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ITR CANADA

CONFIDENTIAL

FINAL REPORT

InSea2®: In vivo Rat Micronucleus Test Using Peripheral Blood Reticulocytes

ITR Study Number: 55484

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.: 55484 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 23,2017 and was completed on June 16, 2017.

Study Director: ~~L aridâ Merah, MSc

Study Director, ITR Laboratories Canada Inc.

Date

Reviewer: jUPtL III tt/Il Date sen Z aryan, PhD

Scien st, Genetic Toxicology ITR Laboratories Canada Inc.

ITR Management Representative: Ginett

Senio ice President, Facility Management ITR Laboratories Canada Inc.

Final Report June 16,2017 20f65


Final Report June 16, 2017

PERSONNEL

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

Toxicology Operations: Adrian Bocan, Manager

Clinical Veterinarian: Pini Zvionow

Pharmacy: Rogy Markose, Manager

Genetic Toxicology: Alicja Golos, Laboratory Services Manager

Flow cytometry: Gabriel Marceau, Immunology Scientist

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 55484 performed at ITR Leborarories 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 Sludl: Plon ond AmendmenlS January 19,2017 Study plan January 19,2017 January 20,2017 Study plan (Post QA) January 20, 2017 January 24, 2017 Study plan amendment 1 January 24,2017 In-Life In§lu~£ti!!ni January 25, 2017 Dosing January 26, 2017 February 02, 2017 Flow cytometry analysis February 03,2017 Data and R~D!!rl March 20,21,2017 Data and report March 21,2017 March 14, 15,21,22,2017 Data March 22, 2017 March 14 to 16,21,22, Report March 23, 2017 2017 March 29,2017 Report March 29,2017 March 31, 2017 Report March31,2017 May 18,2017 Report May 18,2017 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 4of65



TABLE OF CONTENTS

Section Page

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

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

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

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

INDEX TO TABLE ........................................................................................................... 7

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

1 Summary ................................................................................................................ 8

2 Introduction .......................................................................................................... 10

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

3 Justification Of Model, Species, Dose Level And Route Of Administration ...... 11

3.1 Justification of Model and Species Selection ...................................................... 11

3.2 Justification of Route of Administration Selection .............................................. 11

3.3 Justification of Dose Level Selection .................................................................. 11

3.4 Study Schedule..................................................................................................... 11

4 Test, Negative/Vehicle Control And Positive Control Items ............................... 11

4.1 Negative Control .................................................................................................. 12

4.2 Positive Control ................................................................................................... 12

4.3 Information on Test, Negative/Vehicle and Positive Control Items .................... 12

4.4 Preparation and Stability of the Positive Control Formulation ............................ 12

4.5 Preparation of Test Item Formulation .................................................................. 13

4.6 Analysis of Test Item Formulations ..................................................................... 13

4.7 Archive Sampling ................................................................................................ 13

5 Experimental Procedures ..................................................................................... 13

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

5.2 Animal Care Committee ...................................................................................... 13

5.3 Health Status ........................................................................................................ 14

5.4 Housing ................................................................................................................ 14

5.5 Identification ........................................................................................................ 14

5.6 Room Environment .............................................................................................. 14

5.7 Diet/Water ............................................................................................................ 14

5.8 Environmental Enrichment .................................................................................. 14

5.9 Acclimation .......................................................................................................... 15

5.10 Allocation to Study Groups ................................................................................. 15

5.11 Animal Replacement ............................................................................................ 15

5.12 Administration of the Test and Negative/Vehicle Control Items ........................ 15

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6 In-Life Observations ............................................................................................ 15

6.1 Mortality .............................................................................................................. 15

6.2 Clinical Observations ........................................................................................... 15

6.3 Body Weights ....................................................................................................... 15

7 Collection And Processing of Peripheral Blood Erythrocytes ............................. 16

7.1 Peripheral Blood Collection ................................................................................. 16

7.2 Flow Cytometry ................................................................................................... 16

8 Data Capture ........................................................................................................ 16

9 Data Presentation ................................................................................................. 17

10 Data Analysis And Interpretation of Results ....................................................... 17

10.1 Laboratory Proficiency ........................................................................................ 17

10.2 Statistical Analysis ............................................................................................... 17

10.3 Validity of the Assay ........................................................................................... 17

10.4 Basis for Interpretation of the Results ................................................................. 18

11 Quality Assurance ................................................................................................ 18

12 Standard Operating Procedures ............................................................................ 18

13 Archiving ............................................................................................................. 18

14 Results .................................................................................................................. 19

14.1 Analysis of Achieved Concentration ................................................................... 19

14.2 Mortality .............................................................................................................. 19

14.3 Clinical Signs ....................................................................................................... 19

14.4 Body Weight ........................................................................................................ 19

14.5 Flow Cytometry Results ...................................................................................... 19

14.6 Bone Marrow Toxicity / Proportion of Immature Erythrocytes (RET) ............... 19

14.7 Micronucleated Immature Erythrocytes (MN-RET) ........................................... 19

15 CONCLUSION .................................................................................................... 20

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INDEX TO TABLE

Table Description Page

Table 1 Proportion of Immature Erythrocytes and Micronucleated Erythrocytes ............................................................................................. 20

INDEX TO APPENDICES

Appendix Description Page

Appendix 1 Body Weights ........................................................................................... 21

Appendix 2 Flow Cytometry Immunology Report ...................................................... 23

Appendix 3 Laboratory Historical Data ...................................................................... 41

Appendix 4 Certificate of Analysis ............................................................................. 43

Appendix 5 Final Study Plan and Amendment .......................................................... 44

Appendix 6 Minor Deviations ..................................................................................... 65

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1 SUMMARY The purpose of the study was to evaluate the potential cytogenetic damage that could be induced by the test item InSea2® using the in vivo micronucleus test with erythrocytes collected from rat peripheral blood.

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.

During this study, 5 groups with 5 animals per group/sex, except group 5 which had 3 animals/sex, were treated once as described in the table below. Negative control animals received only water while the test item treated animals were treated with one of the three dose levels of the test item, InSea2® (at 2000, 1000 or 500 mg/kg). The vehicle and the test item were administered by oral gavage (p.o) at 20 mL/kg. Animals of Group 5 were treated only once with the positive control, Cyclophosphamide (20 mg/kg), administered by intra-peritoneal route (i.p).

Groups Treatment

Dose Level

(mg/kg)

Dose Concentration

(mg/mL)

Dose Volume(mL/kg) Route

Sampling Time

(hours)

No. of Animals

Males Females

1 Vehicle/Negative control 0 - 20 p.o 48, 72 5 5

2 test item – Low dose 500 25 20 p.o 48 5 5

3 test item – Mid dose 1000 50 20 p.o 48 5 5

4 test item – High dose 2000 100 20 p.o 48, 72 5 5

5 Cyclophosphamide/

positive control 20 2 10 i.p 48 3 3

The animals were treated up to the standard limit dose 2000 mg/kg/day of InSea2®, which is the highest dose recommended by ICH S2 R1 and OECD 474 guidelines.

Parameters monitored during this study included daily mortality, detailed clinical signs (at arrival and on the day of dosing between 1 to 4 hours post dosing), cage side observations for the rest of days (post dosing and on day before dosing) and body weight on arrival, randomization, and one day before dosing and at euthanasia.

The peripheral blood samples were collected from the animals at termination: Negative control, test item and positive controls at 48 hours ± 30 minutes post dose. A second blood sample collected from the test item high dose and negative control at 72 hours ± 30 minutes post dose.

There was no change in animal health status following a single treatment of test item InSea2®, at all tested doses.

Where practical, at least 4000 immature erythrocytes per animal were examined for the presence of micronuclei indicative of chromosome damage. In addition, the proportion of

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immature erythrocytes was assessed for each animal as a measure of potential bone marrow toxicity.

Animals treated with InSea2® showed no decrease in the proportion of immature erythrocyte (RET) in the high dose group 4 (2000 mg/kg) at both sampling time 48 and 72 hours post treatment. There are no statistically significant changes in the incidence of micronucleated immature erythrocytes (MN-RET) in all test item treated groups (2-4) in both sampling time and gender.

The positive control, cyclophosphamide induced highly significant increases in the incidence of micronucleated immature erythrocytes. The results of the negative and positive controls were as expected confirming the sensitivity of the test system and the validity of the assay.

In conclusion, under the conditions used in this study, the test item InSea2® did not induce any chromosomal damage in erythrocytes collected from rats’ peripheral blood.

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2 INTRODUCTION The purpose of the study was to evaluate the potential cytogenetic damage that could be induced by the test item InSea2® using the in vivo micronucleus test on reticulocytes collected from rat peripheral blood.

Regulatory test guidelines from the OECD, EPA, FDA and ICH were used as reference material for preparation of this study.

OECD Guideline 474. OECD Guideline for Testing of Chemicals –Mammalian erythrocyte micronucleus test.

EPA Health Effects Test Guideline OPPTS 870.5395: Mammalian erythrocyte micronucleus 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. Short-Term Tests for Genetic Toxicity.

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

2.1 Experimental Design During this study, 5 groups with 5 animals per group/sex, except group 5 which had 3 animals/sex, were treated once as described in the table below. Negative control animals received only water while the test item treated animals were treated with one of the three dose levels of the test item, InSea2® (at 2000, 1000 or 500 mg/kg). The vehicle and the test item were administered by oral gavage (p.o) at 20 mL/kg. Animals of Group 5 were treated only once with the positive control, Cyclophosphamide (20 mg/kg), administered by intra-peritoneal route (i.p).

