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ITR CANADA

CONFIDENTIAL

FINAL REPORT

InSea2® : Bacterial Reverse Mutation Test

ITR Study Number: 55482

Test Facility: ITR Laboratories Canada Inc 19601 Clark Graham Blvd Baie d’Urfe, Quebec Canada H9X 3T1

Sponsor: InnoVactiv, Inc.

265 2nd Street East Rimouski (QC) Canada, G5L 9H3

Issue Date: June 19, 2017

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ITR Study No. 55482 2

COMPLIANCE STATEMENT

The study was performed in compliance with the OECD Principles on Good Laboratory Practice (Issued Jan 1998) ENV /MC/CHEM(98) 17 and United States Food and Drug Administration Title 21 Code of Federal Regulations Part 58, Good Laboratory Practice for Non-clinical studies issued 22 December 1978 Federal Register plus subsequent amendments with the following exception:

• The test item formulations were not analysed for achieved concentrations, stability and homogeneity.

• The Test Item characterisation and stability were not performed to GLP guidelines.

This study was initiated on February 3, 2017 and completed on June 19,2017.

Study Director: MélaJie G~oussoub, BSc Study Director, Genetic Toxicology ITR Laboratories Canada Inc.

Reviewer: Ars Scientist, enetic Toxicology ITR Laboratories Canada Inc.

Date

ITR Management Representative:

Final Report June 19,2017 20f35


Final Report June 19, 2017

PERSONNEL

The following ITR personnel were responsible for the conduct of the study.

Pharmacy: Rogy Markose, Manager

Genetic Toxicology Laboratory Services: Alicja Golos, Manager

Report Production: Julieta Focsa, Head Laboratory Systems

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QUALITY ASSURANCE STATEMENT

In compliance with the Good Laboratory Practice regulations, the phases of study 55482 performed at ITR Laboratories Canada Inc, have been inspected by the ITR Quality Assurance Unit as indicated below.

The inspection of the final report confirms that the methods, procedures and observations are accurately and completely described and that the reported results accurately and completely reflect the raw data generated during the study.

Date Reported to the Date of Inspection Phase Inspected Study Director and

Management Studl: Plan and Amendments

January 30, 2017 Study plan February 03, 2017 February 02, 2017

In-Life Inspections

February 7, 2017 Dosing February 08, 2017

February 10,2017 Plate reading February 10,2017

Data and Report

March 30, 31, 2017 Data and report April 05, 2017 April 04, 05, 2017

April 07,2017 Report April 07, 2017

May 23, 2017 Report May 24, 2017

June 15,2017 Report June 15,2017

In addition to the study-based inspections above, inspections of facilities and routine non study-related procedures are also carried out. These inspections are reported to Management and are maintained on file at ITR.

Joanne Tyas, BSc (Hons), MRQA, RQAP-GLP Director, Quality Assurance ITR Laboratories Canada, Inc.

Date

Final Report June 19,2017 40f35



TABLE OF CONTENTS

Section Page

COMPLIANCE STATEMENT ......................................................................................... 2

PERSONNEL .................................................................................................................... 3

QUALITY ASSURANCE STATEMENT ........................................................................ 4

TABLE OF CONTENTS ................................................................................................... 5

INDEX TO APPENDICES ................................................................................................ 7

1 SUMMARY ........................................................................................................... 8

2 INTRODUCTION ................................................................................................. 9

2.1 Experimental Design .............................................................................................. 9

2.2 Justification for Model, Bacterial Strains and Dose Levels Selection ................... 9

2.2.1 Justification for Model and Bacterial Strains Selection ......................................... 9

2.2.2 Justification of Dose Level Selection .................................................................. 10

2.3 Proposed Study Schedule ..................................................................................... 10

3 TEST, VEHICLE/NEGATIVE AND POSITIVE CONTROLS ITEMS ............ 10

3.1 Test Item Action .................................................................................................. 10

3.2 Positive Controls .................................................................................................. 10

3.3 Vehicle/Negative Control .................................................................................... 11

3.4 Information of Test and Vehicle/Negative Control Items ................................... 11

3.5 Preparation of Positive Controls Formulations .................................................... 12

3.6 Preparation of Test Item Formulation .................................................................. 12

3.7 Analysis of Formulation Accuracy, Stability and Homogeneity ......................... 12

3.8 Archive Sampling ................................................................................................ 12

4 EXPERIMENTAL PROCEDURES .................................................................... 12

4.1 Test System .......................................................................................................... 12

4.2 Preparation of the Strains ..................................................................................... 13

4.3 Metabolic Activation System ............................................................................... 13

4.4 General Reagents ................................................................................................. 13

4.5 Route of Administration ...................................................................................... 13

4.6 Treatment of Test System (Pre-Incubation method) ............................................ 13

4.7 Sterility Check and Spontaneous Mutation Rates ................................................ 14

5 DATA CAPTURE ............................................................................................... 14

6 EVALUATION AND INTERPRETATION OF RESULTS .............................. 14

6.1 Evaluation of Results ........................................................................................... 14

6.2 Criteria for Valid Test .......................................................................................... 14

6.3 Basis for Interpretation of the Results ................................................................. 15

6.3.1 Laboratory Proficiency ........................................................................................ 15

6.3.2 Interpretation of the Results ................................................................................. 15

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7 QUALITY ASSURANCE ................................................................................... 15

8 STANDARD OPERATING PROCEDURES ..................................................... 15

9 ARCHIVING ....................................................................................................... 15

10 RESULTS AND DISCUSSION .......................................................................... 16

11 CONCLUSION .................................................................................................... 18

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INDEX TO APPENDICES

Appendix Description Page

Appendix 1 Historical Control Data .............................................................................. 19

Appendix 2 Certificate of Analysis ............................................................................... 21

Appendix 3 Final Study Plan ......................................................................................... 22

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1 Summary The purpose of the study was to evaluate the genotoxicity potential of the Test Item InSea2® using the bacterial reverse mutation test.

The InSea2® test material was supplied by the sponsor, InnoVactiv. InSea2® is a demineralized polyphenol extract derived from Ascophyllum nodosum and Fucus vesiculosus, and it is intended to be used as an ingredient in food and supplement products.

Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and Escherichia coli strain (WP2 uvrA) were treated with the Test Item at a range of concentrations up to a standard limit of 5000 µg/plate, in the presence and absence of S9, using pre-incubation method as summarized in the following study design:

Dose Conc.

Number Material

Formulation Conc.

(µg/mL)

Final Conc.

