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ITR CANADA

CONFIDENTIAL

FINAL REPORT

InSea2®: In Vitro Mammalian Chromosome Aberration Test Using Human Peripheral Blood Lymphocytes

ITR Study Number: 55483

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: June 16, 2017

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ITR Study No.: 55483 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 exceptions:

• 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 January 30, 2017 and completed on June 16, 2017.

Study Director: %.~Gl rida Merah, MSc

ITR Laboratories Canada Inc.

Reviewer: Date

Scientist, Genetic Toxicology ITR Laboratories Canada Inc.

ITR Management Representative:

Final Report June 16,2017

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Final Report June 16, 2017

PERSONNEL

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

Pharmacy: Rogy Markose, Manager

Genetic Toxicology: 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 55483 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 26, 2017 Study plan January 30, 2017

In-Life Inspections

February 02, 2017 Dosing February 02,2017

February 15,2017 Slide examination (Mitotic February 16,2017 Index)

Data and Report

April OS, 10, 2017 Data Aprill0,2017

April 04 to 06, 10, 2017 Data and report April II, 2017

April 13,2017 Report April 13,2017

May 19,2017 Report May 19,2017

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 16,2017

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TABLE OF CONTENTS

Section Page

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

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

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

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

INDEX TO TABLES ......................................................................................................... 7

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

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

2 INTRODUCTION ............................................................................................... 10

2.1 Experimental Design ............................................................................................ 10

2.2 Justification for the Model Selection ................................................................... 11

2.3 Justification of Dose Level Selection .................................................................. 11

2.4 Study Schedule..................................................................................................... 11

3 TEST ITEM, VEHICLE/NEGATIVE, AND POSITIVE CONTROLS ............. 11

3.1 Test Item Action .................................................................................................. 11

3.2 Vehicle/Negative Control .................................................................................... 11

3.3 Positive Controls .................................................................................................. 11

4 INFORMATION ON TEST ITEM, VEHICLE/NEGATIVE AND POSITIVE CONTROLS ...................................................................................... 12

4.1 Test Item and Vehicle/Negative Controls ............................................................ 12

4.2 Positive Controls .................................................................................................. 12

4.2.1 Positive Controls Formulations............................................................................ 12

4.2.2 Dose Formulation Analysis of Positive Controls................................................. 13

4.3 Preparation of Test Item Formulations ................................................................ 13

4.4 Analysis of Test Item Formulations ..................................................................... 13

4.5 Archive Sampling ................................................................................................ 13

5 EXPERIMENTAL PROCEDURES .................................................................... 13

5.1 Test System .......................................................................................................... 13

5.1.1 Blood Sampling ................................................................................................... 13

5.1.2 Culture Medium ................................................................................................... 14

5.1.3 Lymphocyte Culture ............................................................................................ 14

5.2 Metabolic Activation System ............................................................................... 14

5.3 General Reagents ................................................................................................. 14

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5.4 Route of Administration ...................................................................................... 14

5.5 Treatment of Test System .................................................................................... 14

5.6 Medium Change (4-hour Treatment only) ........................................................... 15

5.7 Harvesting, Metaphase Preparation and Staining ................................................ 15

6 SLIDE EXAMINATION ..................................................................................... 15

6.1 Mitotic Index (MI) ............................................................................................... 15

6.2 Selection of Slides for Detailed Examination ...................................................... 15

6.3 Detailed Examination for Chromosome Aberrations .......................................... 15

7 BASES FOR DATA ANALYSIS AND INTERPRETATION OF THE RESULTS ............................................................................................................ 16

7.1 Statistical Analysis ............................................................................................... 16

7.2 Validity of the Assay ........................................................................................... 16

7.3 Basis for the Interpretation of the Results ............................................................ 17

8 DATA CAPTURE ............................................................................................... 17

9 QUALITY ASSURANCE ................................................................................... 17

10 STANDARD OPERATING PROCEDURES ..................................................... 17

11 ARCHIVING ....................................................................................................... 17

12 RESULTS AND DISCUSSION .......................................................................... 18

12.1 Formulation Analysis ........................................................................................... 18

12.2 Microscopic Examination .................................................................................... 18

12.2.1 Dose Selection ..................................................................................................... 19

12.2.2 Chromosome Aberrations .................................................................................... 20

13 CONCLUSION .................................................................................................... 20

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INDEX TO TABLES

Table Description Page

Table 1: Summary Results ............................................................................................... 19

INDEX TO APPENDICES

Appendix Description Page

Appendix 1: Historical Control Data .............................................................................. 21

Appendix 2: Mitotic Index Results ................................................................................. 23

Appendix 3: Results of Individual Cultures .................................................................... 27

Appendix 4: Certificate of Analysis ............................................................................... 31

Appendix 5: IRB Approval information ......................................................................... 32

Appendix 6: Final Study Plan ......................................................................................... 33

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1 SUMMARY The purpose of the study was to evaluate the genotoxicity potential of the test item, InSea2®, using the in vitro chromosome aberration test in human peripheral blood lymphocytes.

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.

The test item, InSea2®, was tested in the chromosome aberration test using human peripheral blood lymphocytes. The cells were stimulated into division in culture then treated with the test item at a range of concentrations up to the standard limit of 5 mg/mL. Cultures were treated for 4 hours in the absence and presence of rat liver S9 mixture and for 21 hours in the absence of rat liver S9 mix. Appropriate concurrent vehicle/negative and positive controls were used as well.

Dose Numbe

r Material

FormulationConc.

(µg/mL)

Final Conc.

(µg/mL)

Dosing Volume

(µL)

Culture Number ID 4 Hours

(-S9) 4 Hours

(+S9) 21 Hours (-

S9)

- Vehicle - 500 a01, a02 b01, b02 c01, c02

1 2 3 4 5 6 7 8 9

10

Test Item 90 190 380 750 1500 3000 6000

12500 25000

50000¥

9 19 38 75 150 300 600

1250 2500

5000*

500 500 500 500 500 500 500 500 500 500

a11, a12 a21, a22 a31, a32 a41, a42 a51, a52 a61, a62 a71, a72 a81, a82 a91, a92

a101, a102

b11, b12 b21, b22 b31, b32 b41, b42 b51, b52 b61, b62 b71, b72 b81, b82 b91, b92

b101, b102

c11, c12 c21, c22 c31, c32 c41, c42 c51, c52 c61, c62 c71, c72 c81, c82 c91, c92

c101, c102 1 2 3

Mitomycin C

10.0 20.0 30.0

0.10 0.20 0.30

50 50 50

a111, a112 a121, a122 a131, a132

c111, c112 c121, c122 c131, c132

1 2 3

Cyclophosphamide

400 800 1200

4.0 8.0 12

50 50 50

b111, b112 b121, b122 b131, b132

* x (high level) as per OECD 473 and FDA Redbook IVC1 Guidelines = 5 mg/mL. 5 L/mL or 10 mM, whichever is the lowest (Chemicals). The 21 hour incubation ('confirmatory phase') is required by regulatory authorities where the initial phase does not show any indication of genotoxicity for the test item; however, it is routinely performed at the same time as the initial phase to minimize potential delays in performance of the study. Conc. = Concentration ¥: Stock solution

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Metaphases from cultures treated with at least three dose levels of InSea2®, together with negative and positive control cultures, were subjected to detailed examination for the presence of chromosomal aberrations using light microscopy. There was a heavy precipitate on the slide at the highest three levels in the three regimes, therefore, the slides prepared from these cultures could not be evaluated. Thus, the test item lowest precipitating level (600µg/mL) was selected as high dose for detailed analysis in the three regimes followed by two adjacent dose levels.

