Randomized Non-inferiority Trial of Nano-hydroxyapatite-Containing Toothpastes for Relief of Dentin Hypersensitivity
Phase: NA
Conditions: Dentin Sensitivity
Sponsor: The University of Texas Health Science Center at San Antonio

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Official Study Title:  
Trial of Nano-hydroxyapatite-Containing Toothpastes for Relief of 
Dentin Hypersensitivity 
 
 
NCT number: NCT04590040 
 
IRB Approval Date: 05.04.2020 
 
Unique Protocol ID: HSC20190535H 
 
 
 
 
 
 
 

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EXHIBIT A 
CLINICAL STUDY AGREEMENT BETWEEN  
The University of Texas Health Science Center at San Antonio 
AND  
SANGI Co., Ltd, Tokyo, JAPAN 104-8440 
 
Randomized Non-inferiority Trial of Nano-hydroxyapatite-Containing Toothpastes for 
Relief of Dentin Hypersensitivity 
 
Principal Investigator and Affiliations 
Bennett T. Amaechi, BDS, MS, PhD, MFDS RCPS (Glasg), FADI 
Professor and Director of Cariology 
Department of Comprehensive Dentistry 
University of Texas Health Science Center at San Antonio 
7703 Floyd Curl Drive, San Antonio, Texas 78229-3900, USA 
Tel: +1 210 567 3185  
Fax: +1 210 567 3443 
Cellphone: +1 210 834 7675 
E-mail: amaechi@uthscsa.edu 
 
 
Sponsoring Company Representative ('Sponsor'): 
Roslyn Hayman 
President 
SANGI Co., Ltd 
3-11-6 Tsukiji, Chuo-ku 
Tokyo 
JAPAN 104-8440 
 
Version: 21 February 2020 
 

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INDEX 
OBJECTIVE 
 
SUMMARY OF STUDY DESIGN 
 
POPULATION AND INITIAL SCREENING PROCEDURES 
Inclusion Criteria 
Exclusion Criteria 
 
RECRUITMENT SCREENING AND STUDY PROCEDURES 
(1) Preliminary Examination for Recruitment 
(2) Washout Period 
(3) Final Selection: Baseline Sensitivity Measurement 
Test Products 
Randomization/Allotment to Test Product Groups 
Clinical Procedures 
 
EFFICACY AND SAFETY ASSESSMENT PROCEDURES 
Application of the Stimulus to Trigger Sensitivity: 
Cold Air Stimulus 
Tactile Stimulus 
Visual Analog Scale (VAS) 
Calibration of the Clinical Examiner 
Oral Soft and Hard Tissue Assessment 
Adverse Events 
 
STATISTICAL PROCEDURES 
Product Efficacy Data Analysis 
(1) Data Analysis for Efficacy of the Main Evaluation Item 
(2)Data Analysis for Efficacy of the Secondary Evaluation Item 
Sample Size Calculation 
Non-inferiority Tests for the Ratio of Two Means 
Criteria for Test Aborting and Drop-out 
Criteria for Inclusion of Subjects as Appropriate for Efficacy Analysis 
Period of Data Storage 
Schematization of Design 
 
References 

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OBJECTIVE 
Dentinal hypersensitivity (DHS) is the incidence of short, sharp pain near the base of a 
tooth, which arises because of dentin being exposed to external stimuli by cervical 
erosion/abrasion or gingival recession, and which cannot be attributed to any other form 
of dental defect or pathology such as caries, broken or leaking restorations, or a chipped 
or cracked tooth. The objective of the present study is to determine whether or not the 
regular use of a nano-hydroxyapatite(nano-HAP)-containing toothpaste is non-inferior to 
the regular use of a potassium nitrate(KNO3)-containing toothpaste in the relief of DHS. 
Non-inferiority would be declared if the mean percentage reduction of sensitivity for the 
nano-HAP toothpaste was no worse than the mean percentage reduction for KNO3-
containing toothpaste, within statistical variability, by a margin (Δ) of 20%(at 95% 
probability). Our null hypothesis is that the nano-HAP toothpaste is not similar to the 
KNO3 toothpaste, using anon-inferiority margin of Δ = 20% for the primary outcome 
measure. Our choice of a non-inferiority trial design was based on the abundant and well-
established evidence for the sensitivity relief efficacy of KNO3 toothpaste and the 
expectation that non-inferiority of nano-HAP toothpaste to KNO3 toothpaste would be 
sufficient to place nano-HAP toothpaste in the same class of quasi-drug as the KNO3 
toothpaste for the relief of DHS. A further study objective is to show whether or not, 
statistically, the regular use of a nano-HAP-containing toothpaste is significantly more 
effective in the relief of DHS than the regular use of a placebo toothpaste not containing 
nano-HAP.  
 