(µg/plate)

Dose Volume

(µL)

Number of Replicates Number of

Strains* Absence of

S9 (-S9) Presence

of S9(+S9) Pre-incubation method

- Vehicle - - 100 3 3 5

1

Test Item

16 1.6 100 3 3 5

2 50 5 100 3 3 5

3 160 16 100 3 3 5

4 500 50 100 3 3 5

5 1600 160 100 3 3 5

6 5000 500 100 3 3 5

7 16000 1600 100 3 3 5

8 50000¥ 5000† 100 3 3 5

- Positive controls

‡ ‡ ‡ 3 3 5

†The OECD/ICH S2(R1) standard limit dose. ‡ Dose depends on the test system and the positive controls. *The strains are S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli (WP2 uvrA). ¥ Stock solution.

Overall, the results obtained with negative and positive controls with all the strains were as expected. The mean revertants obtained with the vehicle/negative control were within or close to the historical control data. Appropriate positive controls, with or without S9, produced increases in revertant colony numbers to at least twice the concurrent negative control levels with the appropriate bacterial strain (1.5× for strain TA100), confirming the sensitivity of the test system and the activity of the S9 mix.

No significant increases in revertant colony numbers were obtained with any of the tester strains following exposure to InSea2® at any dose level in the presence or absence of S9 mix.

Under the conditions used in this study, it is concluded that InSea2® did not show genotoxic activity in the bacterial reverse mutation assay.

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2 Introduction The purpose of the study is to evaluate the genotoxicity potential of the test item InSea2® using the bacterial reverse mutation test.

The study was performed in compliance with the following test guidelines:

OECD Guideline 471. OECD Guideline for Testing of Chemicals – Bacterial Reverse Mutation Test.

EPA Health Effects Test Guideline OPPTS 870.5100: Bacterial reverse mutation test.

ICH Harmonised Tripartite Guideline S2 (R1), Step 4. Guidance on Genotoxicity. Testing and Data Interpretation for Pharmaceuticals Intended for Human Use.

US FDA Redbook Chapter IV.C.1. Short-Term Tests for Genetic Toxicity.

ICH Harmonised Tripartite Guideline M3 (R2). Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals.

2.1 Experimental Design

Dose Conc.

Number Material

Formulation Conc.

(µg/mL)

Final Conc.

(µg/plate)

Dose Volume

(µL)

Number of Replicates Number of

Strains* Absence of

S9 (-S9) Presence

of S9(+S9) Pre-incubation method

- Vehicle - - 100 3 3 5

1

Test Item

16 1.6 100 3 3 5

2 50 5 100 3 3 5

3 160 16 100 3 3 5

4 500 50 100 3 3 5

5 1600 160 100 3 3 5

6 5000 500 100 3 3 5

7 16000 1600 100 3 3 5

8 50000¥ 5000† 100 3 3 5

- Positive controls

‡ ‡ ‡ 3 3 5

†The OECD/ICH S2(R1) standard limit dose. ‡ Dose depends on the test system and the positive controls. *The strains are S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli (WP2 uvrA). ¥ Stock solution.

2.2 Justification for Model, Bacterial Strains and Dose Levels Selection 2.2.1 Justification for Model and Bacterial Strains Selection The bacterial reverse mutation test is one of the main in vitro tests in a battery of genotoxicity tests and it is requested by all international Regulatory Agencies to be conducted as part of product safety/risk assessment (ICH S2 (R1); OECD/471). The strains used in the study are chosen because of their high sensitivity to mutagens.

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2.2.2 Justification of Dose Level Selection The test item was dosed at a range of concentrations but was only assessed up to the standard maximum limit of 5000 µg/plate. The concentrations were separated by a factor of approximately half log dilution (3.16 fold).

2.3 Proposed Study Schedule

Study initiation date: February 3, 2017

Experimental start date: (Bacteria growth initiation date)

February 6, 2017

First treatment: (Dosing date)

February 7, 2017

Experimental completion date: (Last date of data collection from bacterial colony counting)

February 10, 2017

Study completion date:

Date the Study Director signs the final report

3 Test, Vehicle/Negative and Positive Controls Items 3.1 Test Item Action The InSea2® test material was supplied by the sponsor, InnoVactiv. InSea2® is a demineralized polyphenol extract derived from Ascophyllum nodosum and Fucus vesiculosus, and it is intended to be used as an ingredient in food and supplement products.

3.2 Positive Controls As recommended by Regulatory Guidelines, the following mutagens were used as positive controls.

Strains Without metabolic activation With metabolic activation Positive

control Item Concentration

(μg/plate) Positive

control Item Concentration

(μg/plate) S. typhimurium TA1535 SAZ 0.5 2AA 5

S. typhimurium TA1537 9AC 20 B[a]P 50 S. typhimurium TA98 2NF 1.25 B[a]P 2 S. typhimurium TA100 SAZ 1.0 B[a]P 5 E. coli WP2 uvrA NQO 0.5 2AA 10

Positive Control -1 Positive Control -2 Abbreviation SAZ 9AC Identity Sodium Azide 9-Aminoacridine Hemihydrate Description White crystals Yellow crystalline powder Batch/Lot No. BCBF9700V A0302188

Expiry/Retest Date 31-05-17 10-07-18

Supplier Sigma Aldrich Acros Organics Vehicle Sterile Water for Injection/Irrigation Dimethyl Sulfoxide (DMSO) Handling Precautions Carcinogen handled as per SOP H.S 5.0 Carcinogen handled as per SOP H.S 5.0CAS Number 26628-22-8 65944-23-2

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POSITIVE CONTROL -3 POSITIVE CONTROL -4 Abbreviation 2NF NQO Identity 2-Nitrofluorene 4-Nitroquinoline N-oxide Description Light yellow to beige powder Yellow powder Batch/Lot No S43858V WXBC1554V


Supplier Sigma Aldrich Sigma Aldrich Vehicle Dimethyl Sulfoxide (DMSO) Dimethyl Sulfoxide (DMSO) Handling Precautions Carcinogen handled as per SOP H.S 5.0 Carcinogen handled as per SOP H.S 5.0CAS Number 607-57-8 56-57-5

POSITIVE CONTROL -5 POSITIVE CONTROL -6 Abbreviation 2AA B[a]P Identity 2-Aminoanthracene Benzo[a]pyrene Description Olive green powder Yellow powder Batch/Lot No STBB1901V SLBM2972V


Supplier Sigma Aldrich Sigma Aldrich Vehicle Dimethyl Sulfoxide (DMSO) Dimethyl Sulfoxide (DMSO) Handling Precautions Carcinogen handled as per SOP H.S 5.0 Carcinogen handled as per SOP H.S 5.0CAS Number 613-13-8 50-32-8

3.3 Vehicle/Negative Control Based on the information provided by the sponsor, the water is the vehicle of choice. This vehicle is compatible with the test system and is considered as the negative control.