The results obtained with the test item did not show any statistically significant increase in the incidence of cells with aberrant chromosomes (p≥0.01) in all conditions tested (4 hours with and without S9 mix and at 21 hours without S9mix).

There were no increases in numerical aberrations in the conditions tested.

The results obtained with vehicle/negative and positive controls were as expected and fell within or close to the historical control data, confirming the sensitivity of the test system, the effectiveness of the S9 mix, and the validity of the assay.

Under the conditions used in this study, it is concluded that InSea2® did not induce chromosome damage in human lymphocytes.

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2 INTRODUCTION The purpose of the study was to evaluate the genotoxicity potential of the test item InSea2® using the in vitro chromosome aberration test in human peripheral blood lymphocytes, in compliance with GLP regulations.

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

OECD Guideline 473. OECD Guideline for Testing of Chemicals – In vitro Mammalian chromosome aberration test.

EPA Health Effects Test Guideline OPPTS 870.5375: In vitro Mammalian chromosome aberration 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 Whole blood was taken from healthy, non-smoking male donor. Mixture of blood/media was used to prepare duplicate cultures. The cultures were treated with appropriate quantity of InSea2®, vehicle/negative and positive control items as described in the table below.

Dose Number Material

Formulationconc.

(µg/mL)

Final conc.

(µg/mL)

Dosing volume

(µL)

Culture Number ID 4 Hours

(-S9) 4 Hours

(+S9) 21 Hours

(-S9)

- Vehicle - 500 a01, a02 b01, b02 c01, c02

1 2 3 4 5 6 7 8 9

10

Test Item 90 190 380 750

1500 3000 6000

12500 25000 50000¥

9 19 38 75

150 300 600 1250 2500

5000*

500 500 500 500 500 500 500 500 500 500

a11, a12 a21, a22 a31, a32 a41, a42 a51, a52 a61, a62 a71, a72 a81, a82 a91, a92

a101, a102

b11, b12 b21, b22 b31, b32 b41, b42 b51, b52 b61, b62 b71, b72 b81, b82 b91, b92

b101, b102

c11, c12 c21, c22 c31, c32 c41, c42 c51, c52 c61, c62 c71, c72 c81, c82 c91, c92

c101, c1021 2 3

Mitomycin C

10.0 20.0 30.0

0.10 0.20 0.30

50 50 50

a111, a112a121, a122a131, a132

c111, c112c121, c122c131, c132

1 2 3

Cyclophosphamide

400 800

1200

4.0 8.0 12

50 50 50

b111, b112 b121, b122 b131, b132

* x (high level) as per OECD 473 and FDA Redbook IVC1 Guidelines = 5 mg/mL. 5 L/mL or 10 mM, whichever is the lowest. The 21 hour incubation ('confirmatory phase') is required by regulatory authorities where the initial phase does not show any indication of genotoxicity for the test item; however, it is routinely performed at the same time as the initial phase to minimize potential delays in performance of the study. Conc. = Concentration ¥: Stock solution

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2.2 Justification for the Model Selection Primary cultures of human peripheral lymphocytes freshly collected were preferred because of their stable chromosome number and structure (karyotype) compared with cell lines, and their low and stable background rate of aberrations. In addition, human cells are generally more relevant for risk assessment than rodent cell lines.

2.3 Justification of Dose Level Selection Based on the information provided by the sponsor and according to the ICH guidelines, the standard limit dose of 5 mg/mL was used as the high dose. The test item was dosed at a range of concentrations but was only assessed at the lowest precipitating level. The concentrations were separated by a factor of approximately 2.

The dose levels of the positive controls were selected based on the ITR Laboratories internal validations.

2.4 Study Schedule

Study initiation date: (The date the Study Director signed the study plan)

January 30, 2017

Experimental start date : (Cell initiation date)

January 31, 2017

First treatment: (Dosing date)

February 02, 2017

Experimental completion date: (Last date of data collection from slide reading)

March 01, 2017

Study completion date: Date the Study Director signs the final report

3 TEST ITEM, VEHICLE/NEGATIVE, AND POSITIVE CONTROLS 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 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.

3.3 Positive Controls Cyclophosphamide monohydrate and mitomycin C, which are known from the literature to induce a clear positive response (in presence or absence of S9 isoenzymes-containing fraction, respectively), were used as positive controls.

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4 INFORMATION ON TEST ITEM, VEHICLE/NEGATIVE AND POSITIVE CONTROLS

4.1 Test Item and Vehicle/Negative Controls TEST ITEM* VEHICLE/NEGATIVE CONTROL

Identity: InSea2® Sterile water for injection USP

Description: Fine brown powder Clear liquid

Lot/Batch No.: INS2-006-TAC W6K03A0

Purity: Assumed 100% Not applicable

Expiry Date: 31-05-19 30-11-17/15-02-17 once opened Storage Conditions:

Room temperature Room temperature/2-8°C after opening

Stability: 36 months (unopened) Not applicable

Handling Precautions:

Standard precautions Standard precautions

Supplier: InnoVactiv. Inc Baxter

*Test item Certificate of analysis was included in the report Appendix 4.

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.

4.2 Positive Controls

CYCLOPHOSPHAMIDE MONOHYDRATE MITOMYCIN C

Description White powder Grey Powder

Batch No MKBS0021V SLBN5647V

Retest Date 31-10-17 31-10-19

Storage Conditions Refrigerated (2-8°C) Refrigerated (2-8°C)

Handling Precautions

Carcinogen handled as per SOP H.S 5.0 Carcinogen handled as per SOP H.S 5.0

Supplier Sigma-Aldrich (cat no C0768) Sigma-Aldrich (cat no M0503)

CAS Number 6055-19-2 50-07-7

4.2.1 Positive Controls Formulations Mitomycin C was reconstituted in the vial as supplied with water, diluted and all the lower concentrations were prepared by mixing appropriate amount of intermediate dilution with sterile water.

A Cyclophosphamide stock solution was prepared by mixing appropriate amount of powder in water and all subsequent dilutions were prepared from this stock solution.

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4.2.2 Dose Formulation Analysis of Positive Controls The positive control formulations were not subjected to dose formulation analysis because the biological response of the test system is considered to be the best measure of the quality of the formulations.

4.3 Preparation of Test Item Formulations 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 stock formulation was homogenized at 5000 rpm to reduce the particles to a minimum.

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.

4.4 Analysis of Test Item Formulations The test item formulations were not analysed for achieved concentration, homogeneity and stability.

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

5 EXPERIMENTAL PROCEDURES 5.1 Test System

5.1.1 Blood Sampling A peripheral blood sample was taken from a donor whose lymphocytes were verified for: normal karyotype, level of chromosome aberrations within the historical control data range, suitable response to known mutagens and mitogens (i.e. phytoagglutinin).

The donors met the following criteria:

Inclusions: young between 18 to 35 years old and healthy without any fever.

Exclusion: non-smoking, no viral diseases such as Hepatitis C and human immunodeficiency virus (HIV), no recent exposures to radiations or hazardous chemicals and non-departmental employee.

A donor’s decision to contribute blood or not for this assay may not, under any circumstance, affect his/her employment with ITR.

The blood was collected into blood collecting tubes containing sodium heparin and was held at room temperature for up to 2 hours prior to culture initiation. The 60 mL of blood volume needed was indicated in genetic worksheet 3.0. Blood tubes were labeled appropriately as per ITR procedures.

