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### ASSESSMENT OF OCULAR IRRITATION POTENTIAL USING THE SKINETHIC RECONSTITUTED HUMAN CORNEAL EPITHELIUM MODEL

PROJECT NUMBER:

AUTHOR: N Warren

STUDY SPONSOR: TEST FACILITY: Harlan Laboratories Ltd Shardlow Business Park Shardlow Derbyshire DE72 2GD UK Telephone: +44 (0) 1332 792896 Facsimile: +44 (0) 1332 799018PROJECT NUMBER ~ PAGE 2


### QUALITY ASSURANCE REPORT This study type is classed as short-term. The standard test method for this study type ("General Study Plan° in OECD terminology) was reviewed for compliance once only on initial production. Inspection of the routine and repetitive procedures that constitute the study is carried out as a continuous process designed to encompass the major phases at or about the time this study was in progress. In addition, inspection of general facilities not specifically related to this study are done monthly or annually in accordance with QA Standard Procedure. This report has been audited by the Quality Assurance Unit, and is considered to be an accurate account of the data generated and of the procedures followed. In each case, the outcome of QA evaluation is reported to the Study Director and Management on the day of evaluation. Audits of study documentation, and process inspections appropriate to the type and schedule of this study were as foliows:

19 November 2008 04 November 2009 04 November 2009 04 November 2009 09 November 2009 Standard Test Method Compliance Audit Test Material Preparation Test System Preparation Exposure Assessment of Response § 08 December 2009 Draft Report Audit § Date of QA Signature Final Report Audit § Evaluation specific to this study

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*Authorised QA Signatures: Senior Audit Staff: J G Riley BSc (Hons) MRQA, J M Crowther MIScT MRQA, G Wren ONC MRQA, S Bevan BSc (Hons) MRQA, L Blaney MRQAPROJECTNUMBER:S PAGE 3


### GLP C®IVIPLIANCE STATEMENT The work described was perFormed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC1CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004110/EC. This report fully and accurately reflects the procedures used and data generated.

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### N Warren MIAT Study Director

This report may be presented in finai form as a digital (pdt) document. Such documents are prepared by scanning the paper original, and are considered of equivalent integrity and authenticity to versions produced by optical photocopy. However, in all cases the hand-signed paper original, held in secure archives, is the definitive document.PROJECT NUMBER: PAGE 4


### CONTENTS QUALITY ASSURANCE REPORT 2 GLP COMPLIANCE STATEMENT 3 CONTENTS 4 SUMMARY 5 1. INTRODUCTION 7 2. TEST MATERIAL 8 2.1 Description, Identification and Storage Conditions 8 2.2 Preparation of Test Material 8 3. NEGATIVE AND POSITIVE CONTROLS 8 4. SKINETHIC HCE MODEL 8 5. METHODS 9 5.1 Pre-Test 9 5.2 Main Test 10 6. INTERPRETATION OF RESULTS 11 7. ASSAY ACCEPTANCE CRITERIA 11 8. ARCHIVES 12 9. RESULTS 13 9.1 Assessment of Direct Test Material Reduction of MTT 13 9.2 Assessment of Eye Irritation Potential 13 9.3 Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment) 13 9.4 Assay Acceptance Criterion 13 10. CONCLUSION 13 11. REFERENCES 14 Table 1 Assessment of Eye Irritation Potential

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### Viability of RHC Tissues 15 Table 2 Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation) 16 Appendix 1 Solution A Composition for 1 Litre 17 Appendix 2 Certificate of Analysis 18 Appendix 3 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 19PROJECT NUMBER: PAGE5

ASSESSMENT OF OCULAR IRRITATION POTENTIAL USING THE SKiNETHIC 

### RECONSTITUTED HUMAN CORNEAL EPITHELIUM MODEL SUMMARY introduction. The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.
Methods. The experimental design of the study consists of a test for direct reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) by the test material followed by the main test. For the main test, triplicate SkinEthic tissues were treated with 30 NI of the test material for 10 minutes. Triplicate tissues treated with 30 NI of Solution A served as the negative control and triplicate tissues treated with 30 NI of 1%w/v Sodium Dodecyl Sulphate served as the positive control. At the end of the exposure period each SkinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues. After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD5 40 ). Data are presented in the form of percentage viability (MTT conversion relative to negative controls). The test material was classified according to the following criteria: i) If the percentage relative mean tissue viability was ?60% the test material was considered to be non-irritant. ii) If the percentage relative mean tissue viability was <60% the test material was considered to be an irritant.PROJECT NUMBER; PAGE 6 Results. The relative mean viability of the test material treated tissues after a 10 minute exposure was 86.3%. It was considered unnecessary to proceed with tissue histopathology. 

### Quality criteria. The quality criteria required for acceptance of results in the test were satisfied. 

### Conclusion. According to the protocol followed the test material was considered to be a Non-Irritant (NI).PROJECT NUMBER: PAGE 7

ASSESSMENT OF OCULAR IRRITATION POTENTIAL USING THE SKINETHIC RECONSTITUTED HUMAN CORNEAL EPITHELIUM MODEL
1. 