SUMMARY OF STUDY DESIGN 
This study is a double-blind, randomized, placebo-and positive-controlled, stratified 
parallel group clinical trial. A total of 192 patients diagnosed with DHS and meeting all 
necessary requirements for selection as subjects will be stratified into three balanced 
groups (64 per group) according to age, gender, and their mean sensitivity to cold air 
stimulus as scored on a Visual Analog Scale (VAS) at the time of baseline examination for 
final selection during the recruitment screening process. Males and females aged between 
20 and 80 years will be enrolled. The three groups will be randomly assigned to use one of 
three test toothpastes having an identical base formulation but containing respectively 
either 0% nano-HAP (placebo), 15% nano-HAP or 5% KNO3 (positive control). Subjects will 
be instructed to use their assigned test toothpaste as their sole oral hygiene product for 
the 8 weeks treatment duration. Subjects will be instructed to brush their teeth twice daily 
for 3 minutes, in the morning and before bed at night, applying on each occasion a one-
inch strip of their assigned toothpaste on a soft-bristled toothbrush which will be supplied. 
Examination for DHS will include response to cold air, as the primary stimulus, and to 
tactile pressure, as a secondary stimulus, and will be conducted at the commencement of 

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recruitment screening (preliminary examination), then subsequently at baseline, 4 and 8 
weeks. All dental examinations for data collection in all 192 subjects will be conducted by 
the same Clinical Examiner throughout the study. Subjects will be examined and queried 
on adverse events at each study visit. 
 
POPULATION AND INITIAL SCREENING PROCEDURES 
This study will be conducted at the clinical research facility of the school of dentistry of 
the University of Texas Health Science Center at San Antonio (UTHSCSA) following 
approval by the UTHSCSA Institutional Review Board (IRB). UTHSCSA IRB will also 
approve the Human Assurance Documents as well as perform the compliance assessment 
(study monitor). All documents for this study will be used with Japanese language 
superimposed and will be approved by the IRB. This study will be conducted according to 
the "Recommendations for Evaluating Agents for the Reduction of Dentinal 
Hypersensitivity" as proposed by the Council on Dental Therapeutics of the American 
Dental Association, and in compliance with International Conference on Harmonization 
(ICH) Good Clinical Practice Guidelines including ICH E6, the Abbreviated Requirements 
of the Investigational Device Exemption Regulations (21CFR Part 812), and with 21 CFR 
Parts 50, 54, and 56. 
Patients presenting to any of the dental school clinics, either with a primary complaint of 
tooth hypersensitivity or whom a clinician discovers to have a hypersensitive tooth or teeth 
during other routine dental treatment or examination will be screened to determine 
whether they meet study entry criteria. 
 
Patients can be referred to the study Investigators by any of the dental school clinics or 
from surrounding private clinics. Initial screening of patients to confirm eligibility to 
participate in the study will be conducted by the Study Coordinator, and will include a 
medical history and information on medication use, and a review of the rest of the 
inclusion/exclusion criteria. 192 adult male and female patients, who are in good general 
health except for the symptoms of DHS, and who meet specific inclusion/exclusion criteria 
and the subsequent requirements of recruitment screening will be enrolled as subjects in 
the clinical study and randomized into the three test groups. The enrolment objective will 
be to have at least 57 subjects complete the study in each of the three test groups. 
Therefore to allow for about 10% dropout in each test group, 192 subjects (64 per group) 
will be enrolled. All relevant medical and non-medical conditions will be taken into 
consideration when deciding whether this protocol is appropriate for a particular subject. 
The following eligibility criteria are designed to select subjects for whom protocol 
treatment is considered appropriate. 
 

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Inclusion Criteria 
・ Males or females between the age of 20 and 80 years, of any socio-economic status 
・ Diagnosed as having DHS, i.e. having at least one sensitive tooth with demonstrated 
cervical erosion/abrasion or gingival recession 
・ Showing a sensitivity response to both tactile and cold air stimulus delivered by a 
Yeaple probe and a one-second blast of cold air respectively (see detailed description 
below) 
・ Patients must be reliable, cooperative and of adequate intelligence to read and 
understand the rating scales and other study instructions 
・ Patients must be able to read, comprehend, and sign the informed consent form 
・ The teeth and sites to be tested should be on the buccal/labial surfaces of incisors, 
canines, premolars or molars where the affected sites are accessible 
・ Teeth selected for testing should have a plaque index of ≤ 2 
 
