Conditions: Caries
Keywords: Caries Remineralization Fluoride Dentifrice
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 52
Healthy volunteers: yes

Brief summary:
The purpose of this study is to compare the remineralization potential of an optimized fluoride dentifrice to a control fluoride dentifrice in an in situ caries model.

Detailed description:
This will be a double blind, single center, 3-way crossover design study. 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 bovine enamel specimens will be placed in the buccal surface of two posterior denture teeth (the specimen site may extend into the buccal flange area, if needed) 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. Specimens will be removed after two weeks, and the subjects will undergo at least 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 three test products. Changes in the mineral content of the enamel specimens will be assessed using surface microhardness (SMH) and transverse microradiography (TMR). Enamel fluoride uptake (EFU) will be determined using the microdrill enamel biopsy technique. In addition, the net acid resistance (NAR) and the comparative acid resistance (CAR) of the demineralized enamel specimens will be determined.

Arms:
arm 1: Crossover design - Cleaning, 2-3 day non-Fluoride (F) dentifrice, Period 1 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 2 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 3 product for 2 weeks. Product sequence: 1100 ppm F as sodium fluoride Test Product,1100 ppm F as sodium fluoride (positive control) , 0 ppm F (placebo, negative control) arm 2: Crossover design - Cleaning, 2-3 day non-F dentifrice, Period 1 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 2 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 3 product for 2 weeks. Product sequence: 1100 ppm F as sodium fluoride Test Product, 0 ppm F (placebo, negative control),1100 ppm F as sodium fluoride (positive control) arm 3: Crossover design - Cleaning, 2-3 day non-F dentifrice, Period 1 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 2 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 3 product for 2 weeks. Product sequence: 1100 ppm F as sodium fluoride (positive control), 1100 ppm F as sodium fluoride Test Product, 0 ppm F (placebo, negative control) arm 4: Crossover design - Cleaning, 2-3 day non-F dentifrice, Period 1 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 2 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 3 product for 2 weeks. Product sequence: 1100 ppm F as sodium fluoride (positive control), 0 ppm F (placebo, negative control), 1100 ppm F as sodium fluoride Test Product arm 5: Crossover design - Cleaning, 2-3 day non-F dentifrice, Period 1 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 2 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 3 product for 2 weeks. Product sequence: 0 ppm F (placebo, negative control), 1100 ppm F as sodium fluoride Test Product,1100 ppm F as sodium fluoride (positive control) arm 6: Crossover design - Cleaning, 2-3 day non-F dentifrice, Period 1 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 2 product for 2 weeks, 4-5 day washout, cleaning, 2-3 day non-F dentifrice, Period 3 product for 2 weeks. Product sequence: 0 ppm F (placebo, negative control),1100 ppm F as sodium fluoride (positive control), 1100 ppm F as sodium fluoride Test Product

Interventions: intervention 1: 0 ppm F (placebo, negative control) intervention 2: 1100 ppm F as sodium fluoride (positive control) intervention 3: 1100 ppm F as sodium fluoride Test Product
Intervention descriptions: intervention 1: • Each subject will use this product during one of the three treatment periods in the crossover study design. intervention 2: • Each subject will use this product during one of the three treatment periods in the crossover study design. intervention 3: • Each subject will use this product during one of the three treatment periods in the crossover study design.

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
 
 
1 
  
  
 
 
 
 
 
 
 
IN SITU RESEARCH PROTOCOL 
 
Study Number: 
 
23-I-121 
 
Title: 
Comparison of the remineralization potential of an optimized 
fluoride dentifrice with a control fluoride dentifrice using an in situ 
caries model 
 
Principal Investigator: 
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 
 
 
Study Manager: 
 
Lorena Galvez 
 
Study Coordinator:  
To be determined 
 
Co-Investigators:  
Anderson Hara, DDS, PhD 
 
 
 
 
Frank Lippert, PhD 
 
Investigation Site: 
 
Oral Health Research Institute 
 
 
 
 
Indiana University School of Dentistry 
 
Version: Final  
  
July 19, 2023  
 
 
 
 
NOTE: Haleon (formerly GSK) is funding this study as an Investigator Sponsor Studies (ISS) 
Grant.  The sponsor has reviewed this protocol via email correspondence prior to submission to the 
IRB. 