3.4 Information of Test and Vehicle/Negative Control Items TEST ITEM* VEHICLE/NEGATIVE CONTROL

Identity: InSea2® Sterile water for injection USP

Description: Fine brown powder Clear liquid, colorless

Batch No.: INS2-006-TAC W6K03A0

Purity: Assumed 100% Not applicable

Expiry Date: 31-05-19 30-11-17 (21-02-17 after opening)Storage Conditions: Room temperature RmT/2-8°C after opening

Handling Precautions:

Standard precautions Standard precautions

Supplier: InnoVactiv. Inc Baxter

* Test item Certificate of analysis is included in the report in Appendix 2 Supporting documentation to confirm test item characterization was provided by the Sponsor in a certificate of analysis.

The Sponsor supplied safety information which may affect the handling of the test material or may preclude some personnel from contact with the material.

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3.5 Preparation of Positive Controls Formulations Positive control formulations and dilutions (as appropriate) were prepared in advance and stored frozen at -20ºC (± 10ºC). 2NF, 9AC, NQO, 2AA, and B[a]P were formulated in DMSO while SAZ was formulated in water.

The stock solutions of each positive control were prepared by weighing an appropriate amount of the positive control powder into a sterile container and dissolving it in the appropriate volume of vehicle. Once the powder was dissolved, dilutions were prepared from the stocks.

The positive control formulations were not subjected to analysis because the biological response of the test system is considered to be the best measure of the quality of each control formulations.

3.6 Preparation of Test Item Formulation The test item InSea2® dose formulations were prepared fresh on the day of dosing by dissolving the appropriate amount of the test item in vehicle to achieve the concentrations indicated in the study design. The formulations were homogenized at 5000 rpm to reduce the particles to a minimum. The formulations were stirred and kept at room temperature pending dosing.

All lower level formulations were made directly from the stock solution. All formulations were prepared in glass vials, stirred and stored at room temperature until the dosing time.

3.7 Analysis of Formulation Accuracy, Stability and Homogeneity The test item formulations were not analysed for achieved concentration, homogeneity and stability.

3.8 Archive Sampling No archive sampling was required for studies of less than 4-week duration. The remaining test item was returned to the supplier.

4 Experimental Procedures 4.1 Test System The bacteria were originally supplied by Moltox, NC, USA. Each bacterial culture was identified by its name (TA1535, TA1537, TA98, TA100, WP2 uvrA) and batch number. Each batch of frozen bacteria was tested for appropriate phenotype characteristics, spontaneous reversion rate and response to diagnostic mutagens. The following bacterial strains were employed:

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Strains Mutation types

S. typhimurium TA1535 hisG46 rfa ΔuvrB Base pair substitution

S. typhimurium TA1537 hisC3076 rfa ΔuvrB Frameshift

S. typhimurium TA98 hisD3052 rfa ΔuvrB pKM101 Frameshift

S. typhimurium TA100 hisG46 rfa ΔuvrB pKM101 Base pair substitution

E. coli WP2 trpE uvrA Base pair substitution

Fresh bacterial cultures were prepared and were in the late-log phase of growth at the time of use. The density of the cultures was confirmed using a bacterial counting chamber to be approximately at or above of 1×109 bacteria/mL before the cultures were used in the test.

4.2 Preparation of the Strains Cultures were prepared the day before the assay by inoculating flasks containing approximately 25 mL of culture media from the appropriate frozen permanent strains (stored at ≤-60ºC). Following inoculation, the flasks were placed overnight in a shaking incubator set at approximately 37ºC and 100 rpm. The shaking was programmed to start and function for approximately 5 hours. Cultures were stored refrigerated until treatment.

4.3 Metabolic Activation System Phenobarbital/5, 6-benzoflavone induced male Sprague-Dawley rat liver fraction (S9), which was obtained from a commercial supplier, was used as a metabolism activation system. Before use, the S9 fraction was thawed and mixed at a concentration of 10 % (v/v) with the following sterile cofactors: 8 mM MgCl2, 33 mM KCl, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP. The S9 mixture was kept at ca. 4°C or on ice until required. A copy of the S9 fraction certificate of analysis was retained in the study file.

4.4 General Reagents The identity, lot/batch no. and expiry dates of all general laboratory reagents used in the study were documented in the raw data.

4.5 Route of Administration Test Item, vehicle/negative and positive control formulations were added directly into the tubes containing bacteria and Phosphate buffer/S9 mix.

4.6 Treatment of Test System (Pre-Incubation method) A 0.5 mL aliquot of S9 mix (+S9) or phosphate buffer 0.1M pH 7.4 (0S9) were combined with 0.1 mL bacterial culture. An appropriate volume, as indicated in the study design, of the test/negative and positive control items were added. The samples were placed in an incubator set to maintain 37°C for 30 minutes with shaking at 180 rpm (nominal value). After pre-incubation period molten top agar (approximately 2 mL) supplemented with 0.05 mM biotin and minimal (0.05 mM) histidine and minimal (0.05 mM) tryptophan were added to the sample. The solution were mixed and overlaid on a minimal glucose plate (1.5% agar, Vogel-Bonner medium E, 2% glucose). After the

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overlay solidifies, the plates were inverted and placed in an incubator set to maintain 37°C for 72 hours. The sterile containers and the plates were labeled with the ITR study number and the plate identification number (ID) that refers to treatment conditions, dose level and strain ID.

4.7 Sterility Check and Spontaneous Mutation Rates The sterility of the buffer, S9 mix, vehicles and the highest concentration of positive controls and test item was assessed on the day of the test by plating the appropriate preparations plus top agar without bacteria on the minimal glucose plates.

The spontaneous mutation rates of the bacterial strains were assessed using the negative control samples.

5 Data Capture The following computerized systems was used during the conduct of this study:

Automated colony counter ProtoCOL3 plus

Version number of the system is held on file at ITR.