Exclusion Criteria 
・ The sensitive tooth is associated with concomitant oral pain due to any other 
condition such as soft-tissue lesions, tooth-ache ascribable to dental caries, tooth 
fracture, or cracked tooth syndrome, or pain due to other surgical procedures or 
injuries. 
・ The sensitive tooth is associated with a periodontal abscess as diagnosed from an X-
ray or clinical examination of the tooth 
・ The sensitive tooth is associated with mobility >1 
・ The sensitive tooth is associated with gum pain from gingivitis, occlusal trauma, 
thermal or chemical burns 
・ Patients having pain from periodontal-related causes but not DHS 
・ Previous professional desensitizing treatment 
・ Patients using medication which could interfere with the perception of pain 
・ Medical histories marked by chronic use of anti-inflammatory agents, daily 
analgesics, anticonvulsants, antihistamines, antidepressants, sedatives and/or other 
psychotropic drugs 
・ Pregnancy or breastfeeding 
・ Allergies and idiosyncratic responses to toothpaste ingredients 
・ Eating disorders or conditions associated with vomiting 
・ Systemic conditions that are etiologic or predisposing to DHS 
・ Excessive dietary or environmental exposure to acids 
・ The sensitive tooth was restored in the preceding three months 
・ The sensitive tooth is an abutment tooth for fixed or removable prostheses 

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・ The sensitive tooth has extensive restoration or restorations extending into the test 
area 
・ Patients below 20 years or above 80 years of age 
・ Smokers 
 
RECRUITMENT SCREENING AND STUDY PROCEDURES 
Only patients who meet all initial screening criteria and provide written informed consent 
will be invited to proceed to recruitment screening for participation in the trial. 
Recruitment screening will consist of three steps, as described below: (1) a preliminary 
examination; (2) a 4-week washout period (for patients who meet the entry criteria during 
the preliminary examination); then (3) at the end of the washout period, evaluation and 
recording of each patient’s baseline sensitivity level, as the basis for final subject selection. 
 
(1) Preliminary Examination for Recruitment 
Patients eligible for recruitment screening will report to the clinical research facility 
having refrained from 1) oral hygiene procedures or use of chewing gum for 8 hours, and 
2) eating or drinking for 4 hours, except for drinking water, prior to the visit. The 
examination will include checking the patient’s medical history and medication use, and 
a thorough oral examination by the Clinical Examiner, with evaluation of sensitive teeth.  
Tooth sensitivity will be assessed by a positive response to tactile and cold air stimuli, and 
the entry criteria for parameters assessed at this preliminary examination for recruitment 
will be 1) a positive response to tactile stimulus using a Yeaple probe at 30g force 
(standardized application for every subject), and 2) a VAS score of 30mm or more assessed 
using a one-second cold air blast from a standard dental unit air syringe. (The procedures 
for each of these tests are described below). 
 
(2) Washout Period 
Only patients who meet the entry criteria in the preliminary examination will proceed to 
the washout period. They will be assigned to use a toothpaste not containing either 
fluoride, hydroxyapatite or KNO3, with an adult soft-bristled toothbrush, for a period of 
four weeks. The toothpaste and toothbrush will be used in place of the patient's normally 
used toothpaste and toothbrush, and will be supplied by the Sponsor. The patient will be 
instructed, after wetting the brush, to apply one inch (2.5cm) of the supplied toothpaste 
per brushing and to brush for 3 minutes by the scrubbing method twice daily, in the 
morning and last thing before bed at night, rinsing out the toothpaste with 10ml of water 
for 10 seconds after brushing. Patients will be asked to return to the clinical research 
facility for assessment at 4 weeks. 
 

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(3) Final Selection : Baseline Sensitivity Measurement 
As for the preliminary examination for recruitment, patients reporting for baseline 
examination will be required, after brushing their teeth with the supplied toothpaste and 
toothbrush on the morning of the visit, to have refrained from 1) any other oral hygiene 
procedures or use of chewing gum, and 2) eating or drinking, except for drinking water, 
prior to the visit. The baseline examination will assess tactile and cold air sensitivity. (The 
procedures for each of these tests are described below). For each patient eligible to 
participate in the study, one or more hypersensitive teeth that satisfy the tactile and cold 
air hypersensitivity enrolment criteria will be identified for evaluation throughout the 
study. For each assessment visit, including the baseline examination, sensitivity must be 
triggered by application of a tactile stimulus and a cold air blast stimulus, and the level of 
sensitivity recorded.  
For each patient completing the washout period, the result obtained according to the 
selection criteria at the preliminary examination for recruitment shall be compared with 
the result obtained at baseline examination. Only patients who 1) show a worsening of 
DHS or 2) maintain the same level of DHS over the duration of the washout period shall 
be selected and enrolled as subjects to participate in the trial, i.e. only patients who at the 
baseline examination meet the qualifying criteria, namely a Yeaple value of 30g or less 
and a VAS score in response to cold air stimulus of 30mm or more, will be accepted as 
subjects. As shown in the section explaining tactile stimulus, however, though the Yeaple 
criterion for acceptance in the study will be sensitivity at a pressure of 30g or less, in 
carrying out the baseline examination, the Clinical Examiner will apply pressure starting 
from a load of 10g, and increasing by increments of 10g, up to a maximum of 80g. 
 