Groups Treatment

Dose Level

(mg/kg)

Dose Concentration

(mg/mL)

Dose Volume(mL/kg) Route

Sampling Time

(hours)

No. of Animals

Males Females

1 Vehicle/Negative control 0 - 20 p.o 48, 72 5 5

2 test item – Low dose 500 25 20 p.o 48 5 5

3 test item – Mid dose 1000 50 20 p.o 48 5 5

4 test item – High dose 2000 100 20 p.o 48, 72 5 5

5 Cyclophosphamide/


Animals’ peripheral blood was collected at the time points indicated in the study design. The presence of micronuclei within immature reticulocytes (RETs) was assessed by a flow cytometry method described in Section 7.2. Micronuclei were quantified and results were analyzed statistically as described in Sections 10.2 and 10.3.

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3 JUSTIFICATION OF MODEL, SPECIES, DOSE LEVEL AND ROUTE OF ADMINISTRATION

3.1 Justification of Model and Species Selection The in vivo micronucleus assay has been widely used for the detection of damages induced by a test item to the chromosomes or the mitotic apparatus of erythroblasts by analysis of rodent erythrocytes from bone marrow or peripheral blood. More specifically the assay allows the identification of cytogenetic damages resulting in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes.

Rodents are often chosen for the micronucleus test because they show adequate sensitivity to detect agents that cause structural or numerical chromosome aberrations. Rats are chosen because of the availability of background information on acute toxicity, metabolism and toxicokinetics of the test item in this species. Young adult animals are chosen for use because of the high rate of cell division in the erythropoietic system and because of their general suitability for toxicological investigations.

3.2 Justification of Route of Administration Selection The oral gavage route has been chosen because it is a human administration route.

The positive control (Cyclophosphamide) was administered intraperitoneally to provide a measure of the sensitivity of the test system.

3.3 Justification of Dose Level Selection The sponsor performed a study where the test item was administered up to 750 mg/kg/day in rats for 30 consecutive days by oral gavage and it was well tolerated. Therefore, the standard limit dose recommended by the Regulatory guidelines of 2000 mg/kg/day was chosen as high dose.

3.4 Study Schedule Study initiation date January 23, 2017 Animal arrival date January 16, 2017 Experimental start date Date of animal randomization

January 23, 2017

First treatment January 25, 2017 Experimental completion date Last date of data collection from flow cytometry

February 02, 2017

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

4 TEST, NEGATIVE/VEHICLE CONTROL AND POSITIVE CONTROL ITEMS

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 lot/batch number for the test material is INS2-006-TAC.

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4.1 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.

4.2 Positive Control Cyclophosphamide is a nitrogen mustard alkylating agent forming DNA crosslinks and known to induce formation of micronuclei. Cyclophosphamide was administered at 20 mg/kg (i.p.). This dose was shown to induce quantifiable chromosomal aberrations in validations studies and was used as a positive control to validate the assay system.

4.3 Information on Test, Negative/Vehicle and Positive Control Items

Test Item* Vehicle/negative control Identity: InSea2® Purified water Description: Fine brown powder (as per C of A) Clear liquid Lot/Batch No.: INS2-006-TAC NA Purity: Assumed 100% Not applicable Expiry Date: 31-05-19 Fresh daily Stability: 36 months (unopened) Not applicable Storage Conditions: Room temperature (protected from long

exposure to light) Room temperature

Handling Precautions: Standard precautions Standard precautions Supplier: InnoVactiv. Inc ITR Laboratories

*A certificate of analysis is included in the final report.

POSITIVE CONTROL VEHICLE FOR THE CYCLOPHOSPHAMIDE

Identity Cyclophosphamide monohydrate Sterile water for injection, USP

Description White powder Clear colorless liquid

Batch/Lot No MKBS 0021V W6K03AO

Expiry Date Retest date: 31-10-17 30-11-17/07-02-17 once opened

CAS No 6055-19-2 Not applicable

Storage Conditions 2 to 8ºC Room temperature/ 2 to 8ºC once

opened

Handling Precautions Carcinogen handle as per SOP H.S 5.0 Standard laboratory precautions

Supplier Sigma-Aldrich Baxter

Supporting documentation to confirm test item characterization was provided by the sponsor in a certificate of analysis.

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

4.4 Preparation and Stability of the Positive Control Formulation Cyclophosphamide (2 mg/mL) was prepared fresh on dosing day in a sterile container by dissolving the appropriate amount of cyclophosphamide powder in the appropriate volume of sterile water for injection and kept at room temperature pending dosing.

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

4.5 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.

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

4.7 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

Species Rat (Rattus norvegicus) Strain Sprague-Dawley Crl:CD (SD)

Source Charles River Canada Inc 324 St-Regis North CP400, J5A 2E7 St-Constant, Québec, Canada

Total Animal No. on Study 23 Males and 23 females, 3 spares for each sex

Body Weight Range 193 to 300 g at onset of treatment

Age Range Approximately 7 weeks at onset of treatment

Acclimation Period 10 days

5.2 Animal Care Committee The study plan was reviewed and assessed by the Animal Care Committee (ACC) of ITR. ACC acceptance of the study plan, and routine inspections reports were maintained on file at ITR.

All animals used on this study were cared for in accordance with the principles outlined in the current "Guide to the Care and Use of Experimental Animals" as published by the Canadian Council on Animal Care and the "Guide for the Care and Use of Laboratory Animals", an NIH publication. The study described in this report does not unnecessarily duplicate previous experiments.

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5.3 Health Status On arrival at ITR, all animals were weighed and then subjected to a detailed clinical examination (DCE) by a designate, to ensure satisfactory health status. Three days after arrival rats were weighed again to allow an assessment of health status. The health status data are not to be reported but retained in the study file.

5.4 Housing Rats were housed in groups of up to 3 in transparent plastic bins with dimensions of 25.4 cm x 48.3 cm x 20.3 cm equipped with an automatic watering system.

The bottoms of the plastic bins were covered with at least 2 cm deep of betachip bedding. The bedding was changed at appropriate intervals to maintain hygienic conditions. Following randomization, all cages were labelled with a color-coded cage card indicating study number, group, animal and cage number, sex and dose level.

5.5 Identification Each animal was uniquely identified by permanent marker followed by ear tag following arrival.

5.6 Room Environment

The animal room environment was controlled (targeted ranges: temperature 21 ± 3C, relative humidity 50 ± 20 %, 12 hours light, 12 hours dark, 10 to 15 air changes per hour) except during designated procedures. Temperature and relative humidity were monitored continuously and records are maintained at ITR.

5.7 Diet/Water A standard certified commercial rodent chow (Teklad Certified Rodent Diet (W) #2018C) was provided to the animals ad libitum except during designated procedures. Concentrations of the constituents of the diet and contaminants (e.g., heavy metals, aflatoxin, organophosphate, pesticides and chlorinated hydrocarbons) are routinely measured by the manufacturer (Batch certificate(s) on file at ITR).

Municipal tap water (which has been purified by reverse osmosis, ultraviolet light and further filtered with a 0.2-µm filter) was provided to the animals ad libitum except during designated procedures. Periodic analyses of municipal tap water (collected by the city) and reverse osmosis water from the animal rooms (collected by ITR) are performed by Exova Canada, Pointe-Claire, Quebec, Canada and the results are retained on file at ITR.

Batch certificates and analytical results were reviewed by the Clinical Veterinarian or designate. It was considered that there were no known contaminants in the diet and water that would had interfered with the assessment of the objectives of the study.

5.8 Environmental Enrichment During the study, the animals were offered non-dietary items (e.g. nylabones) and certified treats (e.g. veggie bites) as part of the ITR environmental enrichment program at appropriate intervals.

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5.9 Acclimation Acclimation period of 10 days were allowed between reception of the animals and the start of treatment to accustom the rats to the laboratory environment.

5.10 Allocation to Study Groups During the acclimation period, 23 Males and 23 females’ rats were assigned to their respective dose groups by block randomization based on body weights.

5.11 Animal Replacement None of the animals selected for the study was replaced by a spare animal.

5.12 Administration of the Test and Negative/Vehicle Control Items The test and vehicle control items were administered once by oral gavage using a gavage needle attached to a syringe. The dose volume was 20 mL/kg for all animals, including the negative controls.

The positive control (Cyclophosphamide) was administered once by intra-peritoneal route (i.p.) at 10 mL/kg.

The actual volume administered to each rat was calculated and adjusted based on the most recent practical body weight of each animal.

6 IN-LIFE OBSERVATIONS 6.1 Mortality Mortality checks were performed at least once a day during the pre-dosing and twice a day during dosing period.

6.2 Clinical Observations For all animals, a detailed clinical examination (DCE) was performed on the day of arrival and on the day of dosing (between 1 to 4 hours after dosing). For the rest of the study (post dosing and on day before dosing), cage-side clinical signs were recorded once a day.

The animals’ health status was acceptable throughout the study and no additional examination was required by a Clinical Veterinarian, and/or a technician working under the supervision of the Clinical Veterinarian.

6.3 Body Weights Body weights were recorded for all animals at arrival, randomization, and at one day before treatment and at euthanasia. The animal’s body weights taken before dosing were within ± 20% of the group mean.

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7 COLLECTION AND PROCESSING OF PERIPHERAL BLOOD ERYTHROCYTES

7.1 Peripheral Blood Collection Approximately 0.3 mL of peripheral blood was collected in K2EDTA from the jugular vein at the time points indicated in the study design table. The samples were inverted to ensure homogeneity and kept on ice. On completion of blood collection, the animals were euthanized by CO2 asphyxiation followed by cervical dislocation and discarded without further examination. For the group 1 and 4, the animals were only euthanized after the second time point 72 hours after treatment.

7.2 Flow Cytometry On collection day, one aliquot of 0.1 mL were removed from each peripheral blood sample and were diluted into 0.35 mL of Anticoagulant/Diluent (provided with the MicroFlow Plus kit). Diluted samples were further split into two aliquots and fixed. Each set of aliquots was stored at -80 ± 5°C in two independent freezers for at least 3 days before being washed and transferred into tubes containing the Long Term Storage Solution (LTSS) and stored at ≤ −60°C pending analysis.

