## FINAL REPORT

Study Title



## EVALUATION OF THE MU TAGENIC ACTIVITY OF EXP1313100

IN THE SALMONELLA TYPHIMURI UM REVERSE MUTATION
ASSAY AND THE ESCHERICHIA COLI REVERSE MUTATION
ASSAY

Author

Test Facility

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

- Hertogenbosch The Netherlands

Laboratory Project Identification

Project 505689 Substance 205622

/A

- Page 1 of 30

-EXP1313100 Project 505689

- Page 2 - 1. CONTENTS

1. CONTENTS

.................................................................................................................................. 2 2. STATEMENT OF GLP COMPLIANCE

.......................................................................................... 3 3. QUALITY ASSURANCE STATEMENT

......................................................................................... 4 4. SUMMARY

................................................................................................................................... 5 5. INTRODUCTION

.......................................................................................................................... 6 5.1. Preface

................................................................................................................................ . 6 5.2. Aims of the study

................................................................................................................... 6 5.3. Guidelines

............................................................................................................................. 6 5.4. Storage and retention of records and materials

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

...................................................................................................... 7 6.1. Test substance

...................................................................................................................... 7 6.1.1. Test substance information

......................................................................................... 7 6.1.2. Study specific test substance information

.................................................................... 7 6.1.3. Test substance preparation

......................................................................................... 7 6.2. Reference substances

.......................................................................................................... 7 6.2.1. Negative control

.......................................................................................................... 7 6.2.2. Positive controls

.......................................................................................................... 7 6.3. Test system

........................................................................................................................... 8 6.4. Cell culture

............................................................................................................................ 9 6.5. Metabolic activation system

.................................................................................................. 9 6.5.1. Preparation of S9-mix

................................................................................................ . 9 6.6. Study design

......................................................................................................................... 9 6.6.1. Dose range finding test

............................................................................................... 9 6.6.2. Mutation assay

............................................................................................................ 9 6.6.3. Colony counting

......................................................................................................... 10 6.7. Electronic data capture

......................................................................................................... 10 6.8. Interpretation

........................................................................................................................ 10 6.8.1. Acceptability of the assay

........................................................................................... 10 6.8.2. Data evaluation and statistical procedures

................................................................ . 10 6.9. List of deviations

.................................................................................................................. 11 6.9.1. List of protocol deviations

........................................................................................... 11 6.9.2. List of standard operating procedures deviations

....................................................... 11 7. RESULTS

.................................................................................................................................... 11 7.1. Dose range finding test/first mutation experiment

................................................................ . 11 7.2. Experiment 2

........................................................................................................................ 12 8. DISCUSSION AND CONCLUSI ON

............................................................................................. 12 9. REFERENCES

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

TABLES Table 1 Dose range finding test: Mutagenic response of EXP1313100 in the Salmonella

typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

......................................................................................................................... 13 Table 2 Experiment 1: Mutagenic response of EXP1313100 in the Salmonella

typhimurium reverse mutation assay

.......................................................................... 14 Table 3 Experiment 2: Mutagenic response of EXP1313100 in the Salmonella

typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

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

APPENDICES APPENDIX 1 SUMMARY TABLES

.................................................................................................. 13 APPENDIX 2 SUPPORTING MATERIALS AND METHOD

.............................................................. 16 APPENDIX 3 DETAILED TABLES

................................................................................................... 17 APPENDIX 4 HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL

............................... 28 APPENDIX 5 HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL SUBSTANCES

....... 29 APPENDIX 6 CERTIFICATE OF ANALYSIS

................................................................................... 30EXP1313100 Project 505689

- Page 5 - 4. SUMMARY

Evaluation of the mutagenic activity of EXP1313100 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

EXP1313100 was tested in the Salmonella typhimuriumreverse mutation assay with four histidinerequiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli

reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP 2 uvrA). The test was performed in two independent experiments in the presence and absence of S9 -mix (rat liver S9-mix induced by Aroclor 1254 ).

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 dissolved in dimethyl sulfoxi de

. In the dose range finding test, EXP1313100 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP 2 uvrA. EXP1313100 did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the mutation assay.

Based on the results of the dose range finding test, EXP1313100 was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, EXP1313100 was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP 2 uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

EXP1313100 did not induce a significant dose-related increase in the number of revertant (His + ) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp + ) colonies in tester strain WP 2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that EXP1313100 is not mutagenic in the Salmonella typhimurium

reverse mutation assay and in the Escherichia coli reverse mutation assay.