 
 
 
 
 
2 
  
TABLE OF CONTENTS 
1.  
RATIONALE AND STUDY OBJECTIVE .............................................................. 4 
2. 
STUDY DESIGN ................................................................................................... 5 
2.1 Study Schedule: ........................................................................................................ 5 
3. 
STUDY POPULATION ......................................................................................... 6 
3.1 Source and Number of Subjects ................................................................................ 6 
3.2 Subject-Selection Criteria .......................................................................................... 6 
4. 
TEST TREATMENTS ........................................................................................... 7 
4.1 Product Use Instructions............................................................................................ 8 
4.2 Treatment Compliance .............................................................................................. 8 
4.3 Randomization Procedures ....................................................................................... 8 
4.4 Blinding Procedures and Code Breaks ...................................................................... 9 
5. 
STUDY METHODOLOGY .................................................................................... 9 
5.1 Conduct of Study ....................................................................................................... 9 
5.2 Clinical Procedures .................................................................................................... 9 
5.3 Lifestyle Requirements ............................................................................................ 10 
5.4 Informed Consent Process ...................................................................................... 10 
5.5 Oral/Hard Soft Tissue Examination ......................................................................... 11 
5.6 Salivary Flow Assessment ....................................................................................... 11 
5.7 Diary for Home Use ................................................................................................. 11 
5.8 Intra-oral Appliance ................................................................................................. 11 
5.9 Adverse Events Assessment ................................................................................... 12 
6.0 
LABORATORY METHODS ................................................................................ 13 
6.1 Specimen Preparation ............................................................................................. 13 
6.2 Lesion Creation ....................................................................................................... 14 
6.3 Lesion Quality .......................................................................................................... 14 
6.4 Efficacy Measurements and Evaluations ................................................................. 14 
6.5 Laboratory Data ....................................................................................................... 16 
6.6 Specimen Retention ................................................................................................ 16 
7. 
STATISTICAL ANALYSES AND SAMPLE SIZE JUSTIFICATION................... 16 
7.1 Statistical Analyses .................................................................................................. 16 
7.2 Sample Size Justification ......................................................................................... 17 
8. 
DATA QUALITY ASSURANCE ......................................................................... 17 


 
 
 
 
 
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9. 
OBLIGATION OF THE INVESTIGATOR ........................................................... 17 
9.1 
Advertising .......................................................................................................... 17 
9.2 
Institutional Review ............................................................................................. 17 
9.3 
Subject Consent ................................................................................................. 18 
9.4 
Data Collection ................................................................................................... 18 
9.5 
Adherence to Protocol ........................................................................................ 18 
9.6 
Records Retention .............................................................................................. 18 
9.7 
Investigator’s Final Report .................................................................................. 18 
10. 
REFERENCES ................................................................................................... 19 
 


 
 
 
 
 
4 
  
1.  
RATIONALE AND STUDY OBJECTIVE  
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 toothpastes sold in the US are 
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.  
Due to the high cost involved in conducting clinical caries trials a number of 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.  
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. 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 


 
 
 
 
 
5 
  
dentifrice products that also included fluoride dose response controls (unpublished data). 
An additional capability of the model involves testing the acid resistance of remineralized 
enamel specimens. This test provides the capability to compare the acid resistance of the 
mineral deposited during the in situ remineralization phase after treatment with different test 
products.  
The purpose of this study is to compare the remineralization potential of an optimized 
fluoride dentifrice to a control fluoride dentifrice in an in situ caries model.  
 2. 
STUDY DESIGN 
This will be a double blind, single center, 3-way crossover design study. 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 bovine enamel specimens will be placed in the buccal surface of two 
posterior denture teeth (the specimen site may extend into the buccal flange area, if needed) 
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. Specimens will be removed after two weeks, and the subjects 
will undergo at least 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 
three test products. Changes in the mineral content of the enamel specimens will be 
assessed using surface microhardness (SMH) and transverse microradiography (TMR). 
Enamel fluoride uptake (EFU) will be determined using the microdrill enamel biopsy 
technique. In addition, the net acid resistance (NAR) and the comparative acid resistance 
(CAR) of the remineralized enamel specimens will be determined.  
 