Sample analysis was performed using a validated method (MD-320) using the Rat Microflow kit (Litron Laboratories). Briefly, one aliquot for each sample was washed out of the LTSS and incubated with RNase solution, rat anti-CD71 antibody and anti-platelets antibody before staining with the DNA staining solution. Data acquisition was performed on a BD FACSCanto II with FACSDiva software. For each individual animal, the following parameters were evaluated:

The number of reticulocytes (RET)

The number of micronucleated reticulocytes (MN-RET)

The number of normochromatic erythrocytes (NCE)

The number of micronucleated normochromatic erythrocytes (MN-NCE)

The proportion of immature and immature among total erythrocytes and the percentage of MN-RETs and MN-NCE for each animal.

A report was prepared by the Contributing Scientist (Immunology Department) for inclusion in the study report.

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

Flow cytometry FACSCanto II/FACSDiva. FCS Express Statistical analysis Sigmastat Version numbers of the systems are held on file at ITR.

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9 DATA PRESENTATION Where practical, at least 4000 immature erythrocytes were scored for each animal for the micronucleus frequency evaluation. In addition, the proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes per animal. Individual animal data were presented in tabular form.

The number of reticulocytes (RET) scored and the number of micronucleated immature erythrocytes (MN-RETs) are listed separately for each animal analyzed.

The proportion of reticulocytes (RET) among total erythrocytes (RETs+NCE) and the percentage of micronucleated reticulocytes (MN-RET) are reported for each animal.

10 DATA ANALYSIS AND INTERPRETATION OF RESULTS 10.1 Laboratory Proficiency Laboratory proficiency was demonstrated by comparison with accumulated historical control data which are included in the report.

10.2 Statistical Analysis Numerical data obtained during the conduct of the study, including the proportions of immature erythrocytes (RET) and micronucleated immature erythrocytes (MN-RET) counted for each animal were subjected to calculation of group means and standard deviations. In addition, statistical analysis of the variance was performed.

All the data were transformed to arcsine [square root (proportion of % MN-RETs or RETs (as appropriate) and the new transformed data were tested for normality and homogeneity of variance. When the variance is not homogenous, non-parametric analysis (Kruskal-Wallis test) would be applied.

The resulting data were analyzed for mean variance. The test item and positive groups were compared with the vehicle/negative control group. Either a t-test or a one way ANOVA test were used for comparing two or more than two groups, respectively, as shown below:

Group 1 vs. Groups 2, 3 and 4 (48 h): one way ANOVA Group 1 vs. 4 (72 h): t-test Group 1 vs. 5: t-test

A significance level of p < 0.01 was reported.

10.3 Validity of the Assay

The incidence of micronucleated immature erythrocytes (MN-RET) for the Negative/vehicle control group fell close to or within the laboratory historical Negative/vehicle control range (< 99 % confidence interval).

The positive control group showed a statistically significant increase (p 0.01) in the incidence of micronucleated immature erythrocytes (MN-RET) as compared to the Negative/vehicle control group and the results were close or within the positive historical control data.

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Appropriate number of doses and cells has been analyzed.

The criteria for selection of the highest dose are consistent with those described in OECD 474 and ICH S2 (R1).

10.4 Basis for Interpretation of the Results

Results were considered to be positive if a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes (MN-RET, p 0.01) was observed or in case of a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. In addition, individual and/or group mean values should also exceed the laboratory historical control range (> 99 % confidence interval).

Results were considered to be negative if there was no dose-related increase in the incidence of micronucleated immature erythrocytes (MN-RET, p > 0.01) and when individual and group mean values fall within (or close to) the historical control range.

Results were considered as equivocal if the results did not meet the criteria specified for a positive or negative response. Further investigation was not needed in this study.

Bone marrow cell toxicity was normally indicated by a substantial and statistically significant dose-related (where appropriate) decrease in the proportion of reticulocytes of test item treated groups when compared with negative control (p 0.01).

11 QUALITY ASSURANCE ITR Quality Assurance Unit (QAU) audited the study plan, amendment, 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.

12 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.

13 ARCHIVING All data that were generated during this study at ITR, together with the original copy of the study plan, amendment and the report were 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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14 RESULTS 14.1 Analysis of Achieved Concentration The test item formulations were not analyzed in this study for achieved concentrations.

The positive response obtained with the positive control indicated that its concentration and formulation was prepared appropriately.

14.2 Mortality There was no mortality during this study.

14.3 Clinical Signs No findings were observed during the study therefore, no tabulated clinical sign’s data are presented in this report.

14.4 Body Weight (Appendix 1)

Body weights of all animals were recorded at arrival. The body weight increased during the acclimation and dosing period up to termination. The individual body weight collected before the dosing day ranged from 193 to 300 g. The individual and mean (±SD) values of the body weight of each group recorded before the dosing and on termination day are shown in Appendix 1.

14.5 Flow Cytometry Results (Appendix 2)

The results from individual animals are presented in Appendix 2. The groups mean values are presented in Table 1.

14.6 Bone Marrow Toxicity / Proportion of Immature Erythrocytes (RET) There was no decrease in the proportion of immature erythrocyte (RET) in the high dose group 4 (2000 mg/kg) at both sampling time 48 and 72 hours indicating that there was no depression on the development of the immature erythrocytes (RET).

14.7 Micronucleated Immature Erythrocytes (MN-RET) There was no significant increase in the proportion of micronucleated immature erythrocytes (MN-RET) following the administration of 500, 1000 or 2000 mg/kg/day of test item InSea2® in both peripheral blood sampling time 48 and 72 hours post treatment and in both males and females. In addition, individual and/or group mean values of InSea2® treated animals, as well as the vehicle/negative control results, all fell within the historical control range which is presented in Appendix 3. Based on these results, the potential of InSea2® to induce chromosomal damages was considered negative.

As expected, the positive control (cyclophosphamide) induced a clear and statistically significant increase (p ≤ 0.01) in the proportion of micronuclei in immature erythrocytes (MN-RET) as compared to the vehicle/negative control treated animals.

The vehicle/negative and the positive control results were as expected confirming the validity of the assay and the sensitivity of the test system.

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Table 1 Proportion of Immature Erythrocytes and Micronucleated Erythrocytes

Group Treatment Parameters Males Females

% RET %MN-RET % RET %MN-RET

1 (48 h)

Vehicle/Negative Control

0 mg/kg/day

Mean 4.89 0.14 2.53 0.12

SD 0.832 0.044 0.604 0.045

No. of Animals 5 5 5 5

Min 3.98 0.09 2.05 0.06

Max 6.24 0.20 3.57 0.18

1 (72h)

Vehicle/Negative Control

0 mg/kg/day

Mean 5.41 0.15 2.71 0.12

SD 0.631 0.031 0.530 0.028


Min 4.66 0.11 2.27 0.09

Max 6.39 0.18 3.50 0.15

2 InSea2®

500 mg/kg/day

Mean 4.73 0.13 3.01 0.11

SD 0.596 0.054 0.520 0.040


Min 4.10 0.08 2.61 0.07

Max 5.70 0.20 3.89 0.17

3 InSea2®

1000 mg/kg/day

Mean 4.18 0.13 2.07 0.15

SD 0.790 0.050 0.504 0.069


Min 3.28 0.05 1.59 0.10

Max 5.01 0.18 2.87 0.27

4 (48 h) InSea2®

2000 mg/kg/day

Mean 4.21 0.16 2.52 0.09

SD 0.663 0.067 0.631 0.038


Min 3.39 0.11 1.78 0.05

Max 4.95 0.27 3.36 0.13

4 (72h) InSea2®

2000 mg/kg/day

Mean 4.94 0.17 2.95 0.12

SD 0.637 0.038 0.767 0.037


Min 4.19 0.11 2.08 0.06

Max 5.71 0.21 3.81 0.15

5 Cyclophosphamide

20 mg/kg

Mean 0.87 2.82* 0.34 2.62*

SD 0.192 0.288 0.072 0.378


Min 0.70 2.56 0.26 2.22

Max 1.08 3.13 0.40 2.97

*: Statistically significant (p≤0.01) RET: immature erythrocytes MN-RET: micronucleated immature erythrocytes

15 CONCLUSION Under the conditions used in this study, the test item InSea2® did not induce any chromosomal damage in erythrocytes collected from rats’ peripheral blood.

