## FINAL REPORT

Study Title



## IN VITRO SKIN IRRITATION TEST WITH EXP1313100 USING A

HUMAN SKIN MODEL

Author

Test Facility

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

- Hertogenbosch The Netherlands

Laboratory Project Identification

Project 505700 Substance 205622/A

- Page 1 of 16

-EXP1313100 Pr oject 505700

- 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

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

........................................................................................................................... 7 6.4. Preparation and preincubation

.............................................................................................. 8 6.5. Study design

......................................................................................................................... 8 6.5.1. Test for reduction of MTT by the test substance

.......................................................... 8 6.5.2. Application/Treatment of the test substance

................................................................ 8 6.5.3. Cell viability measurement

.......................................................................................... 9 6.6. Electronic data capture

.......................................................................................................... 9 6.7. Interpretation

......................................................................................................................... 9 6.7.1. Acceptability of the assay

............................................................................................ 9 6.7.2. Data evaluation and statistical procedures

.................................................................. 9 6.8. List of deviations

.................................................................................................................. 10 6.8.1. List of protocol deviations

........................................................................................... 10 6.8.2. List of standard operating procedures deviations

....................................................... 10 7. RESULTS

.................................................................................................................................... 10 8. CONCLUSION

............................................................................................................................. 10 9. REFERENCES

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

TABLES Table 1 Mean absorption in the in vitro skin irritation test with EXP1313100

........................... 12 Table 2 Mean tissue viability in the in vitro skin irritation test with EXP1313100

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

FIGURES Figure 1 A diagram of the application.

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

APPENDICES APPENDIX 1 TABLES

..................................................................................................................... 12 APPENDIX 2 INDIVIDUAL OD MEASUREMENTS AT 570 NM

....................................................... 13 APPENDIX 3 HISTORICAL CONTROL DATA FOR IN VITRO SKIN IRRITATION STUDIES

.......... 14 APPENDIX 4 TECHNICAL DATA, SAFETY SHEET AND CERTIFICATE OF ANALYSIS

RECONSTRUCTED HUMAN EPIDERMIS

................................................................ 15 APPENDIX 5 CERTIFICATE OF ANALYSIS

................................................................................... 16EXP1313100 Pr oject 505700

- Page 5 - 4. SUMMARY

In vitro skin irritation test with EXP1313100 using a human skin model.

This report describes the ability of EXP1313100 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN- SM TM )). The possible skin irritation potential of EXP1313100 was tested through topical application for 15 minutes.

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

Batch E00873- 025 -001 of EXP1313100 was a brown solid. Skin tissue was moistened with 5 μl of Milli-Q water and 10.2 to 10.9 mg of EXP1313100 was applied directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with EXP1313100 compared to the negative control tissues was 120 %. Since the mean relative tissue viability for EXP1313100 was above 50% after 15 minutes treatment EXP1313100 is considered to be non-irritant.

The positive control had a mean cell viability of 10 % after 15 minutes exposure. The absolute mean OD 570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that EXP1313100 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.