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The use of human material in this study was approved by an institutional review board (IRB) and the approval document is included in the study report (Appendix 5).

5.1.2 Culture Medium Complete RPMI 1640 medium was supplemented with the following filter-sterilized components: fetal calf serum 10% (v/v), penicillin–streptomycin (100 IU/mL and 100 µg/mL, respectively) and 4 units heparin per mL.

5.1.3 Lymphocyte Culture Phytohemagglutinin (PHA, 2%) was added to whole blood mixed with medium to stimulate lymphocyte division. Aliquots of cell suspension were incubated at approximately 37°C in a humidified atmosphere containing 5% CO2.

5.2 Metabolic Activation System Phenobarbital-5, 6-benzoflavone induced male Sprague-Dawley rat liver fraction (S9) obtained from a commercial supplier was used as a metabolism activation system. The S9 fraction was thawed and mixed at 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 is retained in the raw data.

5.3 General Reagents All the information including identity, lot/batch, and expiry date of the general laboratory reagents used in this study were included in the raw data.

5.4 Route of Administration The test item InSea2®, vehicle/negative and positive control formulations were added directly into the culture media tubes.

5.5 Treatment of Test System Treatment with InSea2®, and positive and negative controls were performed approximately 48 hours after culture initiation.

The test item was evaluated in duplicate over a wide range of dose levels so that analyzable cells were available for at least 3 dose levels for each treatment regime in case of cell toxicity. The positive controls were tested at three dose levels.

In order to reach the final concentration indicated in the experimental designs, InSea2®, vehicle/negative controls were dosed at 500 µL and positive controls were dosed at 50 µL per 5 mL of culture.

Cultures tested in the absence of S9 mix were treated as indicated in the study design then returned to the incubator for either 4 or 21 hours as appropriate. For cultures tested in the presence of S9 mix, 1 mL of culture medium was removed and replaced with 1 mL of

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S9 mix; each culture was then treated as indicated in the study design before being returned to the incubator for 4 hours.

5.6 Medium Change (4-hour Treatment only) After the 4-hour treatment (with or without S9), the culture media was replaced with fresh complete medium and then incubated for a further 17 hours before harvesting of cells in metaphase.

5.7 Harvesting, Metaphase Preparation and Staining Colcemid was added at 0.1 µg/mL to all cultures approximately 2 hours prior to cell harvest. The cultures were centrifuged and cell pellet was re-suspended in a hypotonic solution of 0.075 M KCl and incubated for 12 minutes at ca. 37°C to allow the cells to swell. The cells were then washed 2 times with 5 mL of a fixative solution (methanol: glacial acetic acid, 3:1) and stored refrigerated in fixative before use. After at least overnight fixation, cells were centrifuged again and the cell pellet was re-suspended in the fixative solution at an appropriate density for slide preparation.

The fixed cells were dropped onto at least overnight methanol-washed slides and air-dried before staining. At least 2 slides were prepared from each culture. Slides were washed, stained with 10% Giemsa, rinsed, air-dried, and then mounted with coverslips. Fixed cells not used for slide preparation were discarded after completion of the experimental phase of the study.

6 SLIDE EXAMINATION 6.1 Mitotic Index (MI) Lymphocyte toxicity is normally indicated by a decreased MI compared to the concurrent control group (≤ 50%). The MI was determined by examination of at least 500 cells (if available) from selected treatment groups, i.e. relevant dose levels not showing extreme toxic effects. The relative mitotic index (RMI) for each treated group was calculated as a percentage compared to the concurrent negative control group.

6.2 Selection of Slides for Detailed Examination The slides could not be evaluated at the three highest dose levels due to heavy precipitate and there was no sign of toxicity at these dose levels. Therefore, the highest concentration selected for chromosome aberration evaluation was the lowest precipitating level, 600 µg/mL, along with additional two dose levels from adjacent concentrations.

The slides from the negative control and all dose levels of positive controls, were examined in detail for the presence of different types of chromosome aberration.

6.3 Detailed Examination for Chromosome Aberrations Slides were randomized then encoded to minimize potential operator bias. They were examined by light microscopy, a total of 300 readable metaphases per experimental point were examined for the presence of chromosome aberrations.

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The location (Vernier coordinates) of observed aberrant metaphases was recorded for potential peer review.

Readable metaphases were identified by the following criteria:

Chromosome number between 44 and 48 in a single stage of condensation

Well-spread with minimal overlap of chromosomes and chromosome arms

Chromatids separate with centromere intact

Structure of chromosomes clear and well-defined

The International System for Chromosome Aberration Nomenclature (1995) was followed to designate the observed aberrations. Since the nature of chromosomal and chromatid gaps is uncertain (they may or may not represent true breaks in chromatid structure), these 2 types of aberration were recorded but they were not included in statistical analysis of aberrations. The incidences of numerical types of aberration, such as polyploidy and endoreduplication, were recorded as well.

7 BASES FOR DATA ANALYSIS AND INTERPRETATION OF THE RESULTS

7.1 Statistical Analysis Results from replicate cultures were combined to facilitate interpretation and maximize the power of statistical analysis. Numerical data obtained during the conduct of the study were subjected to calculation of means and standard deviations by dose number for each occasion. These descriptive statistics are reported herein along with all individual numerical and non-numerical results.

The statistical analysis of the cells with structural aberrations per group and per condition was based on a pairwise comparison of the cells with structural aberrations at each dose level of the test item or positive control with the vehicle /negative control group using Chi-Square test.

7.2 Validity of the Assay

The vehicle/negative controls were within or close to the historical control range (< 99% confidence limit).

The positive control produced a significant increase in the incidence of aberrant cells compared with the concurrent control as described in a positive response.

All three experimental conditions were tested.

Adequate number of cells and concentrations were analyzable.

The criteria for the selection of top concentrations were consistent with ICH S2 (R1) and OECD 473 test guidelines.

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7.3 Basis for the Interpretation of the Results

A positive response is indicated by a dose-related and statistically significant (p ≤ 0.01) increase in the incidence of aberrant cells for the treatment group compared with the concurrent control group (p ≤ 0.01); individual and/or group mean values should have exceeded the laboratory historical control range (˃ 99% confidence limit).

A negative result is indicated when the group mean incidences of aberrant metaphase cells for the group treated with the test item are not significantly greater than incidences for the concurrent control group (p > 0.01) and when these values fell within or close to the historical control range.

An equivocal response is suggested when the results do not meet the criteria specified for a positive or negative response.

8 DATA CAPTURE The following computerized systems were used during the conduct of this study:

Statistical Analysis Sigmastat

Version numbers of the systems are held on file at ITR.

9 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.

ITR agrees to notify the Sponsor promptly of any government inquiry about, or proposed inspection of, this study.

10 STANDARD OPERATING PROCEDURES All procedures were performed in accordance with the ITR Standard Operating Procedures and these were kept on file at ITR. Deviations to the ITR Standard Operating Procedures were documented in the raw data.

11 ARCHIVING All data and read slides 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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12 RESULTS AND DISCUSSION 12.1 Formulation Analysis The formulation analysis was not performed in this study.

The positive response obtained with the positive controls indicated that concentrations and formulations were acceptable.

12.2 Microscopic Examination The results of the chromosome aberrations are summarized in Table 1. Results of the individual cultures are presented in Appendix 2.