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APPENDIX 1 ITR STUDY NO. 55484BODY WEIGHTS (g)

INDIVIDUAL VALUES, MEAN AND SDMALES

GROUP 1: Vehicle / Negative ControlGROUP 2: InSea 2® (500 mg/kg) GROUP 4: InSea 2® (2000 mg/kg)GROUP 3: InSea 2® (1000 mg/kg) GROUP 5: Cyclophosphamide / positive controlGROUP ANIMAL DAY

NO. NO. -1 4

1 1001A 288 3301 1002A 285 3191 1003A 276 3171 1004A 273 3151 1005A 269 310

MEAN 278 318SD 8.0 7.4

2 2001A 291 3222 2002A 286 3272 2003A 279 3262 2004A 271 3102 2005A 271 311

MEAN 280 319SD 8.9 8.2

3 3001A 290 3213 3002A 285 3343 3003A 283 3273 3004A 276 3213 3005A 273 3133 3005A 273 313

MEAN 281 323SD 6.9 7.8

4 4001A 287 3404 4002A 277 3214 4003A 280 3184 4004A 275 3154 4005A 271 315

MEAN 278 322SD 6.0 10.5

5 5001A 300 3215 5002A 295 3105 5003A 255 322

MEAN 283 318SD 24.7 6.7

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APPENDIX 1 ITR STUDY NO. 55484BODY WEIGHTS (g)

INDIVIDUAL VALUES, MEAN AND SDFEMALES

GROUP 1: Vehicle / Negative ControlGROUP 2: InSea 2® (500 mg/kg) GROUP 4: InSea 2® (2000 mg/kg)GROUP 3: InSea 2® (1000 mg/kg) GROUP 5: Cyclophosphamide / positive controlGROUP ANIMAL DAY

NO. NO. -1 4

1 1501A 220 2491 1502A 210 2341 1503A 200 2261 1504A 202 2251 1505A 199 218

MEAN 206 230SD 8.8 11.8

2 2501A 213 2392 2502A 211 2322 2503A 199 2242 2504A 203 2322 2505A 197 217

MEAN 205 229SD 7.1 8.5

3 3501A 212 2363 3502A 208 2293 3503A 206 2143 3504A 198 2233 3505A 197 2193 3505A 197 219

MEAN 204 224SD 6.5 8.6

4 4501A 212 2334 4502A 203 2214 4503A 202 2334 4504A 193 2144 4505A 193 212

MEAN 201 223SD 8.0 10.1

5 5501A 220 2305 5502A 215 2245 5503A 209 221

MEAN 215 225SD 5.5 4.6

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APPENDIX 2 ITR Study No.55484 1

Final Micronucleus Flow Cytometry Report

FLOW CYTOMETRY REPORT

Quantification of Micronuclei in Rat Blood

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APPENDIX2

SIGNATURE PAGE

Contributing Scientist:

Peer Reviewer:

îêntist, Immunology ITR Laboratories Canada Inc.

essica Godin-Ethier, Senior Scientist, Immunology ITR Laboratories Canada, Inc.

Final Micronucleus Flow Cytometry Report 24of65

ITR Study No. 55484 2

APPENDIX 2 ITR Study No. 55484 3


TABLE OF CONTENTS

Section Page

SIGNATURE PAGE ......................................................................................................... 2

TABLE OF CONTENTS ................................................................................................... 3

INDEX TO FIGURES ....................................................................................................... 4

INDEX TO TABLES ......................................................................................................... 4

ABBREVIATIONS LIST .................................................................................................. 5

1 INTRODUCTION ................................................................................................. 6

2 MATERIALS AND METHOD ............................................................................. 6

2.1 Control Samples and Micronucleus Kit ................................................................. 6

2.2 Study Sample Collection, Processing and Storage ................................................ 6

2.3 Method ................................................................................................................... 7

2.3.1 Study Samples Analysis ......................................................................................... 8

2.3.2 Data Capture System and Calculations .................................................................. 8

3 RESULTS .............................................................................................................. 8

3.1 Study Sample Results ............................................................................................ 8

3.2 In-Study Method Performance ............................................................................... 9

4 DEVIATIONS ....................................................................................................... 9

4.1 Study Plan .............................................................................................................. 9

4.2 Method SOP ........................................................................................................... 9

5 CONCLUSION ...................................................................................................... 9

ATTACHMENT 1 TABULATED RESULTS ............................................................ 10

ATTACHMENT 2 REPRESENTATIVE GATING ................................................... 17

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INDEX TO FIGURES

Figure Description Page Figure 1. Representative Gating Strategy..................................................................... 18

INDEX TO TABLES

Table Description Page Table 1. Summary of Sample Analysis Runs ............................................................. 11

Table 2. Micronuclei Individual Results - Males ........................................................ 12

Table 3. Micronuclei Individual Results - Females .................................................... 13

Table 4. Micronuclei Summary Results – Males ........................................................ 14

Table 5. Micronuclei Summary Results – Females .................................................... 15

Table 6. Summary of Kit-Supplied Control Samples Data ......................................... 16

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ABBREVIATIONS LIST

Cy Cyanine

FITC Fluorescein Isothiocyanate

FSC Forward Scatter

MN-NCE Micronucleated NCE

MN-RET Micronucleated RET

NA Not applicable

NC Negative Control

NCE Normochromatic erythrocytes

PC Positive Control

PE Phycoerythrin

PerCP Peridinin Chlorophyll Protein

RET Reticulocytes

SD Standard Deviation

SOP Standard Operating Procedures

SSC Side Scatter

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1 INTRODUCTION This report presents the analysis results for micronuclei quantification in rat blood samples collected as part of ITR Study No. 55484 entitled “InSea2®: In vivo Rat Micronucleus Test Using Peripheral Blood Reticulocytes”. This report only presents the flow cytometry analysis of micronuclei content. The data interpretation is presented in the study main report.

The sample analyses were performed using a Flow cytometry method validated at ITR Laboratories Canada Inc under ITR Study Nos. MD-320, and in accordance with ITR SOP’s.

The experimental sample preparation and analysis work in the Immunology Department started on January 24, 2017 and was completed on February 2, 2017.

2 MATERIALS AND METHOD

2.1 Control Samples and Micronucleus Kit A positive and negative control sample was provided in the commercial micronuclei kit used for the quantification of micronuclei in rat blood. Additional details about the controls and micronuclei kit are found in the method SOP # IMM BIOM 27.0, which is kept on file at ITR. Certificates of analysis of the kit lots used are available in the raw data of the study.

When not in use the micronuclei kit components were stored, as instructed, at approximately 2-8C (-30 to -10C for RNase, ≤ -60°C for kit control samples).

On completion of the study, the remaining controls were retained until their retest date.

2.2 Study Sample Collection, Processing and Storage

Dates of Sample Collection: January 27, 2017 (Rep A Gr1-4, 48 hours). January 28, 2017 (Rep A Gr5, 48 hours). January 28, 2017 (Rep A Gr1+4, 72 hours).

Storage Temperature: 2 - 8°C

Dates of Sample Fixation January 27, 2017 (Rep A Gr1-4, 48 hours). January 28, 2017 (Rep A Gr5, 48 hours). January 28, 2017 (Rep A Gr1+4, 72 hours).

Storage Temperature: Between −85°C and −75°C

Dates of Transfer into LTSS January 31, 2017 (Rep A Gr1-4, 48 hours). February 1, 2017 (Rep A Gr5, 48 hours). February 1, 2017 (Rep A Gr1+4, 72 hours).

Storage Temperature: ≤ −60°C

Number of Samples Received: 66 samples, 1 aliquot per sample.

Number of Samples Processed 66 samples, 2 aliquots per sample in fixative. 66 samples, 2 aliquots per sample in LTSS.

Number of Samples Analyzed: 66 samples.

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Experimental Start and End Dates: January 27, 2017 (date at which the blood samples were diluted in anticoagulant and fixation started), February 2, 2017 (date at which the last study sample was acquired on the flow cytometer)

Longest Duration of Sample Storage:(first collection date to last analysis):

Less than 1 day at 2 - 8°C4 days in fixative. 2 days in LTSS.

Stability of samples in LTSS Up to 6 months in LTSS at ≤ -60°C

Blood sampling of study animals was performed according to the Study Plan, its subsequent Amendment and general ITR SOP’s.

The peripheral blood was collected 48 hours post-dosing (5/sex/group for Groups 1-4, 3/sex/group for Groups 5) or 72 hours following treatment (Groups 1 and 4). A sample of approximately 0.3 mL of blood was collected via the jugular vein (Gr 1-5) into K2EDTA tubes. Samples were inverted to ensure homogeneity and kept on wet ice pending transfer to Immunology department. The samples were stored refrigerated (2 to 8°C) pending the processing. Within 48 hours of collection, one aliquot of approximately 0.1 mL was removed from each sample and were diluted into 0.35 mL Anticoagulant/Diluent (provided with commercial kit). Samples were then fixed in 2 aliquots each and stored frozen (−80°C ± 5°C) for at least 3 days before being transferred into tubes containing the Long Term Storage Solution (LTSS) and stored frozen (≤ −60°C) pending analysis. Aliquots of each sample were stored in separate freezers to protect samples from a freezer malfunction or other problems.

Any unfixed whole blood sample was discarded at completion of the dilution in anticoagulant step. Any samples, in fixative or LTSS, not employed in the primary analysis were stored at ≤ −60°C and retained at ITR until it was determined by the Contributing Scientist and Study Director that they were not required for confirmatory analysis. These samples were then discarded and their disposition was recorded in the raw data.

2.3 Method The flow cytometry method for quantification of micronucleated erythrocytes in rat blood was validated in ITR Study No. MD-320. The equipment, reagents and methodology used during sample analysis were incorporated into the analytical method SOP #IMM BIOM 27.0, which is included in the raw data. The SOP provides the following information relating to the analysis:

1. General equipment and reagent list, 2. Solution preparation and storage, 3. Negative and Positive Controls biological standards, 4. Data acquisition software programming, flow cytometer programming and

operating conditions, 5. Data analysis, 6. Micronucleus/Flow Cytometry assay procedure and acceptance criteria.

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2.3.1 Study Samples Analysis Study samples were analyzed along with one set of control samples (NC and PC). Study samples were processed, labelled and acquired in a single run of 66 samples and NC/PC.

For a study sample to be deemed reportable, the number of RET had to be above 4000 as per method SOP. Furthermore, the contributing and reviewing scientists evaluated the shape and position of cell clusters, for each sample, and agree on their acceptability.

2.3.2 Data Capture System and Calculations The raw data acquisition was performed using the BD FacsDiva version 6.1.3 Software installed on the BD FACSCanto II flow cytometer with analysis performed with DeNovo FCS Express version 4 software.

The data calculation was performed using the spreadsheet WKS Genetic 1.8 (Flow Cytometric Analysis of Micronuclei Frequency Calculation Spreadsheet) which was subjected to a 100% quality control.