2.1 Study Schedule: 
 
Visit 
1 
Visit  
2, 5, 8  
Visit  
3, 6, 9  
Visit  
4, 7, 10 
 
Screening 
Prophy 
 Begin Tx 
 End Tx 
Informed Consent 
X 
 
 
 
Medical history review 
X 
X 
X 
X 
Oral Soft Tissue exam 
X 
X  
X 
X 
Oral Hard Tissue exam 
X 
X (v2 only)   
X (v10 only) 
Salivary Flow Rate assessment X 
 
 
 
Inclusion/Exclusion criteria 
X 
 
 
 
Continuance Criteria 
 
X  
X  
X 
Randomization 
 
X 
 
 
Adverse Event monitoring 
 
X 
X 
X 


 
 
 
 
 
6 
  
Placement of specimens  
 
 
X 
  
Removal of specimens  
 
 
  
X 
Issue washout toothpaste  
X 
 
 
Return washout toothpaste 
 
 
X 
 
Issue study products and diary 
 
 
X 
  
Return study products and diary   
 
  
X 
Study close-out 
 
 
 
X (v 10 only) 
 
3. 
STUDY POPULATION 
3.1 Source and Number of Subjects 
Potential subjects will be selected from the OHRI’s IRB approved databases of persons 
previously accepted into the partial denture panel (IRB #1110007150 and IRB #18515). 
Potential subjects will be screened to determine eligibility to participate in this study. 65 adult 
subjects, between the ages of 18 and 85 years, will be accepted and randomized in the 
study so that at least 58 subjects can complete the study. 
 
3.2 Subject-Selection Criteria 
3.2.1 Inclusion Criteria 
In order to participate subjects must: 
1. provide voluntary, written informed consent; 
2. be between 18 and 85 years old;  
3. understand and be willing, able and likely to comply with all study procedures and 
restrictions;  
4. be wearing a removable mandibular partial denture with sufficient room to accommodate 
two 4 mm round specimens in the buccal surface of two posterior denture teeth on the 
same side;  
5. be willing and capable of wearing their removable partial denture 24 hours a day for three 
(3), two-week treatment periods; 
6. be willing to allow study personnel to drill specimen sites in two denture teeth in the 
posterior section of one side of their lower partial denture, which may extend into the 
buccal flange area below the teeth;  
7. 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; and 
8. 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). 
 
3.2.2 Exclusion Criteria 
No subject may: 
1. currently be pregnant, intending to become pregnant during the study period or breast 
feeding; 


 
 
 
 
 
7 
  
2. currently have any medical condition that could be expected to interfere with the subject’s 
safety during the study period;  
3. currently be taking antibiotics or have taken antibiotics in the two weeks prior to the 
beginning treatment 1; 
4. known or suspected intolerance or hypersensitivity to the study materials (or closely 
related compounds) or any of their stated ingredients; 
5. have participated in another clinical study or receipt of an investigational drug within 30 
days of beginning treatment 1; or 
6. be taking fluoride supplements, required to use a fluoride mouthrinse or have received a 
professional fluoride treatment in the two weeks preceding specimen placement. 
 
3.2.3 Continuance Criteria 
Each subject must meet the following criteria at each visit to continue in the study. Subjects 
may be dropped from the study and/or excluded from the efficacy analyses if there is 
evidence of: 
1. 
use of a non-study dentifrice or other oral care products during the study; or 
2. 
development of any of the other exclusion criteria. 
 
3.2.4 Removal of Subjects from the Study 
Subjects may withdraw from the study at any time for any reason. The Investigator may also 
remove subjects from the study at any time. The Investigator will document the reason for 
withdrawal for any discontinued subjects. Subjects withdrawn for medical reasons will be 
referred to a physician/dentist by the study personnel and will have their condition monitored 
to resolution or until deemed clinically non-significant. All subjects who discontinue 
participation before the completion of the study will be encouraged to return for an exit oral 
soft tissue examination and to have the specimen sites in their lower partial denture repaired. 
4. 
TEST TREATMENTS 
The following products applied with a provided, marketed toothbrush will be used in this 
study: 
1. 0 ppm F (placebo, negative control): Tom’s of Maine Silly Strawberry 
2. 1100 ppm F as NaF (positive control): Crest Cavity Protection Toothpaste 
3. 1100 ppm F as NaF Test Product: Pronamel Daily Protection 
All three test products will be purchased by Haleon and supplied to OHRI as part of the ISS 
process.    
The toothpaste tubes will be wrapped in opaque adhesive label to conceal product identity 
containing the study number, a code letter that is assigned by the Study Statistician for 
identifying the product, a caution statement, and a phone number to call in the case of an 
adverse response. Subjects will also receive an Oral-B Indicator 35 Soft Toothbrush, rinsing 
cups, and a timer. 
 
