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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:'
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### 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 additiori, 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 follows:

19 November 2008 

### 13 May 2009 19 May 2009 13 May 2009 21 May 2009 § 11 June 2009 § Date of QA Signature Test Material Preparation Test System Preparation 

## Exposure Assessment of Response Draft Report Audit 

### Final Report Audit Standard Test Method ComplianceAudit

§ Evaluation specific to this study



# ~

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# ~~

.. ................... For the Quality Assurance Unit* 

## 2 3
~~ 

## IN 2009 DATE: ................................................

*Authorised QA Signatures: Manager, Quality Assurance: J G Riley BSc (Hons) MRQA Deputy Head of Department JM Crowther MIScT MRQA SeniorAudit Staff: G Wren ONC MRQAPROJECT NUMBER:

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### GLP C®MPLIANCE 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 

### 200410994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 200419/EC and 2004/10/EC. 

### This report fully and accurately reflects the procedures used and data generated.



# ,~ ° _. .. . 

# , N Warren MIAT Study Director DATE: ........... 

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# ....

This report may be presented in final fonn as a dig'rNa1(pdf) tlocument. Such documents are prepared by scanning the paper original, and are considered of equivalent integrity and authenticity to versions produced by optical photowpy. 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 COMPLfANCE STATEMENT 3 

### CONTENTS 4 SUMMARY 5 

## 1. INTRODUCT[ON 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

— 

### Viability of RHC Tissues 15 Table 2 Quafitative 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 20PROJECTNUMBER:. PAGE5



### ASSESSMENT OF OCULAR IRRITATlON POTENTIAL USING THE SKINETHfC 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-yla-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 pi 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 

## ul of 1°/a 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 (01354o). Data are presented in the form of % viability (MTT conversion relative to negative controls). The test material was classified according to the following criteria: i) If the % relative mean tissue viability was z 60% the test material was considered to be non-irritant. ii) If the % relative mean tissue viability was < 60% the test materia[ was considered to be an irritant...
. .. .. . . . .. . .. ... .. .._...

~ PROJECT NUfJIBER: PAGE 6
Resu/ts. 

### The relative mean viability of the test material treated tissues after a 10 minute exposure was 108.0%. 

### 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 tesfmaterial was considered to be a Non-Irritant (NI).PRO.IECT NUMBER: " PAGE 7

ASSESSMENT OF OCULAR IRRITATION POTENTIAL USING THE SKlNETHIC 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 (9,Z)
The SkinEthic RHC model consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a comeal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. Test materials are applied directly to the cufture surFace, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists ofan airlifted, fiving, corneal tissue construct, produced in polycarbonate inserts in serumfree and chemically defined medium. The test is based on the hypothesis that irritant chemicals are abfe to penetrate the stratum corneum of the SkinEthic RHC model and are sufflciently cytotoxic to cause cell death in the underlying cel[ layers. Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the 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 control groups served as common controls with Harlan Laboratories Ltd Project number 272410005. The study was performed between 26 May 2009 and 28 May 2009.PROJECT NUh76ER: ` PAGE 8
2. TEST MATERIAL 2.4 Description, Identification and Storage Conditions Sponsors identification : Description : amber coloured liquid Batch number : Date received 11 December 2008 Storage conditions room temperature in the dark The integrity of supplied data relating to the identity, purity and stabiiity 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 materiaf was used as supplied.
3. NEGATIVE AND POSITIVE CONTROLS 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 solutioh in sterile distilled water.
4. SKINETHIC HCE MODEL Supplier SkinEthic Laboratories, Nlce, France Date Received 26 May 2009PROJECT NUMBER:' pAGE g



### 5. METHODS 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 rnimicking dehydrogenase activity of the cellular mitochondria of viable cells. This property of the test material is only a problem, if at the time of the MTT test (after the chemical has been rinsed ofl) 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 abiiity to reduce MTT directfy. 30 NI of test material was added to a 0.5 mgtml MTT solution and incubated at room temperature in the dark for 60 minutes. Untreated MTT solution was used as a control. If the MTT so4ution tumed 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% CO 2 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 NUMBER PAGE 10

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### 5.2 Main Test Triplicate tissues were treated with 30 pl 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 equai exposure time. Triplicate tissues were treated with 30 Nl of soiution A to serve as negative controls and triplicate tissues were treated with 30 pl of 1% w/v SDS to serve as positive controls. The plates were incubated at 37°C, 5% COZ 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 blotting the bottom of the insert with absorbent paper. Each tissue was 

