additional statistical techniques may also be applied in order to more fully 
understand the data.  
 
The SnF2 product used in Period 5 will be compared to the negative control treatment used in other 
periods using a paired t-test. 
 
Safety Analyses 
Adverse events reported during the study will be listed in the final report. 
 
5.2 
Hypotheses 
The following hypothesis will be tested for each index and each time point: 
 
Null: there is no difference between the treatments using pastes with different fluoride level.  
 
Alternative: there is a difference between at least two treatments with different fluoride level. 


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Period 5 Hypotheses: 
Null: there is no difference between SnF2 and the negative control.  
 
Alternative: there is a difference between SnF2 and the negative control. 
 
5.3 
Number of Subjects 
The number of subjects (16) was chosen for logistical reasons for this study.   
 
5.4 
Selection of Subjects  
5.4.1 
Intent-to Treat (ITT) Population 
The subjects will be selected from the OHRI’s IRB approved database of persons previously accepted 
into the partial denture panel (IRB #1110007150). Potential subjects will be screened to determine 
eligibility to participate in this study. Sixteen adult subjects, between the ages of 18 and 85 years, will 
be accepted and randomized in the study so at least 10 subjects can complete the study. The ITT 
population will include those subjects randomized and who had at least one exposure to product.  
 
5.4.2 
Per Protocol (PP) Population 
 
The PP population will be a subset from the ITT population without any major protocol deviations. 
 
 
 
6. 
DATA MANAGEMENT PLAN 
The following steps will be taken to ensure the accuracy, consistency, completeness, and reliability 
of the data: 
• documenting Clinical Trial Risk Assessment and Risk Management Plan; 
• site initiation meeting; 
• routine site and/or data monitoring; 
• ECRF review against source documents; 
• data management quality control checks; 
• statistical quality control (QC) checks; 
• continuous data acquisition and cleaning; 
• internal review of data. 
 
In addition, a Sponsor representative may conduct periodic quality assurance audits of the study  
processes, including, but not limited to, clinical site visits, laboratories, vendors and contract research 
organizations, the study database, and P&G's final study report. Data monitoring will ensure quality of 
collected information by detection of inconsistent and missing information. The investigational site will 
follow up on any queries from P&G Data Manager. If a query arises from a subject that was lost to follow-
up, the site will take action to contact the subject and document any attempts made. The study monitor 
will ensure that the study was conducted in accordance with the protocol and any amendments, good 
epidemiological practice, and applicable laws and regulations. 
 
Case Report Forms 
The Data Manager will supply the paper and/or electronic CRFs to be used in this study. It is the 
responsibility of the Investigator to maintain and submit accurate and timely CRFs to the Sponsor. All 
hard copy CRFs will be filled out legibly in ink. 
  
All questions should be answered. For paper CRFs, if an entry requires correction, a single line will be 
placed through the entry so as not to obscure the original record, the corrected entry will be initialed and 
dated by the individual making the change, and a reason will be given for the change. There will be no 
whiteouts or erasures. For electronic CRFs, if an entry requires correction, the change is made directly 
to the CRF in the database, the user is prompted to provide a reason for the change, and the correction 
is logged in by an electronic audit trail. 
 


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As necessary, the Data Manager may make specified allowable changes to the database without issuing 
a query to the site, as agreed upon by study site per this protocol. Examples of allowable changes 
include incorrect date formats, incorrect current year recorded (as in the start of a new year), and 
unambiguous spelling errors. Changes to common abbreviations and symbols to equivalent text to meet 
system or coding constraints (e.g., @ = at, ~ = approximately), may also be allowable. Values that are 
ambiguous or open to interpretation will be queried to the sites. It is the responsibility of the Data 
Manager to ensure all changes are supported by information contained elsewhere and/or are 
unambiguous. 
 
6.1 
Access to Source Data/Documents 
The Investigator has the responsibility for ensuring that all source documents (i.e., study and/or medical 
records) and CRFs are completed and maintained according to the study protocol and are available at 
the site. Any CRF used as a source document must be identified as such in the Investigator Notebook. 
 