Test Products 
The test products shall contain respectively 0% nano-HAP (placebo), 15% nano-HAP, and 
5% KNO3 (positive control). 
 
Randomization/Allotment to Test Product Groups 
A total of 192 subjects diagnosed with DHS and who have met all necessary requirements 
for selection as subjects in the trial will be stratified into three balanced groups according 
to age, gender and VAS score in response to cold air stimulus at the time of baseline 
examination for final selection of subjects eligible to participate in the trial (64 per group). 
One of the three test products shall be randomly assigned to each of the three groups. The 
0% and 15% nano-HAP-containing toothpastes and the 5% KNO3 toothpaste shall be 
randomly coded as either A, B, or C by the manufacturer or Sponsor, and the code shall 
not be disclosed until all statistical analysis is complete. 
 

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Clinical Procedures 
Immediately following the baseline sensitivity examination, each patient who qualifies to 
become a subject in the trial will be given and make the first use of his/her allotted test 
toothpaste, under the supervision of the Study Coordinator, after the latter has confirmed 
and recorded the weight of the tube. During the next 8 weeks, subjects will be required to 
brush their teeth twice daily with their allotted test product, as instructed, and not to take 
any food or drink for at least 30 minutes after brushing except for drinking water. A diary 
covering the duration of the study will be provided to each subject to (1) keep a record of 
number of times of brushing each day, and (2) record the name of any medication taken 
during the study period. All subjects will be asked to maintain their normal dietary habits. 
However, the use of oral hygiene products that are not related to this clinical trial, such 
as mouthwash and prescription medicine, along with dental clinic visits, dental treatment, 
professional plaque removal, whitening, use of pain relievers or commercial hypoallergenic 
products as well as use of narcotics, anti-inflammatory drugs, conventional analgesics, 
anticonvulsants, antihistamines, antidepressants, sedatives and other psychotropic drugs 
shall be prohibited throughout the course of the study. 
 
Each subject will be given an appointment 4 weeks and 8 weeks after the baseline 
examination, for post-treatment sensitivity assessment. On the day of each sensitivity 
assessment, subjects shall be prohibited, after morning tooth-brushing, from (1) the use of 
any other oral hygiene procedures or chewing gum and (2) from eating or drinking, except 
for drinking water, prior to the examination. The same sensitivity scoring procedures and 
restrictions used at baseline will be repeated for the examinations at 4 and 8 weeks. To 
ensure standardization, all dental examinations in all the 192 subjects will be performed 
by the same Clinical Examiner throughout the study. Subjects will be instructed to bring 
their allotted test toothpaste for weighing and their diary for inspection at the time of each 
assessment visit, and at the 4 week visit each subject will be given a fresh tube of his/her 
allotted test toothpaste whose weight has also been confirmed and recorded. This 
information will be used for monitoring adherence to the protocol. At each visit, subjects 
will also be interviewed with respect to the occurrence of adverse events and the use of 
concomitant medications. 
 
EFFICACY AND SAFETY ASSESSMENT PROCEDURES 
 
Application of the Stimulus to Trigger Sensitivity 
Before stimulus application, all psychological and physiological factors that may alter the 
subject's degree of perception (for example—anticipation) should be controlled to the 
extent possible by standardization of procedure, training of the subject, demonstration of 

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the stimulus, and environmental controls (for example—noise, room temperature) 
including familiarization of the subject with the entire system. 
DHS will be triggered by two stimuli, spaced at least 10 minutes apart to prevent the 
stimuli affecting each other. The two stimuli will be applied in the following order: first 
tactile, then cold air blast. If considered necessary, in the Clinical Examiner's judgement, 
each stimulus may be applied twice and/or a fake test included (in which no stimulus is 
delivered) to ensure that the subject is not responding erroneously.A1 
 
Cold Air Stimulus 
Assessment of cold air sensitivity will be conducted using air delivered from a standard 
dental unit air syringe at room temperature (~70°F±3°F)(~21°C±2°C) and a pressure of 
60psi (± 5psi). The air will be directed perpendicular to the exposed root surface of the 
sensitive tooth for one second from a distance of approximately one centimeter, and to 
prevent false results, the sensitive tooth to be tested will be isolated from the adjacent 
teeth (mesial and distal) using cotton rolls held in place by the examiner's gloved fingers.A2 
Assessment of sensitivity will be carried out using VAS.A3 Within 2 minutes from 
application of the stimulus, the Clinical Examiner shall ask the subject to record the 
intensity of the sensitivity he/she experienced, using the VAS scale. (See explanation 
below).  
 
