Clinical Protocol RH02217 (202423)

Intra-oral kinetics of fluoride containing dentifrices in modified oral fluoride retention study

GlaxoSmithKline Consumer Healthcare St George’s Avenue, Weybridge Surrey KT13 0DE United Kingdom

1. Protocol approval date: 25-Mar-2014 2. Final Amendment to Protocol approval date: 31-Mar-2014

Copyright: GlaxoSmithKline. This document contains confidentiality statements that are not relevant for this publicly available version Clinical Protocol RH02217

Copyright: GlaxoSmithKline. This document contains confidentiality statements that are not relevant for this publicly available version Confidential RH02217 Intra-oral kinetics of fluoride containing dentifrices in modified oral fluoride retention study GlaxoSmithKline Consumer Healthcare St George’s Avenue, Weybridge Surrey KT13 0DE United Kingdom

Protocol Amendments

(Record details of amendment in table below, mark the sections/pages affected “See Amendment” and attach amendment to this protocol) Number Date of Issue Section(s) & Page(s) Amended

4 Confidential Summary Information

Title: Intra-oral kinetics of fluoride containing dentifrices in a modified oral fluoride retention study Protocol Number: RH02217 Sponsor: GlaxoSmithKline Consumer Healthcare (GSKCH) St George’s Avenue, Weybridge, Surrey, KT13 0DE, United Kingdom (UK) Protocol Author (Clinical): PPD , MSc GSKCH, St George’s Avenue, Weybridge, Surrey, KT13 0DE, UK Telephone: PPD Protocol Author PPD , MSc (Biostatistical): GSKCH, Plot No. 67, Sector 32, Gurgaon, (Haryana), India – 122001 Telephone: PPD Protocol Author (Medical): PPD , DDS, MSc GSKCH, St George’s Avenue, Weybridge, Surrey, KT13 0DE, UK Telephone: PPD Protocol Author (New PPD , MSc, PhD Product Research) GSKCH, St George’s Avenue, Weybridge, Surrey, KT13 0DE, UK Telephone: PPD 453

PRIMARY CONTACT/ Study PPD , MSc Manager: GSKCH, St George’s Avenue, Weybridge, Surrey, KT13 0DE, UK Telephone: PPD Medical Lead: PPD , PhD, MRSc GSKCH, St George’s Avenue, Weybridge, Surrey, KT13 0DE, UK Telephone: PPD Expert dental advice outside Weybridge Telephone: PPD normal office hours: Principal Investigator: Dr David Payne, BDS

Study Site: Intertek Clinical Research Services (CRS) 6 Brindley Road, City Village Business Park Old Trafford, Manchester, M16 9HQ Tel: PPD

Product Name: Dentifrice containing 5% potassium nitrate (KNO3) and 1450parts per million (ppm) fluoride as sodium fluoride (NaF) (Sensodyne® Pronamel® Daily Protection)

Dentifrice containing 5% KNO3 and 1450ppm fluoride as NaF (Colgate® Sensitive Enamel Protect) IND/EUDRACT No: N/A Phase of study: Exploratory

® Sensodyne is a registered trademark of GlaxoSmithKline Consumer Healthcare, LP. ® Pronamel is a registered trademark of GlaxoSmithKline Consumer Healthcare, LP. ® Colgate is a registered trademark of Colgate-Palmolive. Protocol Agreement Intra-oral kinetics of fluoride containing dentifrices in a modified oral fluoride retention study RH02217

The signature of the investigator below constitutes his/her approval of this protocol and provides the necessary assurances that this study will be conducted according to Good Clinical Practice [ICH 1996] and to all stipulations, clinically and administratively, as stated in the protocol, including all statements as to confidentiality. It is agreed that the conduct and results of this study will be kept confidential and that the case report forms and other pertinent data will become the property of GlaxoSmithKline Consumer Healthcare (GSKCH). It is agreed that the protocol contains all necessary information required to conduct the study and that the study will not be initiated without the approval of an appropriate Independent Ethics Committee (IEC) It is agreed that all participants in this study will provide written informed consent in accordance with the requirements specified in the Declaration of Helsinki [World Medical Association Declaration of Helsinki, 59th General Assembly, Seoul 2008]. All participants will also be informed that their medical records will be kept confidential except for review by representatives of GSKCH and/or appropriate IEC representatives and regulatory authorities. In some instances, a summary of the protocol and study results, along with the names of the principal investigator from each study site, and details of the institutions with which the investigator is affiliated will be posted in one or more publicly accessible worldwide registers at any time after the commencement of the study. The signature of the investigator below constitutes his consent to have his name and institution disclosed should this study be made publicly available on a register. In addition, in order to avoid confusing or conflicting information, the signature of the investigator below constitutes his agreement not to post information about the study on any clinical trials registry without first obtaining the prior written consent of GSK.

Dr David Payne, BDS PPD PPD

Principal Investigator Signature Date Table of Contents Summary Information ...... 3 Protocol Agreement...... 5 Study Synopsis...... 9 List of Abbreviations...... 15 Study Schedule...... 16 1 Introduction...... 17 2 Exploratory Objectives...... 18 3 Investigational Plan...... 19 3.1 Study Design...... 19 3.2 Rationale for Study Design...... 22 4 Study Population...... 23 4.1 Source and Number of Subjects...... 23 4.2 Inclusion Criteria...... 23 4.3 Exclusion Criteria ...... 24 4.4 Subject Withdrawal Criteria...... 25 4.5 Subject Replacement ...... 26 4.6 Subject Restrictions ...... 26 4.6.1 Lifestyle...... 26 4.6.2 Medications and treatments...... 27 5 Study Products, Assignment, and Supply Management ...... 27 5.1 Study Products...... 27 5.1.1 Identity of study products...... 27 5.1.2 Selection of doses ...... 28 5.1.3 Administration ...... 28 5.1.4 Dose schedule ...... 29 5.1.5 Dose modification...... 29 5.1.6 Product compliance...... 29 5.1.7 Precautions...... 29 5.1.8 Overdosage ...... 29 5.2 Study Product Assignment ...... 30 5.2.1 Randomisation procedure...... 30 5.2.2 Blinding procedure and code breaks...... 30 5.3 Study Product Supplies Management...... 31 5.3.1 Packaging and labelling ...... 31 5.3.2 Accountability of study supplies ...... 31 5.3.3 Storage of study product supplies...... 32 6 Study Schedule ...... 32 7 Screening and Baseline Methods, Measurements and Evaluations and Study Conclusion ...... 32 7.1 Screening ...... 32 7.1.1 Informed consent ...... 32 7.1.2 Demographics ...... 33 7.1.3 Medical history ...... 33 7.1.4 Full Oral soft tissue examination...... 33 7.1.5 Salivary flow rates ...... 33 7.1.6 Study Conclusion...... 34 8 Outcome Measurements and Evaluations ...... 34 8.1 Saliva Sample Collection Procedure ...... 34 8.2 Sample Analysis ...... 35 9 Safety Measurements and Evaluations...... 35 9.1 Adverse Events and Serious Adverse Events ...... 35 9.1.1 Definitions ...... 35 9.1.2 Reporting adverse events and serious adverse events ...... 38 9.1.3 Adverse event grading and assessments ...... 41 9.2 Pregnancy...... 42 9.2.1 Time period for collecting pregnancy information...... 42 9.2.2 Action to be taken if pregnancy occurs...... 42 10 Data Analysis Methods ...... 43 10.1 Sample Size Determination ...... 43 10.2 General Considerations ...... 43 10.2.1 Analysis populations ...... 43 10.2.2 Criteria for evaluation ...... 44 10.2.3 Handling of dropouts and missing data...... 44 10.3 Statistical Methods and Analytical Plan...... 44 10.3.1 Demographic and baseline characteristics ...... 44 10.3.2 Outcomes...... 45 10.3.3 Safety...... 47 11 Ethical and Regulatory Aspects ...... 47 11.1 Local Regulations/Declaration of Helsinki ...... 47 11.2 Informed Consent ...... 47 11.3 Independent Ethics Committee (IEC) ...... 48 12 Monitoring of the Study...... 48 13 Study Documentation, eCRFs, and Record Keeping .. 49 13.1 Investigator’s Files/Retention of Documents...... 49 13.2 Source Documents/Data ...... 50 13.3 Case Report Forms (CRF)...... 50 13.4 Data Handling ...... 51 13.4.1 Data queries ...... 51 13.5 External Data ...... 52 14 Process for Amending the Protocol...... 52 15 Conditions for Terminating the Study...... 53 16 Confidentiality of Study Documents and Subject Records ...... 53 17 Publication of Data and Protection of Trade Secrets.. 53 18 Audits/Inspections...... 53 19 References...... 55 Study Synopsis

Title

Intra-oral kinetics of fluoride containing dentifrices in a modified oral fluoride retention study.

Brief Summary

This is a single centre, single blind (with respect to the laboratory analyst who will be blinded to treatment allocation), three treatment arm, crossover design study in healthy adult volunteers to explore the intra-oral kinetics of fluoride containing dentifrices following a dietary acid challenge in a modified oral fluoride retention study. The standard oral fluoride retention study design has been modified to explore the effect of a dietary acid challenge on fluoride and calcium concentrations in the oral cavity 60 minutes after brushing with a fluoride dentifrice.

In this exploratory study the concentration of fluoride and calcium in the oral cavity 60 minutes after brushing with a marketed fluoridated dentifrice (Sensodyne® Pronamel® Daily Protection Dentifrice (UK)) followed by a dietary acid challenge (orange juice, 60 minutes post brushing) will be compared to brushing with the same dentifrice followed by a non-acidic (deionised water, 60 minutes post brushing) challenge. In order to comment on the potential relevance of any differences observed in this study, a reference fluoride formulation has also been included (Colgate® Sensitive Enamel Protect Dentifrice (UK).

The study will be funded by GlaxoSmithKline Consumer Healthcare (GSKCH). It will take place in the UK managed by Intertek CRS (contract research organisation (CRO)). Subjects will be recruited primarily from the CRO’s volunteer database.

Exploratory Objectives

To evaluate, and compare between treatments, the concentration of fluoride and calcium ions in saliva following a rinse with either de-ionised water or orange juice 60 minutes after a single brushing with a fluoride dentifrice.

® Sensodyne is a registered trademark of GlaxoSmithKline Consumer Healthcare, LP. ® Pronamel is a registered trademark of GlaxoSmithKline Consumer Healthcare, LP. ® Colgate is a registered trademark of Colgate-Palmolive. To evaluate, and compare between treatments, the concentration of fluoride and calcium ions in saliva, 1, 5, 10, 15, 30, and 60 minutes after a single brushing with a fluoride dentifrice.