6 Evaluation and Interpretation of Results 6.1 Evaluation of Results After the incubation period, plates were removed from the incubator. The plates were examined for the quality of the background lawn and the number of revertant colonies. The background lawn was assessed visually for any presence of toxicity, precipitate or microbial contamination. An inverted microscope was used to facilitate the evaluation of the background lawn. Revertant colony counts were scored by using the automated colony counter ProtoCOL3 system. The plates were discarded at the completion of the study.

The Test Item was non-toxic, therefore the five levels up to the standard limit of 5000 µg/plate were reported. Cytotoxicity of the test item was indicated by the partial or complete absence of a background lawn (colony counts, if any, was not reported) or a substantial dose-related reduction in revertant colony counts compared with lower dose levels and concurrent vehicle control (i.e., fold response < 0.5) taking into account the laboratory historical control range. A thinning of the background lawn which was not accompanied with reduction in revertant colonies was not considered as toxicity.

6.2 Criteria for Valid Test

The results obtained with the concurrent vehicle/negative control fall within or close to the laboratory historical control data range.

All positive controls +/-S9 produce increases in revertant colony numbers to at least twice the concurrent vehicle control level with the appropriate bacterial strain (1.5x for strain TA100).

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6.3 Basis for Interpretation of the Results 6.3.1 Laboratory Proficiency Laboratory proficiency is demonstrated by accumulated historical control data which is included in the report and presented in Appendix 1.

6.3.2 Interpretation of the Results Means and standard deviations for appropriate dose concentrations was calculated and presented in the report along with triplicate results. The mean number of revertant colonies obtained with test item and positive controls was compared with those obtained for vehicle/negative control cultures. The mutagenic activity is routinely assessed by applying the following criteria:

1. The results are considered positive if the group mean values of the test item show a substantial and dose-related increase in revertant colony counts, i.e., response ≥ 2 times the vehicle/negative culture values for TA98, TA1535, TA1537 and WP2 uvrA, or ≥ 1.5 times for TA100, with mean value(s) outside the historical control range.

2. If the above criterion is not met, the response was considered as negative.

3. When neither of the above criteria is clearly met (equivocal), further testing may be appropriate to clarify the results using an appropriately modified study design, e.g., a narrower dose interval with the appropriate strain. In such cases, if no substantial increase is obtained in the confirmatory test, the results were considered negative.

7 Quality Assurance ITR Quality Assurance Unit (QAU) audited the study plan, the raw data and the report, and inspected critical phases of those portions of the study conducted at the facility in accordance with the standard operating procedures of the test facility.

8 Standard Operating Procedures All procedures were performed in accordance with the ITR Standard Operating Procedures and these were kept on file at ITR. No deviations occurred during the study. Deviations to the ITR Standard Operating Procedures were documented in the raw data.

9 Archiving All data that are generated during this study at ITR, together with the original copy of the study plan and the report will be retained for approximately 1 year, in the scientific archives of ITR. The archiving period will commence from the date of study finalization. For studies where finalization does not occur within six months of draft report, the archiving period will start at that point.

ITR agrees to give the Sponsor sufficient advance notification of any intended disposal of such materials after the 1-year holding period, to allow the Sponsor to secure alternative storage facilities.

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10 Results and Discussion The results obtained in the study are summarized in Tables 1, 2 and 3.

The absence of colonies on sterility check plates of vehicle and positive control items confirmed absence of microbial contamination. These results of the sterility check are not reported herein but kept in study raw data.

Table 1: InSea2® - Pre-Incubation in the Absence of S9 Mix

Strains Dose level (µg/plate)

Number of revertants Mean SD Background observations

Fold response†

x1 x2 x3 x1 x2 x3

TA1535 Water 20 15 23 19.3 4.0

50 14 23 26 21.0 6.2 1.1

160 17 18 22 19.0 2.6 1.0

500 18 22 18 19.3 2.3 1.0

1600 17 16 18 17.0 1.0 0.9

5000 9 16 20 15.0 5.6 0.8

TA1537 Water 10 15 15 13.3 2.9

50 7 16 16 13.0 5.2 1.0

160 14 13 23 16.7 5.5 1.3

500 16 19 16 17.0 1.7 1.3

1600 19 26 12 19.0 7.0 1.4

5000 21 13 21 18.3 4.6 1.4

TA98 Water 19 20 16 18.3 2.1

50 27 23 28 26.0 2.6 1.4

160 23 28 28 26.3 2.9 1.4

500 18 20 26 21.3 4.2 1.2

1600 15 15 29 19.7 8.1 1.1

5000 19 24 23 22.0 2.6 1.2

TA100 Water 97 102 81 93.3 11.0

50 104 115 112 110.3 5.7 1.2

160 132 129 133 131.3 2.1 1.4

500 86 132 109 109.0 23.0 1.2

1600 105 102 120 109.0 9.6 1.2

5000 111 105 96 104.0 7.5 1.1

WP2 uvrA Water 15 12 14 13.7 1.5

50 20 27 21 22.7 3.8 1.7

160 12 19 17 16.0 3.6 1.2

500 19 21 11 17.0 5.3 1.2

1600 17 19 17 17.7 1.2 1.3

5000 20 20 26 22.0 3.5 1.6

Comments on plate and/or background lawn (as appropriate): Normal (no comment), slightly reduced (2). †: Fold response in mean revertants compared to concurrent negative control; SD: Sample standard deviation

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Table 2: InSea2® -Pre-Incubation in the Presence of S9 Mix

Strains Dose level (µg/plate)

Number of revertants Mean SD Background observations

Fold response†

x1 x2 x3 x1 x2 x3

TA1535 Water 10 14 19 14.3 4.5

50 15 12 16 14.3 2.1 1.0

160 12 17 18 15.7 3.2 1.1

500 20 14 17 17.0 3.0 1.2

1600 16 13 11 13.3 2.5 0.9

5000 21 19 16 18.7 2.5 1.3

TA1537 Water 27 17 16 20.0 6.1

50 17 17 15 16.3 1.2 0.8

160 15 19 22 18.7 3.5 0.9

500 12 23 11 18.7 6.7 0.8

1600 14 17 12 14.3 2.5 0.7

5000 17 9 20 15.3 5.7 0.8

TA98 Water 18 27 13 19.3 7.1

50 22 30 28 26.7 4.2 1.4

160 24 34 31 29.7 5.1 1.5

500 27 28 36 30.3 4.9 1.6

1600 33 31 30 31.3 1.5 1.6

5000 31 33 34 32.7 1.5 1.7

TA100 Water 77 106 81 88.0 15.7

50 89 69 95 84.3 13.6 1.0

160 114 110 109 111.0 2.6 1.3

500 111 89 104 101.3 11.2 1.2

1600 110 116 112 112.7 3.1 1.3

5000 88 114 104 102.0 13.1 1.2

WP2 uvrA Water 17 9 18 14.7 4.9

50 17 14 17 16.0 1.7 1.1

160 13 10 24 15.7 7.4 1.1

500 22 16 26 21.3 5.0 1.5

1600 21 19 21 20.3 1.2 1.4

5000 20 20 17 19.0 1.7 1.3

Comments on plate and/or background lawn (as appropriate): Normal (no comment), slightly reduced (2). †: Fold response in mean revertants compared to concurrent negative control; SD: Sample standard deviation