6.2 
Data Handling and Recordkeeping  
The Investigator must retain the subject identification codes, informed consent documentation, clinical 
materials inventory, CRFs (paper or electronic media), medical records and other source data for a 
minimum of 2 years after the last regulatory approval has been received or the discontinuation of the 
study. The Investigator must receive written authorization from the Sponsor before destroying any study 
document. The Investigator will make the records available for inspection and copying upon the request 
of an authorized employee of a government authority or the Sponsor, at reasonable times. In the event 
the Investigator retires, relocates, or for any other reason withdraws from the responsibility for 
maintaining records for the period of time required, custody of the records may be transferred to another 
person who will accept responsibility for the records. Notice of such a transfer must be given in writing 
to the Sponsor. 
 
The subject will be identified with a unique identification code assigned by the Principal Investigator to 
each trial subject to protect privacy. The identification codes are used in lieu of the subject’s name when 
the Principal Investigator reports all adverse events and other trial related data. These codes will be 
used on all study documents for the subject’s confidentiality, as stated in section 2.11 of the ICH-GCP. 
 
Any advertisements used in recruitment of subjects must receive prior approval from P&G and/or the 
Investigator’s IRB. A copy of the IRB-approved advertising and the documentation thereof must be 
provided to P&G. 
 
Following completion of the study, the Investigator shall submit a final report to the Sponsor describing 
the conduct of the study, deviations from planned conduct, early withdrawals and subject accountability, 
adverse events, and other information on study conduct. The Investigator’s IRB may require more 
frequent status reports. 
 
7. 
ETHICS 
Subject participation is voluntary. Subjects have a right to refuse participation in the research study at 
any stage. The study will be conducted in accordance with all international laws and regulations, and 
also national laws and regulations, and in accordance with any applicable guidelines. Informed consent 
will be obtained from potential participants of the study. Beforehand the study site should fully inform 
the subject regarding all the aspects of the clinical study, including objectives, collected data, expected 
risks and benefits of participation, and voluntary participation in the study.  
 
Conduction of the study will comply with the principles of ICH-GCP. Before study start an approval of 
Independent ethical committee for conduction of the present study will be obtained. A list of investigators 
and investigational sites will be approved. All documents and data related to the study are strictly 
confidential. Protocol contents cannot be disclosed to third parties without written permission of the 
Sponsor.  
 


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Sponsor of the study is obliged to adhere to confidentiality of the study participants’ personal data. Any 
information identifying the subject’s identity should not be disclosed. The necessary personal data of 
the subjects as part of the study (for example, gender, age) will be collected only for achieving study 
purposes and in minimum quantity. The study site will maintain an identification list of all screened and 
enrolled subjects. Therefore, names and surnames of subjects will not be disclosed or reported to the 
Sponsor. If subject name and surname are mentioned in any document, then before sending a copy of 
such document to the Sponsor these data will be erased. Results of the study stored in electronic format 
will be kept in accordance with current information protection laws. Sponsor employees or regulatory 
authorities’ representatives should not contact the subject directly. Before enrollment into the study, 
subjects will be informed regarding confidentiality provisions and use of their personal data, including 
necessity of access to them of the monitor and other authorized people (in case of auditing, inspection 
and etc.). 
 
8. 
FINANCING AND INSURANCE  
Financing and insurance can be found in signed and executed clinical study agreement.  
 
9. 
PUBLICATION POLICY  
The results of this study may be published in oral presentations, written abstracts, written manuscripts, 
and/or with regulatory agency.  
 
 


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APPENDIX 
STUDY RESTRICTIONS: 
• 
During the study, do not use any oral hygiene products other than the products given to you 
unless you have been told so by the study staff. You may use floss if this is your normal 
practice. 
• 
Your lower partial denture and upper partial or full denture, if applicable, may be cleaned outside 
of your mouth with water only.  
• 
Do not use any denture cleaning products.  
• 
You may remove your lower partial denture for short periods (to rinse your mouth with water after 
meals and snacks), however, you will have to wear it 24 hours a day, every day, including when 
you sleep, during each two-week test period. 
• 
Do not use any denture adhesive on your lower partial denture. If you need an adhesive for your 
upper denture, you must use a zinc free adhesive like Poligrip®. 
• 
Please refrain from eating canned sardines during the course of the study. 
• 
Please refrain from having hard candy or nuts when the enamel specimens are in place. 
 