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Table 1: Summary Results

Treatment (µg/mL) Mean Mitotic

Index Cells

Scored

Structural aberrations / Cell Cells with Aberrations

Mean ±SD Numerical Structural

4 h incubation, -S9 activation 0.9% Saline

0.00 5.3 300 0.00 0.000 0.5 1.0

InSea2®

150 4.6 300 0.00 0.000 0.0 0.0 300 5.0 300 0.00 0.000 0.0 0.0 600 4.1 300 0.00 0.000 0.0 0.5

Mitomycin C 0.10 5.8 300 0.03 0.005 0.0 4.5 0.20 5.2 300 0.04 0.005 0.5 6.5¹ 0.30 3.2 300 0.06 0.005 0.0 8.5*

4 h incubation, +S9 activation 0.9% Saline

0.00 5.8 300 0.00 0.000 0.0 0.0

InSea2® 150 5.7 300 0.00 0.000 0.0 0.0 300 5.3 300 0.00 0.000 0.0 0.0 600 5.5 300 0.00 0.000 0.0 0.0

Cyclophosphamide 4.0 3.0 300 0.05 0.000 0.0 8.0* 8.0 2.9 300 0.05 0.053 0.5 7.5* 12 2.3 300 0.07 0.005 0.0 10.5*

21 h incubation, -S9 activation

0.9% Saline

0.00 4.8 300 0.00 0.000 0.0 0.0

InSea2®

150 4.0 300 0.00 0.000 0.0 0.0

300 3.7 300 0.00 0.000 0.0 0.0

600 4.0 300 0.00 0.000 0.0 0.0

Mitomycin C

0.10 4.0 300 0.05 0.000 0.0 8.0*

0.20 3.2 300 0.07 0.009 0.0 9.5*

0.30 2.7 300 0.08 0.009 0.5 12.0*

SD: Standard deviation * Statistically significant (at least p≤0.01), ¹ The statistical significance p =0.014

12.2.1 Dose Selection There was a heavy precipitate on the slide at the highest three levels in the three regimes, therefore, the slides prepared from these cultures could not be evaluated. Thus, the test item lowest precipitating level was selected for detailed analysis in the three regimes followed by two adjacent dose levels. The metaphases of these doses levels were assessed for any presence of chromosomal damage and reported as recommended in the OECD guidelines.

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All the positive control concentrations along with vehicle were selected for chromosome aberration analysis.

12.2.2 Chromosome Aberrations As shown in Table 1, the results obtained with vehicle/negative control groups were negative and the values fall within or close to the laboratory historical control range (see Appendix 1) for negative and positive historical control results). The results of negative and positive controls were as expected confirming the sensitivity of the test system, the effectiveness of the S9 mix and the validity of the assay.

Cultures treated with the test item, InSea2®, up to the lowest precipitating level did not show any statistically significant increase in the incidence of cells with aberrant chromosomes (p ≥ 0.01).

There were no increases in numerical aberrations in either the 4 hour treatment in the absence and presence of S9 activation, or in the 21 hour treatment in the absence of S9 activation.

13 CONCLUSION Under the conditions used in this study, it is concluded that InSea2® did not induce chromosome damage in human lymphocytes.

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APPENDIX 1 ITR Study No. 55483 1

In vitro Chromosome Aberration Test Using Human Lymphocytes

Laboratory historical control data collected from GLP and non-GLP studies

performed since July 2012 to prior to this study.

Vehicles/Negative Control

Mean incidence of aberrant metaphase cells is 0.14%, standard deviation (SD) 0.27 with

a minimum of 0 and maximum of 1.5.

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Positive Control Data‐Structural aberrations

Positive Control Data-Structural aberrations

4 h incubation without S9 mix

Historical data 0.10 µg/mL 0.20 µg/mL 0.30 µg/mL

Mean 4.4 9.0 14.5

SD 2.41 2.40 3.47

Minimum 2 3 10

Maximum 12 13 21

Number of Experiments

16 16 15

4 h incubation with S9 mix

Historical data 4 µg/mL 8 µg/mL 12 µg/mL

Mean 7.2 10.9 16.6

SD 3.48 4.45 4.47

Minimum 0.5 0.5 6.5

Maximum 13 18 24.5


13 17 16

21 h incubation without S9 mix

Historical data 0.10µg/mL 0.20 µg/mL 0.30 µg/mL

Mean 8.1 13.1 19.6

SD 2.74 3.84 5.67

Minimum 3.5 8.5 11

Maximum 14 21.5 30


16 16 16

SD: Standard deviation The positive controls used in the studies are Cyclophosphamide and Mitomycin C with and without S9 mix, respectively.

22 of 47


FORMULAS

- %AMI = (Number of cells with abnormal metaphases / Total number of cells) per

individual replicate (x100)

- %MI = (Number of cells with normal metaphases / Total number of cells) per individual replicate (x100)

- MI mean = Sum of MI of each individual replicate / Number of replicates

- % RMI = (MI mean of treated cultures / MI mean of vehicle cultures) x100

23 of 47

APPENDIX 2 ITR Study No. 55483 2 Table 1: Mitotic Index Results: 4 Hour Treatment in Absence of S9 Mix

Culture ID

Nucleated cells

Cells with Normal

Metaphase

Cells with Abnormal Metaphase Total of Normal + Abnormal

Metaphases

Total of Abnormal

Metaphases %AMI MI Mean MI % RMI

Polyploidy Endoreduplication

a01 484 28 0 0 512 0 0.0 5.5 5.3 100.0 a02 488 26 0 0 514 0 0.0 5.1 a11 489 32 0 0 521 0 0.0 6.1 5.6 106.2 a12 519 28 0 0 547 0 0.0 5.1 a21 535 21 0 0 556 0 0.0 3.8 4.1 77.6 a22 516 24 0 0 540 0 0.0 4.4 a31 525 26 0 0 551 0 0.0 4.7 4.9 92.4 a32 505 27 0 0 532 0 0.0 5.1 a41 545 25 0 0 570 0 0.0 4.4 4.2 79.0 a42 482 20 0 0 502 0 0.0 4.0 a51 501 23 0 0 524 0 0.0 4.4 4.6 87.0 a52 512 26 0 0 538 0 0.0 4.8 a61 523 27 0 0 550 0 0.0 4.9 5.0 94.5 a62 520 28 0 0 548 0 0.0 5.1 a71 509 21 0 0 530 0 0.0 4.0 4.1 76.8 a72 528 23 0 0 551 0 0.0 4.2 a81 - - - - - - - - Heavy

precipitate Heavy

precipitate a82 - - - - - - - -a91 - - - - - - - - Heavy

precipitate Heavy

precipitate a92 - - - - - - - -a101 - - - - - - - - Heavy

precipitate Heavy

precipitate a102 - - - - - - - -a111 510 35 0 0 545 0 0.0 6.4 5.8 110.3 a112 539 30 0 0 569 0 0.0 5.3 a121 511 28 0 0 539 0 0.0 5.2 5.2 98.0 a122 548 30 0 0 578 0 0.0 5.2 a131 509 17 0 0 526 0 0.0 3.2 3.2 61.3 a132 504 17 0 0 521 0 0.0 3.3

24 of 47

APPENDIX 2 ITR Study No. 55483 3 Table 2: Mitotic Index Results: 4 Hour Treatment in Presence of S9 Mix