 


 
 
 
 
 
8 
  
Label: 
Study # 23-I-121 
Directions for use:  Use only this toothpaste & toothbrush provided, brush daily after 
breakfast and before going to bed for one minute using a full ribbon of toothpaste. Rinse 
with 15 ml of water for 10 seconds using the marked cup provided.    
Keep out of the Reach of Children 
For Clinical Trial Use Only 
Oral Health Research Institute, 415 Lansing Street, Indianapolis, IN  46202 
Emergency Contact # (317) 278-9095 
 
Subjects will also receive a placebo toothpaste and standard toothbrush for home use during 
the 2- to 3-day lead in period. 
 
4.1 Product Use Instructions 
Subjects will first clean their natural teeth with the partial removed with a toothbrush and 
water only. They may also brush their partial denture outside of their mouth with a brush and 
water to reach the areas not accessible when the partial denture is in place, while being very 
careful not to touch the specimen sites. For the test dentifrice they will place their partial 
denture in their mouth and apply a full ribbon of test dentifrice onto the toothbrush. Subjects 
will be instructed to brush the biting surfaces of their back teeth in all four quadrants of their 
mouth for a total of one timed minute taking care to not brush the specimen sites. They will 
then expectorate the toothpaste slurry and rinse with 15 mL of water for 10 seconds and 
expectorate.  
 
4.2 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 two-week 
treatment period will be considered non-compliant. The reason for non- compliance will be 
noted in the subject’s study records. 
 
4.3 Randomization Procedures 
A unique study number will identify all subjects screened for study participation. Screening 
numbers will be assigned in ascending numerical order according to appearance at the study 
site. Subjects who meet all inclusion and exclusion criteria will be randomized into the study. 
At the first treatment visit, randomization numbers will also be assigned in ascending 
numerical order according to appearance at the study site. 


 
 
 
 
 
9 
  
A randomization schedule will indicate the treatment order sequence. Each subject will 
complete all three treatment regimens, one treatment regimen in each of the three treatment 
periods. The randomization schedule will be provided by the statistician, IU Department of 
Biostatistics. 
 
4.4 Blinding Procedures and Code Breaks 
The blind will only be broken in an emergency where it is essential to know which treatment 
a subject received in order to give the appropriate medical care.  
The subject, the study dentist and the laboratory technicians responsible for performing the 
surface microhardness, TMR and fluoride analyses will be blinded. Only specimens and their 
tracking sheets without product identifiers will be sent to laboratory personnel.  
  
5. 
STUDY METHODOLOGY 
5.1 Conduct of Study 
This study will be performed according to GLP and GCP. SOP’s for all procedures are on 
file at the Oral Health Research Institute. 
  
5.2 Clinical Procedures 
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, they will complete 
an informed consent statement and demographic form. Upon review of these documents, 
an update of their medical 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 two posterior 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.  
Visit 2: Dental Cleaning Treatment Period 1 – Two to three days prior to the beginning of 
Treatment Period 1, subjects will return for a professional dental prophylaxis. The subject’s 
mandibular partial denture will be prepared to hold the enamel specimens. Specimen sites 
will be drilled in the buccal surface of two posterior denture teeth on the same side of the 
partial denture. This area may extend into the buccal flange area, if needed. A temporary 
filling (DentuSilTM) 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. Subjects will receive an OST and OHT exam at the end of their dental 
cleaning and continuance criteria will be reviewed. 
Visit 3: Begin Treatment Period 1 – Each subject will receive an OST examination, answer 
continuation questions, and have their medical history/medication information updated. The 
two enamel partially demineralized specimens will be placed in the buccal posterior 
teeth/buccal flange 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 


 
 
 
 
 
10 
  
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. Each subject will be dispensed 
their assigned treatment product according to the randomization scheme and will be 
instructed on the brushing method and perform their first brushing under supervision at the 
Institute. 
Visit 4: End Treatment Period 1 – Two weeks following Visit 3, the subjects will return to the 
Institute and receive an oral soft tissue examination, answer continuance criteria questions, 
and update their medical history/medication information. The enamel specimens will be 
removed from their partial denture and the hollows will be filled with a temporary dental filling 
until the next treatment visit. They will be told to use their regular toothpaste until they return 
and encouraged to remove their lower partial denture at night. 
Visits 5-10 – The procedures outlined above will be repeated until each subject has used 
each of the three test products. At visit 10, subjects will receive an oral hard tissue 
examination, have their mandibular partial denture cleaned through sonication, if applicable, 
have their partial denture repaired either that day or on a future day (if not going on to another 
in situ study) and their participation in the study will end. 
 