IMPORTANT TO REMEMBER 
• 
Record any changes in your health, medications (prescription and over the counter medications) 
or treatments. 
• 
Bring your diary and remaining product tubes to your next study visit. 
 
 
WASHOUT PERIOD INSTRUCTIONS 
1) Remove your lower partial denture and upper partial or full denture, if applicable.  
2) Brush your natural teeth with provided wash out toothpaste and toothbrush. 
3) Brush your lower partial/denture with water only.   
4) Place the partial/denture back in your mouth. 
5) Please remove your partial denture at night. 
 
TEST PERIOD INSTRUCTIONS 
PRE-STUDY PRODUCT USE INSTRUCTIONS (in the morning after breakfast and at night just 
before bedtime) 
Because you are brushing your teeth with your lower partial denture in place, it is important that you 
take care of your partial denture and your natural teeth in the places that might be missed during 
study product brushing. The following instructions should be followed before brushing with study 
product: 
 
1) Remove your lower partial denture and upper partial or full denture, if applicable.  
2) Brush your natural teeth with water only, paying close attention to the teeth surfaces that might be 
covered by the partial/denture. 
3) Brush your lower partial/denture with water only. Be sure to brush the areas that can’t be reached 
when the partial is in your mouth. DO NOT BRUSH THE SPECIMEN SITES. 
4) Place the partial/denture back in your mouth and follow the instructions below for study product 
use. 
 
 


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STUDY PRODUCT USE INSTRUCTIONS  
5) Put a strip of toothpaste on the toothbrush, covering the full head of the brush. 
 
6) Set your timer for 1 minute. 
 
7) Measure 15mL of tap water into the dosing cup provided.  
 
8) Brush the biting surfaces of your back teeth in all four quadrants of your mouth (do not brush the 
specimen sites) for a total of one timed minute and then spit out slurry.  
 
9) Rinse your mouth with the 15mL of measured water for 10 seconds and spit out. 
 
10) Record each brushing in the diary.  
 
 
 


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HOME USE DIARY 
a) Shaded area shows you an example of how to complete your diary.  
b) Make an entry for each day of the study starting with the first brushing.  
c) Tick the box each time you brush.  
d) Add a comment if you have not brushed according to the instructions provided. 
 
Treatment 
Day 
Date 
Brushing 
Time 
AM / PM 
Brushed with 
Full Ribbon of 
Toothpaste for 
1 Minute 
Spit Out 
Slurry 
Rinsed with 15 
ml Tap Water for 
10 Seconds 
0 
01/12/2021 
8:00am 
✓ 
✓ 
✓ 
10:00pm 
✓ 
✓ 
✓ 
1 
 
 
 
 
 
 
 
 
 
2 
 
 
 
 
 
 
 
 
 
3 
 
 
 
 
 
 
 
 
 
4 
 
 
 
 
 
 
 
 
 
5 
 
 
 
 
 
 
 
 
 
6 
 
 
 
 
 
 
 
 
 
7 
 
 
 
 
 
 
 
 
 
8 
 
 
 
 
 
 
 
 
 
9 
 
 
 
 
 
 
 
 
 
10 
 
 
 
 
 


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11 
 
 
 
 
 
 
 
 
 
 
 
 


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Treatment 
Day 
Date 
Brushing 
Time 
AM / PM 
Brushed with 
Full Ribbon of 
Toothpaste for 
1 Minute 
Spit Out 
Slurry 
Rinsed with 15 
ml Tap Water for 
10 Seconds 
12 
 
 
 
 
 
 
 
 
 
13 
 
 
 
 
 
 
 
 
 
14 
 
 
 
 
 
 
 
 
 
15 
 
 
 
 
 
 
 
 
 
16 
 
 
 
 
 
 
 
 
 
17 
 
 
 
 
 
 
 
 
 
18 
 
 
 
 
 
 
 
 
 
19 
 
 
 
 
 
 
 
 
 
20 
 
 
 
 
 
 
 
 
 
21 
 
 
 
 
 
 
 
 
 
 
 
 


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Date 
Comments related to changes in your health, medications or brushing procedures 
 
Date 
Comments related to changes in your health, medications or brushing procedures