Tactile Stimulus 
Tactile sensitivity will be assessed using the Model 200A Yeaple Electronic Force Sensing 
Probe (Yeaple Research, Pittsford, NY). The probe will be calibrated prior to use. The 
explorer tip of the Yeaple probe will be placed adjacent to the exposed dentin on the 
buccal/labial surface of the sensitive tooth, predominantly at the cement-enamel junction 
(CEJ). The explorer tip will be applied perpendicular to the tooth surface, starting from a 
load of 10g, and increasing its pressure by increments of 10g, up to a maximum of 80g, for 
all sensitivity examinations except the preliminary screening for recruitment, which shall 
be conducted using a standard pressure of 30g for all patients. 
 
The explorer tip will be drawn slowly across the tooth surface in a distal to medial direction 
for approximately 3-5 seconds, to ensure application of the stimulus across all exposed 
tubules. The interval between applications shall be 60 seconds. The subject will be asked 
to say "yes" at the point where he/she feels the pressure is uncomfortable, and at that time 
point the Clinical Examiner will apply the explorer tip again at the same load for 
confirmation, and the load at which the subject again says "yes" will be recorded.A4 
 
 

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Visual Analog Scale (VAS)A3 
VAS will be used within 2 minutes of stimulus application to evaluate the intensity of 
sensitivity on each measurement occasion: preliminary examination for recruitment, 
baseline examination, and examination after 4 and 8 weeks. The VAS scale, which must 
be explained to the subject, consists of an unmarked horizontal straight line, 100mm long, 
drawn on a sheet of paper, with the beginning and end representing no pain and maximum 
pain respectively. Following stimulus application as described above, the Clinical 
Examiner shall ask the subject to draw a vertical mark on the line, indicating how much 
pain he/she just felt, with “no pain” being on the far left of the scale at 0mm and “pain as 
bad as it can be,” i.e. the maximum pain the subject could experience, being on the far 
right at 100mm.The distance between 0mm and the mark made by the subject shall then 
be measured in mm by the Clinical Examiner and recorded as the subject's VAS score, i.e. 
his/her indicated level of pain. 
 
Calibration of the Clinical Examiner 
To ensure standardization and repeatability in all sensitivity assessment procedures, 
including the manufacturers’ instructions, as well as to provide accurate interpretation 
and analysis of the sensitivity response, the Clinical Examiner will be calibrated on the 
sensitivity assessment techniques described above, for both the Yeaple probe and delivery 
of cold air stimulus. The calibration will be conducted by a benchmark calibrator who is 
experienced in sensitivity assessment using cold air and the Yeaple probe. The method of 
calibration will be determined by the benchmark calibrator. This will be done using about 
10 patients attending the clinic and diagnosed as having DHS. The sensitivity evaluation 
data collected during calibration will be recorded, and the agreement between the Clinical 
Examiner and benchmark calibrator for objective evaluation of sensitivity (inter-
examiner) and between the Clinical Examiner’s own repeated sensitivity evaluations 
(intra-examiner) will be evaluated using Bland-Altman plots and intra-class correlation 
coefficients. Midway during the trial, duplicate examinations will be carried out on 10% of 
the study subjects to confirm maintenance of repeatability by the Clinical Examiner. 
Calculations of agreement between the Clinical Examiner and the calibrator and between 
the Clinical Examiner's own repeated measures will clearly educate the Clinical Examiner 
on the importance of keeping to one standard to maintain repeatability in the sensitivity 
assessment procedures. 
Oral Soft and Hard Tissue Assessment 
At the time of each sensitivity assessment, the Clinical Examiner shall visually examine 
each subject’s oral cavity and perioral area using a dental light and dental mirror. This 
examination will include an evaluation of the soft and hard palate, gingival mucosa, buccal 

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mucosa, mucogingival fold areas, tongue, sublingual and submandibular areas, salivary 
glands, and the tonsillar and pharyngeal areas. 
 
Adverse Events 
Information concerning any adverse events will be obtained by the Clinical Examiner from 
the subjects at the time of sensitivity assessment. All observed or voluntarily reported 
adverse events regardless of test group or suspected causal relationship with a test 
product shall require immediate notification within 24 hours to the IRB and the Sponsor 
or its designated representative, during a reporting period beginning from the time that 
the subject first provides informed consent, which is obtained prior to the start of the 
clinical study, i.e. prior to the subject undergoing any study-related procedure and/or 
receiving any test product, through to and including 30 calendar days after the last 
administration of the test product. Any adverse event occurring any time after the 
reporting period must be promptly reported to assess whether it meets the criteria for 
classification as a serious adverse event requiring immediate notification to the Sponsor 
or its designated representative. The Principal Investigator is required to assess causality, 
and for all adverse events, sufficient information should be obtained by the Principal 
Investigator to try to determine the causality of the adverse event. For adverse events 
with a suspected causal relationship to a test product, follow-up by the Principal 
Investigator is required until the event or its sequelae are resolved or stabilized at a level 
acceptable to the Principal Investigator, and the Sponsor concurs with that assessment. 
 
