## FINAL REPORT

Study Title



## REPEATED DOSE 28- DAY ORAL TOXICITY ST UDY WITH

EXP1313100

BY DAILY GAVAGE

IN THE RAT

FOLLOWED BY A 14

- DAY RECOVERY PERIOD

Author

Test Facility

WIL Research Europe B.V. Hambakenwetering 7

5231 DD ‘ s- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 505705

Substance 205622 /AEXP1313100 Project 505705

- Page 2 - 1. CONTENTS

1. CONTENTS

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

2. STATEMENT OF GLP COM PLIANCE

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

3. QUALITY ASSURANCE ST ATEMENT

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

4. SUMMARY

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

5. INTRODUCTION

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

5.1. Preface

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

5.2. Study plan

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

5.3. Aim of the study

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

5.4. Guidelines

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

5.5. Storage and retention of records and materials

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

6. MATERIALS AND METHOD S

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

6.1. Test substance

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

6.1.1. Test su bstance information

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

6.1.2. Study specific test substance information

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

6.1.3. Test substance formulation

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

6.1.4. Chemical analysis of dose preparations

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

6.2. Test system

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

6.3. Allocatio n

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

6.4. Animal husbandry

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

6.5. Treatment

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

6.6. Observation s

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

6.7. Clinical laboratory investigations

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

6.8. Pathology

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

6.8.1. Necropsy

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

6.8.2. Organ weights

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

6.8.3. Histotechnology

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

6.8.4. Histopathology

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

6.9. Electronic data capture

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

6.10. Interpretation

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

6.11. List of deviations

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

6.11.1. List of protocol deviations

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

6.11.2. List of standard operating procedures deviations

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

7. RESULTS

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

7.1. Analysis of dose preparations

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

7.2. Observations

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

7.2.1. Mortality

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

7.2.2. Clinical signs

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

7.2.3. Functional observations

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

7.2.4. Body weights

................................................................................................................. 19

7.2.5. Food consumption

........................................................................................................ 19

7.3. Clinical laboratory investigations

............................................................................................ 19

7.3.1. Haematology

................................................................................................................. 19

7.3.2. Clinical biochemistry

..................................................................................................... 19

7.4. Pathology

............................................................................................................................... 20

7.4.1. Macroscopic examination

............................................................................................. 20

7.4.2. Org an weights

............................................................................................................... 20

7.4.3. Microscopic examination

.............................................................................................. 20

8. DISCUSSION AND CONCL USION

................................................................................................ 21

9. REFERENCES

............................................................................................................................... 22

APPENDICES Appendix 1 FIGURES AND SUMMARY TABLES Appendix 2 INDIVIDUAL DATA TABLES Appendix 3 CERTIFICATE OF ANALYSIS Appendix 4 PHASE REPORT FORMULATION ANALYSIS Appendix 5 PHASE REPORT HISTOPATHOLOGY Appendix 6 HISTORICAL DATA HAEMATOLOGY AND CLINICAL BIOCHEMISTRYEXP1313100 Project 505705

- Page 4 - 3. QUALITY ASSURANCE ST ATEMENT

WIL Research Europe B.V., ‘s

- Hertogenbosc h, 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 505705

Type of Inspections Phase/Process Start Inspection

date End Inspection

date Reporting

date

Study Protocol 20

- Aug

- 2014 20

- Aug

- 2014 20

- A ug

- 2014
Exposure 18

- Sep

- 2014 18

- Sep

- 2014 18

- Sep

- 2014

Protocol Amendment 01 13

- Oct

- 2014 13

- Oct

- 2014 13

- Oct

- 2014

Protocol Amendment 02 16

- Oct

- 2014 16

- Oct

- 2014 16

- Oct

- 2014

Protocol Amendment 03 03

- Nov

- 2014 03

- Nov

- 2014 03

- Nov

- 2014
Report 09

- Feb

- 2015 12

- Feb

- 2015 12

- Feb

- 2015

Process Animal Facilities 07

- Jul

- 2014 18

- Jul

- 2014 18

- Jul

- 2014 Test Substance Handling
Exposure

Observations/Measurements
Specimen Handling

Test Substance Receipt 24

- Jul

- 2014 31

- Jul

- 2014 01

- Aug

- 2014 Test Substance Handling

Test Substance Formulation 25

- Jul

- 2014 29

- Jul

- 2014 01

- Aug

- 2014 Test Substance Handling

Clinical pathology 04

- Aug

- 2014 07

- Aug

- 2014 07

- Aug

- 2014

Observations/Measurements
Specimen Handling

A nalytical and physical chemistry 05

- Aug

- 2014 12

- Aug

- 2014 13

- Aug

- 2014 Test Substance Handling
Exposure

Observations/Measurements
Specimen Handling

Necropsy 15

- Sep

- 2014 26

- Sep

- 2014 30

- Sep

- 2014

Observations/Measurements
Spec imen HandlingEXP1313100 Project 505705

- Page 6 - 4. SUMMARY

Title Repeated dose 28

- day oral toxicity study with EXP1313100 by daily gavage in the rat, followed by a

14

- day recovery period.