To evaluate, and compare between treatments, the concentration of fluoride and calcium ions in dentifrice and rinse expectorates immediately following application of the test dentifrice and following rinsing with either de-ionised water or orange juice 60 minutes after brushing application in vivo.

To explore saliva and rinse expectorate pH values 60 minutes after a single brushing application of the test dentifrice, and following rinsing with either de-ionised water or orange juice in vivo.

Study Design and Methodology

A single centre, randomised, controlled, single blind (with respect to the laboratory analyst who will be blinded to treatment allocation), three treatment arm, crossover study design in healthy subjects.

Eligible subjects will be supplied with a washout toothpaste (non-fluoride), and toothbrush to use twice a day in place of their normal oral hygiene products for the duration of the study (with the exception of washout period between Visits 3 and 4 during which subjects will be required to use their own toothbrush and toothpaste for the first 7 days; study site staff will remind subjects 7 days (±1 day) prior to Visit 4 to use the provided home use Washout toothpaste and toothbrush provided to them at screening).

Subjects will return to the site (Visit 2) following a washout period of 7 days (±1 day). Subjects will have abstained from brushing their teeth the morning of each treatment visit. Compliance with the dietary restrictions and washout toothpaste use will be checked. A full OST will be conducted and eligibility to continue will be assessed. Eligible subjects will be randomised and allocated to study treatment and rinse regimen according to the randomisation schedule. Each subject will use each study treatment once under supervision at the site (one at each Visits 2, 3, 4). At each treatment visit, following an OST examination, a pre- brushing (baseline) unstimulated saliva sample will be collected (the accepted time to sample this will be up to 3 minutes). This will be followed by supervised brushing with allocated test dentifrice for 1 timed minute followed by an immediate rinse with 10ml of de-ionised water for 5 timed seconds only.

The initial dentifrice expectorate (toothpaste slurry obtained immediately after the supervised brushing), and the immediate post-brushing rinse with de-ionised water will be collected. Unstimulated saliva samples will be collected at 1, 5, 10, 15, 30 and 60 minutes post supervised brushing. At the 60 minute post brushing time point post saliva sample collection, subjects will rinse with 10ml of either orange juice or de- ionised water (depending on the randomisation schedule) for 30 timed seconds. The orange juice or de-ionised water rinse expectorate will be collected. 1 minute post orange juice or de-ionised water rinse, a final unstimulated saliva sample will be collected.

Subjects will be required to remain at site until 60 minutes post brushing post rinsing saliva sample is collected. Sampling tolerance for the 1 minute time point will be + 20 seconds and for 5, 10, 15, 30, and 60 minute time points will be +1 minute. Saliva will be collected for up to 2 minutes at each collection time point post brushing (including the 1 minute time point) and will then be analysed for fluoride, calcium and pH (according to the RH02217 Analytical Protocol).

After collecting all saliva and expectorate samples, a full OST will be performed at the end of each treatment visit (Visit 2, 3 and 4).

Subjects will be reminded by the study staff to use the washout toothpaste and toothbrush provided for 7 days prior to Visits 2, 3, 4.

At the final test visit, subjects will return the washout product (used and unused) and then will be discharged from the study. Adverse events will be recorded throughout the study period.

For the laboratory analysis, samples will be placed in a labelled capped sterile tube and stored on ice until processed according to the analytical protocol. Collected samples will be analysed for fluoride and calcium ions and pH as detailed in the RH02217 analytical protocol. Details of sample storage, preparation and analysis will be provided in the RH02217 Analytical Protocol). Planned Number of Subjects

A sufficient number of subjects will be screened so that a maximum of 20 subjects will be randomised to ensure that at least 16 subjects complete the study.

Diagnosis and Main Criteria for Inclusion

Healthy subjects aged 18 to 65 years, with good general and oral health. Eligible subjects must have a minimum of 20 permanent natural teeth, a stimulated whole saliva flow rate ≥ 0.8 mL/min and an unstimulated whole saliva flow rate ≥ 0.5 mL/min.

Test Treatment, Dose and Mode of Administration

 Test Treatment: Sensodyne® Pronamel® Daily Protection Dentifrice (UK) and orange juice rinse 60 minutes after a single brushing application of the test dentifrice.

A single use of study treatment will take place at the study site under supervision. 1.5g (± 0.05g) of toothpaste will be weighed onto a dry brush by a member of the site staff. The subject will brush for 1 timed minute (without expectorating during brushing) and then expectorate dentifrice slurry into a labelled container. Subjects will subsequently rinse with an accurate measure of 10ml de-ionised water for 5 timed seconds and expectorate into another labelled container. 60 minutes post brushing and rinsing with de-ionised water, subjects will be asked to rinse with 10ml of orange juice for 30 timed seconds.

Reference Treatments, Dose and Mode of Administration

 Reference Treatment: Sensodyne Pronamel Daily Protection Dentifrice (UK) and de-ionised water rinse 60 minutes after a single brushing application of the test dentifrice.

® Sensodyne is a registered trademark of GlaxoSmithKline Consumer Healthcare, LP. ® Pronamel is a registered trademark of GlaxoSmithKline Consumer Healthcare, LP. Subjects will subsequently rinse with an accurate measure of 10ml de-ionised water for 5 timed seconds, and expectorate into another labelled container. At 60 minutes post brushing, and rinsing with de-ionised water, subjects will be asked to rinse with 10ml of de-ionised water for 30 timed seconds.

 Comparator Treatment: Colgate® Sensitive Enamel Protect Dentifrice (UK) and orange juice rinse 60 minutes after a single brushing application of the test dentifrice.

A single use of each of the reference treatments will take place at the study site under supervision. 1.5g (± 0.05g) of toothpaste will be weighed onto a dry brush by a member of the site staff. The subject will brush for 1 timed minute (without expectorating during brushing) and then expectorate dentifrice slurry into a labelled container. Subjects will subsequently rinse with an accurate measure of 10ml de- ionised water for 5 timed seconds and expectorate into another labelled container. At 60 minutes post brushing and rinsing with de-ionised water, subjects will be asked to rinse with 10ml of orange juice for 30 timed seconds.

Duration of Product use

At each of the three treatment visits, subjects will brush once for 1 timed minute with one of the treatments, and rinse with either orange juice or de-ionised water for 30 timed seconds, 60 minutes post supervised brushing as per the randomisation schedule.

Study Duration

Each individual’s expected participation in the study from screening to the final visit is expected to be approximately 4 weeks.

The total study duration from first subject visit to last subject visit will be approximately 6 weeks.

Criteria for Evaluation

Success Criterion The primary success criterion for this exploratory study is to observe greater fluoride and calcium ion concentrations in saliva following an orange juice rinse compared to saliva following a de-ionised water rinse, 60 minutes after a single brushing of the

® Colgate is a registered trademark of Colgate-Palmolive. Sensodyne product. However, observance of a statistically significant difference is not anticipated in this exploratory study.

Safety Safety will be assessed with respect to Adverse Events (AEs) and OST abnormalities (oral tolerability).

Statistical Methods

Fluoride and calcium ion concentrations in saliva at different time points (including post dietary acid challenge) will be analysed using analysis of covariance (ANCOVA). The model terms will include subject as a random effect, treatment, and period as fixed factors, and two baseline concentration covariates: (i) the subject-level baseline concentration calculated as the mean baseline concentration across all periods within a subject, and (ii) the period level baseline concentration minus the subject-level baseline concentration. If the baseline fluoride concentrations are negligible, then analysis of variance (ANOVA) will be performed instead for post- rinsing fluoride concentration analysis.

Concentrations of fluoride and calcium ions in expectorate, saliva pH values, and expectorate pH values will be analysed using ANOVA. The model terms will include subject as random effect, treatment and period as fixed factors.

For expectorate samples collected 60 minutes after rinsing with de-ionised water post brushing, following rinsing with either de-ionised water or orange juice, the analysis will be conducted in two ways:

1. The concentrations in the expectorate samples will be used in the analysis.

2. The estimated concentrations of fluoride and calcium ions in orange juice will be subtracted from the concentrations of fluoride and calcium in the post- rinsing expectorate respectively. These values will be used in the analysis. Estimates of fluoride and calcium ion concentrations in the orange juice will be determined as described in the RH02217 Analytical Protocol.

For all the analysis stated above, treatment differences and 95% confidence intervals will be presented. All tests will be 2-sided and at the 5% level of significance. No adjustments for multiplicity will be made. List of Abbreviations

AE Adverse Event ANCOVA Analysis of Covariance ANOVA Analysis of Variance CD Compact Disc CRF Case Report Form CRO Clinical Research Organisation EAP External Alliance Portal ECG Electrocardiogram EDC Electronic Data Capture GCP Good Clinical Practice GSKCH GlaxoSmithKline Consumer Healthcare HBS Human Biological Samples HTA Human Tissue Act ICH International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use IEC Independent Ethics Committee ITT Intention to Treat ml/min Millilitre/minute ºC Degree Centigrade OST Oral Soft Tissue PII Personally Identifiable Information PP Per Protocol ppm Parts Per Million SAE Serious Adverse Event SAS Statistical Analysis Software Study Schedule

VISIT 1 VISIT 2 VISIT 3 VISIT 4 Procedure Screening Informed consent X

Demographics, medical history X d Current/concomitant medications X X X X Full Oral Soft Tissue examination X X a X a X a Confirm salivary flow rates (unstimulated and stimulated) X Inclusion/exclusion X Subject eligibility X Dispense washout products and instruct subjects in oral hygiene, X dietary & lifestyle restrictions Confirmation of compliance with oral hygiene, dietary & lifestyle X X X restrictions. Randomisation X Supervised use of study treatment at study site X X X Washout Period of 7 days (± 1 day) Washout Period Washout Period of 7 days (± 1 day) Collect of saliva/expectorate samples b X X of 15Washout Period days (± 1 day) X Adverse events c X X X Return home use products X Study completion X a OST examinations will be performed twice; pre-supervised brushing and after the 60 minutes sampling time point following all other assessments. b Unstimulated pre-brushing saliva sample, post brushing expectorate (toothpaste slurry), post brushing de-ionised water rinse expectorate, post brushing unstimulated saliva time points at 1, 5, 10, 15, 30 and 60 minutes, orange juice or de-ionised water rinse expectorate at 60 mins post brushing saliva and saliva sample 1 minute post orange juice or de-ionised water rinse. C Adverse events can be any time after washout toothpaste dispensed. d Subject will be asked to use their own toothpaste and toothbrush for the first week of the washout period between Visit 3 and Visit 4, Study staff will remind subjects to use the Study Washout toothpaste for 7 days prior to Visit 4. 1 Introduction

Dental erosion may be defined as the loss of tooth structure, enamel and dentine, from chemical (acid) attack without the bacterial involvement [Layer, 2009; Chadwick, 2006]. Intra-orally, when the acid exposure is sufficient, tooth surface demineralisation may occur resulting in a softened tooth surface which is more prone to mechanical forces leading to tooth wear [Eisenburger, 2003].