STATISTICAL PROCEDURES: 
Product Efficacy Data Analysis: 
Efficacy outcomes: The primary efficacy outcome will be the decrease in cold air stimulus 
sensitivity (VAS score) from baseline to 8 weeks. The secondary efficacy outcome will be 
the increase in tactile stimulus force from baseline to 8 weeks. Additional outcomes will 
include changes in cold air stimulus sensitivity and tactile stimulus force from baseline to 
4 weeks. 
(1) Data Analysis for Efficacy of the Primary Evaluation Item 
Comparisons among the three groups for differences in the primary and secondary efficacy 
outcomes at each time point will be analyzed using analysis of covariance; because of the 
stratified randomization, age, gender, and baseline VAS cold air sensitivity will be 
included as covariates. Pre-planned post-hoc comparisons include: 1) pair-wise 
comparisons of the test product and the positive control against the placebo, to 
demonstrate effectiveness and 2) 95% confidence intervals (CI) for the ratio of means for 
the nano-HAP test product to the positive control to demonstrate non-inferiority of the 
test product with the positive control. Non-inferiority will be demonstrated if the lower 

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bound of the 95% CI falls within the 20% non-inferiority margin. If the data are log-
normally distributed, then the analyses will be done on the log-transformed data. If the 
95% CI for the difference between the test paste and the positive control is transformed 
back to the original scale, the back-transformed CI is a 95% CI for the ratio of means. If 
data are normally distributed, the 95% CI will be computed using Fieller's theorem, and 
the other condition that must be met to show non-inferiority is that the reference (i.e. 
positive control) mean must be significantly different from zero using a one-sided test. A 
5% significance level will be used for all tests. From the results obtained for the placebo 
and positive control groups, it will be confirmed whether the non-inferiority margin used 
in the trial (20%) was appropriate or not. 
(2) Data Analysis for the Efficacy of the Secondary Evaluation Item 
Repeated measures analysis of covariance will be used for comparison between the 4-week 
and 8-week outcomes within each group. Interactions of the treatment effect with age, 
gender, and tactile sensitivity will be examined to evaluate significance of differential 
effects of the treatment by patient characteristics. 
Sample Size Calculation 
Based on data from prior studies, A2, A5, A6 the coefficients of variation (the ratio of the 
standard deviation to the mean (SD/Mean)) at 8 weeks in cold air stimulus sensitivity and 
tactile stimulus force are estimated to be 0.500 and 0.400, respectively. A7 Based on this 
COV, the power and sample size for non-inferiority t-tests from a parallel-groups design 
is calculated with the statistical hypotheses expressed in terms of mean ratios (Treatment 
Mean / Reference Mean) instead of mean differences. Using the software PASS 2019, 
v19.0.1, the sample size for Non-Inferiority Tests for the Ratio of Two Means is calculated 
from R Package of Chow A8 by inputting the following information;  
Option  
 
 
Value 
Design Tab 
Solve For: 
 
Sample Size 
Higher Means Are: 
 
Better (H1: R > 1 - NIM) 
Power: 
 
0.80 
Alpha: 
 
.050 
Group Allocation: 
 
Equal (N1 = N2) 
NIM (Non-Inferiority Margin): 
0.2 
R1 (Actual Ratio): 
 
1.0 
COV (Coefficient of Variation): 0.5 0.4 
 
 

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The following results were generated by the software. 
Non-Inferiority Tests for the Ratio of Two Means 
Numeric Results for a T-Test 
R = Treatment Mean / Reference Mean 
Higher Means are Better 
Hypotheses:    H0: R ≤ 1 - NIM   vs.   H1: R > 1 - NIM 
Target 
Power 
Actual 
Power 
N1 
N2 
N 
-NIM 
Bound 
R0 
R1 
COV 
Alpha 
0.80 
0.80419 38 
38 
76 
-0.200 
0.800 
1.000 
0.400 
0.050 
0.80 
0.80557 57 
57 
114 
-0.200 
0.800 
1.000 
0.500 
0.050 
The highlighted numbers are the sample sizes if primary outcome measure is based on 
cold air (green) or tactile stimuli (yellow). The sample sizes were calculated for nano-
HAP (N1) and KNO3 (N2) groups. (By default the placebo group will have the same 
sample size). 
Report Definitions 
Target power is the desired power value (or values) entered in the procedure. Power is 
the probability of rejecting a false null hypothesis. 
Actual power is the power obtained in this scenario. Because N1 and N2 are discrete, 
this value is often (slightly) larger than the target power. 
N1 and N2 are the number of items sampled from each population. 
N is the total sample size, N1 + N2. 
-NIM is the magnitude and direction of the margin of non-inferiority. Since higher 
means are better, this value is negative and is the distance below one that is still 
considered non-inferior.  
R0 is the corresponding bound to the non-inferiority margin and equals 1 - NIM. 
R1 is the mean ratio (treatment/reference) at which the power is computed. 
COV is the coefficient of variation on the original scale. 
Alpha is the probability of rejecting a true null hypothesis. 
 