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Table 3: Positive control Pre-Incubation in Presence and Absence of S9 mix

Strains S9 Compound Dose level Number of revertants Mean SD Fold response† (µg/plate) x1 x2 x3

TA1535 - SAZ 0.5 320 359 348 342.3 20.1 17.7* TA1537 - 9AC 20 578 535 519 544.0 30.5 40.8* TA98 - 2NF 1.25 79 99 114 97.3 17.6 5.3* TA100 - SAZ 1.0 252 243 226 240.3 13.2 2.6*

WP2 uvrA - NQO 0.5 339 520 529 462.7 107.2 33.9* TA1535 + 2AA 5 109 95 112 105.3 9.1 7.3* TA1537 + B[a]P 50 85 73 83 80.3 6.4 4.0* TA98 + B[a]P 2 121 137 118 125.3 10.2 6.5* TA100 + B[a]P 5 145 139 138 140.7 3.8 1.6*

WP2 uvrA + 2AA 10 96 82 90 89.3 7.0 6.1*

†: Fold response in mean revertants compared to concurrent vehicle control SD: Sample standard deviation *: Increase in revertant colony Overall, the results obtained with negative and positive controls with all the strains were as expected. The mean revertants obtained with the vehicle/negative control were within or close to the historical control data. Appropriate positive controls, with or without S9, produced increases in revertant colony numbers to at least twice the concurrent negative control levels with the appropriate bacterial strain (1.5× for strain TA100), confirming the sensitivity of the test system and the activity of the S9 mix.

No significant increases in revertant colony numbers were obtained with any of the tester strains following exposure to InSea2® at any dose level in the presence or absence of S9 mix.

11 Conclusion Under the conditions used in this study, it is concluded that InSea2® did not show genotoxic activity in the bacterial reverse mutation assay.

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APPENDIX 1 ITR Study No. 55482 1

Laboratory historical control data collected from GLP and non-GLP studies performed between September 2013 and the onset of this study.

Pre-Incubation Method

Vehicle controls, without S9Mix

Strains TA1535 TA1537 TA98 TA100 WP2 uvrA Mean revertants per plate 14.2 15.8 27.2 114.4 20.2 Standard deviation 4.5 7.6 11.8 32.7 10.0 Minimum 5 2 7 67 7 Maximum 24 36 67 210 48 Number of experiments 22 22 22 22 22

Vehicle controls, with S9Mix


Positive controls, without S9Mix


Positive controls, with S9Mix


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APPENDIX 1 ITR Study No. 55482 2

Positive controls without S9 Mix

Positive controls with S9 Mix

TA1535 SAZ ( 0.5 µg/plate)

TA1535 2AA (5 µg/plate) TA1537 9AC ( 20 µg/plate)

TA1537 B[a]P ( 50 µg/plate)

TA98 2NF ( 1.25 µg/plate)

TA98 B[a]P ( 2 µg/plate) TA100 SAZ ( 1 µg/plate)

TA100 B[a]P ( 5 µg/plate)

WP2 uvrA NQO ( 0.5 µg/plate)

WP2 uvrA 2AA ( 10 µg/plate)

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APPENDIX 2 ITR Study No. 55482

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ITR CANADA

CONFIDENTIAL

FINAL STUDY PLAN

InSea2® : Bacterial Reverse Mutation Test

ITR Study Number: 55482

Test Facility: ITR Laboratories Canada Inc (ITR)

19601 Clark Graham Blvd

Baie d’Urfe, Quebec

Canada H9X 3T1

Sponsor: InnoVactiv Inc.

265 2nd Street East

Rimouski (QC)

Canada, G5L 9H3

Issue Date:

February 3, 2017

Page Number:

1 of 14

APPENDIX 3

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ITR Study No. 55482

Page 2 of 14

Draft Study Plan February 3, 2017

PREFACE PAGE

Study Title: InSea2® : Bacterial Reverse Mutation Test

Study GLP Status: GLP

Study Director (SD): Mélanie Ghoussoub, BSc.

Genetic Toxicology Department

ITR Laboratories Canada Inc.

19601 Clark Graham

Baie d’Urfé, Québec

H9X 3T1, Canada

Tel: 514 457 7400 ext 263

Fax: 514 457 7303

E-mail: [email protected]

Sponsor’s Monitor: Jocelyn Bérubé, MSc

InnoVactiv, Inc.

265 2nd Street East

Rimouski (QC)

Canada, G5L 9H3

Tel : (418) 721-2308 x 221

Fax : (418) 721-2318

Email: [email protected]

Note: the naming conventions used in this study plan meet those prescribed by the OECD

but they are considered to be synonymous with those used by other regulatory bodies.

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TABLE OF CONTENTS

Section Page

COVER PAGE ................................................................................................................ 1

PREFACE PAGE ............................................................................................................ 2 TABLE OF CONTENTS ................................................................................................ 3 1 INTRODUCTION ............................................................................................... 4 2 TEST, VEHICLE/NEGATIVE AND POSITIVE CONTROL ITEMS ................. 5 3 EXPERIMENTAL PROCEDURES ..................................................................... 8 4 DATA CAPTURE ............................................................................................. 10 5 EVALUATION AND INTERPRETATION OF RESULTS ............................... 10 6 CHANGES TO THE STUDY PLAN ................................................................. 11 7 REPORTING ..................................................................................................... 11 8 GLP COMPLIANCE ......................................................................................... 12 9 QUALITY ASSURANCE ................................................................................. 13 10 STANDARD OPERATING PROCEDURES .................................................... 13 11 ARCHIVING ..................................................................................................... 13 12 STUDY PLAN APPROVAL PAGE .................................................................. 14

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1 INTRODUCTION

The purpose of the study is to evaluate the genotoxicity potential of the Test Item InSea2® using the bacterial reverse mutation test.

Regulatory test guidelines from the OECD, EPA, FDA and ICH were used as reference

material for preparation of this study plan.