Most commonly the acid will be of dietary origin, such as citric acid from fruit juices or carbonated drinks, phosphoric acid from cola drinks and acetic acid such as from vinaigrette.

Dental erosion may also be caused by repeated contact with hydrochloric acid produced in the stomach, which may enter the oral cavity through an involuntary response such as gastroesophageal reflux, or through an induced response as may be encountered in sufferers of bulimia [Lussi, 2004].

Treatment of dental erosion generally involves the use of fluoride which, when applied topically from toothpaste, can accelerate the natural enamel remineralisation process. There is substantial evidence that fluoride encourages remineralisation of enamel by adsorbing to the surface of enamel and reacting with calcium and phosphate ions found in saliva to form a mineral lower in solubility than the original enamel. The net result is that the tooth surface becomes harder and more resistant to subsequent acid attack [ten Cate 1999, Featherstone 1990].

In order to realise the full remineralisation benefits of fluoride it is generally agreed that the dentition needs to be exposed to slightly elevated concentrations of fluoride on a continuous basis. Saliva fluoride clearance studies have demonstrated that fluoride found within the oral cavity is primarily derived from fluoride-containing dentifrices and mouthrinses and that the fluoride concentration in saliva is biphasic in nature. In the first phase, the concentration of fluoride ions decreases rapidly after the initial post-brushing peak. The second phase clearance profile is much slower, with slightly elevated levels being detected several hours after toothbrushing. This clearance profile is generally explained as comprising an initial rapid clearance of loosely bound fluoride entrapped within the oral cavity, with a subsequent slower release of fluoride that is more strongly bound to the oral surfaces. The net result is that regular use of a fluoridated dentifrice leads to an overall increase in resting levels of fluoride in the saliva [Edgar, 1992; Duckworth1989/92]. Importantly, fluoride present in an acid labile form is considered likely to be beneficial in inhibiting the erosive potential of a dietary acid challenge. To date, the majority of short-term (~120 minutes) fluoride clearance studies have been conducted under “resting” conditions, whereby the influence of eating and drinking, particularly acidic foods has been excluded.

It is hypothesised that under dietary acid challenge conditions the concentration of fluoride and calcium in saliva will increase as a result of solubilisation of acid labile calcium fluoride reservoirs formed in the oral cavity.

The aim of this study is to explore the concentrations of dietary acid-labile fluoride and calcium ions in saliva following a dietary acid challenge 60 minutes after brushing with marketed fluoridated dentifrices. The effect of an acidic challenge (orange juice) on salivary fluoride and calcium ions at time point 60 minutes post brushing will be compared to a non-acidic challenge (de-ionised water).

This sponsored study by GlaxoSmithKline Consumer Healthcare (GSKCH) will take place in the UK and will be managed by Intertek Clinical Research Services (contract research organisation (CRO)). 2 Exploratory Objectives

To evaluate, and compare between treatments, the concentration of fluoride and calcium ions in saliva, 1, 5, 10, 15, 30, and 60 minutes after a single brushing with a fluoride dentifrice.

To explore saliva and rinse expectorate pH values 60 minutes after a single brushing application of the test dentifrice, and following rinsing with either de-ionised water or orange juice in vivo. 3 Investigational Plan

3.1 Study Design

A single centre, randomised, controlled, single blind (with respect to the laboratory analyst who will be blinded to treatment allocation), three treatment arm, and crossover study design in healthy subjects.

At the screening visit (Visit 1), subjects in good general and oral health, with at least 20 natural permanent teeth and 18-65 years of age who give written informed consent will receive a full oral soft tissue (OST) examination. Salivary flow rates (unstimulated and stimulated) will be measured and eligibility to participate in the study will be determined according to the inclusion/exclusion criteria. Eligible subjects will be supplied with a washout toothpaste (non-fluoride) and toothbrush to use twice a day in place of their normal oral hygiene products for the duration of the study (with the exception of washout period between Visits 3 and 4 during which subjects will be required to use their own toothbrush and toothpaste for the first 7 days; study site staff will remind subjects 7 days (±1 day) prior to Visit 4 to use the provided home use Washout toothpaste and toothbrush provided to them at screening).

Eligible subjects will be randomised and allocated to study treatment and rinse regimen according to the randomisation schedule. The study treatments are as follows:

 Test Treatment: Sensodyne® Pronamel® Daily Protection Dentifrice (UK) and orange juice rinse 60 minutes after a single brushing application of the test dentifrice.

 Reference Treatment: Sensodyne Pronamel Daily Protection Dentifrice (UK) and de-ionised water rinse 60 minutes after a single brushing application of the test dentifrice.

® Sensodyne is a registered trademark of GlaxoSmithKline Consumer Healthcare, LP. ® Pronamel is a registered trademark of GlaxoSmithKline Consumer Healthcare, LP.  Comparator Treatment: Colgate® Sensitive Enamel Protect Dentifrice (UK) and orange juice rinse 60 minutes after a single brushing application of the test dentifrice.

Each subject will use each study treatment once under supervision at the site (one at each Visits 2, 3, 4). At each treatment visit, following an OST examination, a pre- brushing unstimulated saliva sample will be collected (the accepted time to sample this will be up to 3 minutes). This will be followed by supervised brushing with allocated test dentifrice for 1 timed minute followed by an immediate rinse with 10ml of de-ionised water for 5 timed seconds only. The initial dentifrice expectorate (toothpaste slurry obtained immediately after the supervised brushing) and the immediate post-brushing rinse with de-ionised water will be collected.

Unstimulated saliva samples will be collected at 1, 5, 10, 15, 30 and 60 minutes post supervised brushing. At the 60 minute post brushing time point, post saliva sample collection, subjects will rinse with 10ml of either orange juice or de-ionised water (depending on the randomisation schedule) for 30 timed seconds. The orange juice or de-ionised water rinse expectorate will be collected. Approximately 1 minute post orange juice or de-ionised water rinse, a final unstimulated saliva sample will be collected.

Subjects will be required to remain at site until 60 minutes post brushing and a post rinsing saliva sample is collected. Sampling tolerance for the 1 minute time point will be + 20 seconds and for 5, 10, 15, 30, and 60 minute time points will be +1 minute. Saliva will be collected for up to 2 minutes at each collection time point post brushing (including the 1 minute time point) and will then be analysed for fluoride, calcium and pH (according to the RH02217 Analytical Protocol).

After collecting all saliva and expectorate samples, a full OST will be performed at the end of each treatment visit (Visit 2, 3 and 4).

Subjects will be reminded by the study staff to use the washout toothpaste and toothbrush provided for 7 days prior to Visits 2, 3, 4.

At the final test visit, subjects will return the washout product (used and unused), and then will be discharged from the study. Adverse events will be recorded throughout the study period.

® Colgate is a registered trademark of Colgate-Palmolive. For the laboratory analysis, samples will be placed in a labelled capped sterile tube and stored on ice until processed according to the analytical protocol. Collected samples will be analysed for fluoride and calcium ions and pH as detailed in the RH02217 analytical protocol. Analysis of the samples will be completed for each sample within 24hours of collection. Details of sample storage, preparation and analysis will be provided in the RH02217 Analytical Protocol).

Information covering the type of samples collected, the time and the analytical test required are summarised in the table below:

Sampling Time Type of samples collected Analyses Calcium + Pre-brushing Baseline Unstimulated saliva samples Fluoride Brushing with allocated treatment per randomisation schedule Calcium + Immediate post-brushing Expectorate (paste slurry) Fluoride 5 Seconds rinsing with 5ml de-ionised water post supervised brushing

Immediate post-rinsing with de- Expectorate of de-ionised Calcium + ionised water water rinse Fluoride 1 minute (+20 seconds) post- Calcium + rinsing and after previous Unstimulated saliva samples Fluoride expectorate sample Calcium + 5 mins (+1 min) post-rinsing Unstimulated saliva samples Fluoride Calcium + 10 mins (+1 min) post-rinsing Unstimulated saliva samples Fluoride Calcium + 15 mins (+1 min) post-rinsing Unstimulated saliva samples Fluoride Calcium + 30 mins (+1 min) post-rinsing Unstimulated saliva samples Fluoride 60 mins (+1 min) pre-rinsing Calcium + orange juice or de-ionised water Unstimulated saliva samples Fluoride + pH rinse 30 Seconds rinsing with 10ml de-ionised water or orange juice based on randomisation schedule Immediate post orange juice or Expectorate of orange juice Calcium + de-ionised water rinse or de-ionised water Fluoride + pH 1 minute post orange juice or Calcium + Unstimulated saliva samples de-ionised water rinse Fluoride + pH 3.2 Rationale for Study Design

The study design utilises a standard methodology to investigate the clearance of actives such as fluoride from the oral cavity after the use of dentifrices, [Gilbert 1987, Cummins, 1992].

The standard salivary retention model has been modified to explore the effect of rinsing with an acidic challenge 60 minutes post brushing on the concentration of fluoride and calcium in the oral cavity.Fluoride and calcium present in an acid labile form is considered likely to be beneficial in inhibiting the erosive potential of a dietary acid challenge. To date, the majority of short-term (~120 minutes) fluoride clearance studies have been conducted under “resting” conditions, whereby the influence of eating and drinking, particularly acidic foods has been excluded.

The buffering capacity of saliva is also an important determinant in inhibiting the erosive potential of f saliva. To investigate the response of saliva to a dietary acid challenge, this study will also measure the pH of saliva at a number of time points.

In order to comment on the potential relevance of any differences observed in this study, a marketed fluoride formulation has also been included (Colgate® Sensitive Enamel Protect Dentifrice (UK). The fluoride delivery by Sensodyne Pronamel Daily Protection dentifrice (Test Treatment) will be compared to a marketed dentifrice: Colgate Sensitive Enamel Protect (Comparator Treatment).

In order to assess the addition of an acidic challenge post 60minutes of brushing, a reference treatment, comprising of the same fluoride dentifrice: Sensodyne Pronamel however with de-ionised water rinse, has been included in this study.

Both Pronamel and Colgate sensitive have been clinically tested in an in situ demineralisation model. The respective formulations were observed to provide different levels of demineralisation prevention that was attributed to the ingredients in the formulation inhibiting fluoride retention in the oral cavity. This is a single centre, single blind (with respect to the laboratory analyst who will be blinded to treatment allocation), randomised, three treatment crossover study design in healthy subjects. Subjects will dose with each of the treatments under site supervision at each treatment visit as per the randomisation schedule.