5.3 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 14 days of each treatment period subjects may use only the study product 
assigned to them and toothbrush provided 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 14-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 when the specimens are in place. 
 
5.4 Informed Consent Process 
Written informed consent will be obtained at the screening visit from each subject after they 
have had the opportunity to read the document, ask questions and had adequate time to 
decide about their participation. A study representative trained and delegated by the 
Principal Investigator will review the purpose, procedures risk and benefits of the study prior 
to the subject’s signing of the document. The study representative will sign and date (and 
give the time) the consent form to confirm the consent process was completed prior to 
initiation of any study procedures. The subject will be given a copy of the signed document. 
 


 
 
 
 
 
11 
  
5.5 Oral/Hard Soft Tissue Examination 
The study dentist will complete an oral soft and hard tissue (OSHT) examination at 
screening, oral hard tissue (OHT) at Visit 1, 2 and 10 and an oral soft tissue (OST) 
examination at each study visit. The exams will be conducted via a visual examination of the 
oral cavity and perioral area utilizing a light source, dental mirror, gauze, 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, 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, residual roots, faulty restorations and 
implants. Observations will be listed as “Absent” or “Present” and conditions noted as 
present will be described 
 
5.6 Salivary Flow Rate Assessment 
Salivary flow rate 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. 
 
5.7 Diary for Home Use 
At the start of each treatment period, subjects will be provided with a diary 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 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 Card with the subject to confirm treatment compliance and clarify 
listed medical conditions, medications, and treatments. 
 
5.8 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 hollows in two teeth on the same side large enough to 
accommodate the enamel specimens. The opening may include the buccal flange area 
below the denture tooth, as needed. Two gauze-covered enamel specimens (4 mm round) 


 
 
 
 
 
12 
  
will be mounted on the partial denture as described previously. The specimens placed in the 
buccal surface of the denture teeth 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 buccal surface of the denture teeth when 
fully seated. Again, 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 is not a functional part 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 surface of 
posterior denture teeth 
 
 
 
5.9 Adverse Events Assessment 
Subjects will be questioned at each visit regarding any general health or oral complaints and 
symptoms they have experienced since baseline. Any findings will be documented on the 
AE CRF. In the event of subjects reporting AEs outside the scheduled clinical visit, they will 
be assessed at the earliest opportunity by the study dentist and/or Principal Investigator. 
All AEs, regardless of severity or relationship to the test product, will be recorded. Serious 
AEs include any events resulting in death, decreased life expectancy, life-threatening 
situations, persistent or permanent disability/incapacity, hospitalization, or congenital 
anomaly/birth defect. Within 24 hours, the Investigator will submit a written report 
documenting the circumstances of the serious AE. The IRB will be notified within five days 
of the incident. 
 


 
 
 
 
 
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6.0 
LABORATORY METHODS 
6.1 Specimen Preparation 
Bovine teeth will be used as the hard tissue test substrate for the preparation of the 4 mm 
round specimens in the modified in situ caries model. Upon receipt at OHRI, the teeth will 
be sorted, cleaned and stored in 0.1% thymol solution during sample preparation 
procedures. 
Teeth will be selected based on the following criteria: 
• Have 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. 
Approximately two specimens will be obtained from the buccal surface of each bovine tooth. 
A core of enamel 4 mm in diameter will be prepared from each bovine tooth by cutting 
perpendicularly to the buccal surface with a hollow-core diamond drill bit.  
The 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 sonicated in deionized 
water (dw) for two 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, sonicated for 
two 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 Bovine Enamel Specimen 
 


 
 
 
 
 
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6.2 Lesion Creation 
The enamel specimens will be partially demineralized using a modification of the method 
described by White [1987]. The 4 mm round bovine specimens will be immersed for 16 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), and then rinsed thoroughly with deionized water. 
The demineralized enamel specimens will then be stored in a moist environment to prevent 
dehydration. 
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. 
6.4 Efficacy Measurements and Evaluations 
6.4.1 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 ± 3 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 be 120 ± 20 µm with a standard deviation of ≤ 10µm. After 14 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. 


