PAGE 1 OF 19 PAGES 

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 799018 
PROJECT 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 

~_ ) \' ~1 ~ ~~~ V ............................ DATE:..........Z. ~Y..D % 2009 

.......................... ..... .......... 

For the Quality Assurance Unit' 

*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 MRQA 
PROJECTNUMBER: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. 

.................... /..V..................... ................. DATE: .......... zL.~I~~~~.7... 

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 — 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 19 
PROJECT 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 

(OD540). 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 2009 
PROJECT 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 OD540 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 OD54° 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 17 
PROJECT NUMBER: PAGE 18 

Appendix 2 Certificate of Analysis 

E • A " f 

13pte 2211012009 

Tesr f9 R39"9 

I#ttici€ 

F'rnduet 

Test IL}'rthnd Test Ilescreptiun 

OP&5 pH 

6P2J1 B .4sls 

BP'tIVs Gcalow 

UPhIS6 Totw $WAs 

(JC2?2 Nitresgen 

C7rs#amer,'Vexnae It & Lr 

G"uslamer f#srBrr ;1'o. 

,4ffn. 7M'aac. F'est ReSulf 

6.2 

2, i 

5.0 

24,0 

24 

3igner7 

d, F- PPak'f+°Ry At9 ffCarotab) C. CYiem. 3fRSC 

abrb+ucnf :Fiamagsrl f°we J erf f 
PROJECT NUMBER: PAGE 19 

Appendfx 3 Statement of GLP Compliance in Accordance wtith Directive 

2004/9/EC 

pjy Department 

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. 

zG ~ ,11o°i 

DnAndcew J. Gray 

Iiead,UKGLPMonitoringAuthorit.y .. ..