### placed into a pre-labelled 24-well plate designated 'holding plate' containing 300 pI of maintenance medium (at room temperature) until all the tissues were rinsed. Following 

### rinsing, the tissues (two per group) were transferred to a pre-iabelled 24-well plate designated 'MTT Loading plate' containing 300 ul 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% COZ 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 mi 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 fltted 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 pI samples were transferred to the appropriate wells of a pre-labelled 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.PR03ECT 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 OD540 values of the duplicate tissues were calculated. Each of these ODeao values had already been corrected for bfanks by the microplate reader. The relative mean tissue viabilities (% of the negative control) were calculated as follows:


### Relative mean tissue viabifity = mean OD $4o of test material 

### x 100 mean OD s , o of negative control The mean tissue viabirities for the test material was compared to the respective untreated negative control and classified according to the following table: Relative Mean tissue viability Prediction (% 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 foflowing assay acceptance criterion was achieved: 

### AssayAcceptance Criterion: Positive Control 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 NUMBER
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### 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 ftve 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 OD 540 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 108.0%. 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. CONCLUS[ON 

### According to the protocol followed the test material was considered to be a Non-lrritant (NI)•PROJECT NUflflBER:' PAGE 14

11. REFERENCES 1. Van Goethem F., Adriaens E., Afepee N., Straube F., De Wever B., Cappadoro M., Catoire S., Hansen E., Wolf A. and Vanparys P. Prevafidation of a new 

### in vitro reconstituted human comea 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 vitm 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 0D 5 , o % Viability

0.960 Negative Control® 0.909 100* 0.857

0.704 Positive Control® 0.682 75.0 0.660

0.964 Test Material 0.982 108.0 0.999

»= The mean viability of the negative control tissues is set at 100% ®= Control group shared with Harlan Laboratories Ltd Project number 2724/0005PROJECT NUMBER:' PAGE16
ASSESSMENT OF OCULAR IRRITATIOIt[ POTENTIAL USING THE SKINETHIC RECONSTITUTEf} HUMAN CORNEAL EPITHELIUM MODEL 

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

evaluation)

Material Score Tissue 1 Tissue 2
Negative Control®

Positive Control® + +

Test Matenal

MTT Yisual Scoring Scheme of SkinEthic Tissues

- = Blue tissue (viable) + = Blue/White tissue (semi viable) ++ = Tissue completely white (dead)

®= Control group shared with Harlan Laboratories Ltd Project number 2724l0005PROJECT NUWSBER:'

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

### Appendix 1 Sofution A Composition for 1 Litre

9 

### Na2HPO4 0.142 g/I ® Glucose 1.802 g!I ® HEPES 7.149 g/I • KCI 0.224 g/I ® NaCI 7.597 g/I PAGE 17PROJECT NUMBER:: PAGE 18



### Appendix 2 Certificate of Analysis

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### Appendix 2(continued) Certificate of Analysis

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### Appendix 3 Staterrtent of GLP Compliance in Accordance with Directive

2004/9/EC ; 

## I Department

~ 

### of Heafth

THE I3EPARTMEINT UF IiEALTH OF 1'HE GOVERNMENT

OF THE UMTED KIPTGDOM

- GOOD LABORATORY PRACTICE - - STATEMENTOFCOMPLL4NCE
. INACCORDANCER7TFTDIRECTIVE20Dd/91EC

iFSTFACIWTY TFSTTYPE

. HarintiLaboratocieSLtd. Analyfical/ClinicalChemistiT

Shardlow Business Park Enadronmental Fute London Road Environmental Tovcnlogy

. Shardlor Mutagenieity

Derby - - Phys7Chem

. DE722GD Toxicolugy

DATEOFtNSPECTION ' I9th.zlugust 2008 , A general inspection for compfiauee with the Priuciples of Good Laboratory Pactice was carried out at the above test.faci&ty as patt of tlie tiK GLP Complfance Programme. - At the tinte of inspection no deviations were found of sufficient magnitude to affect the.vatidity of non-clinicai studies performed at these facilities.

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• 'Dr.Andre»7.Gray

_, Reed, IIK GLP Monitoring Autfiority