For each individual animal, the following parameters were evaluated:

the number of immature erythrocytes (reticulocytes, RET)

the number of micronucleated immature erythrocytes (micronucleated reticulocytes, MN-RET)

the number of mature erythrocytes (normochromatic erythrocytes, NCE)

the number of micronucleated mature erythrocytes (micronucleated normochromatic erythrocytes, MN-NCE)

In addition to these values, the proportion of RET among total erythrocytes and the percentage of MN-NCE and MN-RETs are reported for each animal.

Additional tabulation and calculations were performed with Microsoft Excel spreadsheet program using unrounded data, which at certain stages were rounded for reporting. Consequently, due to differences in decimal rounding, some minor discrepancies may be found between the electronic raw data on the FCS Express software and the data displayed in this report. This has no impact on the conclusions of this report.

3 RESULTS

3.1 Study Sample Results A total of 66 rat blood samples from the main toxicology animals were collected and analyzed for micronuclei content. The samples were analyzed in one batch. Refer to Table 1 for a summary of sample analysis runs.

The individual results for the number of RETs, MN-RETs, NCE and MN-NCE as well as the %MN-NCE, %RETs, and %MN-RETs of sample collected at termination are presented in Attachment 1, Table 2 and Table 3.

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From the 66 main study samples analyzed, values are reported for 66 samples. The group means and standard deviations (SD) of the %MN-NCE, %RET and %MN-RET are summarized in Attachment 1, Table 4 and Table 5. Data interpretation and conclusions from these values are presented in the study main report.

3.2 In-Study Method Performance In every sample analysis run, a kit-supplied negative and positive control was included. The controls expected values of %RETs and %MN-RETs, provided by the kit supplier, were used to calibrate the gating strategy (refer to Attachment 2, Figure 1) for study samples analysis (Table 6).

For the accuracy to be considered acceptable, %RETs of NC and PC samples had to be between 2.12 and 3.32% and between 1.12 and 1.74% respectively. The %RETs in run 1 were 2.92% for the NC and 1.46% for the PC.

For the accuracy to be considered acceptable, %MN-RETs of NC and PC samples had to be between 0.08 and 0.17% and between 0.94 and 2.11% respectively. The %MN-RETs in run 1 were 0.14% for the NC and 1.52% for the PC.

From the data presented in Table 6, the %RETs and %MN-RETs of all NC and PC samples from the sample analysis run met the acceptance criteria. Therefore, the sample analysis run was considered acceptable.

4 DEVIATIONS

4.1 Study Plan There were no Study Plan deviations from this section of the study.

4.2 Method SOP There were no method SOP deviations from this section of the study.

5 CONCLUSION In conclusion, a total of 66 rat blood samples were analyzed by Flow cytometry for micronuclei content. All 66 study samples from main animals met the SOP acceptance criteria and were reported.

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ATTACHMENT 1 Tabulated Results

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APPENDIX 2 ITR Study No. 55484 11 ATTACHMENT 1


Table 1. Summary of Sample Analysis Runs

Run ID Date * Objective Pass/Fail Comments

01 02-02-17 Analysis of Study Samples 48 hrs post-dosing Rep A Gr 1-5 (46 samples), 72 hours post-treatment Rep A Gr 1&4 (20 samples)

Pass NA

* Refers to Flow Cytometry data acquisition date.

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Table 2. Micronuclei Individual Results - Males

Group 1: Vehicle/Negative Control Group 2: InSea2® Low Dose (500 mg/kg) Group 3: InSea2® Mid Dose (1000 mg/kg) Group 4: InSea2® High Dose (2000 mg/kg) Group 5: Positive Control

Group Animal ID

Number of events Frequency (%)

NCE MN-NCE RET MN-

RET MN-NCE RET MN-

RET 48 hours post-dosing

1 1001A 238861 21 11978 18 0.01 4.78 0.15 1002A 197741 33 13143 26 0.02 6.24 0.20 1003A 287784 21 11919 12 0.01 3.98 0.10 1004A 231253 11 11873 11 0.00 4.89 0.09 1005A 254199 23 12127 17 0.01 4.56 0.14

2 2001A 287176 14 12254 10 0.00 4.10 0.08 2002A 244081 29 11557 23 0.01 4.53 0.20 2003A 195783 22 11828 13 0.01 5.70 0.11 2004A 234170 46 11788 10 0.02 4.80 0.08 2005A 254449 21 12056 20 0.01 4.53 0.17

3 3001A 316971 26 11483 17 0.01 3.50 0.15 3002A 221582 14 11446 6 0.01 4.91 0.05 3003A 264812 14 11588 18 0.01 4.20 0.16 3004A 368403 35 12474 22 0.01 3.28 0.18 3005A 226892 32 11945 15 0.01 5.01 0.13

4 4001A 266278 43 12603 14 0.02 4.52 0.11 4002A 354191 58 13380 15 0.02 3.64 0.11 4003A 259111 59 12316 33 0.02 4.55 0.27 4004A 360001 66 12602 22 0.02 3.39 0.17 4005A 244259 58 12708 17 0.02 4.95 0.13

5 5001A 2282594 602 15620 505 0.03 0.70 3.13 5002A 1845090 276 15195 433 0.01 0.84 2.77 5003A 1256013 185 13303 349 0.01 1.08 2.56

72 hours post-dosing 1 1001A 198327 18 11410 19 0.01 5.45 0.17

1002A 177834 16 12117 22 0.01 6.39 0.18 1003A 249307 80 12180 19 0.03 4.66 0.16 1004A 211489 16 12102 14 0.01 5.42 0.12 1005A 210271 13 11416 13 0.01 5.15 0.11

4 4001A 212846 17 11629 22 0.01 5.19 0.19 4002A 259910 16 11851 13 0.01 4.37 0.11 4003A 209462 21 11510 19 0.01 5.22 0.16 4004A 267667 46 11689 19 0.02 4.19 0.16 4005A 187515 13 11329 24 0.01 5.71 0.21

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Table 3. Micronuclei Individual Results - Females

Group 1: Vehicle/Negative Control Group 2: InSea2® Low Dose (500 mg/kg) Group 3: InSea2® Mid Dose (1000 mg/kg) Group 4: InSea2® High Dose (2000 mg/kg)Group 5: Positive Control

Group Animal ID

Number of events Frequency (%)

NCE MN-NCE RET MN-

RET MN-NCE RET MN-

RET 48 hours post-dosing

1 1501A 553644 35 12629 14 0.01 2.23 0.11 1502A 594961 21 12424 18 0.00 2.05 0.14 1503A 318327 16 11763 21 0.01 3.57 0.18 1504A 506724 41 13027 13 0.01 2.51 0.10 1505A 525148 45 12311 8 0.01 2.29 0.06

2 2501A 453940 18 13145 12 0.00 2.82 0.09 2502A 443771 13 12212 11 0.00 2.68 0.09 2503A 453466 21 12129 9 0.00 2.61 0.07 2504A 286099 12 11568 20 0.00 3.89 0.17 2505A 381822 15 12063 16 0.00 3.07 0.13

3 3501A 545417 22 12082 13 0.00 2.17 0.11 3502A 727651 32 12598 34 0.00 1.71 0.27 3503A 585625 55 11885 16 0.01 1.99 0.13 3504A 409476 18 12096 19 0.00 2.87 0.16 3505A 830058 71 13411 14 0.01 1.59 0.10

4 4501A 739556 71 13384 17 0.01 1.78 0.13 4502A 574751 74 13392 11 0.01 2.28 0.08 4503A 366248 74 12747 6 0.02 3.36 0.05 4504A 411273 36 12516 15 0.01 2.96 0.12 4505A 619220 44 14050 7 0.01 2.22 0.05

5 5501A 4437500 355 11503 261 0.01 0.26 2.22 5502A 4440813 586 15777 435 0.01 0.36 2.68 5503A 3841706 280 14888 455 0.01 0.40 2.97

72 hours post-dosing 1 1501A 531645 32 12322 11 0.01 2.27 0.09

1502A 587644 102 15241 21 0.02 2.53 0.14 1503A 321423 14 11649 10 0.00 3.50 0.09 1504A 384818 12 11842 15 0.00 2.99 0.13 1505A 546619 61 12681 19 0.01 2.27 0.15

4 4501A 545214 40 11563 17 0.01 2.08 0.15 4502A 432839 21 11733 7 0.00 2.64 0.06 4503A 321506 24 12723 15 0.01 3.81 0.12 4504A 316773 20 12183 18 0.01 3.71 0.15 4505A 469067 63 12091 14 0.01 2.52 0.12

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Table 4. Micronuclei Summary Results – Males


Group Frequencies (%), means Group 48 hours post-dosing ID Designation MN-NCE RET MN-RET

Mean SD n Mean SD n Mean SD n

Main

1 Vehicle Control 0.01 0.007 5 4.89 0.832 5 0.14 0.044 5

2 Low Dose 0.01 0.007 5 4.73 0.596 5 0.13 0.054 5

3 Mid Dose 0.01 0.000 5 4.18 0.790 5 0.13 0.050 5

4 High Dose 0.02 0.000 5 4.21 0.663 5 0.16 0.067 5

5 Positive Control 0.02 0.012 3 0.87 0.192 3 2.82 0.288 3

72 hours post-dosing 1 Low Dose 0.01 0.009 5 5.41 0.631 5 0.15 0.031 5

4 High Dose 0.01 0.004 5 4.94 0.637 5 0.17 0.038 5

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Table 5. Micronuclei Summary Results – Females


Group Frequencies (%), means and SD Group 48 hours post-dosing ID Designation MN-NCE RET MN-RET