OECD Guideline 471. OECD Guideline for Testing of Chemicals – Bacterial

Reverse Mutation Test.

EPA Health Effects Test Guideline OPPTS 870.5100: Bacterial reverse mutation test

ICH Harmonised Tripartite Guideline S2 (R1), Step 4. Guidance on Genotoxicity

Testing and Data Interpretation for Pharmaceuticals Intended for Human Use.

US FDA Redbook Chapter IV.C.1. Short-Term Tests for Genetic Toxicity.

ICH Harmonised Tripartite Guideline M3 (R2). Nonclinical Safety Studies for the

Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals.

1.1 Experimental Design

Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and Escherichia

coli strain (WP2 uvrA) will be treated with the test item at a range of concentrations up to

a standard limit of 5000 µg/plate, in the presence and absence of S9, using modified

bacterial reverse mutation pre-incubation method as summarized in the study design below:

Dose

Conc.

Number

Material

Formulation

Conc.

(µg/mL)

Final

Conc.

(µg/plate)

Dose

volume

(µL)

Number of Replicates Number

of

Strains* Absence of

S9 (-S9)

Presence

of S9(+S9)

Pre-incubation method

- Vehicle - - 100 3 3 5

1

Test Item

16 1.6 100 3 3 5

2 50 5 100 3 3 5

3 160 16 100 3 3 5

4 500 50 100 3 3 5

5 1600 160 100 3 3 5

6 5000 500 100 3 3 5

7 16000 1600 100 3 3 5

8 50000¥ 5000† 100 3 3 5

- Positive

controls ‡ ‡ ‡ 3 3 5

†The OECD/ICH S2(R1) standard limit dose.

‡ Dose depends on the test system and the positive controls. *The strains are S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli (WP2 uvrA). ¥ Stock solution.

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1.2 Justification for Model, Bacterial Strains and Dose Levels Selection

1.2.1 Justification for Model and Bacterial Strains Selection

The bacterial reverse mutation test is one of the main in vitro tests in a battery of

genotoxicity tests and it is requested by all international Regulatory Agencies to be

conducted as part of product hazard/risk assessment (ICH S2 (R1); OECD/471). The strains

used in the study are chosen because of their high sensitivity to mutagens.

1.2.2 Justification of Dose Levels

The Test Item is dosed at a range of concentrations but is only assessed at the highest levels

below the toxic level or, if non-toxic, at levels up to the standard maximum limit of

5000 µg/plate or 5 µL/plate, whichever is the lowest. The concentrations are separated by

a factor of approximately half log dilution (3.16 fold).

1.3 Proposed Study Schedule

Study initiation date:

Date the Study Director signs the

study plan

Experimental start date:

(Bacteria growth initiation date) February 06, 2017

First treatment:

(Dosing date) February 07, 2017

Experimental completion date:

(Last date of data collection from bacteria colony

counting)

February 17, 2017

Audited draft report date: April 6, 2017

Study completion date:

Date the Study Director signs the

final report (Normally within 3

months of issue of draft report.

Please also reference the

Reporting section).

2 TEST, VEHICLE/NEGATIVE AND POSITIVE CONTROL ITEMS

2.1 Test Item Action

The test item is a demineralized polyphenol extracted from Ascophyllum nodosum and

Fucus vesiculosus, and it is used as dietary supplement.

2.2 Positive Controls

As recommended by Regulatory Guidelines, the following mutagens are used as positive

controls.

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Strains

Without metabolic activation With metabolic activation

Positive control

Item

Concentration

(μg/plate)

Positive control

Item

Concentration

(μg/plate)

S. typhimurium TA1535 SAZ 0.5 2AA 5

S. typhimurium TA1537 9AC 20 B[a]P 50

S. typhimurium TA98 2NF 1.25 B[a]P 2

S. typhimurium TA100 SAZ 1 B[a]P 5

E. coli WP2 uvrA NQO 0.5 2AA 10

POSITIVE CONTROL -1 POSITIVE CONTROL -2

Abreviation SAZ 9AC

Identity Sodium Azide 9-Aminoacridine Hemihydrate

Description To be documented in raw data and report To be documented in raw data and report

Batch/Lot No To be documented in raw data and report To be documented in raw data and report

Expiry/Retest

Date

To be documented in raw data and report To be documented in raw data and report

Supplier To be documented in raw data and report To be documented in raw data and report

Vehicle Sterile Water for Injection/Irrigation Dimethyl Sulfoxide (DMSO)

Handling

Precautions Carcinogen handle as per SOP H.S 5.0 Carcinogen handle as per SOP H.S 5.0

CAS Number 26628-22-8 65944-23-2


Abreviation 2NF NQO

Identity 2-Nitrofluorene 4-Nitroquinoline N-oxide



Expiry/Retest

Date



Vehicle Dimethyl Sulfoxide (DMSO) Dimethyl Sulfoxide (DMSO)

Handling


CAS Number 607-57-8 56-57-5


Abreviation 2AA B[a]P

Identity 2-Aminoanthracene Benzo[a]pyrene



Expiry/Retest

Date



Vehicle Dimethyl Sulfoxide (DMSO) Dimethyl Sulfoxide (DMSO)

Handling


CAS Number 613-13-8 50-32-8

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2.3 Vehicle/Negative Control

Based on the information provided by the sponsor, water is selected as the vehicle. This

vehicle is compatible with the test system and is considered as the negative control.

2.4 Information of Test and Vehicle/Negative Control Items

TEST ITEM* VEHICLE/NEGATIVE CONTROL Identity InSea2® Sterile water for injection USP

Description Fine Brown Powder Clear liquid

Batch/Lot No INS2-006-TAC To be documented in raw data and report

Purity Assumed 100% Not applicable

Expiry Date 31-05-2019 To be documented in raw data and report

Storage Conditions Room Temperature To be documented in raw data and report

Handling Precautions Standard laboratory precautions Standard laboratory precautions

Supplier InnoVactiv. Inc ITR Laboratories

*Test Item Certificate of analysis will be included in the final report.

Supporting documentation to confirm test item characterization was provided by the

Sponsor in a certificate of analysis. However, the certificate did not clearly state if the

characterization and stability under the storage conditions of the bulk test item was

performed in compliance with GLP or other regulations.

The Sponsor supplied safety information which may affect the handling of the test material

or may preclude any personnel from contact with the material.