Culture ID

Nucleated cells

Cells with Normal

Metaphase

Cells with Abnormal Metaphase

Total of Normal + Abnormal

Metaphases

Total of Abnormal


Polyploidy Endo-

reduplication b01 513 29 0 0 542 0 0.0 5.4 5.8 100.0 b02 498 33 0 0 531 0 0.0 6.2 b11 501 34 0 0 535 0 0.0 6.4 5.3 91.8 b12 490 22 0 0 512 0 0.0 4.3 b21 536 33 0 0 569 0 0.0 5.8 5.5 95.4 b22 504 28 0 0 532 0 0.0 5.3 b31 495 25 0 0 520 0 0.0 4.8 5.3 91.7 b32 485 30 0 0 515 0 0.0 5.8 b41 500 26 0 0 526 0 0.0 4.9 5.6 97.0 b42 520 35 0 0 555 0 0.0 6.3 b51 551 39 0 0 590 0 0.0 6.6 5.7 98.0 b52 580 29 0 0 609 0 0.0 4.8 b61 513 29 0 0 542 0 0.0 5.4 5.3 91.5 b62 504 28 0 0 532 0 0.0 5.3 b71 534 29 0 0 563 0 0.0 5.2 5.5 95.4 b72 557 35 0 0 592 0 0.0 5.9 b81 - - - - - - - - Heavy

precipitate Heavy

precipitate b82 - - - - - - - -b91 - - - - - - - - Heavy

precipitate Heavy

precipitate b92 - - - - - - - -b101 - - - - - - - - Heavy

precipitate Heavy

precipitate b102 - - - - - - - -b111 502 17 0 0 519 0 0.0 3.3 3.0 51.4 b112 506 14 0 0 520 0 0.0 2.7 b121 518 14 0 0 532 0 0.0 2.6 2.9 50.3 b122 513 17 0 0 530 0 0.0 3.2 b131 516 14 0 0 530 0 0.0 2.6 2.3 39.3 b132 513 10 0 0 523 0 0.0 1.9

25 of 47

APPENDIX 2 ITR Study No. 55483 4 Table 3: Mitotic Index Results: 21 Hour Treatment in Absence of S9 Mix

Culture ID

Nucleated cells

Cells with Normal

Metaphase

Cells with Abnormal Metaphase

Total of Normal + Abnormal

Metaphases

Total of Abnormal


Polyploidy Endo-

reduplication c01 493 28 0 0 521 0 0.0 5.4 4.8 100.0 c02 484 21 0 0 505 0 0.0 4.2 c11 491 22 0 0 513 0 0.0 4.3 4.8 100.7 c12 528 30 0 0 558 0 0.0 5.4 c21 486 18 0 0 504 0 0.0 3.6 3.7 77.0 c22 528 21 0 0 549 0 0.0 3.8 c31 498 28 0 0 526 0 0.0 5.3 4.3 88.9 c32 512 17 0 0 529 0 0.0 3.2 c41 479 22 0 0 501 0 0.0 4.4 4.5 93.8 c42 537 26 0 0 563 0 0.0 4.6 c51 533 23 0 0 556 0 0.0 4.1 4.0 82.7 c52 531 21 0 0 552 0 0.0 3.8 c61 499 17 0 0 516 0 0.0 3.3 3.7 77.3 c62 534 23 0 0 557 0 0.0 4.1 c71 513 24 0 0 537 0 0.0 4.5 4.0 82.5 c72 503 18 0 0 521 0 0.0 3.5 c81 - - - - - - - - Heavy

precipitate Heavy

precipitate c82 - - - - - - - -c91 - - - - - - - - Heavy

precipitate Heavy

precipitate c92 - - - - - - - -c101 - - - - - - - - Heavy

precipitate Heavy

precipitate c102 - - - - - - - -c111 496 19 0 0 515 0 0.0 3.7 4.0 83.7 c112 506 23 0 0 529 0 0.0 4.3 c121 502 17 0 0 519 0 0.0 3.3 3.2 67.5 c122 484 16 0 0 500 0 0.0 3.2 c131 508 13 0 0 521 0 0.0 2.5 2.7 56.8 c132 492 15 0 0 507 0 0.0 3.0

26 of 47


Final Report

ABBREVIATIONS page 1

Poly polyploidy cs chromosome

Endo endoreduplication ctg chromatid gap

ctb chromatid break csg chromosome gap

cte chromatid exchange cse chromosome exchange

csb chromosome break mult >8 aberrations

pvz pulverised FORMULAS

- % RMI = (MI mean of treated cultures / MI mean of vehicle cultures) x100

- Total number of structural aberrations / culture = Sum of all types of structural aberrations

- Total number of numerical aberrations / culture = Sum of all types of numerical aberrations

- Average number of structural aberrations / cell = Total number of structural type aberrations per individual replicate / Total number of cells scored

27 of 47


Final Report

Table 1: Individual Results for Regimen 4 Hours, -S9

Culture ID

Treatment (g/mL) MI %

RMI Cells

Scored

% Aberrant Cells (≥1 aberration/cell)

Total Number of Numerical Aberrations

Total Number of Structural Aberrations Total

Number of structural

aberrations / culture

Average Structural

aberrations / cell Struc-

tural Num-erical Poly Endo

Chromatid-type aberrations

Chromosome-type aberrations Others

Gaps

ctb cte csb cse mult pvz

cell cs ctg csg

sterile water

a01 0.00

5.5 100.0

150 1 1 1 0 0 1 0 0 0 0 0 0 0 1 0.001

a02 5.1 150 1 0 0 0 0 0 1 0 0 0 0 0 0 1 0.001

InSea2®

a51 150

4.4 87.0

150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

a52 4.8 150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

a61 300

4.9 94.5

150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

a62 5.1 150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

a71 600

4.0 76.8

150 1 0 0 0 0 0 1 0 0 0 0 0 0 1 0.007

a72 4.2 150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

Mitomycin C

a111 0.10

6.4 110.3

150 4 0 0 0 3 0 1 0 0 0 0 0 0 4 0.027

a112 5.3 150 5 0 0 0 2 0 3 0 0 0 0 1 1 5 0.033

a121 0.20

5.2 98.0

150 6 1 1 0 3 2 1 0 0 0 0 0 0 6 0.040

a122 5.2 150 7 0 0 0 4 1 2 0 0 0 0 0 1 7 0.047

a131 0.30

3.2 61.3

150 9 0 0 0 3 2 4 0 0 0 0 0 0 9 0.060

a132 3.3 150 8 0 0 0 1 4 3 0 0 0 0 0 0 8 0.053 Refer to page 127 for clarifications of formulas and abbreviations.

28 of 47


Final Report

Table 2: Individual Results for Regimen 4 Hours, +S9

Culture ID

Treatment (g/mL) MI % RMI Cells

Scored


Total Number of Numerical

Aberrations Total Number of Structural Aberrations

Total Number of structural


Average Structural

aberrations / Cell Struc-


Chromatid-type aberrations

Chromosome-type aberrations Others

Gaps


cell cs ctg csg

sterile water

b01 0.00

5.4 100.0

150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

b02 6.2 150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

InSea2®

b51 150

6.6 98.0

150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

b52 4.8 150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

b61 300

5.4 91.5

150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

b62 5.3 150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

b71 600

5.2 95.4

150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

b72 5.9 150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

Cyclophosphamide

b111 4.0

3.3 51.4

150 8 0 0 0 3 2 3 0 0 0 0 1 1 8 0.053

b112 2.7 150 8 0 0 0 3 3 2 0 0 0 0 0 0 8 0.053

b121 8.0

2.6 50.3

150 9 0 0 0 3 4 2 0 0 0 0 1 0 9 0.060

b122 3.2 150 6 1 1 0 4 1 2 0 0 0 0 0 0 7 0.047

b131 12

2.6 39.3

150 11 0 0 0 8 2 1 0 0 0 0 0 0 11 0.073

b132 1.9 150 10 0 0 0 3 3 4 0 0 0 0 0 0 10 0.067

Refer to page 127 for clarifications of formulas and abbreviations.