 
 
 
 
 
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6.4.2 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 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.  
6.4.3 Net Acid Resistance test 
To test whether the dentifrice imparts acid resistance to the enamel after 14 days of intra-
oral exposure, a second in vitro demineralization will be repeated following the same 
protocol as described above on the two enamel specimens. SMH will be then evaluated by 
placing 5 indentations 100µm to the right of the indentations placed in situ remineralization 
(See Figure 2). The %NAR will be calculated by the method of Corpron [Corpron et al., 
1986]: 
 
% Net Acid Resistance = [(D1-D2) / (D1-B)] * 100 
 
B= Indentation length (µm) of sound enamel at baseline 
D1= Indentation length (µm) after first in vitro demineralization 
D2= Indentation length (µm) after second in vitro demineralization 
Acid resistance is indicative of any protection that the treatments and intra-oral exposure 
may afford the enamel specimens. The net loss of enamel due to clinical caries is the result 
of multiple cycles of demineralization and repair (remineralization). It is well established that 
repaired enamel is more resistant to subsequent acid challenges. 
6.4.4 Comparative Acid Resistance test 
This measure takes a different approach to understanding whether the enamel formed 
during remineralization is more resistant to acid than the original enamel. Using the data 
from the two enamel specimens, the equation used will compare explicitly the reduction in 
SMH brought by the first and second acid challenges:  


 
 
 
 
 
16 
  
 
% Comparative Acid Resistance = [(D2-R) / (D1-B)] * 100 
 
B= Indentation length (µm) of sound enamel at baseline 
R= Indentation length (µm) of enamel after in situ remineralization  
D1= Indentation length (µm) after first in vitro demineralization 
D2= Indentation length (µm) after second in vitro demineralization 
6.4.5 Transverse Microradiography 
Sections, approximately 100 µm in thickness, will be cut parallel to the orientation cut (as 
shown in Figure 2), after completion of the SMH analysis, using a Silverstone-Taylor Hard 
Tissue Microtome (Scientific Fabrications Laboratories, USA). The sections will be placed in 
the TMR-D system and X-rayed at 45 kV and 45 mA at a fixed distance for 12 s. An 
aluminium step wedge will be X-rayed under identical conditions. The digital images will be 
analyzed using the TMR software v.3.0.0.18. Sound enamel will be assumed to be 87% v/v 
mineral. 
Lesions will be analyzed after in situ demineralization and the following three parameters 
calculated: 
1. Integrated Mineral Loss - ∆Z= [(lesion depth x 87) - area under the curve*] 
2. Lesion Depth – L (83% mineral i.e. 95% of the mineral content of sound enamel) 
3. Maximum mineral density at the surface-zone – SZmax 
Note: *area under the curve which relates volume % mineral at distances from the specimen 
surface with respect to section thickness. 
 
6.5 Laboratory Data 
After the analyses are completed, all enamel specimen surface microhardness, transverse 
microradiography and enamel fluoride uptake data will be reviewed by the principal 
investigator before transfer to the statistician. The investigator will document this review and 
documentation will be filed with the laboratory study files. 
6.6 Specimen Retention 
Laboratory specimens will be retained by the study site for twelve months following database 
lock.  
7. 
STATISTICAL ANALYSES AND SAMPLE SIZE JUSTIFICATION 
7.1 Statistical Analyses 
The mean % SMH recovery will be calculated using the four 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, mean fluoride uptake, mean acid 
resistance and mean ∆Z, L, SZmax will be computed for the two enamel specimens within a 
subject by treatment; if a subject is missing an enamel specimen, the available enamel 


 
 
 
 