® Colgate is a registered trademark of Colgate-Palmolive. There will be a washout period where subjects will use a placebo non-fluoride (0ppm) dentifrice between each treatment visit avoid any carry-over effect of the fluoride into treatment visit days and the analysis of samples.

Subjects will be asked to refrain from oral hygiene in the mornings of treatment days in order to avoid any interference between the ingredients contained in oral hygiene products and the test products.

Saliva and expectorate samples are classed as Human Biological Samples (HBS), as they carry DNA information. To protect subject confidentiality the samples will be coded. The code will not be directly traceable to the subject, and will be used only to track the samples until their destruction. 4 Study Population

4.1 Source and Number of Subjects

Subjects will be recruited by Intertek CRS (a Contract Research Organisation), primarily from their volunteer database. Subjects may also be recruited via advertisements placed in the local press, by use of leaflets and posters or by word of mouth. A sufficient number of subjects will be screened so that a maximum of 20 subjects will be randomised to ensure that at least 16 subjects complete the study.

4.2 Inclusion Criteria

1. Consent Demonstrates understanding of the study and willingness to participate as evidenced by voluntary written informed consent and has received a signed and dated copy of the informed consent form.

2. Age Aged 18-65 years.

3. Compliance Understands and is willing, able and likely to comply with all study procedures and restrictions.

4. General Health Good general and mental health with, in the opinion of the investigator or medically qualified designee: a) No clinically significant and relevant abnormalities of medical history or oral examination. b) Absence of any condition that would impact on the subject’s safety or wellbeing or affect the individual’s ability to understand and follow study procedures and requirements. 5. Oral Requirements a) A minimum of 20 permanent natural teeth b) A gum-base stimulated whole saliva flow rate ≥ 0.8 mL/min and an unstimulated whole saliva flow rate ≥ 0.5 mL/min.

4.3 Exclusion Criteria

1. Pregnancy Women who are known to be pregnant or who are intending to become pregnant over the duration of the study.

2. Breast-feeding Women who are breast–feeding.

3. Disease a) Presence of chronic debilitating disease. b) Any condition that causes xerostomia as determined by the Investigator. c) Diabetes 4. Dentition Exclusions a) Evidence of untreated caries. b) Gross periodontal disease. c) Tongue or lip piercing or presence of dental implants. d) Professional tooth cleaning or dental treatment during study. e) Oral surgery or extraction within 6 weeks of the screening visit. f) Self reported oral symptoms including lesions, sores or inflammation 5. Allergy/Intolerance Known or suspected intolerance or hypersensitivity to the study materials (or closely related compounds) or any of their stated ingredients. 6. Concomitant Medication a) Currently taking antibiotics or have taken antibiotics within 2 weeks of the screening visit. b) Any medication that could affect salivary flow or cause xerostomia as determined by the Investigator. c) Use of multivitamins, calcium supplements and or fluoride supplements within 7 days of treatment phase. 7. Smoking Subject unwilling to abstain from smoking for at least 4 hours on the day of each test visit.

8. Clinical Study/Experimental Medication a) Participation in another clinical study or receipt of an investigational drug within 30 days of the screening visit. b) Previous participation in this study. 9. Substance abuse Recent history (within the last year) of alcohol or other substance abuse.

10. Employment/Associations a) Employee of the sponsor or the study site or member of their immediate family. b) Subject (or a member of their household) is associated with development, manufacture or marketing of oral care products. 11. Any subject who, in the judgment of the investigator, should not participate in the study (for example, excessive oral hygiene regimen).

4.4 Subject Withdrawal Criteria

Subjects have the right to withdraw from the study at any time for any reason. The investigator also has the right to withdraw subjects from the study in the event of intercurrent illness, adverse events (AEs) or product failure after a prescribed procedure, protocol deviations, administrative reasons or other reasons. It is understood by all concerned that an excessive rate of withdrawals can render the study uninterpretable; therefore, unnecessary withdrawal of subjects should be avoided. Should a subject decide to withdraw, all efforts will be made to complete and report the observations as thoroughly as possible. Whatever the reason for withdrawal, a complete final evaluation at the time of the subject’s withdrawal should be made with an explanation of why the subject is withdrawing from the study.

If the reason for removal of a subject from the study is an AE or an abnormal laboratory test result, the principal specific event or test will also be recorded on the case report form (CRF). If a subject is withdrawn from the study because of a product-limiting AE, thorough efforts should be clearly made to document the outcome. Any AEs ongoing at the final visit will be followed up until resolved, the condition stabilises, is otherwise explained, or the subject is lost to follow-up.

If a subject withdraws or is withdrawn from the study all of the human biological samples (HBS) collected before they left may be analysed and reported unless the subject requests otherwise. A subject may request that their samples must be destroyed and no further research is conducted,

4.5 Subject Replacement

Subjects who withdraw from the study post-randomisation will not be replaced.

4.6 Subject Restrictions

4.6.1 Lifestyle For the entire duration of the study:

 Subjects should only use the dentifrice and toothbrush provided to them, and must abstain from use of all other oral hygiene products including mouthwashes. Subjects should abstain from interproximal cleaning (using dental floss, water picks and toothpicks), except for the removal of impacted food.

For each test visit:

 Subjects will be required to abstain from the consumption of tea for a period of 12 hours prior to each treatment visit. If a subject attends a visit having drunk tea then their treatment visit may be rescheduled.

 Subjects will be required to abstain from using any antacids from 11pm the night before a treatment visit.  Subjects will be required to abstain from eating or drinking from 11pm the night before a treatment visit.

 Subjects may drink a glass of water up to 1 hour prior to attending the study site (prescribed, permitted medications may be taken with a sip of water).

 Subjects will not be permitted to brush their teeth at home on the morning of a treatment visit.

 Smoking is prohibited within 2 hours of attending the study site for a test visit and for the duration of the treatment visit.

4.6.2 Medications and treatments The following restrictions apply for the duration of the study from screening to study completion.

 Subjects who enter the study will be requested to delay having any non- emergency dental treatment until after study completion (including prophylaxis).

 If concomitant medications and traditional herbal ingredients/treatments are used during the study, their identity, as well as their dosage and frequency, start and stop dates must be reported to the Investigator and recorded in the eCRF. Should a randomised subject embark on a course of treatment during the study which includes a prohibited medication, the identity of that medication/treatment, dosage and frequency and start date will be recorded. The subject will not be withdrawn. 5 Study Products, Assignment, and Supply Management

5.1 Study Products

5.1.1 Identity of study products The following study products will be supplied by the Clinical Supplies Department, GSKCH:

 Test Dentifrice: Sensodyne Pronamel Daily Protection (UK) (CCI ) containing 1450ppm fluoride as NaF and 5% potassium nitrate.

 Marketed Dentifrice: Colgate Sensitive Enamel Protect (UK) containing 1450ppm fluoride as NaF and 5% potassium nitrate Other items to be supplied by the Clinical Supplies Department, GSKCH:

 Washout Toothpaste: Placebo toothpaste, 0ppm fluoride, 5% potassium nitrate ()CCI

 Aquafresh® Clean Control (Everyday Clean) toothbrushes (for home use and on site supervised brushings)

 Countdown timers

 De-Ionised water - The de-ionised water will be free of fluoride and calcium minerals. This will be confirmed by the site as part of the pre-study validation process.

 Orange Juice: Tesco Smooth (from concentrate) - The baseline concentrations of fluoride and calcium in the orange juice will need to be collected as described in the RH02217 Analytical Protocol. If multiple batches are needed then a baseline level would be required for each new batch used. In any case, study site will provide the batch number of the orange juice used for each subject.

 Unflavoured Gum

 Graduated Dosing Cups

In addition saliva collection pots will be supplied by the study site.

5.1.2 Selection of doses The dose was selected to replicate the expected daily treatment regimen for individuals following normal oral hygiene habits and practices.

5.1.3 Administration A single use of each study treatment will take place at the study site under supervision. For the dentifrice treatments, 1.50g (± 0.05g) of toothpaste will be weighed onto a dry brush by a member of the site staff. The subject will brush for 1 timed minute without expectorating, and then expectorate excess dentifrice slurry into a labelled container. Subjects will subsequently rinse with an accurate measure of 10ml potable de-ionised water for 5 seconds and expectorate into a labelled container.

® Aquafresh is a registered trademark of GlaxoSmithKline Consumer Healthcare, LP. Subjects will be asked to swish for 30 timed seconds with 10ml of orange juice in the mouth, following the saliva sample collection at the 60 minute post-rinsing time point. The orange juice expectorate will be collected for fluoride and calcium ions and pH determination.

Subjects will also be supplied with a washout toothpaste and toothbrush to use twice a day in place of their normal oral hygiene products for the duration of the study.

5.1.4 Dose schedule At each test visit, subjects will brush only once with one of the test treatments at the study site under supervision. There will be a washout period of 7 days (± 1 day) following the screening visit and before randomisation at the first treatment visit. The washout toothpaste should be used for 7 days (± 1 day) post first treatment between treatments. Subjects will be required to use their own toothbrush and toothpaste between Visit 3 and Visit 4 for 7 days; study site staff will remind subjects 7 days prior to Visit 4 to use the provided home use Washout toothpaste and toothbrush provided to them at screening.

5.1.5 Dose modification No dose modification will be allowed.

5.1.6 Product compliance Subjects will perform all the test treatment brushings under the supervision of study site staff. Each test treatment will be weighed and recorded on a dispensing log; any discrepancies will be documented in the eCRF. Any violation of compliance will require evaluation by the investigator or designee to determine whether the subject should continue in the study.

5.1.7 Precautions No special precautions are necessary provided the study is carried out in accordance with this protocol. Study products will be labelled “For Clinical Trial Use Only”.

5.1.8 Overdosage An overdose is a deliberate or inadvertent administration of a treatment at a dose higher than specified in the protocol. 5.2 Study Product Assignment

5.2.1 Randomisation procedure A unique screening number will identify each subject screened for study participation. Screening numbers will be assigned in ascending numerical order as each subject signs their consent form. Subjects who meet all inclusion and exclusion criteria will be randomised according to the randomisation schedule. Randomisation numbers will be assigned in ascending numerical order as each subject is determined to be fully eligible.

The randomisation schedule will be provided by the Biostatistics Department, GSKCH.

5.2.2 Blinding procedure and code breaks The study statistician, data management staff and other employees of the Sponsor who may influence study outcomes are blinded to the product allocation.