29 of 47


Final Report

Table 3: Individual Results for Regimen 21 Hours, -S9

Culture ID

Treatment (g/mL) MI % RMI Cells

Scored


Total Number of Numerical

Aberrations Total Number of Structural Aberrations

Total Number of structural


Average Structural

aberrations / Cell Struc-


Chromatid-type

aberrations Chromosome-

type aberrations Others Gaps


cell cs ctg csg

sterile water

c01 0.00

5.4 100.0

150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

c02 4.2 150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

InSea2®

c51 150

4.1 82.7

150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

c52 3.8 150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

c61 300

3.3 77.3

150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

c62 4.1 150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

c71 600

4.5 82.5

150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

c72 3.5 150 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.000

Mitomycin C c111

0.10 3.7

83.7 150 8 0 0 0 3 1 4 0 0 0 0 0 0 8 0.053

c112 4.3 150 8 0 0 0 3 0 5 0 0 0 0 0 0 8 0.053

c121 0.20

3.3 67.5

150 9 0 0 0 2 3 4 0 0 0 0 0 0 9 0.060

c122 3.2 150 10 0 0 0 1 5 5 0 0 0 0 0 0 11 0.073

c131 0.30

2.5 56.8

150 11 1 1 0 3 3 5 0 0 0 0 0 0 11 0.073

c132 3.0 150 13 0 0 0 7 0 6 0 0 0 0 0 1 13 0.087

Refer to page 127 for clarifications of formulas and abbreviations.

30 of 47

APPENDIX 4 ITR Study No. 55483

31 of 47

Telephone 514.337.0442 • Facsimile 514.336.1142 • www.veritasIRB.com

A member of the ethica Clinical Research Inc. group of companies.

8555 Transcanada Hwy., Suite 201, Saint-Laurent (Montreal), Quebec H4S 1Z6

Page 1 of 1

Research ethics simplified™

INDEPENDENT REVIEW BOARD COMMUNICATION

IRB Review Date: January 30, 2017

IRB Tracking Number: 15058-11:12:4325-01-2017

Sponsor: ITR Laboratories Inc.

Study Name: In Vitro Mammalian Chromosome Aberration Test Using Human Peripheral Blood Lymphocytes

Sub-Study Number: 55483 Principal Investigator: Ms. Farida Merah Study Expiration Date: August 31, 2017

RE: Changes to Research – Sub-Study Protocol – Unconditionally Approved

The following document was reviewed and acknowledged by the Independent Review Board Chair in compliance with normative documents governing research with humans:

! ‘InSea2®: In Vitro Mammalian Chromosome Aberration Test Using Human Peripheral Blood

Lymphocytes’ – Sub-Study Protocol 55438, dated January 25, 2017.

NOTE ! The purpose and scope of the Sub-Study mentioned above has been approved. As per the Independent

Review Board Communication dated November 20, 2015, the above-referenced Sub-Study may commence.

Should you have any questions or require clarification of any issues, please do not hesitate to contact us.

Thank you.

Veritas IRB Inc.

Verified by Kevin Emery on 30-Jan-2017 @ 11:39

APPENDIX 5 ITR Study No. 55483

32 of 47

ITR CANADA

CONFIDENTIAL

FINAL STUDY PLAN

InSea2®: In Vitro Mammalian Chromosome Aberration Test

Using Human Peripheral Blood Lymphocytes

ITR Study Number: 55483

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: January 30, 2017

Page Number: 1 of 15

APPENDIX 6

33 of 47

ITR Study No. 55483

Page 2 of 15

Final Study plan January 30, 2017

PREFACE PAGE

Study Title: InSea2®: In Vitro Mammalian

Chromosome Aberration Test Using

Human Peripheral Blood Lymphocytes

Study GLP Status: GLP

Study Director (SD): Farida Merah, MSc

Genetic Toxicology Department

ITR Laboratories Canada Inc.

19601 Clark Graham blvd

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.

APPENDIX 6

34 of 47

mailto:[email protected]�

ITR Study No. 55483

Page 3 of 15


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 ................................................ 6 3 Experimental Procedures ........................................................................................ 7 4 Slide Examination ................................................................................................... 9 5 Data Analysis and Interpretation of Results ......................................................... 11 6 Data Capture ......................................................................................................... 12 7 Changes to the Study Plan .................................................................................... 12 8 Reporting............................................................................................................... 12 9 GLP Compliance ................................................................................................... 14 10 Quality Assurance ................................................................................................. 14 11 Standard Operating Procedures............................................................................. 14 12 Archiving .............................................................................................................. 14 13 Study Plan Approval Page .................................................................................... 15

APPENDIX 6

35 of 47

ITR Study No. 55483

Page 4 of 15


1 Introduction

The purpose of the study is to evaluate the genotoxicity potential of the test item InSea2®

using the in vitro chromosome aberration test in human peripheral blood lymphocytes.

The study will be performed in compliance with GLP regulations.

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

material for preparation of this study plan.

OECD Guideline 473. OECD Guideline for Testing of Chemicals – In vitro

Mammalian chromosome aberration test.

EPA Health Effects Test Guideline OPPTS 870.5375: In vitro Mammalian

chromosome aberration 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 Harmonized Tripartite Guideline M3 (R2). Nonclinical Safety Studies for the

Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals.

1.1 Experimental Design

Human peripheral blood lymphocytes will be treated as per the table below:

Dose

Number Material

Formulation

conc.

(µg/mL)

Final

conc.

(µg/mL)

Dosing

volume

(µL)

Culture Number ID

4 Hours

(-S9)

4 Hours

(+S9)

21 Hours

(-S9)

- Vehicle - 500 a01, a02 b01, b02 c01, c02

1

2

3

4

5

6

7

8

9

10

Test Item 90

190

380

750

1500

3000

6000

12500

25000

50000¥

9

19

38

75

150

300

600

1250

2500

5000*

500

500

500

500

500

500

500

500

500

500

a11, a12

a21, a22

a31, a32

a41, a42

a51, a52

a61, a62

a71, a72

a81, a82

a91, a92

a101, a102

b11, b12

b21, b22

b31, b32

b41, b42

b51, b52

b61, b62

b71, b72

b81, b82

b91, b92

b101, b102

c11, c12

c21, c22

c31, c32

c41, c42

c51, c52

c61, c62

c71, c72

c81, c82

c91, c92

c101, c102

1

2

3

Mitomycin

C

10.0

20.0

30.0

0.10

0.20

0.30

50

50

50

a111, a112

a121, a122

a131, a132

c111, c112

c121, c122

c131, c132

1

2

3

Cyclophosp

hamide

400

800

1200

4.0

8.0

12

50

50

50

b111, b112

b121, b122

b131, b132

* x (high level) as per OECD 473 and FDA Redbook IVC1 Guidelines = 5 mg/mL. 5 L/mL or 10 mM, whichever is

the lowest (Chemicals). The 21 hour incubation ('confirmatory phase') is required by regulatory authorities where

the initial phase does not show any indication of genotoxicity for the test item; however, it is routinely performed at

the same time as the initial phase to minimize potential delays in performance of the study.