 
17 
  
specimen will be used. Analyses of fluoride uptake will be performed after using a natural 
logarithm transformation because the measurements generally are not normally distributed. 
The treatments will be compared using analysis of variance models (ANOVA) suitable for a 
crossover study. The models will include random effects for subject and fixed effects for 
study period, and product. The random effects will model the correlation and variances over 
time between treatments. The primary comparison of interest will be to determine if the test 
treatment to be statistically significant compared to the positive control treatment. Secondary 
comparisons will be to determine if the test treatment to be statistically significant compared 
to the negative control treatment and if the positive control treatment to be statistically 
significant compared to the negative control treatment. A 5% significance level will be used 
for all tests. No alpha-level adjustments for multiple comparisons adjustments will be 
applied.  
7.2 Sample Size Justification 
Based on previous studies we estimate the standard deviation of the differences between 
treatments in the crossover model to be 20 for % SMH recovery. With a sample size of 58 
subjects completing the study, the study will have 80% power to detect a difference between 
any two treatments of 7.5 for % SMH recovery, assuming two-sided tests each conducted 
at a 5% significance level. Due to expected dropouts during the crossover study, the study 
will enroll 65 subjects.  
8. 
DATA QUALITY ASSURANCE 
This study will be conducted in compliance with the US Code of Federal Regulations (CRF) 
governing informed consent (21 CFR 50), Institutional Review Board (IRB) (21 CFR 56), and 
with applicable regulations governing Investigator conduct (21 CFR 312). 
  
9. 
OBLIGATION OF THE INVESTIGATOR 
9.1 
Advertising 
All potential advertising materials used to recruit subjects for this study will be submitted to 
the relevant IRB.  
 
9.2 
Institutional Review 
The IRB approval for this study will be obtained from: 
Human Research Protection Program (HRPP) 
Office of Research Compliance 
Indiana University 
986 Indiana Avenue, 5th Floor 
Indianapolis, Indiana 46202 
 


 
 
 
 
 
18 
  
9.3 
Subject Consent 
Written informed consent will be obtained from all subjects prior to their enrollment into the 
study. The consent form will comply with all applicable regulations governing protection of 
the participants in the study, and include basic elements specified in the US. Code of Federal 
Regulations, 12 CFR 50.25(a). A signed copy of the consent form will be given to the subject 
and the original one will be retained by the Investigator. In order to ensure confidentially, the 
Investigator will store the consent forms separate from the CRFs. 
 
9.4 
Data Collection 
Data will be collected on source documents/CRFs created by the Investigator. 
 
9.4.1 Case Report Forms 
Case report forms will be used for recording all data not entered directly into computers. The 
Investigator will be responsible for maintaining original consent forms, CRFs and other 
source documentation. 
The CRFs that will be used, unless otherwise indicated are: 
• Inclusion/Exclusion Criteria 
• Continuance Criteria 
• Oral soft tissue 
• Oral hard tissue 
• Adverse event 
• Serious adverse event 
• Subject Accountability 
 
9.5 
Adherence to Protocol 
The final protocol constitutes the conduct of the study. The Investigator is required to adhere 
to this final protocol. Any reasonable alternatives, variations, or deviations from the protocol 
must first be approved by the IRB. Any clarification to the protocol will be documented in the 
study file.  
 
9.6 
Records Retention 
At the conclusion of the study, all records, relevant medical/dental records, copies of CRFs, 
and informed consent forms for all subjects treated with the drug or employed as a control 
in the study are retained by the Investigator for 15 years.  
 
9.7 
Investigator’s Final Report 
Following the completion of the study the Investigator shall prepare an integrated clinical 
and final study report. The final report will include a general description of the conduct of the 
study including protocol deviations, subject withdrawals, a discussion of AEs, safety data, 
and laboratory analysis. This report will be approved and signed by the Investigator. 
 


 
 
 
 
 
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10. 
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Intra-oral effects of a fluoride-releasing device on acid-softened enamel. J Am Dent 
Assoc 1986;113:383-388. 
Featherstone JD, Mellberg JR. Relative rates of progress of artificial carious lesions in 
bovine, ovine and human enamel. Caries Res 1981;15(1):109–14. 
Gelhard TBFM, ten Cate JM, Arends J: Rehardening of artificial enamel lesions in vivo. 
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Koulourides, T, Phantumvanit, P, Munksgaard, EC, Housch, T. An intraoral model used 
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Marinho VC, Higgins JP, Sheiham A, Logan S (2003c). Fluoride toothpastes for 
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Mellberg JR: Hard-tissue substrates for evaluation of cariogenic and anti-cariogenic 
activity in situ. J Dent Res 1992; 71: 913–919. 
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Walsh T, Worthington HV, Glenny AM, Appelbe P, Marinho VC, Shi X. Fluoride 
toothpastes of different concentrations for preventing dental caries in children and 
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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, 
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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 
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