The study site will receive two versions of the randomisation schedule, each in a sealed envelope and clearly marked as “For Dispensing” or “Emergency Use Only”. The dispensing schedule will be used by site for subject randomisation purposes and will not identify the treatments by name (treatments will be identified as A, B etc. corresponding to the labelled study supplies). The other randomisation schedule will only be removed from the sealed envelope in an emergency situation. This schedule however will be footnoted with a key identifying the content of treatments A, B etc. However, to maintain the blinding of the study as far as possible, all treatment allocations for all randomisation numbers on this randomisation schedule will be masked with scratch off panels. Only the panels required for the subject unblinding should be removed. The lab analyst will be blinded to the study treatments, but not the type of sample (saliva, expectorate, rinse).

The blind must 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. Wherever possible the Investigator (or designee) must contact the Sponsor prior to breaking the blind. The investigator must document the reason for breaking the code, and also sign and date the appropriate document. 5.3 Study Product Supplies Management

5.3.1 Packaging and labelling Packaging and labelling of all test products will be carried out according to ICH GCP guidelines and will be the responsibility of the Clinical Supplies Department, GSKCH.

The treatment dentifrices will be supplied in their commercial packaging with a study label affixed to each. The washout dentifrice will be supplied in plain white tubes with a study label affixed to each.

The orange juice and de-ionised water will be supplied in their commercial packaging with a study label affixed to each sample.

All other sundry items will be supplied in their commercial packaging.

Each product label will contain, but not be limited to, protocol number, product code letter (for the treatment products only), directions for storage, emergency contact telephone number and a statement “For Clinical Trial Use Only”.

Care should be taken with the supplied study products and their labels so that they are maintained in good condition. It is important that all labels remain intact and legible for the duration of the trial. Subjects should be instructed to not remove or deface any part of the study label.

5.3.2 Accountability of study supplies All material supplied is for use only in this clinical study and should not be used for any other purpose.

The investigator or designee will maintain a full record of study product accountability. An Investigational Product Dispensing Log must be kept current and will contain the following information:

 The identification of the subject to whom the study product was dispensed.

 The date(s) and quantity of the study product dispensed to the subject.

 The date(s) and quantity of the study product returned by the subject (if applicable). The inventory must be available for inspection by the study monitor during the study. At the end of the study, study product supplies will be verified by the monitor. Study product supplies will then be either collected by the study monitor or returned by the investigator or designee to the GSKCH Clinical Supplies Department or designated vendor.

5.3.3 Storage of study product supplies Study product supplies must be stored in compliance with the label requirements in a secure place with limited or controlled access. 6 Study Schedule

Please refer to the Study Schedule for a complete listing of all assessments to be performed. 7 Screening and Baseline Methods, Measurements and Evaluations and Study Conclusion

7.1 Screening

7.1.1 Informed consent The investigator, or designee, must obtain written (signed and dated by the subject) informed consent from each subject, participating in this study after adequate explanation of the aims, methods, objectives, and potential hazards of the study. The investigator, or designee, must also explain to the subjects that they are completely free to refuse to enter the study or to withdraw from it at any time. Appropriate forms for documenting a written consent will be provided by the investigator or by GSKCH. The investigator, or designee, should sign and date the consent form to confirm that the consent process was completed correctly. The subject will be provided with a copy of their signed and dated consent form and any other written information which they should be instructed to retain.

If, during a subject’s participation in the study, any new information becomes available that may affect the subject’s willingness to participate in the study, each ongoing subject should receive a copy of this new information and be re-consented into the study. Subjects should be provided with a copy of the signed and dated amended consent form. 7.1.2 Demographics The Investigator, or designee, will record each subject's year of birth, gender and race.

7.1.3 Medical history For each subject, the medical history will be taken and reviewed by the Investigator or medically qualified designee. Details of any relevant medical or surgical history, including allergies or drug sensitivity, will be recorded. Any concomitant therapy taken in the 30 days prior to the Screening Visit and throughout the study will also be recorded.

7.1.4 Full Oral soft tissue examination This procedure is conducted at Screening and at each treatment visit, before supervised brushing and following the final saliva sampling time point. It should be conducted by a single dental examiner. The examination will be accomplished by direct observation and palpation with retraction aids as appropriate. The examiner will include examination of the Labial Mucosa (including lips), Buccal Mucosa, Mucogingival folds, Gingival Mucosa, Hard Palate, Soft Palate, Tonsilar Area, Pharyngeal Area, Tongue, Sublingual Area, Submandibular Area and Salivary Glands. The results of the examination will be recorded in the eCRF as either normal or abnormal with details of any abnormalities. A brief description of any post- treatment abnormality observed by the examiner or reported by the subject at the application site will be recorded as an AE.

7.1.5 Salivary flow rates For the unstimulated collection, subjects will be asked to sit quietly with their head tilted down for 1 timed minute. After 1 minute, subjects will be instructed to swallow any pooled saliva. They will then sit quietly with their head tilted down for 2 timed minutes with the exception for the pre-brushing unstimulated saliva sample, where subjects will be permitted to have up to 3 minutes to collect the saliva sample, during which time they will keep any pooled saliva in their mouth and only empty it into a collection tube at the end of the time period. They may empty into the collection tube before the end of the collection time only if they are unable to hold the pooled saliva in their mouth.

For the stimulated saliva collection, subjects will chew on a piece of unflavoured gum for 1 timed minute. After 1 minute, subjects will be instructed to swallow any pooled saliva. They will then chew the unflavoured gum for a further 2 minutes, timed, during which time they will empty any pooled saliva into a collection tube. They may empty into the collection tube before the end of the collection time only if they are unable to hold the pooled saliva in their mouth.

The flow rate for each sample will be calculated as millilitres/minute (with the assumption that 1g of saliva approximates to 1ml). Samples will be weighed to establish the weight of saliva provided and the flow rate calculated.

7.1.6 Study Conclusion Subjects will be evaluated to determine if they completed all study procedures or if they were discontinued from the study early. If the subject discontinued at any point during the study, the primary reason for withdrawal should be recorded on the Study Conclusion eCRF by selecting one of the options below.

 Subject did not meet study criteria

 Adverse Event

 Lost to Follow Up

 Protocol Violation

 Withdrawal of Consent

 Other 8 Outcome Measurements and Evaluations

8.1 Saliva Sample Collection Procedure

At each test visit, a pre-brushing saliva sample will be collected followed by the supervised brushing with the test treatment. The initial expectorant (the slurry immediately after supervised brushing), the 5ml de-ionised water rinse expectorate and saliva samples at 1, 5, 10, 15, 30 and 60 minutes post-rinsing will then be collected.

For the saliva samples, the saliva will be collected for 2 minutes at each collection time. Subjects will be instructed at the start of each time point to sit without talking and with their heads bowed forward to start pooling saliva in their mouth only when the collection time point is reached and not to start pooling saliva in advance of a sampling time point. The sampling tolerance will be + 20 seconds for 1 minute time point and + 1 minute up to 60 minutes time point. The samples will be collected into labelled pots with a sample identification.

If a subject should produce insufficient saliva at any time point to accommodate the fluoride and calcium analyses, the fluoride analyses will take precedence.

8.2 Sample Analysis

Salivary fluoride and calcium ion concentrations and pH values in saliva will be analysed at Intertek CRS according to the analytical protocol. Samples will be placed in a labelled capped sterile tube and stored on ice until processed according to the analytical protocol. Analysis of the samples will commence within 3 hours of collection. Saliva and expectorated samples will be analysed for fluoride and calcium ion concentration and pH by trained technicians, the results will be recorded into an Excel spreadsheet which will be transferred to the Biostatistics Department, GSKCH for statistical analysis using External Alliance Portal (EAP).

The saliva samples will be disposed of once the laboratory analysis has been completed, (the Human Biological Samples (HBS) GSK protocol will be followed for all saliva sample management up to destruction to conform to Human Tissue Act (HTA) legislation). 9 Safety Measurements and Evaluations

9.1 Adverse Events and Serious Adverse Events

The investigator or site staff will be responsible for detecting, documenting, and reporting events that meet the definition of an AE or SAE.

9.1.1 Definitions 9.1.1.1 Adverse event Any untoward medical occurrence in a patient or clinical investigation subject, temporally associated with the use of an investigational product (or washout product) whether or not considered related to the investigational product (or washout product). An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use of an investigational product (or washout product). Events meeting the definition of an AE include:

 Exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency and/or intensity of the condition.

 New condition(s) detected or diagnosed after investigational product administration even though it may have been present prior to the start of the study.

 Signs, symptoms, or the clinical sequelae of a suspected interaction.

 Signs, symptoms, or the clinical sequelae of a suspected overdose of either investigational product or a concomitant medication (overdose per se should not be reported as an AE/SAE).

Events that do not meet the definition of an AE include:

 Medical or surgical procedure (e.g., endoscopy, appendectomy); the condition that leads to the procedure is an AE.

 Situations where an untoward medical occurrence did not occur (social and/or convenience admission to a hospital).

 Anticipated day-to-day fluctuations of pre-existing disease(s) or condition(s) present or detected at the start of the study that do not worsen.

 The disease/disorder being studied, or expected progression, signs, or symptoms of the disease/disorder being studied, unless more severe than expected for the subject’s condition.

Clinical AEs will be described by diagnosis and not by symptoms when possible (e.g., upper respiratory tract infection, seasonal allergy, etc. instead of runny nose).

Reporting instructions are provided in Section 9.1.2.

9.1.1.2 Serious adverse event An SAE is any untoward medical occurrence that, at any dose: a) Results in death. b) Is life-threatening. NOTE: The term 'life-threatening' in the definition of 'serious' refers to an event in which the subject was at risk of death at the time of the event. It does not refer to an event, which hypothetically might have caused death, if it were more severe. c) Requires hospitalisation or prolongation of existing hospitalisation.

NOTE: In general, hospitalisation signifies that the subject has been detained (usually involving at least an overnight stay) at the hospital or emergency ward for observation and/or treatment that would not have been appropriate in the physician’s office or out-patient setting. Complications that occur during hospitalisation are AEs. If a complication prolongs hospitalisation or fulfils any other serious criteria, the event is serious. When in doubt as to whether “hospitalisation” occurred or was necessary, the AE should be considered serious.

Hospitalisation for elective treatment of a pre-existing condition that did not worsen from baseline is not considered an AE. d) Results in disability/incapacity, or

NOTE: The term disability means a substantial disruption of a person’s ability to conduct normal life functions. This definition is not intended to include experiences of relatively minor medical significance such as uncomplicated headache, nausea, vomiting, diarrhoea, influenza, and accidental trauma (e.g., sprained ankle) which may interfere or prevent everyday life functions but do not constitute a substantial disruption. e) Is a congenital anomaly/birth defect. f) Medical or scientific judgment should be exercised in deciding whether reporting is appropriate in other situations, such as important medical events that may not be immediately life-threatening or result in death or hospitalisation but may jeopardise the subject or may require medical or surgical intervention to prevent one of the other outcomes listed in the above definition. These should also be considered serious. Examples of such events are invasive or malignant cancers, intensive treatment in an emergency room or at home for allergic bronchospasm, blood dyscrasias or convulsions that do not result in hospitalisation, or development of drug dependency or drug abuse or reports of spontaneous abortion. 9.1.2 Reporting adverse events and serious adverse events 9.1.2.1 Time period for reporting adverse events and serious adverse events The investigator or site staff will be responsible for detecting, documenting and reporting events that meet the definition of an AE or SAE.