Mean SD n Mean SD n Mean SD n

Main

1 Vehicle Control 0.01 0.004 5 2.53 0.604 5 0.12 0.045 5

2 Low Dose 0.00 0.000 5 3.01 0.520 5 0.11 0.040 5

3 Mid Dose 0.00 0.005 5 2.07 0.504 5 0.15 0.069 5

4 High Dose 0.01 0.004 5 2.52 0.631 5 0.09 0.038 5

5 Positive Control 0.01 0.000 3 0.34 0.072 3 2.62 0.378 3

72 hours post-dosing 1 Low dose 0.01 0.008 5 2.71 0.530 5 0.12 0.028 5

4 High Dose 0.01 0.004 5 2.95 0.767 5 0.12 0.037 5

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Table 6. Summary of Kit-Supplied Control Samples Data

Biological Standard

NC %RETs

NC %MN-RETs

PC %RETs

PC %MN-RETs

Expected Range 2.12 to 3.32 0.08 to 0.17 1.12 to 1.74 0.94 to 2.11

Run 01 2.92 0.14 1.46 1.52

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ATTACHMENT 2 Representative Gating

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Figure 1. Representative Gating Strategy

Run ID: 01 Date: 02-02-17 Tube name: FCM PC

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HISTORICAL CONTROL DATA

Micronucleus Test

Figure 1 Laboratory Historical Vehicle/Negative Values of Micronucleated Reticulocytes (MN-RETs) collected from GLP studies performed prior to this study

%MN-RETs of Individual Animals

Group Mean of %MN-RETs

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Table 1 Laboratory Historical Positive Values of Micronucleated Reticulocytes (MN-RETs) collected from GLP studies performed prior to this study

Cyclophosphamide 20 mg/kg

Number of data points 66

Mean 2.37

Standard Deviation 0.843

Minimum 0.91

Maximum 4.68

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APPENDIX 4ITR Study No. 55484

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ITR CANADA

CONFIDENTIAL

FINAL STUDY PLAN

InSea2®: In vivo Rat Micronucleus Test

Using Peripheral Blood Reticulocytes


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 23, 2017

Page Number: 1 of 16

APPENDIX 5

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ITR Study No. 55484

Page 2 of 17

Final Study Plan January 23, 2017

PREFACE PAGE

Study Title: InSea2®: In vivo Rat Micronucleus Test


Study GLP Status: GLP

Study Director (SD): Farida Merah, MSc

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.

APPENDIX 5

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mailto:[email protected]�

ITR Study No. 55484

Page 3 of 17


TABLE OF CONTENTS

Section Page

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

PREFACE PAGE................................................................................................................ 2 TABLE OF CONTENTS .................................................................................................... 3 1 Introduction ............................................................................................................. 4 2 Justification of Model, Species, Dose Level and Route of Administration ............ 5 3 Test, Negative/vehicle control and Positive Control items ..................................... 6 4 Experimental Procedures ........................................................................................ 8 5 In-life Observations .............................................................................................. 10 6 Collection and Processing of Peripheral Blood Erythrocytes ............................... 11 7 Data Capture ......................................................................................................... 11 8 Data Presentation .................................................................................................. 12 9 Data Analysis and Interpretation of Results ......................................................... 12 10 Changes to the Study Plan .................................................................................... 14 11 Reporting............................................................................................................... 14 12 GLP Compliance ................................................................................................... 15 13 Quality Assurance ................................................................................................. 15 14 Standard Operating Procedures............................................................................. 16 15 Archiving .............................................................................................................. 16 16 Study Plan Approval Page .................................................................................... 17

APPENDIX 5

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ITR Study No. 55484

Page 4 of 17


1 Introduction

The purpose of the study is to evaluate the potential cytogenetic damage that could be

induced by the Test Item InSea2® using the in vivo micronucleus test on reticulocytes

collected from rat peripheral blood.

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

material for preparation of this study plan:

OECD Guideline 474. OECD Guideline for Testing of Chemicals –Mammalian

erythrocyte micronucleus test.

EPA Health Effects Test Guideline OPPTS 870.5395: Mammalian erythrocyte

micronucleus 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. 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

During this study, 5 groups with 5 animals per group/sex, except group 5 which will have

3 animals/sex, will be treated once as described in the table below. Negative control

animals will receive only 0.5% methylcellulose while the test item treated animals will be

treated with one of the three dose levels of the Test Item, InSea2® (at 2000, 1000 or 500

mg/kg). The vehicle and the test item will be administered by oral gavage (p.o). Animals

of Group 5 will be treated only once with the positive control, Cyclophosphamide (20

mg/kg), administered by intra-peritoneal route (i.p).

Groups Treatment

Dose

Level

mg/kg)

Dose

Concentration

(mg/mL)

Dose

Volume

(mL/kg)

Route

Sampling

Time

(hours)

Number of

animals

Males Females

1 Vehicle/Negative

control 0 - 10 p.o 48, 72 5 5

2 Test Item – Low

dose 500 50 10 p.o 48 5 5

3 Test Item – Mid

dose 1000 100 10

p.o 48 5 5

4 Test Item – High

dose 2000 200 10

p.o 48, 72 5 5

5 Cyclophosphamide/


Animals’ peripheral blood will be collected at the time points indicated in the study

design. The presence of micronuclei within immature reticulocytes (RETs) will be

assessed by flow cytometry method. Micronuclei will be quantified and results will be

analyzed statistically.

APPENDIX 5

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ITR Study No. 55484

Page 5 of 17


The positive control group will not be subjected to all in life observations except

mortality. The clinical signs will be performed only if needed and the body weight will be

done prior to dosing (at least one day) for dose calculation purpose. The peripheral blood

will be collected from these animals at the time point indicated in the experimental design

and processed for micronucleus evaluation.

2 Justification of Model, Species, Dose Level and Route of Administration

2.1 Justification of Model and Species Selection

The in vivo micronucleus assay has been widely used for the detection of damage induced

by a Test Item to the chromosomes or the mitotic apparatus of erythroblasts by analysis

of erythrocytes as sampled in bone marrow or peripheral blood of rodents. This assay is

used to identify substances that may cause cytogenetic damage which results in the

formation of micronuclei containing lagging chromosome fragments or whole

chromosomes.

Rodents are often chosen for the micronucleus test because they show adequate

sensitivity to detect agents that cause structural or numerical chromosome aberrations.

Rats are chosen because of the availability of background information on acute toxicity,

metabolism and toxicokinetics of the Test Item for that specie. Young adult animals are

chosen for use because of the high rate of cell division in the erythropoietic system and

because of their general suitability for toxicological investigations.

2.2 Justification of Route of Administration Selection

The oral gavage route has been chosen because it is a human administration route. The

positive control (Cyclophosphamide) is administered intraperitoneally as a measure of the

sensitivity of the test system.

2.3 Justification of Dose Level Selection

The sponsor performed a study where the test item was administered up to 750

mg/kg/day in rats for 30 consecutive days by oral gavage and it was well tolerated.

Therefore, the standard limit dose recommended by the Regulatory guidelines of 2000

mg/kg/day was chosen as high dose.

2.4 Proposed Study Schedule

Study initiation date Date the SD signed the study plan

Animal arrival date January 16, 2017

Experimental start date

Date of animal randomization

January 23, 2017

First treatment January 24, 2017

Experimental completion date

Last date of data collection from flow cytometry January 30, 2017

Audited draft report date March 23, 2017

Study completion date

Date the SD signs the final report

Normally within 3 months of issue

of draft reports

APPENDIX 5

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3 Test, Negative/vehicle control and Positive Control items

3.1 Test Item Action

The Test Item is a demineralized polyphenols extracted from Ascophyllum nodosum and

Fucus vesiculosus, and it is used as dietary supplement.

3.2 Control/Vehicle

Based on the information provided by the sponsor, the 0.5% methylcellulose is selected

as the vehicle. This vehicle is compatible with the test system and is considered as the

negative control.

3.3 Positive Control

Cyclophosphamide, a nitrogen mustard alkylating agent forming DNA crosslinks and

known to induce formation of micronuclei will be used as a positive control.

Cyclophosphamide will be administered at 20 mg/kg (i.p) demonstrated in validations

studies.

3.4 Information on Test, Negative/Vehicle and Positive Control Items

Test Item* Vehicle/negative control

Identity: InSea2® 0.5% methylcellulose

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 (protected from long

exposure to light)

To be documented in raw data and report

Handling

Precautions:

Standard precautions Standard precautions

Supplier: InnoVactiv. Inc ITR Laboratories

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

POSITIVE CONTROL VEHICLE FOR THE

CYCLOPHOSPHAMIDE

Identity Cyclophosphamide monohydrate Sterile water for injection

Description White powder Clear colorless liquid

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

report

To be documented in the raw data and

report

Expiry Date To be documented in the raw data and

report


report

CAS No 6055-19-2 Not applicable

Handling

Precautions Carcinogen handle as per SOP H.S 5.0 Standard laboratory precautions

Supplier To be documented in the raw data and

report


report

APPENDIX 5

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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.

Information about any clinical changes or toxicity which may be exhibited following

administration of the test item should be supplied by the Sponsor.

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

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

3.5 Preparation and Analysis of the Positive Control Formulation

3.5.1 Preparation

Cyclophosphamide (2 mg/mL) will be prepared fresh on the day of the dosing by

weighing appropriate amount of the powder in sterile container and mixes it with

appropriate volume of vehicle.

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 the

formulations.

3.6 Preparation of Test and Negative/Vehicle Control Items

The vehicle, 0.5% methylcellulose, formulation will be prepared on the day of dosing or

in advance as per ITR procedures.

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.

3.7 Analysis of Test Item Formulations

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

homogeneity and stability.

3.8 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.

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4 Experimental Procedures

4.1 Test System

Species Rat (Rattus norvegicus)

Strain Sprague-Dawley Crl:CD (SD)

Source Charles River Canada Inc

324 St-Regis North CP400, J5A 2E7

St-Constant, Québec, Canada

Total Animal No. on Study 23 Males and 23 females, 3 spares for each sex

Target Body Weight Range 150 to 250 g at onset of treatment. Weight variation shall not

exceed ± 20% of the mean weight of each sex.

