## FINAL REPORT

Study Title



## MICRONUCLEUS TEST I N BONE MARROW CELLS OF THE RAT

WITH EXP1313100

Author

Test Facility

WIL Research Europe B.V. Hambakenwetering 7
5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Proj ect Identification

Project 505691
Substance 205622/A

- Page 1 of 29

-EXP1313100 Project 505691

- Page 2 - 1. CONTENTS

1. CONTENTS

.................................................................................................................................. 2

2. STATEMENT OF GLP COMPLIANCE

.......................................................................................... 3

3. QUALITY ASSURANCE STATEMENT

......................................................................................... 4

4. SUMMARY

................................................................................................................................... 6

5. INTRODUCTION

.......................................................................................................................... 7
5.1. Preface

................................................................................................................................ . 7
5.2. 7

5.3. Aims of the study

................................................................................................................... 7
5.4. Guidelines

............................................................................................................................. 8

5.5. Storage and retention of records and materials

..................................................................... 8
6. MATERIALS AND METHODS

...................................................................................................... 8

6.1. Test substance

...................................................................................................................... 8
6.1.1. Test substance information

......................................................................................... 8
6.1.2. Study specific test substance information

.................................................................... 8

6.1.3. Test substance preparation

......................................................................................... 9
6.2. Reference substances

.......................................................................................................... 9

6.2.1. Negative control

.......................................................................................................... 9
6.2.2. Positive control

............................................................................................................ 9

6.3. Test system

........................................................................................................................... 9
6.4. Animal husbandry

................................................................................................................ 10
6.5. Treatment

............................................................................................................................ 10

6.6. Observations

........................................................................................................................ 11
6.7. Study design

........................................................................................................................ 11

6.7.1. Dose range finding study

............................................................................................ 11
6.7.2. Micronucleus main test

............................................................................................... 11
6.7.3. Isolation of bone marrow

............................................................................................ 11

6.7.4. Preparation of bone marrow smears

.......................................................................... 11
6.7.5. Staining of the bone marrow smears

.......................................................................... 12

6.7.6. Analysis of the bone marrow smears for micronuclei

.................................................. 12
6.8. Electronic data capture

......................................................................................................... 12

6.9. Interpretation

........................................................................................................................ 12
6.9.1. Acceptability of the assay

........................................................................................... 12
6.9.2. Data evaluation and statistical procedures

................................................................ . 12

6.10. List of deviations

........................................................................................................ 13
6.10.1. List of protocol deviations

...................................................................................... 13

6.10.2. List of standard operating procedures deviations

................................................... 13

7. RESULTS

.................................................................................................................................... 13

7.1. Dose range finding study

...................................................................................................... 13

7.2. Micronucleus main test

......................................................................................................... 13

7.2.1. Mortality and toxic signs

............................................................................................. 13

7.2.2. Micronucleated polychromatic erythrocytes

................................................................ 13
7.2.3. Ratio polychromatic to normochromatic erythrocytes

................................................. 14

7.2.4. Chemical analysis of dose preparations

..................................................................... 14
8. CONCLUSION

............................................................................................................................. 14
9. REFERENCES

............................................................................................................................ 14

TABLES

Table 1 Mean body weight immediately prior to dosing with EXP1313100

.............................. 15
Table 2 Mean number of micronucleated polychromatic erythrocytes and ratio of

polychromatic/normochromatic erythrocytes

.............................................................. 15

APPENDICES

APPENDIX 1 TABLES

..................................................................................................................... 15

APPENDIX 2 INDIVIDUAL DATA

.................................................................................................... 16

APPENDIX 3 STATISTICS

.............................................................................................................. 17

APPENDIX 4 HISTORICAL CONTROL DATA FOR MICRONUCLEUS STUDIES

........................... 18

APPENDIX 5 CERTIFICATE OF ANALYSIS

................................................................................... 19
APPENDIX 6 FORMULATION ANALYSIS

....................................................................................... 20EXP1313100 Project 505691

- Page 4 - 3. QUALITY ASSURANCE STATEMENT

WIL Research Europe B.V., ‘s-Hertogenbosch, The Netherlands

This report was inspected by the WIL Research Europe Quality Assurance Unit to confirm that the methods and results accurately and completely reflect the raw data.

During the on-site process inspections, procedures applicable to this type of study were inspected.

The dates of Quality Assurance inspections are given below.

Project 505691

Type of Inspections Phase/Process Start Inspection

date End Inspection

date Reporting date

Study Protocol 27 -May- 2014 27 -May- 2014 27 -May- 2014

Protocol Amendment 01 04 -Jul- 2014 04 -Jul- 2014 04 -Jul- 2014

Protocol Amendment 02 09 -Jul- 2014 09 -Jul- 2014 09 -Jul- 2014 Specimen processing 10 -Jul- 2014 10 -Jul- 2014 10 -Jul- 2014

Protocol Amendment 03 07 -Aug-2014 07 -Aug-2014 07 -Aug-2014

Protocol Amendment 04 03 -Sep- 2014 03 -Sep- 2014 03 -Sep- 2014

Protocol Amendment 05 08 -Sep- 2014 08 -Sep- 2014 08 -Sep- 2014 Report 24 -Oct- 2014 24 -Oct- 2014 24 -Oct- 2014

Protocol Amendment 06 30-Oct- 2014 30-Oct- 2014 30

- Oct- 2014

Process Genetic and In Vitro

Toxicology 17 -Jun-2014 30 -Jun-2014 30

- Ju n-2014 Test Substance Handling Exposure

Observations/Measurements Specimen Handling

Animal Facilities 07 -Jul- 2014 18 -Jul- 2014 18

- Ju l- 2014 Test Substance Handling Exposure

Observations/Measurements Specimen Handling

Test Substance Receipt 24 -Jul- 2014 31 -Jul- 2014 01

- A ug-2014 Test Substance Handling

Analytical and physical chemistry 05 -Aug-2014 12 -Aug-2014 13

- A ug-2014 Test Substance Handling Exposure

Observations/Measurements Specimen Handling

Necropsy 15 -Sep- 2014 26 -Sep- 2014 30

- S ep- 2014

Observations/Measurements Specimen HandlingEXP1313100 Project 505691

- Page 6 - 4. SUMMARY

Micronucleus test in bone marrow cells of the rat with EXP1313100.

EXP1313100 was tested in the Micronucleus Test in rats, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch E00873- 025 -001 of EXP1313100 was a brown solid. The test substance was suspended in propylene glycol.

The concentrations analysed in the formulations of Group B/C, Group D an Group E were in agreement with target concentrations (i.e. mean accuracies between 85 and 115%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group A formulations

. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks.

Analysis of Group B/C and Group E formulations after storage yielded a relative difference of ≤ 10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

In the dose range finding study 3 males and 3 females were dosed via oral gavage with 2000 mg EXP1313100 per kg body weight. The animals showed no treatment related clinical signs or mortality after dosing. Since there were no substantial differences in toxicity between sexes only males were used in the main study.

In the main study male animals were dosed by oral gavage with vehicle or with 2000

, 1000 and

5 00 mg EXP1313100 per kg body weight. A positive control group was dosed once by oral gavage with 20 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with EXP1313100 or control animals receiving vehicle or cyclophosphamide.

Bone marrow of the groups treated with EXP1313100 was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with EXP1313100 compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with EXP1313100 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

It is concluded that EXP1313100 is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male rats up to a dose of 2000 mg/kg the recommended dose in accordance with current regulatory guidelines under the experimental conditions described in this report.