AEs will be collected from the start of the investigational product (or washout product), and until 5days following last administration of the investigational product.

SAEs will be collected over the same time period as stated above for AEs. However, any SAEs assessed as related to study participation (e.g., investigational product, protocol mandated procedures, invasive tests, or change in existing therapy) or related to a GSK concomitant medication will be recorded from the time a subject consents to participate in the study up to and including any follow-up contact. All SAEs will be reported to GSKCH within 24 hours of the investigator or designee becoming aware of the situation.

Medical conditions reported prior to the time period for reporting AEs/SAEs should be recorded as part of the subject’s medical history.

9.1.2.2 Reporting and reviewing adverse events AEs will be recorded in the AE section of the eCRF.

Medical conditions recorded by the subject on a diary card or similar document that meet the definition of an AE must also be recorded in the AE section of the eCRF, if not previously well-characterized by the investigator in the subject’s medical history.

AEs elicited by the investigator in a standard manner at the study visits should also be recorded in the AE section of the eCRF. The investigator or designee must ask the subject the following question during each visit including any follow-up visits: “Have you felt unwell, experienced any symptoms or taken any medication (since your last visit) (today) (since your last dose) (since the last session)?”

The medically qualified investigator should review adverse events in a timely manner. This review should be documented in writing in the source document, or in the case report form.

9.1.2.3 Reporting serious adverse events A copy of the SAE form provided in the investigator study master file should be completed as fully as possible. It is essential to enter the following information:

 Protocol and subject identifiers

 Subject’s demography

 Description of events, with diagnosis if available

 Investigator opinion of relationship to study product (see Section 9.1.3.2 below)

 Criterion for seriousness.

The following are desirable and are of particular relevance for investigator and GSKCH assessment of the SAE report:

 Date of onset of AE

 Date AE stopped, if relevant

 Study product start date

 Study product end date if relevant

 Action taken on study product

 Outcome if known

The SAE form, completed as fully as possible, and SAE fax cover sheet must be faxed or e-mailed to the appropriate GSKCH Study Manager as soon as possible, but not later than 24 hours after study site personnel learn of the event. The GSKCH Study Manager should be notified of the situation by telephone or email.

Fax Serious Adverse Events to PPD (UK)

The GSKCH Study Manager will forward the SAE form to the Case Management Group Global Clinical Safety and Pharmacovigilance, the Medical Director responsible for the study and other GSKCH personnel as directed/appropriate.

The initial report will be followed up with more information as relevant, or as requested by the GSKCH study manager. This may require the investigator to obtain copies of hospital case reports, autopsy reports and other documents as applicable. 9.1.2.4 Follow-up of adverse events and serious adverse events After the initial report, the investigator is required to proactively follow up with each subject and provide further information on the subject’s condition. All AEs/SAEs will be followed until resolution, until the condition stabilizes, until the event is otherwise explained, or until the subject is lost to follow-up. The investigator may be required to obtain additional laboratory tests or investigations, and/or provide GSKCH with additional documentation, including autopsy reports.

Investigators are not obliged to actively seek AEs or SAEs in former subjects. However, if the investigator learns of any SAE, including the death, at any time after a subject has been discharged from the study, and considers the event reasonably related to the investigational product or study participation, the investigator will promptly notify GSKCH.

9.1.2.5 Regulatory reporting requirements for serious adverse events The investigator will promptly report all SAEs to GSKCH within the designated reporting timeframes (within 24 hours of learning of the event). GSKCH has a legal responsibility to notify, as appropriate, the local regulatory authority and other regulatory authorities about the safety of a product under clinical investigation. Prompt notification of SAEs by the investigator to GSKCH is essential so that legal obligations and ethical responsibilities towards the safety of subjects are met.

GSKCH will comply with country specific regulatory requirements relating to safety reporting to the regulatory authority, IEC and investigators.

Investigator safety reports are prepared according to GSKCH policy and are forwarded to investigators as necessary. An investigator safety report is prepared for a SAE(s) that is both attributable to investigational product and unexpected. The purpose of the report is to fulfil specific regulatory and GCP requirements, regarding the product under investigation.

An investigator who receives an investigator safety report describing a SAE(s) or other specific safety information (e.g., summary of listing of SAEs) from GSKCH will file it with the Investigator Brochure (or safety statement) and will notify IEC, if appropriate according to local requirements. 9.1.3 Adverse event grading and assessments 9.1.3.1 Intensity grading All AEs will be graded on a three-point scale and reported in detail as indicated on the eCRF:

 Mild – easily tolerated, causing minimal discomfort and not interfering with normal everyday activities

 Moderate – sufficiently discomforting to interfere with normal everyday activities

 Severe – any event that prevents normal everyday activities.

9.1.3.2 Relationship assessment The investigator is obligated to assess the relationship between investigational product and the occurrence of each AE/SAE. A "reasonable possibility" is meant to convey that there are facts/evidence or arguments to suggest a causal relationship, rather than a relationship cannot be ruled out. The investigator will use clinical judgement to determine the relationship. Alternative causes, such as natural history of the underlying diseases, concomitant therapy, other risk factors, and the temporal relationship of the event to the investigational product will be considered and investigated. The investigator will also consult the Investigator Brochure and/or Product Information, for marketed products, in the determination of his/her assessment.

There may be situations when an SAE has occurred and the investigator has minimal information to include in the initial report to GSK. However, it is very important that the investigator always make an assessment of causality for every event prior to the initial transmission of the SAE data to GSK. The investigator may change his/her opinion of causality in light of follow-up information, amending the SAE data collection tool accordingly. The causality assessment is one of the criteria used when determining regulatory reporting requirements.

The investigator will provide the assessment of causality as per the instructions for completion of the AE/SAE form. 9.2 Pregnancy

9.2.1 Time period for collecting pregnancy information Pregnancy information will be collected on all pregnancies reported following administration of any investigational product (or washout product) Information on pregnancy identified during the screening phase and prior to investigational product (or washout product) administration does not need to be collected.

9.2.2 Action to be taken if pregnancy occurs The investigator will collect pregnancy information on any subject who becomes pregnant while participating in the study after administration of the investigational product (or washout product). The investigator will record pregnancy information on the appropriate form and submit it to GSKCH within 2 weeks of learning of the subject becoming pregnant. The subject will be followed to determine the outcome of the pregnancy. Information on the status of the mother and infant / neonate (including concomitant medications taken by the mother during the pregnancy) will be forwarded to GSK. Generally, follow-up will be no longer than 6 to 8 weeks following the estimated delivery date. Any termination of the pregnancy will be reported.

While pregnancy itself is not considered to be an AE, any pregnancy complication or elective termination for medical reasons will be recorded as an AE or SAE as defined in Section 9.1.1.

A spontaneous abortion is always considered to be an SAE and will be reported as such. An SAE occurring in association with a pregnancy, brought to the investigator’s attention after the subject completed the study and considered by the investigator as possibly related to the investigational product, must be promptly forwarded to GSK. While the investigator is not obliged to actively seek this information in former study participants, he or she may learn of an SAE through spontaneous reporting.

There is no requirement for the subject to be withdrawn from the study as a result of the pregnancy. However if they are withdrawn, this should be recorded in the appropriate section of the eCRF. 10 Data Analysis Methods

10.1 Sample Size Determination

A sufficient number of subjects will be screened so that a maximum of 20 subjects will be randomised to ensure that at least 16 subjects complete the study. For studies with similar designs, this sample size is considered sufficient to assess the calcium and fluoride concentrations in saliva samples (CCI ).

10.2 General Considerations

10.2.1 Analysis populations 10.2.1.1 Definition of Analysis Populations The Safety population will include subjects who fulfil the study entry criteria and receive at least one dose of the study treatment.

Intent-To-Treat (ITT) population will include all subjects in Safety population who have at least one post-baseline outcome evaluation.

The Per Protocol (PP) population will be a subset of the ITT population. Subjects with a protocol violation that is deemed to affect outcome assessments in all study periods will be excluded from the PP population. Subjects with a protocol violation that is deemed to affect outcome assessments in some (but not all) study periods will be part of the PP population, but their data will be excluded from the period(s) in which the protocol violation occurred.

The primary analysis will be performed on PP population because it is deemed to provide better estimates for oral fluoride retention model.

10.2.1.2 Exclusion of data from analysis Any of the following may be considered as a major protocol violation which may warrant exclusion from the PP analysis:

1. Violation of inclusion or exclusion criteria that are deemed to affect outcomes.

2. Use of prohibited treatment or medication before or during the study, which is felt to affect the assessment of outcome variables.

3. Non-compliance with supervised brushing regimen.

4. Not receiving treatments in the order specified by the randomisation schedule. 5. Subjects having high fluoride concentrations at baseline.

This list may be expanded upon in the Statistical Analysis Plan (SAP).

Protocol violations which warrant exclusion from analysis will be identified between the Biostatistician and Medical Director or designee, prior to database lock and breaking the study blind.

10.2.2 Criteria for evaluation 10.2.2.1 Criteria for assessing success The primary success criterion for this exploratory study is to observe greater fluoride and calcium ion concentrations in saliva following an orange juice rinse compared to saliva following a de-ionised water rinse, 60 minutes after a single brushing of the Sensodyne product. However, observance of a statistically significant difference is not anticipated in this exploratory study.

10.2.2.2 Criteria for assessing safety Safety will be assessed with respect to AEs and OST abnormalities (oral tolerability).

10.2.3 Handling of dropouts and missing data Subjects with data from only one of the study periods will still be used in the analysis.

No data will be imputed in the case of dropouts or missing data.

10.3 Statistical Methods and Analytical Plan

More details of the proposed statistical analysis will be documented in the statistical analysis plan, which will be written following finalisation of the protocol and prior to study unblinding.