Summary Statements 
Group sample sizes of 38 in the first group and 38 in the second group achieve 80% 
power to detect non-inferiority using a one-sided, two-sample t-test. The margin of non-
inferiority is 20%. The true ratio of the means at which the power is evaluated is 1.000. 
The significance level (alpha) of the test is 0.050. The COV of both groups are assumed to 
be 0.400. 
 

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With a sample size of at least 57 subjects per group completing the study, a non-inferiority 
test of means using two one-sided tests achieves 80% power at a 5% significance level for 
cold air stimulus sensitivity when the true ratio of the means is 1.000 and the non-
inferiority limit of the mean ratio is 20%. Because the COV for tactile stimulus force is 
smaller, the study will have higher power for that outcome. To allow for 10% dropout, the 
study will enroll 64 subjects per group (192 subjects in total). Sample size calculations 
were performed using PASS 2019 (Kaysville, UT). 
Dropout-Inflated Sample Size  
 
 
Sample Size 
Dropout-Inflated Enrollment 
sample size 
Expected Number of 
Dropouts 
Dropout 
Rate 
N1 N2 N 
N1' 
N2' 
N' 
D1 
D2 
D 
10% 
38 
38 
76 
43 
43 
86 
5 
5 
10 
10% 
57 
57 
114 64 
64 
128 
7 
7 
14 
 
Definitions 
Dropout Rate (DR) is the percentage of subjects who are expected to be lost at random 
during the course of the study and for whom no response data will be collected (i.e. who 
will be treated as "missing"). 
N1, N2, and N are the evaluable sample sizes at which power is computed. If N1 and N2 
subjects are evaluated out of the N1' and N2' subjects that are enrolled in the study, the 
design will achieve the stated power. 
N1', N2', and N' are the number of subjects that should be enrolled in the study in order 
to end up with N1, N2, and N evaluable subjects, based on the assumed dropout rate. 
After solving for N1 and N2, N1' and N2' are calculated by inflating N1 and N2 using the 
formulas N1' = N1 / (1 - DR) and N2' = N2 / (1 - DR), with N1' and N2' always rounded 
up. (See references A7, pages 52-53, or A8, pages 32-33.) 
D1, D2, and D are the expected number of dropouts. D1 = N1' - N1, D2 = N2' - N2, and D 
= D1 + D2. 
 
 
 
 
 

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16 
 
Chart Section  
 
 
Criteria for Test Aborting and Drop-out 
Subjects may withdraw from the trial at any time at their own request, or they may be 
withdrawn at any time at the discretion of the Principal Investigator or Sponsor for safety, 
behavioral, or administrative reasons. If a subject does not return for a scheduled visit, 
every effort should be made to contact the subject. In any circumstance, every effort shall 
be made to document subject outcome, if possible. The Principal Investigator should 
inquire about the reason for withdrawal, and request that the subject return all unused 

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17 
 
test product, and return for a final visit, if applicable, and should also follow-up with the 
subject regarding any unresolved adverse events. 
If a subject withdraws from the trial and also withdraws his/her consent for disclosure of 
future information, no further evaluations should be performed and no additional data 
should be collected. The Sponsor may retain and continue to use any data collected before 
such withdrawal of consent. 
The Principal Investigator may discontinue a subject's participation if, in the opinion of 
the Principal Investigator, the subject is no longer a suitable candidate for the study. 
Possible reasons for such discontinuation include, but are not limited to: 
・ Adverse event 
・ Deviation from the protocol 
・ Missed appointment(s) 
・ Subject no longer meets eligibility criteria 
・ Use of antibiotic therapy or other prohibited medication during the study period 
 
Criteria for Inclusion of Subjects as Appropriate for Efficacy Analysis 
・ Usage rate of toothpaste was 80% or more 
・ No evidence of events or activity likely to impair the reliability of the examination 
results, such as loss of diary records, etc. 
・ Subject has complied with the required restrictions during the examination period 
 