Conc. = Concentration

¥: Stock solution

APPENDIX 6

36 of 47

ITR Study No. 55483

Page 5 of 15


1.2 Justification for Model and Dose Level Selection

1.2.1 Justification for Model Selection

Primary cultures of human peripheral lymphocytes freshly collected are preferred

because of their stable chromosome number and structure (karyotype) compared with cell

lines, and their low and stable background rate of aberrations. In addition, human cells

are generally the most relevant for risk assessment compared to rodent cell lines.

1.2.2 Justification of Dose Level Selection

The test item, which is a dietary supplement, will be dosed at a range of concentrations

but will only be assessed at the highest levels below the toxic level or, if non-toxic, at

levels up to the standard limit of 5 mg/mL, 5 µL/mL or 0.01 M whichever is the lowest as

required by the OECD 473 sand US FDA Redbook guidelines. Compounds with limited

aqueous solubility are tested up to a level showing visible precipitation in the culture

medium. The concentrations are separated by a factor of approximately 2.

The dose levels of the positive controls were selected based on the ITR Laboratories

internal validations.

1.3 Proposed Study Schedule

Study initiation date:

Date the Study Director signs the study plan

Experimental start date :

(Cell initiation date) January 31, 2017

First treatment:

(Dosing date) February 02, 2017

Experimental completion date:

(Last date of data collection from the slide reading) February 20, 2017

Audited draft report:

April 12, 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).

APPENDIX 6

37 of 47

ITR Study No. 55483

Page 6 of 15


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 a dietary supplement.

2.2 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.

2.3 Positive Controls

Cyclophosphamide and Mitomycin C are positive controls known from the literature to

induce a clear positive response in the presence and absence of S9 isoenzymes-containing

fraction, respectively.

2.4 Information of Test, Vehicle/Negative and Positive Control Items

TEST ITEM* VEHICLE/NEGATIVE CONTROL

Identity: InSea2® Sterile water for injection USP

Description: Fine brown powder Clear liquid

Batch 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 precautions Standard precautions

Supplier: InnoVactiv. Inc To be documented in raw data and report

*A certificate of analysis was supplied for inclusion in the final report.

POSITIVE CONTROL -1 POSITIVE CONTROL -2

Identity Cyclophosphamide monohydrate Mitomycin C

Batch/Lot No To be documented in raw data and

report To be documented in raw data and report

Expiry 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

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

CAS number 6055-19-2 50-07-7

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.

APPENDIX 6

38 of 47

ITR Study No. 55483

Page 7 of 15


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

material or may preclude some personnel from contact with the material.

2.5 Preparation of Positive Controls Formulations

For Mitomycin C, it will be reconstituted in the vial as supplied with water, diluted and

all the lower concentrations will be prepared by mixing appropriate amount of

intermediate dilution with sterile water.

For Cyclophosphamide, a stock solution will be prepared by mixing appropriate amount

of powder in water and all subsequent dilutions will be prepared from this stock solution.

The positive control formulations will not be subjected to analysis for concentration and

stability because the biological response of the test system is considered to be the best

measure of the quality of the formulations.

2.6 Preparation of Test Item Formulation

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(s) 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

3.1.1 Blood Sampling

A peripheral blood sample will be taken from a donor for whom its lymphocytes were

verified for: normal karyotype, level of chromosome aberrations within the historical

control data range, suitable response to known mutagens and mitogens (i.e.

phytoagglutinin). It is also possible to take blood from 2 to 3 donors concurrently for one

study to account for natural variability between individuals.

APPENDIX 6

39 of 47

ITR Study No. 55483

Page 8 of 15


The donors should meet the following criteria:

Inclusions: young between 18 to 35 years old and healthy without any fever.

Exclusion: non-smoking, no viral diseases such as Hepatitis C and human

immunodeficiency virus (HIV), no recent exposures to radiations or hazardous chemicals

and non-departmental employee.

A donor’s decision to contribute blood or not for this assay may not, under any

circumstance, affect his/her employment with ITR.

The blood will be collected into blood collecting tubes containing sodium heparin and

will be held at room temperature for up to 2 hours prior to culture initiation. The volume

of blood needed will be indicated in genetic worksheet 3.0 and may vary from 20 to

60 mL. Blood tubes will be labeled appropriately as per ITR procedures.

3.1.2 Culture Medium

Complete RPMI 1640 medium will be supplemented with the following filter-sterilized

components: fetal calf serum 10% (v/v), Penicillin–Streptomycin (100 IU/mL and

100 µg/mL, respectively) and 4 units heparin per mL.

3.1.3 Lymphocyte Culture

Phytohemagglutinin (PHA, 2%) will be added to whole blood mixed with the culture

medium to stimulate lymphocyte division. Aliquots of cell suspension will be incubated

at approximately 37°C in a humidified atmosphere containing 5% CO2.

3.2 Metabolic Activation System

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

will be used as a metabolism activation system and is obtained from commercial supplier.

Before use, the S9 fraction is thawed and mixed at 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’s certificate of analysis will be

retained in the study file.

3.3 General Reagents

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

the study will be documented in the raw data.

3.4 Route of Administration

test item, positive and vehicle/negative control formulations will be added directly into

the media in culture tubes.

3.5 Treatment of Test System

Treatments with test item, the positive and vehicle/negative controls will be performed

approximately 48 hours after culture initiation.

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The test item and negative control will be dosed at 200 µL per 5 mL of complete culture

media, while the positive controls will be dosed at 50 µL per 5 mL of culture media.

The test item will be tested in duplicate over a wide range of dose levels so that

analyzable cells will be available for at least 3 dose levels for each treatment regime in

case of cell toxicity. The positive controls will be tested at three dose levels. Where the

dosing volume is greater than 50 L/ mL, medium will be removed from the culture to

ensure achievement of the nominal final concentration.

Cultures tested in the absence of S9 mix will be treated as indicated in the study design

then returned to the incubator for either 4 or 21 hours as appropriate. For cultures tested

in the presence of S9 mix, 1 mL culture medium will be removed and replaced with 1 mL

of S9 mix; each culture will then be treated as indicated in the study design before being

returned to the incubator for 4 hours.

3.6 Medium Change (4-hour Treatment only)

After the 4-hour treatment (with or without S9), the culture media will be replaced with

fresh complete medium then incubated for a further 17 hours before metaphase

harvesting.

3.7 Harvesting, Metaphase Preparation and Staining

Colcemid (or equivalent) will be added to all cultures to stop the cell division and the

cells will be harvested approximately two hours later. The cell tubes will be centrifuged

and the pellet will be re-suspended in hypotonic solution and incubated at ca. 37°C to

allow the cells to swell. The cells will be fixed with a mixture of acetic acid and methanol

solution, treated with 2 changes of fixative and stored for at least overnight. On the slide

preparation day, the cell tubes will be centrifuged again and the cell density adjusted

visually by adding a few drops of fixative.

The fixed cells will be dropped onto overnight methanol-washed slides and air-dried

before staining. At least two slides will be prepared from non-toxic cultures. Fixed cells

not used for slide preparation will be discarded after completion of the experimental

phase of study. Slides will be washed, stained with Giemsa, rinsed, air-dried then

mounted with coverslips.

4 Slide Examination

4.1 Mitotic Index (MI)

The MI will be determined by examination of at least 500 cells (if available) from

selected treatment groups, i.e. relevant dose levels not showing extreme toxic effects.