10.3.1 Demographic and baseline characteristics Descriptive statistics (percentages, means, and standard deviations) of demographic and baseline characteristics (fluoride and calcium concentrations in saliva) data will be tabulated. 10.3.2 Outcomes 10.3.2.1 Exploratory outcomes The exploratory outcome variables are:

1. Concentrations of fluoride and calcium ions in saliva after 60 minutes post brushing prior to administration of the orange juice or de-ionised water rinse at 60 minutes

2. Concentrations of fluoride and calcium ions in saliva following administration of the orange juice or de-ionised water rinse (60 minutes post-brushing)

3. Concentrations of fluoride and calcium ions in saliva at baseline, 1, 5, 10, 15, and 30 minutes post brushing prior to administration of orange juice or de- ionised water rinse

4. Concentrations of fluoride and calcium ions in the initial expectorate, de- ionised water rinse post-brushing expectorate, and 60 minutes post-brushing following administration of orange juice or de-ionised water rinse

5. Saliva pH values at 60 minutes post-brushing pre orange juice or water rinse and 60 minutes post-brushing post-rinsing with orange juice or de-ionised water

6. Expectorate pH values at 60 minutes post-brushing following administration of orange juice or de-ionised water rinse

7. Change from baseline in fluoride and calcium ion concentrations in saliva samples at 60 minutes post-brushing following administration of orange juice or de-ionised water rinse.

Baseline is defined as the concentration of fluoride or calcium ions in the saliva sample obtained immediately before supervised brushing.

For all post-baseline outcome variables for saliva prior to administration of orange juice or de-ionised water rinse (pre-brushing baseline, 1, 5, 10, 15, 30, and 60 minutes), and expectorate (initial expectorate and immediate de-ionised water rinse expectorate) samples, the following treatment comparisons will be performed:

 Sensodyne Pronamel dentifrice vs. Colgate Sensitive dentifrice. The data from two Sensodyne Pronamel dentifrice arms (Sensodyne Pronamel dentifrice with de-ionised rinse and Sensodyne Pronamel dentifrice with orange rinse) will be combined for the purpose of this comparison.

For all post-baseline outcome variables following administration of orange juice or de-ionised water rinse at 60 minutes for saliva and expectorate samples, the following treatment comparisons will be performed:

 Sensodyne Pronamel dentifrice and orange juice rinse vs. Sensodyne Pronamel dentifrice and de-ionised water rinse

 Colgate Sensitive dentifrice and orange juice rinse vs. Sensodyne Pronamel dentifrice and de-ionised water rinse

 Sensodyne Pronamel dentifrice and orange juice rinse vs. Colgate Sensitive dentifrice and orange juice rinse.

Fluoride and calcium ion concentrations in saliva at different time points will be analysed using analysis of covariance (ANCOVA). The model terms will include subject as a random effect, treatment, and period as fixed factors, and two baseline concentration covariates: (i) the subject-level baseline concentration calculated as the mean baseline concentration across all periods within a subject, and (ii) the period level baseline concentration minus the subject-level baseline concentration. If the baseline fluoride concentrations are negligible, then ANOVA will be performed instead for post-brushing fluoride concentration analysis.

For expectorate samples collected 60 minutes after the initial rinsing, following rinsing with de-ionised water or orange juice, the analysis will be conducted in two ways:

1. The concentrations in the expectorate samples will be used in the analysis

To further assess the affect of the acid challenge, changes from pre- to post- rinsing at the 60 minute time point in fluoride and calcium ion concentrations in saliva samples and saliva pH values will be summarised by treatment group (N, mean, standard deviation, standard error, median, minimum, and maximum).

For all the analysis stated above, treatment differences and 95% confidence intervals will be presented.

All tests will be 2-sided and at the 5% level of significance.

The assumption of normality and homogeneity of variance will be investigated. Violation of these assumptions may be overcome using suitable transformation or performing a non-parametric test (e.g., Wilcoxon Signed Rank test, Kruskal-Wallis nonparametric ANOVA, Dunn’s post test).

Mean (± standard error) plots by treatment over time will be presented.

10.3.3 Safety AEs and OST abnormalities will be listed by treatment. AEs that occur after the first supervised brushing with experimental product will be termed as treatment-emergent AEs and will be tabulated. AEs occurring between screening and randomisation will be termed as pre-treatment AEs and will be included in the AE listing. Any AEs occurring during the washout period between treatments will be assigned to the last experimental treatment administered. 11 Ethical and Regulatory Aspects

11.1 Local Regulations/Declaration of Helsinki

The Principal Investigator will ensure that this study is conducted in full conformance with the laws and regulations of the country in which the research is conducted and the Declaration of Helsinki.

11.2 Informed Consent

It is the responsibility of the investigator, or designee, to obtain written (signed and dated by the subject) informed consent from each individual participating in this study or his/her legally acceptable representative and/or impartial witness. If the legally acceptable representative or impartial witness consent process will be used, the GSKCH representative and the investigator must agree on the process for obtaining and documenting legally acceptable representative or impartial witness, and ensure that the process is compliant with the local regulatory requirements, prior to the start of the study. Major/substantial amendments to the protocol that affect the scope of the study at the subject level and/or updates to the safety profile of the investigational product (Investigator Brochure) should be reflected in the consent form and active subjects re-consented.

11.3 Independent Ethics Committee (IEC)

It is the understanding of GSKCH that this protocol (and any modifications) as well as appropriate consent procedures, will be reviewed and approved by an IEC. This body must operate in accordance with the current local requirements. A letter or certificate of approval will be sent by the investigator to the sponsor prior to initiation of the study, and also when subsequent modifications to the protocol are made. 12 Monitoring of the Study

In accordance with applicable regulations, GCP, and GSKCH procedures, monitors will contact the site prior to the start of the study to review with the site staff the protocol, study requirements, and their responsibilities to satisfy regulatory, ethical, and GSKCH requirements. When reviewing data collection procedures, the discussion will include identification, agreement and documentation of data items for which the eCRF will serve as the source document.

GSKCH will monitor the study to ensure that the:

 data is authentic, accurate, and complete

 safety and rights of subjects are being protected

 study is conducted in accordance with the currently approved protocol and any other study agreements, GCP, and all applicable regulatory requirements.

The extent and nature of monitoring will be described in a written monitoring plan on file at GSKCH. The monitor will inspect the eCRFs, laboratory test reports and other subject records at regular intervals throughout the study to verify the adherence to the protocol and the completeness, consistency, and accuracy of the data being entered. The monitor will verify that each subject has consented in writing to direct access to study records as well as to the study procedures. The monitor will also ensure that all required regulatory documents are present and up to date and that accountability and reconciliation of study product are performed according to GSKCH procedures. The investigator (or designee) agrees to co-operate with the monitor to ensure that any problems detected in the course of these monitoring visits are resolved. 13 Study Documentation, eCRFs, and Record Keeping

Each subject will be assigned and identified by a unique Screening Number as set out in Section 5.2.1 – ‘Randomisation’. Any reference made to an individual subject within the study must be done using the unique Screening Number.

13.1 Investigator’s Files/Retention of Documents

The investigator must maintain adequate and accurate records to enable the conduct of the study to be fully documented and the study data to be subsequently verified. These documents should be classified into two separate categories: (1) investigator’s study master file, and (2) study/subject clinical source documents.

The investigator’s study master file will contain the protocol/amendments, case report and query forms, IEC and governmental approval with correspondence, informed consent, study product records, staff curriculum vitae, authorisation forms, subject identification, screening and enrolment information and other appropriate documents/correspondence, etc.

Clinical source documents (defined in advance to record key outcome/safety variables independent of the eCRFs) would include patient/subject hospital/clinic records, physician’s and nurse’s notes, appointment book, original laboratory reports, ECG, X-ray, pathology and special assessment reports, consultant letters, screening and enrolment logs, etc. These two categories of documents must be kept on file by the investigator according to local regulations or as specified by the supervisory regulatory agency (GSKCH recommends that documents be kept for 10 years). The investigator is also required to keep subject identification codes on file for at least 15 years after completion or discontinuation of the study.

No study document should be destroyed without a prior written agreement between GSKCH and the investigator. Should the investigator wish to assign the study records to another party or move them to another location, GSKCH must be notified in advance. If the investigator cannot guarantee this archiving requirement at the investigational site for any or all of the documents, special arrangements must be made between the investigator and GSKCH to store these in a sealed container(s) outside of the site so that they can be returned sealed to the investigator in case of a regulatory inspection. Where source documents are required for the continued care of the subject, appropriate copies should be made for storing outside of the site.

The investigator shall supply the sponsor, on request, with any required background data from the study documents or clinic records. In case of special problems and/or governmental queries, it is also necessary to have access to the complete study records provided that subject confidentiality is protected.

13.2 Source Documents/Data

The source documents (e.g. hospital records, clinical and office charts, laboratory notes, memoranda, subjects’ diaries or evaluation checklists, pharmacy dispensing records, recorded data from automated instruments, copies or transcriptions certified after verification as being accurate copies, microfiches, photographic negatives, microfilm or magnetic media, x-rays, subject files and records kept at the pharmacy, at the laboratory and at the medico-technical departments involved in the clinical study) which contain the source of data recorded in the eCRF should be specified in the Source Document Designation Form. In some cases the eCRF can be used as a source document.

13.3 Case Report Forms (CRF)

A CRF is a printed, optical, or electronic document designed to record all of the protocol required information to be reported to the sponsor on each trial subject.

For each subject who has given informed consent/assent and has been screened, a CRF must be completed and signed by the Principal Investigator (or authorised designee) to certify that the data are complete and correct.

Management of clinical data will be performed in accordance with applicable GSKCH standards and data cleaning procedures to ensure the integrity of the data e.g. removing errors and inconsistencies in the data.

In order to protect the privacy of subjects, no Personally Identifiable Information (PII) (including the subject's name or initials or birth date) is to be recorded in the CRF or as part of the query text. AEs and concomitant medications terms (if applicable) will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) and an internal validated medication dictionary, GSKCH Drug.

Subject data will be entered into GSKCH defined CRFs and transmitted electronically to GSKCH in a validated (21 CFR Part 11 compliant) web-based electronic data capture system (InFormTM).

All eCRF pages should be completed during a subject assessment when the eCRF has been designated as the source. Data that is sourced elsewhere should be entered into the eCRF in an agreed upon timeframe between the Investigator and Sponsor.

The eCRFs (including queries, query responses and audit trails) will be retained by GSKCH.Site data archived compact discs (CD(s)) prepared by a third party will be sent to the investigator to maintain as the investigator copy following the decommissioning of the study.

13.4 Data Handling

Documentation of all data management activities should allow step-by-step retrospective assessment of data quality and study performance. This includes maintenance of documentation to support any changes or corrections applied to the eCRFs.

13.4.1 Data queries The study monitor at the study site will review the eCRFs in accordance with the monitoring plan, and any queries will be generated to the Investigator or designee enabling the errors to be addressed prior to Data Management review.

Queries arising during consistency checking will be provided to the site for clarification and approval by the Investigator or designee. Data Management will run reports and listings on the eCRFs, in addition to the queries already programmed and generated by the system, and raise queries if needed for site clarification or correction. The Clinical Dictionary Development and Management Group will raise queries to the site if needed to clarify medical terms to be coded.