2.5 Preparation of Positive Control Item Formulations

Positive control formulations and dilutions (as appropriate) will be prepared in advance

and stored frozen at -20ºC (± 10 ºC). 2NF, 9AC, NQO, 2AA and B[a]P are formulated in

DMSO while SAZ is formulated in water.

The stock solution of each positive control will be prepared by weighing an appropriate

amount of the positive control powder into a sterile container and dissolving it in the

appropriate volume of vehicle. Once the powder is dissolved, dilutions will be prepared

from the stocks.

The positive control formulations will not be subjected to analysis because the biological

response of the test system is considered to be the best measure of the quality of each

control formulations.

2.6 Preparation of Test and Negative/Vehicle Control Items

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The Test Item inSea2® dose formulations will be prepared fresh on the day of dosing by

dissolving the appropriate amount of the Test Item in vehicle to achieve the concentrations

indicated in the study design. The formulations may be homogenized as needed at 5000

rpm to reduce the particles to a minimum. The formulations will be stirred (if particles

remain) and kept at room temperature pending dosing.

All lower level formulations will be made directly from the stock solution. All formulations will

be prepared in glass vials, stirred and stored at room temperature until the dosing time.

2.7 Analysis of Achieved Test Item Formulations

The Test Item formulations will not be analysed for achieved concentration, homogeneity

and stability.

2.8 Archive Sampling

No archive sampling is required for studies of less than 4-week duration. The remaining

Test Item may be returned to the Sponsor on completion of the study or may be retained

for future studies.

3 EXPERIMENTAL PROCEDURES

3.1 Test System

The bacteria were originally supplied by Moltox, NC, USA. Each bacterial culture is

identified by its name (TA1535, TA1537, TA98, TA100, WP2 uvrA) and batch number.

Each batch of frozen bacteria is tested for appropriate phenotype characteristics,

spontaneous reversion rate and response to diagnostic mutagens. The following bacterial

strains will be employed:

Strains Mutation type

S. typhimurium TA1535 hisG46 rfa ΔuvrB Base pair substitution

S. typhimurium TA1537 hisC3076 rfa ΔuvrB Frameshift

S. typhimurium TA98 hisD3052 rfa ΔuvrB pKM101 Frameshift

S. typhimurium TA100 hisG46 rfa ΔuvrB pKM101 Base pair substitution

E. coli WP2 trpE uvrA Base pair substitution

Fresh bacterial cultures will be prepared and will be in the late-log phase of growth at the

time of use. The density of the cultures will be confirmed to be approximate or above of

1x109 bacteria/mL using a bacterial counting chamber before the cultures are used in the

test.

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3.2 Preparation of the Strains

Cultures will be prepared the day before of each assay by inoculating flasks containing

approximately 25 mL of culture media from the appropriate frozen permanent strains

(stored at ≤-60ºC). Following inoculation, the flask will be placed overnight in a resting

shaker/incubator set at approximately 37ºC and 100 rpm. The shaking will be

programmed to start and function for approximately 5 hours. Cultures will be kept at cool

until treatment.

3.3 Metabolic Activation System

Phenobarbital/5, 6-benzoflavone induced male Sprague-Dawley rat liver fraction (S9),

which was obtained from a commercial supplier, will be used as a metabolism activation

system. Just before use, the S9 fraction is thawed and mixed at a concentration of 10 %

(v/v) with the following sterile cofactors: 8 mM MgCl2, 33 mM KCl, 100 mM sodium

phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP. The S9 mixture

will be kept at ca. 4°C or on ice until required. A copy of the S9 fraction certificate of

analysis will be retained in the study file.

3.4 General Reagents

The identity, lot/batch no. and expiry dates of all general laboratory reagents used in the study

will be documented in the raw data.

3.5 Route of Administration

Test Item, vehicle/negative and positive control formulations will be added directly into

the tubes containing bacteria, Phosphate buffer or S9 mixture.

3.6 Treatment of Test System (Pre-incubation Method)

A 0.5 mL aliquot of S9 mix (+S9) or phosphate buffer 0.1M pH 7.4 (0S9) will be combined

with 0.1 mL bacterial culture. An appropriate volume, as indicated in the study design, of

the test/negative and positive control items will be added. The samples will be placed in

an incubator set to maintain 37°C for 30 minutes with shaking at 180 rpm (nominal value).

After pre-incubation period molten top agar (approximately 2 mL) supplemented with

0.05 mM biotin and minimal (0.05 mM) histidine and minimal (0.05 mM) tryptophan will

be added to the sample. The solution will be mixed and overlaid on a minimal glucose

plate (1.5% agar, Vogel-Bonner medium E, 2% glucose). After the overlay solidifies, the

plates will be inverted and placed in an incubator set to maintain 37°C for 48 to 72 hours.

The sterile containers and the plates are labeled with the ITR study number and the plate

identification number (ID) that refers to treatment conditions, dose level and strain ID.

3.7 Sterility Check and Spontaneous Mutation Rates

The sterility of the buffer, S9 mix, vehicles and the highest concentration of test and

positive control items will be assessed on the day of the test by plating the appropriate

preparations plus top agar without bacteria on the minimal glucose plates.

The spontaneous mutation rates of the bacterial strains will be assessed using the negative

control samples.

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4 DATA CAPTURE

The following computerised system may be used during the conduct of this study:

Automated colony counter ProtoCOL3 plus

Version number of the system will be held on file at ITR.

5 EVALUATION AND INTERPRETATION OF RESULTS

5.1 Evaluation of Results

After the incubation period, plates will be removed from the incubator and may be stored

for up to 1 week at 2 to 8ºC prior to examination. Plates will be examined for the quality

of the background lawn and the number of revertant colonies. The background lawn will

be assessed visually for any presence of toxicity, precipitate or microbial contamination.

An inverted microscope may be used to facilitate interpretation. Revertant colony counts

will be evaluated by using the automated colony counter ProtoCOL3 system. The revertant

colony counts could also be evaluated visually by using electronic counting pen and the

results will be recorded in the experimental design worksheets. The plates will be discarded

at the completion of the study.

If available, the plates from at least five non-toxic dose levels of the Test Item will be

assessed, i.e. the five highest levels below the toxic level. If the Test Item is non-toxic, the

five levels up to the standard limit of 5000 µg/plate will be reported. Cytotoxicity of the

Test Item is normally indicated by the partial or complete absence of a background lawn

(colony counts, if any, will not be reported) or a substantial dose-related reduction in

revertant colony counts compared with lower dose levels and concurrent vehicle control

(i.e., fold response < 0.5) taking into account the laboratory historical control range. A

thinning of the background lawn that is not accompanied by reduction in revertant colonies

will not be considered as toxicity.