Guidelines The study was based on the following guideli nes.

- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.

- OECD 407, Repeated Dose 28

- day Or al Toxicity Study in Rodents, 2008.

- OPPTS 870.3050, Repeated dose 28

- day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712 -C- 00

- 366, 2000.

Rationale for dose levels

Based on the results of a 14

- day range finding study ( Project 505704 ) , the dose levels for this 28

- day oral gavage study were selected to be 0, 100

, 3 00 and 1000 mg/kg.

Study outline

The test substance, formulated in propylene glycol, was administered daily for 28 days by oral gavage

to SPF

- bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 1 4 days of recovery.

Evaluated parameters Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 6 hours. The following parameters were evaluated: clinical signs daily; functional observation tests in W eek 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Results

Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were stable over at least 6 hours.

Treatment up to 1000 mg/kg was well tolerated. Only body weight gain was lower in males at 1000 mg/kg during treatment and at initiation of the recovery phase in both sexes. Food intake remained normal and no toxicologically significant clinical signs were noted, and no mortality occurred

. No

treatment

- related changes in functional observation tests occurred

One ma le at 1 000 mg/kg showed a slight multifocal necrosis of the liver, which was considered to be

related to treatment

. Treatment

- related changes in clinical biochemistry parameters at 1000 mg/kg that would support hepatocellular damage included h igher alanine and as partate aminotransferase activity in both sexes (alanine aminotransferase activity was increased with a factor of approximately 12 and 7 for males and females, respectively), and higher alkaline phosphatase activity in males. Other clinical biochemistry changes at the end of treatment that were considered to be related to treatment at 1000 mg/kg consisted of a lower total protein, albumin, cholesterol and calcium level and higher total

bilirubin and bile acid level in both sexes

, and lower creatinine level in males

. At the end of the

r ecovery period, multifocal necrosis of the liver was absent and only total protein level remained lower in males at 1000 mg/kg. Given the magnitude of change of primarily alanine aminotransferase activity at 1000 mg/kg, in combination with the observed liver necrosis in a single male and other clinical biochemistry changes that point to a disturbance in liver function, it is considered that an adverse effect on liver integrity occurred at this dose.EXP1313100 Project 505705

- Page 7 - At 300 mg/kg, clinical biochemistry changes consisted of a h igher alanine and aspartate aminotransferase activity in males (not statistically significant), lower total protein, albumin and calcium

level in both sexes, higher total bilirubin level in females, and higher bile acid lev el in both sexes. At 100 mg/kg, clinical biochemistry changes were confined to lower total protein and albumin level s in
males, h igher total bilirubin levels in females and l ower calcium levels in both sexes

. These clinical biochemistry changes at 100 and 300 mg/kg were not supported histopathologically, and generally

remained within the range considered normal for rats of this age and strain. Also, these changes at 1000 mg/kg were essentially reversible within 14 days (except for the lower total protein le vel that remained lower in males at 1000 mg/kg ). Therefore, these clinical biochemistry changes at 100 and 300 mg/kg were considered not to be adverse in nature.

Haematological assessment showed higher white blood cell counts in both sexes at 1000 mg/kg a t the end of treatment. For males, the higher white blood cell counts were due to higher values of two animals, one of which also showed multifocal necrosis of the liver. Hence, this change in white blood cell counts was considered toxicologically significant for males. No toxicological significance was

ascribed to the higher white blood cell counts in females, since individual white blood cell values remained within the range considered normal for rats of this age and strain

, and the mean of th is
parameter was similar to control levels at the end of recovery. The shorter activated partial thromboplastin time in males at 1000 mg/kg at the end of the treatment and recovery period was considered not toxicologically relevant since the opposite effect (i.e. an extended activated partial thromboplastin time) would be expected in case of target organ toxicity, and since the changes were slight (means remained within the range considered normal for rats of this age and strain),

Histopathological assessment and nec ropsy findings did not support the organ weight changes

recorded at the end of treatment at 1000 mg/kg . Also, given that these changes were slight in nature (i.e. within the range considered normal for rats of this age and strain), and essentially resolved during the recovery period, these were considered to be of no toxicological relevance and may in part be related to the lower terminal body weights. At the end of treatment, these changes consisted of higher relative kidney weights and lower absolute and/ or relative pros tate and seminal vesicle weight in
males, and lower absolute and relative heart and thymus weight and higher relative spleen weight in

females

. At the end of the recovery period, all these organ weights were similar to control levels
except for relative kidney weights in males at 1000 mg/kg that remained slightly higher.

Conclusion

Based on the presence of multifocal necrosis of the liver in one male at 1000 mg/kg, and the magnitude of change of primarily alanine aminotransferase activity at 1000 mg/kg, in combination with other clinical biochemistry changes that point to a disturbance in liver function, a No Observed Adverse Effect Level (NOAEL) for the test material of 300 mg/kg was established.