An audit trail will serve to provide a complete record of the changes and corrections endorsed by the Investigator. 13.5 External Data

External Data are subject data obtained external to the eCRF. These data are transcribed into a format agreed upon by GSKCH to identify the subject and time point referenced in the eCRF and/or protocol. An agreed upon quality control process is performed against the transcribed data to the source to ensure the accuracy of the transcription, since data from the external source will be used for analysis and reporting.

These data will be transmitted in an agreed upon format to GSKCH via secured web portal or CD/DVD via mail carrier with tracking capabilities. 14 Process for Amending the Protocol

Protocol modifications to ongoing studies which could potentially adversely affect the safety of subjects or which alter the scope of the investigation, the scientific quality of the study, the experimental design, dosages, duration of therapy, assessment variables, the number of subjects treated, or subject selection criteria are considered major/substantial amendments and must be made only after appropriate consultation between an appropriate representative of GSKCH and the investigator.

Protocol modifications must be prepared by a representative of GSKCH. All changes must be justified in the Reason for Amendment section of the protocol amendment. Approval of amendments will be made by the original signatories to the protocol agreement, or their appropriate designees.

All major/substantial protocol modifications must be reviewed and approved by the appropriate IEC in accordance with local requirements, before the revised edition can be implemented.

Modifications which eliminate an apparent immediate hazard to subjects do not require pre-approval by the IEC but the IEC will be notified of these amendments. Non-substantial amendments do not require submission to the IEC and will be held on file by the sponsor and investigator.

Amendments to protocols should be recorded on page 2 and attached to all copies of the protocol. 15 Conditions for Terminating the Study

Both GSKCH and the investigator reserve the right to terminate the study at any time. Should this be necessary, the procedures will be arranged on an individual study basis after review and consultation by both parties. In terminating the study, GSKCH and the investigator will assure that adequate consideration is given to the protection of the subject’s interests.

If the study is terminated prematurely or suspended, the IEC will be informed and provided with the reason(s) for termination or suspension by the sponsor or by the investigator/institution, as specified by the applicable regulatory requirement. 16 Confidentiality of Study Documents and Subject Records

The investigator must assure that the subject’s anonymity will be maintained. On CRFs or other documents submitted to GSKCH, subjects should not be identified by their names or initials, but by an identification code.

The investigator should keep a separate log of subjects’ codes, names and addresses. Documents not for submission to GSKCH, e.g. subjects’ written consent forms, should be maintained by the investigator in strict confidence. 17 Publication of Data and Protection of Trade Secrets

The investigator will not seek to arrange for publication of any of the information or results from the study in any scientific journal or other publication or by way of lecture without GSKCH’s prior written consent.

Any formal publication of the study in which input of GSKCH personnel exceeded that of conventional monitoring will be considered as a joint publication by the investigator and the appropriate GSKCH personnel. Authorship will be determined by mutual agreement. 18 Audits/Inspections

The investigator and study subjects should understand that source documents for this study should be made available to appropriately qualified personnel or designee(s) from the GSKCH Regulatory Compliance Group or to regulatory authority inspectors. The verification of the CRF data may be by direct inspection of source documents (where permitted by law) or through an interview exchange. The Regulatory Compliance Group or designee assesses the quality of data with regard to accuracy, adequacy and consistency. In addition, Regulatory Compliance assures that the sponsor’s studies are in accordance with Good Clinical Practices and that relevant regulations/guidelines are being followed.

An inspection is the act by a regulatory authority of conducting an official review of documents, facilities, records and any other resources that are deemed by the authority to be related to the clinical trial and that may be located at the site of the trial, at the sponsor’s and/or contract research organisation’s (CRO’s) facilities, or at other establishments deemed appropriate by the regulatory authority (ICH GCP 1.29).

The focus of an inspection is compliance with Good Clinical Practice legislation, associated guidance documents, and company policies to ensure that data are accurate, complete and valid and to ensure subjects’ welfare and safety are protected.

The sponsor will be available to help investigators prepare for an inspection. 19 References

Chadwick R, Graham R. Dental Erosion in ‘Quintessentials of Dental Practice’. Quintessence, London, p 4, 34, 2006.

D. Cummins and J.E. Creeth. Delivery of Antiplaque Agents from Dentifrices, Gels, and Mouthwashes. J Dent Res1992 71: 1439

Duckworth, RM, Morgan, SN, and Burchell, CK. Fluoride in plaque following use of dentifrices containing sodium monofluorophosphate. J. Dent. Res. 1989: 68;130-133.

Duckworth, RM, Morgan, SN, Ingram, GS, and Page, DJ, Oral fluoride reservoirs and their relationship to anticaries efficacy. In: Clinical and biological aspects of dentifrices. 1992, Pp. 91-104. Ed. G. Embery and G. Rolla, Pub. Oxford University Press, Oxford.

Edgar, WM, Ingram, GS, and Morgan, SN. Fluoride in saliva and plaque in relation to fluoride in drinking water and in dentifrice. In: Clinical and biological aspects of dentifrices. 1992 Pp. 91-104. Ed. G. Embery and G. Rolla, Pub. Oxford University Press, Oxford.

Eisenburger M.,Shellis R.P., Addy M. Comparative Study of Wear of Enamel Induced by Alternating and Simultaneous Combinations of Abrasion and Erosion in vitro. Caries Res; 37:450–455, 2003

Featherstone JDB, Glena R, Shariati M, Shields CP. Dependence of in vitro demineralization and remineralization of dental enamel on fluoride concentration. J Dent Res (1990) 69: 620–5.

Fowler C, Willson R, Rees GD. In vitro microhardness studies on a new anti- erosion desensitizing toothpaste. J Clin Dent. (2006) 17 (4): 100-105.

Gilbert R, Williams PEO. The oral retention and antiplaque efficacy of triclosan in human volunteers. Br. J. Clin. Pharmac. 1987; 23, 579-583.

CCI ICH Topic 6 Guideline for Good Clinical Practice CPMP/ICH/135/95 17th July 1996.

Layer T M. Formulation considerations for developing toothpastes suitable for those at risk from erosive tooth wear. J. Clin. Dent. 20 (Spec Iss): 2009: 199-202.

Lussi A, Jaeggi T, Zero D. The role of diet in the aetiology of dental erosion. Caries Res 38 (suppl 1): 34-44, 2004. ten Cate JM. Current concepts on the theories of the mechanism of action of fluoride. Acta Ondontol Scand (1999) 57, 325-329.

World Medical Association Declaration of Helsinki, 59th General Assembly, Seoul 2008.

Zero DT (1995). In situ caries models. Adv Dent Res, 9, 214-230. Consumer Healthcare Protocol No. RH02217 - Amendment No. 01

Study Title: Intra-oral kinetics of fluoride containing dentifrices in modified oral fluoride retention study

Type of Amendment: Administrative/non- Major/substantial substantial Effect on Subject Safety/Scope of the Investigations/Scientific Quality of Study None Will the Informed Consent Form Require Amendment? Yes - The ICF has been amended in order to clarify wording regarding the subject being on washout product only for 7 days (± 1 day) before visit 2and to provide additional clarification regarding the washout period between visits 3 and 4.

Will the Case Report Form Require Amendment? Yes – The eCRF content does not require updating, however the data checks related to visit 2 window has been updated to reflect clarification provided in the protocol amendment.

Will the Safety Statement Require Amendment? Yes – It has been updated to clarify the wording regarding the period of washout. Reason for Amendment A protocol amendment is required in order to clarify the visit window leading up to visit2. It was expected that prior to the protocol starting that 5 subjects would be screened for each eligible subject. It has been found on starting the screening process that around 1 in 3 have been eligible. As the site has to schedule only 4 subjects per day starting visit 2, this has meant that the 7 ± 1 window for visit 2 was not sufficient and needed to be expanded in order to allow site to schedule 4 subjects per day only The amendment is to clarify that washout product will be used for 7 days prior to next visit and that the washout period can be 6 days at minimum.

The amendment also contains clarifications to exiting text and corrections to typographical errors.

Summary of changes Updates to study table to clarify when exclusion and eligibility to be done. Changing typographical error of study sampling 5ml to 10ml. clarify that washout product will be used for 7 days prior to next visit and that the washout period can be 6 days at minimum.

Original Text (section number and page) Section Study Schedule, page 16:

Study Schedule

Eligibility not ticked for visit 2 only visit 1. (typographical error) Amended Text (section number and page) Section Study Schedule, page 16:

Eligibility to be ticked for visit 2 as well as visit 1. Original Text (section number and page) Section Investigation Plan – Study Design, page19:

Subjects will return to the site (Visit 2) following a washout period of 7 days (±1 day). Amended Text (section number and page) Section Investigation Plan – Study Design, page19:

Subjects will return to the site (Visit 2) following a washout period 7 days, with a minimum of 6 days wash out.

Subjects will return to the site (Visit 2) after a minimum of 6 days wash out. Subjects will be contacted to be informed when to start using the washout product. The washout product should be used ideally for 7 days (± 1 day) prior to next visit.

Original Text (section number and page) Section Investigation Plan – Study Design , page 21: Text in study sampling table – 5 seconds rinsing with 5ml de-ionised water post supervised brushing.

Amended Text (section number and page)

Section Investigation Plan – Study Design , page : 21 Text from study sampling table – 5 seconds rinsing with 10ml de-ionised water post supervised brushing.

Original Text (section number and page) Section Study Products, Assignment, and Supply Management – Dose Schedule, page29: There will be a washout period of 7 days (± 1 day) following the screening visit and before randomisation at the first treatment visit. Amended Text (section number and page) Section Study Products, Assignment, and Supply Management – Dose Schedule, page 29:

There will be a washout period of 7 days (+/- 1 day) (6 days minimum), following the screening visit and before randomisation at the first treatment visit. During that washout period, subjects will be asked to use a non fluoride toothpaste for 7 days (± 1 day) prior to next visit. Subjects will be contacted to be informed when to start using the washout product.

Original Text (section number and page)

Section Outcome measurements and evaluations- Saliva sample collection procedure , page 34: The initial expectorant (the slurry immediately after supervised brushing), the 5ml de-ionised water rinse expectorate and saliva samples at 1, 5, 10, 15, 30 and 60 minutes post-rinsing will then be collected. Amended Text (section number and page)

Section Outcome measurements and evaluations- Saliva sample collection procedure , page 34:

The initial expectorant (the slurry immediately after supervised brushing), the 10ml de-ionised water rinse expectorate and saliva samples at 1, 5, 10, 15, 30 and 60 minutes post-rinsing will then be collected.

Investigator’s signature: Date: PPD PPD