Target Age Range Approximately 6 to 8 weeks at onset of treatment

Acclimation Period Minimum 5 days

The actual age and body weight ranges will be noted in the final report.

4.2 Animal Care Committee

This study plan will be reviewed and assessed by the Animal Care Committee (ACC) of

ITR. ACC acceptance of the study plan will be maintained on file at ITR.

All animals used on this study will be cared for in accordance with the principles outlined

in the current "Guide to the Care and Use of Experimental Animals" as published by the

Canadian Council on Animal Care and the "Guide for the Care and Use of Laboratory

Animals", a NIH publication. The study described in this study plan does not

unnecessarily duplicate previous experiments.

4.3 Health Status

On arrival at ITR, all animals will be weighed and then subjected to a detailed clinical

examination (DCE) by the Clinical Veterinarian or a designate, to ensure satisfactory

health status. Three days after arrival rats will be weighed again to allow an assessment

of health status. The health status data will not be reported but retained in the study file.

4.4 Housing

Rats will be housed in groups of up to 3 in bins made of transparent plastic with

dimensions of 25.4cm x 48.3cm x 20.3cm equipped with an automatic watering system

supplemented by water bottles as appropriate.

The bottom of the plastic bins will be covered by an at least 2 cm layer of bedding (Beta

chips or other materials approved for animal use). Bedding will be changed at

appropriate intervals to maintain hygienic conditions. Following randomization, all

cages will be labeled with a color-coded cage card indicating study number, group,

animal and cage number, sex and dose level.

APPENDIX 5

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ITR Study No. 55484

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4.5 Identification

Each animal will be uniquely identified by permanent marker followed by ear tag or

tattoo following arrival.

4.6 Room Environment

The animal room environment will be controlled (targeted ranges: temperature 21 ± 3C,

relative humidity 30 to 70 %, 12 hours light, 12 hours dark, 10 to 15 air changes per hour)

except during designated procedures. Temperature and relative humidity will be

monitored continuously and records will be maintained at ITR.

4.7 Diet/Water

A standard certified commercial rodent chow (Teklad Certified Rodent Diet (W) # 2018C)

will be provided to the animals ad libitum except during designated procedures.

Concentrations of the constituents of the diet and contaminants (e.g., heavy metals,

aflatoxin, organophosphate, pesticides and chlorinated hydrocarbons) are routinely

measured by the manufacturers (Batch certificate(s) on file at ITR).

Municipal tap water (which has been purified by reverse osmosis, ultraviolet light and

further filtered with a 0.2µm filter) will be provided to the animals ad libitum except

during designated procedures. Periodic analyses of municipal tap water (collected by the

city) and reverse osmosis water from the animal rooms (collected by ITR) are performed

by Exova Canada, Pointe-Claire, Quebec, Canada and the results are retained on file at

ITR.

Batch certificates and analytical results are reviewed by the Clinical Veterinarian or

designate. It is considered that there are no known contaminants in the diet and water

that would interfere with the assessment of the objectives of the study.

4.8 Environmental Enrichment

During the study, the animals may be offered certified treats and other non-dietary items

(e.g. nylabones) as part of the ITR environmental enrichment program at appropriate

intervals.

4.9 Acclimation

A minimum of 5 days acclimation period will be allowed between receipt of the animals

and the start of treatment to accustom the rats to the laboratory environment.

Acclimatization to restraint or dosing procedures may be performed as per ITR SOPs.

4.10 Allocation to Study Groups

During the acclimation period, 23 male and 23 female rats will be assigned to their

respective dose groups by block randomization based on body weights.

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ITR Study No. 55484

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4.11 Animal Replacement

Any animal with unacceptable pre-treatment data or where there are difficulties related to

study procedure (such as dosing or acclimation procedures), will be replaced by a spare

animal from the same shipment and maintained under the same environmental conditions.

During the treatment phase, animals which die accidentally or where the death is clearly

not related to treatment may be replaced. Animals may also be replaced if there are

procedural difficulties after initial dosing has commenced.

4.12 Administration of the Test and Negative/Vehicle Control Items

The test and vehicle control items will be administered once by oral gavage using a

gavage needle attached to a syringe. The dose volume will be 10 mL/kg for all animals,

including the negative controls.

The positive control (Cyclophosphamide) will be administered once by intra-peritoneal

route (i.p) at 10 mL/kg.

The actual volume administered to each rat will be calculated and adjusted based on the

most recent practical body weight of each animal.

5 In-life Observations

5.1 Mortality

Mortality checks will be performed at least once a day before dosing period and at least

twice during the dosing phase.

Moribund animals will be euthanized for humane reasons at the discretion of the Study

Director in consultation with the Clinical Veterinarian and the Sponsor (if possible) and

will be subjected to detailed external and internal necropsy examination. Every effort

will be made to contact the Study Director or Study Director Management outside normal

working hours, but if no such consultation can be made the Clinical Veterinarian has the

responsibility to ensure humane treatment of study animals including euthanasia where

necessary. Blood will not be collected from these animals for micronucleus evaluation.

5.2 Clinical Observations

For all animals a detailed clinical examination (DCE) will be performed on the day of

arrival, approximately 1 to 4 hours after dosing and at termination. During the rest of the

dosing period, cage side observation will be performed. Animals whose health status is

judged to warrant additional evaluation will be examined by a Clinical Veterinarian, or a

technician working under the supervision of the Clinical Veterinarian. Any veterinarian-

recommended treatments will only be performed once agreement has been obtained from

the Study Director. Where possible, the Study Director will be consulted prior to

administration of therapeutic agents.

The positive control group will not be subjected to all in life observations except

mortality. The clinical signs will be performed only if needed.

APPENDIX 5

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ITR Study No. 55484

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5.3 Body Weights

Body weights will be recorded for all animals at arrival, randomization, at least one day before

dosing and at euthanasia. If on the day of dosing, the body weight of the test item animals had

changed more than ± 20% of the mean weight, either the animals are replaced by spares or the

dosing is postponed until the animals recover (in case the weight decreased) and the body weight

returns to normal (within ± 20% of the mean).

The body weight will be performed for the positive control group prior to dosing (at least

one day) for dose calculation purpose.

5.4 Collection and Processing of Peripheral Blood Erythrocytes

5.4.1 Peripheral Blood Collection

Approximately 0.3 mL of peripheral blood will be collected in K2EDTA tubes from

jugular vein at the time point indicated in the study design table. On completion of blood

collection, the animals will be euthanized by CO2 asphyxiation followed by cervical

dislocation and discarded without further examination. The samples will be inverted to

ensure homogeneity and kept on ice and stored refrigerated (2-8°C) pending the

processing.

5.4.2 Flow cytometry

Within 48 hours of blood collection, one aliquot of 0.1 mL will be removed from each

sample and will be diluted into 0.35 mL Anticoagulant/Diluent (provided with the

MicroFlow Plus kit). Diluted samples will then be split into two aliquots and will be fixed.

Each set of aliquots will be stored at ≤-60°C in two independent freezers for at least 3

days before being washed and transferred into tubes containing the Long Term Storage

Solution (LTSS) and stored at ≤-60°C pending analysis.

Sample analysis will be performed using a validated method (MD-320) developed based

on the Rat Microflow kit (from Litron Laboratories). Briefly, one aliquot for each sample

will be washed out of LTSS and will be incubated with RNase solution, rat anti-CD71

antibody and platelets antibody before being stained with the DNA staining solution.

Data acquisition will be performed on a BD FACSCanto II with FACSDiva software.

For each individual animal, the following parameters will be evaluated:

The number of reticulocytes (RET)

The number of micronucleated reticulocytes (MN-RET)

The number of normochromatic erythrocytes (NCE)

The number of micronucleated normochromatic erythrocytes (MN-NCE)

The proportion of immature among total erythrocytes and the percentage of MN-

RETs will be given for each animal.

A report will be prepared by the Contributing Scientist for inclusion in the study report.

6 Data Capture

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

APPENDIX 5

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Flow cytometry FACSCanto II/FACSDiva

Statistical analysis Sigmastat

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

7 Data Presentation

Where practical, at least 4000 immature erythrocytes, will be scored for each animal for

the micronucleus frequency evaluation. In addition, the proportion of immature

erythrocytes will be assessed by examination of at least 1000 erythrocytes per animal.

Individual animal data will be presented in tabular form.

The number of reticulocytes (RET) scored and the number of micronucleated

immature erythrocytes (MN-RETs) should be listed separately for each animal

analyzed.

The proportion of reticulocytes (RET) among total erythrocytes (RETS+NCE)

and the percentage of micronucleated reticulocytes (MN-RET) will be given for

each animal.

8 Data Analysis and Interpretation of Results

8.1 Statistical Analysis

Numerical data obtained during the conduct of the study, including the proportions of

immature erythrocytes and micronucleated immature erythrocytes will be subjected to

calculation of group means and standard deviations. In addition to these calculations,

statistical analysis of the variance will be performed.

All the data will be transformed to arcsine [square root (proportion of % MN-RETs or

RETs (as appropriate) and the new transformed data will be tested for normality and

homogeneity of variance. If the variance is not homogenous non parametric (Kruskal-

Wallis test) will be applied.

The resulting data will be analyzed for mean variance. The Test Item and positive

groups will be compared with the vehicle/negative control. Either a T-test or a one way

ANOVA test will be used for comparing two or more than two groups, respectively, as

shown below:

Group 1 Vs Groups 2, 3 and 4 one way ANOVA

Group 1 Vs Group 4 T-test

Group 1 and 5 T-test

A significance level of p < 0.01 will be reported and p <0.05 for the subsequent follow

up tests.

Additional analysis could be performed and the results will be included in the raw data.