Period of Data Storage： 
The Principal Investigator agrees that all basic documentation used in the study (informed 
consent forms, Case Report Forms (CRFs) and all other relevant documents) shall bear 
superimposed Japanese translation, for reference purposes only, to be provided in advance 
by the Sponsor, and that the Sponsor shall have access to copies of all such documentation, 
if it so requests, upon completion of the study. To enable evaluations and/or audits from 
regulatory authorities or the Sponsor, the Principal Investigator agrees to keep records 
including the identity of all participating subjects (sufficient information to link records, 
e.g. CRFs and hospital records), all original signed informed consent forms, copies of all 
CRFs, serious adverse event forms, source documents, and detailed records of treatment 
abrogation. The records shall be retained by the Principal Investigator according to ICH 
guidelines and local regulations, or as specified in the Clinical Study Agreement, 
whichever is the longer period. 
If the Principal Investigator relocates, retires, or for any reason withdraws from the study, 
the Sponsor must be notified in advance. The study records must be transferred to an 
acceptable designee, such as another investigator, another institution, or to the Sponsor. 

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The Principal Investigator must obtain the Sponsor's written permission before disposing 
of any records, even if retention requirements have been met. 
 
Schematization of Test Design 
 
 
 
References  
A1. Clark GE. Designing hypersensitivity clinical studies. Dental Clinics of North 
America, 34:531-543, 1990 
A2. Pradeep AR, Agarwal E, Naik SB, Bajaj P, Kalra N. Comparison of efficacy of three 
commercially available toothpastes [corrected] on dentinal hypersensitivity: A 
randomized clinical trial. Australian Dental Journal,57:429–34, 2012 
A3. Huskisson EC. Measurement of pain. Lancet 2:1127-1131, 1974 
 
1st 
Visit 
2nd 
Visit 
4 
weeks 
3rd 
Visit 
4  
weeks 
4th 
Visit 
4  
weeks 
5th 
Visit 
Initial Screening: 
selection of candidates eligible for 
recruitment screening and receipt of 
candidates' informed consent 
○ 
 
 
 
 
 
 
 
Recruitment Screening: 
①Preliminary examination: 
selection of candidates eligible to 
proceed to washout period 
 
◎ 
 
 
 
 
 
 
②Washout period(4 weeks) 
 
 
 
 
 
 
 
 
③Baseline measurement: 
final selection of subjects eligible to 
participate in the trial 
 
 
 
◎ 
 
 
 
 
Subject enrolment 
 
 
 
○ 
 
 
 
 
Randomization / assignment to groups 
 
 
 
○ 
 
 
 
 
Use of test toothpaste (4 weeks) 
 
 
 
○ 
 
 
 
 
Examination/measurement after 4weeks 
 
 
 
 
 
◎ 
 
 
Use of test toothpaste (another 4 weeks) 
 
 
 
 
 
 
 
 
Examination/measurement after 8 weeks 
 
 
 
 
 
 
 
◎ 
Record-keeping in diary (8 weeks) 
 
 
 
 
 
 
 
 

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19 
 
A4. Mason S, Kingston R, Shneyer L, Harding M. Clinical study to monitor dentinal 
hypersensitivity with episodic use of a desensitising toothpaste. British Dental 
Journal, Open volume 3, Article number:17011, p.3, 2017 
A5. Silverman G, Berman E, Hanna CB, Salvato A., Fratarcangelo P, Bartizek RD, 
Bollmer BW, Campbell SL, Lanzalaco AC, Mackay BJ, Mcclanahan SF, Perlich MA, 
Shaffer JB. Assessing the Efficacy of Three Dentifrices in the Treatment of Dentinal 
Hypersensitivity. Journal of the American Dental Association, 127:191-201, 1996 
A6. West NX, Addy M, Jackson RJ, Ridge DB: Dentine hypersensitivity and the placebo 
response. A comparison of the effect of strontium acetate, potassium nitrate and 
fluoride toothpastes. Journal of Clinical Periodontology, 24:209-215, 1997 
A7. Julious, SA. Tutorial in Biostatistics. Sample sizes for clinical trials with Normal 
data. Statistics in Medicine, 23:1921-1986, 2004, partially republished as Julious, 
SA, Owen, RJ. A comparison of methods for sample size estimation for non-
inferiority studies with binary outcomes. Statistical Methods in Medical Research, 
20(6):595-612. doi:10.1177/0962280210378945. Epub. Oct 1. Review, 2010 
A8. Chow, SC, Shao, J, Wang, H, and Lokhnygina, Y. Sample Size Calculations in 
Clinical Research, Third Edition. Taylor & Francis/CRC. Boca Raton, Florida, 2018