### INTRODUCTION The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes (' 2)

. The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. The test material is applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium. The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHC model and are sufficiehtly cytotoxic to cause cell death in the underlying cell layers. Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in #he test material treated tissues (quantitative measurement of tissue viability) relative to the negative control. One tissue for each treatment group was retained for possible tissue histopathology. The study was performed between 01 December 2009 and 03 December 2009.PROJECT NUMBER: PAGE8

2. TEST MATERIAL 2.1 Description, Identification and Storage Conditions Sponsor's identification Description pale amber coloured Iiquid Batch number : Date received 26 October 2009 Storage conditions : room temperature in the dark The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor. A Certificate of Analysis supplied by the Sponsor is given in Appendix 2.
2.2 Preparation of Test Material The test material was used as supplied.
3. NEGATIVE AND POSITIVE CONTROL3 The negative control material, Solution A, was used as supplied (composition of Solution A included as Appendix 1). The positive control material, Sodium Dodecyl Sulphate (SDS), was prepared as a 1% w/v solution in sterile distilled water.
4. SKINETHIC HCE MODEL Supplier : SkinEthic Laboratories, Nice, France Date Received 01 December 2009PROJECT NUMBER: PAGE 9

5.1 Pre-Test
5.1.1 Assessment of Direct Test Material Reduction of MTT One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT to formazan, thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. This property of the test substance is only a problem, if at the time of the MTT test (after the chemical has been rinsed off) there are still sufficient amounts of the test material on or in the tissues. To identify this possible interference, the test material was checked for its ability to reduce MTT directly. 30 NI of test material was added to a 0.5 mg/ml MTT solution and incubated at room temperature in the dark for 60 minutes. Untreated MTT solution was used as a control. If the MTT solution turned black/purple, the test material was presumed to have reduced the MTT.
5.1.2 Receipt of Tissues On arrival, the SkinEthic HCE tissues (Day 6 cultures), were stored at room temperature prior to transferring into 24-well plates designated 'arrival plates' containing 300 NI of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37°C, 5% COZ in air.
5.1.3 Preparation of Tissues Using sterile techniques, 1 ml of maintenance medium at room temperature, was dispensed into the appropriate number of wells of 6-well plates designated 'treatment plates'. Each well was labelled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for the test material and negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment, the 7 day old tissues were transferred from the 'arrival plates' into the wells of the 'treatment plates' containing the maintenance medium.PROJECT NUhABERr - PAGE 10 5.2 Maln Test Triplicate tissues were treated with 

### 30 NI of the test material for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 

### 30 pl of solution A to serve as negative controls and triplicate tissues were treated with 

### 30 NI of 1% w/v SDS to serve as positive controls. The plates were incubated at 

### 37°C, 5% CO2 in air during the exposure time. At the end of the relevant exposure period, each tissue insert was removed from the well using forceps and rinsed using a wash boftle containing Dulbecco's Phosphate Buffered Saline (DPBS). Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blofting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated 'holding plate' containing 

### 300 pl of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing, the tissues (two per group) were transferred to a pre-labelled 24-well plate designated 'MTT Loading plate' containing 

### 300 pl of a 0.5 mg/ml MTT solution freshly prepared in maintenance medium. The MTT loading plate was placed into an incubator for approximately three hours at 

### 37°C, 5% CO2 in air. At the end of the incubation period, the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labelled 24-well plate designated 'MTT extraction plate' containing 0.75 ml of Isopropanol in each of a sufficient number of wells. An extra 0.75 ml of Isopropanol was added onto each tissue and the plate sealed to prevent Isopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue. At the end of the extraction period, each tissue insert was pierced with a pipefte fifted with a 1000 

### NI tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 pl samples were transferred to the appropriate wells of a pre-Iabelled 96-well plate. 200 

### NI of Isopropanol alone was added to three wells designated as `blanks'. The optical density was measured (quantitative measurement of tissue viability) at 540nm (OD 540 ) using the Anthos 2001 microplate reader.PROJECT NUMBER:' PAGE 11



### 5.2.1 Tissue Histology One tissue for each treatment group was retained for possible tissue histopathology. The tissues were carefully cut out of the polycarbonate inserts with a sharp scalpel. The tissues were carefully cut in half. Both halves were placed into a pre-labelled 1.5 ml Eppendorf tube containing 1 ml of 10% Formalin and stored at room temperature.
6. INTERPRETATION OF RESULTS The mean OD 540 values of the duplicate tissues were calculated. Each of these OD540 values had already been corrected for blanks by the microplate reader. The relative mean tissue viabilities (percentage of the negative control) were calculated as follows: Relative mean tissue viability (%) = 

### mean 

### OD 54° of test material x 100 mean OD, 4O of negative control The mean tissue viabilities for the test material was compared to the respective untreated negative control and classified according to the following table: Relative Mean tissue viability Predictiori ercenta e of ne ative control Tissue viability <60 Irritant (1) Tissue viability ?60 Non-Irritant (NI) 7. ASSAY ACCEPTANCE CRITERIA The results of the assay are considered acceptable if the following assay acceptance criterion was achieved: 