5.2 Criteria for Valid Test

The results obtained with the concurrent vehicle/negative control should fall within

or close to the laboratory historical control data range.

All positive controls +/-S9 should produce increases in revertant colony numbers

to at least twice the concurrent vehicle control level with the appropriate bacterial

strain (1.5x for strain TA100).

Invalid assay results will normally lead to an automatic repeat of the appropriate part of

the assay. If that is not practical then any values that do not comply with these criteria will

be discussed in the report.

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5.3 Basis for Interpretation of the Results

5.3.1 Laboratory proficiency

Laboratory proficiency is demonstrated by accumulated historical control data which will

be included in the report.

5.3.2 Interpretation of the Results

Means and standard deviations for appropriate dose concentrations will be calculated and

presented in the report along with triplicate results. The mean number of revertant colonies

obtained with the Test Item and positive controls will be compared with those obtained for

vehicle/negative control cultures. The mutagenic activity is routinely assessed by applying

the following criteria:

1. The results will be considered positive if the group mean value of the Test Item

show a substantial increase in revertant colony counts (dose-related, if applicable),

i.e., response ≥ 2 times the vehicle/negative culture values for TA98, TA1535,

TA1537 and WP2 uvrA, or ≥ 1.5 times for TA100, with mean value(s) outside the

historical control range.

2. If the above criteria are not met, the response will be considered as negative.

3. When neither of the above criteria are clearly met (equivocal), further testing may

be appropriate to clarify the results using an appropriately modified study design,

e.g., a narrower dose interval with the appropriate strain. In such cases, if no

substantial increase is obtained in the confirmatory test, the results will be

considered negative.

6 CHANGES TO THE STUDY PLAN

All mutually agreed upon changes in study plan content will be documented in the form of

a Study plan amendment which will be duly authorized by the Study Director of ITR and

by the Sponsor.

7 REPORTING

A complete detailed Quality Assurance audited draft report (PDF and any Word versions

of text portions) will be submitted to the Sponsor as per the contract. Where external

contributions to the report are unavailable the report will be issued as scheduled unless

otherwise directed by the Sponsor. Subsequent to any modifications or corrections (agreed

to by the Sponsor and ITR Study Director), only report sections that were subject to change

will be submitted to the Sponsor by the Study Director for final review. A fully compiled

version of the proposed final report will only be issued if requested by the Sponsor (at

additional cost). Once agreement on the final content of the report has been agreed between

ITR and the Sponsor a searchable PDF version of the final report will be issued. A hard

copy of the final report will be sent to the Sponsor, only if requested.

The report will accurately describe the methods used for the generation and analysis of the

results and will include, but not limited to the following:

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Test Item: identification data and CAS no., if known; physical nature and purity;

physicochemical properties relevant to the conduct of the study; stability of the test

substance, if known.

Solvent/Vehicle: justification for choice of solvent/vehicle; solubility and stability

of the Test Item in solvent/vehicle, if known.

Strains: Strains used; number of cells/mL per culture; strain characteristics.

Test conditions: amount of test substance per plate (mg/plate or g/plate) with

rationale for selection of dose and number of plates per concentration; media used;

type and composition of metabolic activation system, including acceptability

criteria; treatment procedures.

Results: signs of toxicity; signs of precipitation; individual plate counts; the mean

number of revertant colonies per plate and standard deviation; dose-response

relationship, where possible; statistical analyses, if any; concurrent negative

(solvent/vehicle) and positive control data, with ranges, means and standard

deviations;

historical negative (solvent/vehicle) and positive control data, with e.g. ranges,

means and standard deviations.

Discussion of the Results

In the absence of ongoing communications and after notification in writing to the Sponsor,

ITR reserves the right to finalize, sign and issue the final report from this study three

months after issue of the draft. In such an event, all materials will be transferred to the

archive. Any subsequent requests for modifications, corrections or additions to the final

report will be the subject of a formal report amendment and will be subject to additional

cost.

A searchable and linked PDF final report will be dispatched to the Sponsor. A hard copy

of the final report will be sent to the Sponsor, if requested.

8 GLP COMPLIANCE

The study will be performed in compliance with the Organisation for Economic-Co-

operation and Development (OECD) Principles on Good Laboratory Practice (Issued Jan

1998) ENV/MC/CHEM(98)17 and United States Food and Drug Administration Title 21

Code of Federal Regulations Part 58, Good Laboratory Practice for Non-clinical studies

issued 22 December 1978 Federal Register plus subsequent amendments with the

exception that the test item formulations will not be analysed for test item achieved

concentrations, stability and homogeneity.

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9 QUALITY ASSURANCE

ITR Quality Assurance Unit (QAU) will audit the study plan and amendment(s), if any, the

raw data and the report, and will inspect critical phases of those portions of the study

conducted at the facility in accordance with the standard operating procedures of the test

facility.

ITR agrees to notify the Sponsor promptly of any government inquiry about, or proposed

inspection of, this study.

10 STANDARD OPERATING PROCEDURES

All procedures will be performed in accordance with the ITR Standard Operating

Procedures and these will be kept on file at ITR. Deviations to the ITR Standard Operating

Procedures will be documented in the raw data.

11 ARCHIVING

All data that are generated during this study at ITR, together with the original copy of the

study plan, study plan amendment(s), if any, and the report will be retained for

approximately 1-year, in the scientific archives of ITR. The archiving period will

commence from the date of study finalization. For studies where finalization does not

occur within six months of draft report, the archiving period will start at that point.

ITR agrees to give the Sponsor sufficient advance notification of any intended disposal of

such materials after the 1-year holding period, to allow the Sponsor to secure alternative

storage facilities.

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12 SWDY PLAN APPROVAL PAGE

This study plan bas been approved by:

03 - 02 -/ '=l-MelanieCJhO~ Date Study Director lTR Laboratories Canada Inc.

Gillette Senior Vice President ITR Laboratories Canada Inc.

~03.d(2(;tDare I

This study plan has been peer revieWed by:

LI-' -4~"/~Merah.~ Scientist, Genetic Toxicology ITR Laboratories Canada Inc.

This study plan has been reviewed. by the Quality Assurance Department:

Joanne • BSc (Hons). MRQA. RQAP-GLP Quality Assurance Representative ITR Laboratories Canada Inc.

Approved on behalf of the Sponsor by:

Draft Study Plan February 3,2017

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