Lymphocyte toxicity is normally indicated by a decreased MI compared to the concurrent

control group (≤ 50%). The relative mitotic index (RMI) for each treated group will be

calculated as a percentage compared to the concurrent vehicle/negative control group.

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4.2 Selection of Slides for Detailed Examination

Routinely, slides selected for chromosome aberrations examination are chosen based on

the following cases:

When the test item is cytotoxic, the highest concentration will be selected based on the

relative mitotic index. The highest concentration should aim to achieve 55 ± 5% of RMI

when the test item causes reduction in MI (45 ± 5% compared to concurrent negative

control).

When the test item is poorly soluble in culture media and not cytotoxic at concentrations

lower than the lowest insoluble concentration, the highest concentration to be analyzed

will be the one producing turbidity or visible precipitate at the end of the treatment. Even

if toxicity occurs above the lowest insoluble concentration, the lowest insoluble (turbid or

visible precipitate) concentration will be selected as high dose. However, the precipitate

should not interfere with the scoring.

If there is no precipitate or cytotoxicity, the highest test concentration should correspond

to the standard limit 5 mg/mL, 5 μL/mL or 10 mM, whichever is the lowest.

On occasion the treatment procedure may have a clear effect on the quality of metaphases

or cause an extreme reduction in the absolute number of metaphases, in which case a

different test maximal dose for examination may be justified.

At least two additional lower dose levels of the test item will be assessed. Usually these

are the two adjacent lower dose levels, but this number may be extended to cover range

of concentrations showing toxicity, little and no toxicity.

In addition, at least one non-toxic dose levels of each positive and for each regime will be

selected for examination from each of the three treatment regimen/phases along with

negative controls.

4.3 Detailed Examination for Chromosome Aberrations

Slides selected for examination will be randomized then encoded to minimize potential

operator bias. They will be examined by light microscopy, and (where practical) a total

of 300 readable metaphases per experimental point will be examined for the presence of

chromosome aberrations.

The location (Vernier reading) of observed aberrant metaphases will be recorded for

potential peer review.

Readable metaphases are identified by the following criteria:

chromosome number between 44 and 48 in a single stage of condensation

well-spread with minimal overlap of chromosomes and chromosome arms

chromatids separate with centromere intact

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structure of chromosomes clear and well-defined

The International System for Chromosome Aberration Nomenclature (1995) will be

followed to designate the observed aberrations. Since the nature of chromosomal and

chromatid gaps is uncertain (they may or may not represent true breaks in chromatid

structure), these two types of aberration will be recorded but will not be included in

statistical analysis of aberrations. The incidences of numerical types of aberration, such

as polyploidy and endoreduplication, will be recorded.

As required by OECD guideline 473, obvious treatment-related increases in the incidence

of polyploidy (if present) will be described in the report.

5 Data Analysis and Interpretation of Results

5.1 Laboratory proficiency

Laboratory proficiency is demonstrated by accumulated historical control data which will

be included in the report.

5.2 Statistical Analysis

Results from replicate cultures will be combined to facilitate interpretation and maximize

the power of statistical analysis. Numerical data obtained during the conduct of the study,

will be subjected to calculation of group means and standard deviations. These

descriptive statistics will be reported in the final report along with all individual

numerical and non-numerical results.

The statistical analysis of the cells with structural aberrations per group and per condition

will be based on a pairwise comparison of the cells with structural aberrations at each

dose level of the test item with the Vehicle/negative control group using the Fisher’s

Exact Test (or the X2 / Chi-Square test ) using SigmaStat v.3.1.

5.3 Validity of the Assay

The vehicle/negative control results should lie within or close to the historical

control range (< 99% confidence limit) and comparable to the untreated results.

The positive control should produce a significant increase in the incidence of

aberrant cells compared with the concurrent control as described in a positive

response.

All three experimental conditions are tested.

Adequate number of cells and concentrations are analyzable.

The criteria for the selection of top concentrations are consistent with those

described in section 4.2.

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5.4 Basis for Interpretation of the Results:

The following criteria will be used in the interpretation of the results. However, the

biological relevance will be considered first and the conclusion will be based on scientific

judgment especially in case of equivocal results.

A positive response is normally indicated by a statistically significant

(dose-related, if applicable) increase in the incidence of aberrant cells for the

treatment group compared with the concurrent control group (p ≤ 0.01);

individual and/or group mean values should exceed the laboratory historical

control range (>99% confidence limit).

A negative result is indicated where group mean incidences of aberrant metaphase

cells for the group treated with the test item are not significantly greater than

incidences for the concurrent control group (p > 0.01) and where these values fall

within or close to the historical control range.

An equivocal response is obtained when the results do not meet the criteria

specified for a positive or negative response. Additional slide reading or

experimental work may be required by the Study Director to clarify results and

the activity, if any, will be documented in a study plan amendment.

6 Data Capture

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

Statistical Analysis Sigmastat

Version numbers of the systems will be held on file at ITR.

7 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.

8 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.

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

the results and will include, but not limited to:

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.

Cells: type and source of cells; karyotype features (modal number of

chromosomes) and suitability of the cell type used; information on cell cycle

length; sex of blood donors, mitogen used.

Test conditions: identity of metaphase arresting substance, its concentration and

duration of cell exposure; rationale for selection of concentrations and number of

cultures including, e.g. cytotoxicity data and solubility limitations, if available;

composition of media, CO2 concentration if applicable; concentration of test item;

volume of vehicle and test item added; incubation temperature; incubation time;

duration of treatment; type and composition of metabolic activation system,

including acceptability criteria; positive and vehicle/negative controls; methods of

slide preparation; criteria for scoring aberrations; number of metaphases analyzed;

methods for the measurements of toxicity; criteria for considering studies as

positive, negative or equivocal.

Results: signs of toxicity, e.g. mitotic index; signs of precipitation; data on pH

and osmolality of the treatment medium, if determined; definition for aberrations,

including gaps; number of cells with chromosome aberrations and type of

chromosome aberrations (structural and numerical) given separately for each

treated and control culture; dose-response relationship, where possible; statistical

analyses, if any; concurrent negative (solvent/vehicle) and positive control data;

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

and standard deviations.

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 scanned PDF version of the final report will be dispatched at the request of the Sponsor

and will contain, to the best of our knowledge, a full copy of the available information

presented in the original and verified hard copy of the final report.

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9 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

following exceptions:

-The Test Item formulations will not be analysed for achieved concentrations, stability

and homogeneity.

-It was not clearly stated that the Test Item was characterized as per GLP guidelines.

10 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.

11 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.

12 Archiving

All data and read slides 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.

APPENDIX 6

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13 Study Plan Approval Page

This study plan has been approved by:

4i~~~~ Study Director ITR Laboratories Canada Inc

\.. ~~'.I GinetteS Senior Vice ident ITR Laboratories Canada Inc

This study plaD bas been peer reviewed by:

Gabriel Marceau, PhD Scientist, Immunology ITR Laboratories Canada Inc.

JTR Srudy No. 55483 Pagcl50rl 5

3D.hlr-,

::)~ = JO 'J.O/'l-Date ;

This study plan bu been reviewed by the Qua)jty Assurance Depar1mCllt:

Joanne T ~Sc (Hons), MRQA. RQAP·GLP Quality Assurance Representative ITR Laboratories Canada Inc

Approved on bej~o( the S..£9Dsor by:

/.

,-,,~Vactiv, Inc.

h bru..""b L) 20 \ 7 Dale

January 30, 2017

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