### AssayAcceptance Criterion: Positive Contro/ The assay meets the acceptance criterion for an acceptable test if a decrease in relative tissue viability is observed following the 10-minute exposure period.PROJECT M19UMBER:: PAGE 12 8. ARCHIVES Unless instructed otherwise by the Sponsor, all original data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years, after which instructions will be sought as to further retention or disposal.PROJECT NUMBER:' PAGE 13

9. RESULTS 9.1 Assessment of Direct Test Material Reduction of MTT The test material was not able to directly reduce MTT.
9.2 Assessment of Eye Irritation Potential The mean OD540 values and mean viabilities for each treatment group are given in Table 1. The relative mean viability of the test material treated tissues after a 10 minute exposure was 86.3%. It was considered unnecessary to proceed with tissue histopathology.
9.3 Qualitative Evaluation of Tissue Viability (MTT Uptake Visual

Assessment) The qualitative evaluation of tissue viability 

### is presented in Table 2. The test material and negative control material treated tissues appeared blue which was considered to be indicative of viable tissue. The positive control material treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue.
9.4 Assay Acceptance Criterion The quality criterion required for the acceptance of results in the test was satisfied.
10. CONCLUSION According to the protocol followed the test material was considered to be a Non-Irritant (NI).PROJECT NUMBER: ; PAGE 14

11. REFERENCES 1. Van Goethem F., Adriaens E., Alepee N., Straube F., De Wever B., Cappadoro M., Catoire S., Hansen E., Wolf A. and Vanparys P. Prevalidation of a new in vitro reconstituted human cornea model to assess the eye irritating potential of chemicals. Toxicology 

### in vitro 20 (2006), pp.1-17.
2. Nguyen D. H., Beuerman R. W., De Wever B. and Rosdy M. Three-dimensional construct of the human corneal epithelium for 

### in vitro toxicology. In: H. Salem and S.A. Katz, Editors, Alternative Toxicological Methods, CRC Press (2003), pp. 147-159.PROJECT NUMBER: PAGE 15

ASSESSMENT OF OCULAR IRRITATION POTENTIAL USING THE SKINETHIC RECONSTITUTED HUMAN CORNEAL EPITHELIUM MODEL 

### Table 1 Assessment of Eye Irritation Potential - Viability of RHC Tissues

Material Mean Tissue Viability Mean OD

~ o Viability (%)
0.962 Negative Control 0.941 100* 0.920

0.208 Positive Control 0.218 23.2 0.227

0.819 Test Material 0.812 86.3 0.804

*= The mean viability of the negative control tissues is set at 100%PROJECT NUMBER:
. . PAGE 16

. ASSESSMENT OF OCULAR IRRITATION POTENTIAL USING THE SKINETHIC RECONSTITUTED HUMAN CORNEAL EPITHELIUM MODEL. 

### Table 2 Qualitative Evaluation of Tissue Viability (MTT uptake visual

evaluation)

Material Score Tissue 1 Tissue 2
Negative Control

Positive Control

Test Material
MTT Visual Scoring Scheme of SkinEthic Tissues = Blue tissue (viable) + = Blue/White tissue (semi viable) ++ = Tissue completely white (dead)PROJECT NUMBER:

ASSESSMENT OF OCULAR IRRITATION POTENTIAL USIIe1G THE SKINETHIC RECONSTITUTED HUMAN CORNEAL EPITHELIUM MODEL 

### Appendix 1 Solution A Composition for 1 Litre ® Na2HPO4 0.142 g/I ® Glucose 1.802 g/I ® HEPES 7.149 g/I ® KCI 0.224 g/1

m 

### NaCI 7.597 g/I PAGE 17PROJECT NUMBER: PAGE 18



### Appendix 2 Certificate of Analysis

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### Appendfx 3 Statement of GLP Compliance in Accordance wtith Directive

2004/9/EC 

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## of Health

Tf3(E DEPARTMEN 1' OB HF,AI.'I'H OF THE GOi'ERIVIVEFNT

OF TIIE UIVITED KINGDOM GOOD LABORAT'ORY PRP.CTYCE

. ST.ITEMEryTOFCOMIR.IqNCE IN ACCORDANCE WITA DDtF,t°PIYE 200419/EC

TE9TFAGIIITY TES'I'TYPE .

Harian Laboratories Ltd. ShardlowBnsiness Park London Road S)tardlow Derby DE72 2GD AnalytirallCllnical Chemistry Environroental Fate Envlroumental Toxicology Mutagenieity Ptiys/Chem TozicnlogY

DAZEOF/7VSPECTI0N 15tfi September2009 A general.iiaspection fnr compliance with the Principles of Good Laboratory Practice was canied out at the aliove test faciliTy as part of the UR GLP Compliance Programme. At.the time of inspection. no deviations were found of sufficient maguitude to.aftect the validityof non-clinical studies perfoimed at these facilities.



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