APPENDIX 5

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ITR Study No. 55484

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8.2 Validity of the Assay

The incidence of micronucleated immature erythrocytes for the Negative/vehicle

control group should fall close to or within the laboratory historical

Negative/vehicle control range (<99 % confidence interval).

The positive control group should show a statistically significant increase

(p 0.01) in the incidence of micronucleated immature erythrocytes as compared

to the Negative/vehicle control group and the results should be close or within the

positive historical control data.

Appropriate number of doses and cells has been analyzed (as stated in section 7).

The criteria for selection of the highest dose are consistent with those described in OECD

474 and ICH S2 (R1).

8.3 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.

Results will be considered as positive if a statistically significant dose-related

increase in the incidence of micronucleated immature erythrocytes (p 0.01) is

observed or in case of a clear increase in the number of micronucleated cells in a

single dose group at a single sampling time. In addition, individual and/or group

mean values should also exceed the laboratory historical control range (> 99 %

confidence interval).

Results will be considered as negative if there is no dose-related increase in the

incidence of micronucleated immature erythrocytes (p > 0.01) and when

individual and group mean values fall within (or close to) the historical control

range.

Results will be considered as equivocal if the results do not meet the criteria

specified for a positive or negative response. Further investigation may be

required by the Study Director to clarify results and the activity, if any, will be

documented in a study plan amendment.

Bone marrow cell toxicity is normally indicated by a substantial and statistically

significant dose-related (where appropriate) decrease in the proportion of reticulocytes of

Test Item treated groups when compared with negative control (p 0.01). If any treated

group of animals shows evidence of severe bone marrow depression (proportion of CD71

positive less than 5% of the concurrent control), then any apparent increase in the

incidence of micronucleated erythrocytes will be interpreted with caution.

APPENDIX 5

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ITR Study No. 55484

Page 14 of 17


The proportion of the immature erythrocytes (%RETs) of the positive control will not be

considered for the statistical analysis and if there is any decrease (toxicity) it will not be

discussed in the report.

9 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.

10 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:

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 Item, if known; source, lot number, limit date for use, if available; - stability

of the test chemical, if known.

Negative/vehicle control: justification for choice of the Negative/vehicle control

(e.g. solvent); solubility and stability of the Test Item in the vehicle, if known.

Test Animals: species/strain used and justification for use; number, age and sex

of animals; source, housing conditions, diet; method for uniquely identifying the

animals; for short term studies: clinical signs (if any), individual weight of the

animals at the start and end of the test; for studies longer than one week:

individual body weights during the study and food consumption. Body weight

range, mean and standard deviation for each group should be included.

Test Conditions: Negative/vehicle control and positive control data; data from

range-finding study, if conducted; rationale for dose level selection; details of test

substance preparation; details of the administration of the test substance; rationale

for route of administration; methods for verifying that the test substance or

metabolites reached the general circulation or target tissue, if applicable;

conversion from diet/drinking water Test Item concentration (ppm) to the actual

dose (mg/kg body weight/day), if applicable; details of food and water quality;

APPENDIX 5

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ITR Study No. 55484

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detailed description of treatment and sampling schedules; methods of slide

preparation; methods for measurement of toxicity; criteria for scoring

micronucleated immature erythrocytes; number of cells analyzed per animal;

criteria for considering studies as positive, negative or equivocal.

Results: signs of toxicity; proportion of immature erythrocytes among total

erythrocytes; number of micronucleated immature erythrocytes, given separately

for each animal; mean ± standard deviation of micronucleated immature

erythrocytes per group; dose-response relationship, where possible; statistical

analyses and method applied; concurrent and historical vehicle / control data and

99% control limits for the distribution; concurrent positive control data.

Discussion of the Results Conclusion

References

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 automatically 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.

11 GLP Compliance

The study will be performed in compliance with the Organization 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.

12 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.

APPENDIX 5

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ITR Study No. 55484

Page 16 of 17


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

inspection of, this study.

13 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.

14 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 Laboratories Canada Inc. will ship the archived study data to the Sponsor after

completion of the 1-year holding period.

APPENDIX 5

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• ITR Study No. 55484

Page 17 of 17

15 Study Plan Approval Page

Tbis study plan has been approved by:

~~:~Oinette '. C . 1!

ThIs study plan has been peer reviewed by:

~~ Scientist,1mmnnology ITR Laboratori.es Canada Inc..

This study plan has been .reviewoo. by the Quality Assurance Departtnent:

Joann; ~BSc (Hona). MRQA. RQAP-GLP Quality Assurance Representative ITR Laboratories Canada Inc

Approved onbebalfdf the Sponsor by: /"

'2.3 2017 )

Final Study Plan January 23. 2017

APPENDIX 5

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ITR CANADA

Study Plan Amendment No. 1


Study Title: InSea2®: In vivo Rat Micronucleus Test


Study Plan Date: January 23, 2017

Amendment Date: January 24, 2017

The following amendment to the above-referenced Study Plan is effective as of the date

above:

Please note that text to be removed will be identified by a strikethrough and new text will

be presented in bold.

1)

Study Plan Section Number: 1.1

Study Plan Section Title: Experimental Design

During this study, 5 groups with 5 animals per group/sex, except group 5 which will have

3 animals/sex, will be treated once as described in the table below. Negative control

animals will receive only 0.5% methylcellulose water while the test item treated animals

will be treated with one of the three dose levels of the Test Item, InSea2® (at 2000, 1000

or 500 mg/kg). The vehicle and the test item will be administered by oral gavage (p.o) at

20 mL/kg. Animals of Group 5 will be treated only once with the positive control,

Cyclophosphamide (20 mg/kg), administered by intra-peritoneal route (i.p).

Groups Treatment

Dose

Level

mg/kg)

Dose

Concentration

(mg/mL)

Dose

Volume

(mL/kg)

Route

Sampling

Time

(hours)

Number of

animals

Males Females

1 Vehicle/Negative

control 0 - 10 20 p.o 48, 72 5 5

2 Test Item – Low

dose 500 50 25 10 20 p.o 48 5 5

3 Test Item – Mid

dose 1000 100 50 10 20

p.o 48 5 5

4 Test Item – High

dose 2000 200 100 10 20

p.o 48, 72 5 5

5 Cyclophosphamide/


APPENDIX 5

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Study Plan Amendment No. 1 ITR Study No. 55484

Page 2 of 4

January 24, 2017

Reason for amendment: The formulations with 0.5% methylcellulose were too thick

therefore, the vehicle was changed to water, the test item concentrations were reduced

and the dosing volume increased.

2)


Study Plan Section Title: Proposed Study Schedule

Study initiation date Date the SD signed the study plan

Animal arrival date January 16, 2017

Experimental start date

Date of animal randomization

January 23, 2017

First treatment January 24, 2017 January 25,

2017

Experimental completion date

Last date of data collection from flow cytometry January 30, 2017

Audited draft report date March 23, 2017

Study completion date

Date the SD signs the final report

Normally within 3 months of

issue of draft reports

Reason for amendment: To document the change in dosing day.

3)


Study Plan Section Title: Control/Vehicle

Based on the information provided by the sponsor, the 0.5% methylcellulose water is

selected as the vehicle. This vehicle is compatible with the test system and is considered

as the negative control.

Reason for amendment: To document the change in vehicle.

4)


Study Plan Section Title: Information on Test, Negative/Vehicle and Positive Control

Items

Test Item* Vehicle/negative control

Identity: InSea2® 0.5% methylcellulose Purified water

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 (protected from long

exposure to light)

To be documented in raw data and report

APPENDIX 5

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Study Plan Amendment No. 1 ITR Study No. 55484

Page 3 of 4

January 24, 2017

Handling

Precautions:

Standard precautions Standard precautions

Supplier: InnoVactiv. Inc ITR Laboratories

Reason for amendment: To document the change in vehicle.

5)


Study Plan Section Title: Preparation of Test and Negative/Vehicle Control Items

Preparation of the Test and Negative/Vehicle Control Items Item Formulations

The vehicle, 0.5% methylcellulose, formulation will be prepared on the day of dosing or

in advance as per ITR procedures.

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.

Reason for amendment: To remove the preparation of the vehicle control.

6)


Study Plan Section Title: Administration of the Test and Negative/Vehicle Control Items

The test and vehicle control items will be administered once by oral gavage using a

gavage needle attached to a syringe. The dose volume will be 10 20 mL/kg for all

animals, including the negative controls.

The positive control (Cyclophosphamide) will be administered once by intra-peritoneal

route (i.p) at 10 mL/kg.

The actual volume administered to each rat will be calculated and adjusted based on the

most recent practical body weight of each animal.

Reason for amendment: To update the dosing volume.

APPENDIX 5

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Study Plan Amendment No. I

This amendment bas been approved by:

1t;,<4~L

This amendment has been peer reviewed by:

~-Scientist. Immunology ITR Laboratories Canada Inc

ITR Study No. 55484 Page 4 of 4

~f4J..ory J.s-: cH7l / D, I

This amendment bas been reviewed by the Quality Assurance Department:

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

Approved on behalf of the Sponsor by: -~ L~~

January 24, 2017

:rWIYG-~ ,24- I JolJ . Date

2

APPENDIX 5

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APPENDIX 6 ITR Study No.: 55484

MINOR STUDY PLAN DEVIATIONS

Throughout the conduct of the study, there were occasional instances of deviations from

the Study Plan which were very minor and as such were considered, by the Study

Director to be of no impact upon the integrity of the study or its outcome. Such instances

of Study Plan deviations were documented and acknowledged by the Study Director

within the study records. These minor deviations are summarized below:

Activity Nature of Deviation

Mortality check The afternoon mortality check was n - -

Clinical signs - -

- - 17 while it is not required by the study plan.

Cage side observation was done at termination instead of

detailed clinical signs.

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