PAGE 1 OF 19 PAGES 

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DETERMINATION OF SKIN IRRITATION POTENTIAL USING 

THE EPISKINTMRECOhISTITUTED HUMAN EPIDERMIS 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 repoit 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 Standard Test Method Compliance Audit 

08 October 2009 Test Material Preparation 

07 October 2009 Test System Preparation 

08 October 2009 Exposure 

09 October 2009 Assessment of Response 

§ 18 November 2009 Draft Report Audit 

§ Date of QA Signature Final Report Audit 

§ Evaluation specific to this study 

.. DATE: °t4€~~3 

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

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 
PROJECT NUMBER:.. PAGE3 

GLP COMPLIANCE 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 2004/91EC 

and 2004/10/EC. 

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

_ ~~~~ _ DATE: ... &Mv.V..l.. 

N Warren MIAT 

Study Director 

This report may be presented in final form as a digdal (pdf) document. Such documents are prepared by scanning the paper original, 

and are considered of equivalent integrity and authenticity ta 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 

CKo7.-N:1 >YK9 

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 CONTROL MATERIALS 8 

3.1 Preparation of Negative and Positive Control Materials, MTT and Acidified 

Isopropanol 8 

4. EPISKINTMM®d®1 Klt 9 

5. PROCEDURE 9 

5.1 Pre-Test 9 

5.2 Main Test 10 

6. INTERPRETATION OF RESULTS 12 

6.1 Quantitative MTT Assessment (percentage tissue viability) 12 

6.2 Quality Criteria 12 

7. ARCHIVES 13 

8. RESULTS 14 

8.1 Direct MTT Reduction 14 

8.2 Test Material, Posftive Control Material and Negative Control Material 14 

8.3 Quality Criteria 14 

9. CONCLUSION 14 

10. REFERENCE3 15 

Table 1 Mean OD540 Values and Percentage Viabilities for the Negative Control 

Material, Positive Control Material and Test Material 16 

Table 2 Qualitative Evaluation of Tissue Viability (MTT uptake visual 

evaluation) 17 

Appendix 1 Certificate of Analysis 18 

Appendix 2 Statement of GLP Compliance in Accordance with Directive 2004/9/EC 19 
PROJECTNUMBER: __._ PAGE5 

DETERMINATION OF SKIN IRRITATION POTENTIAL USING 

THE EPISKINTM RECONSTITUTED HUMAN EPIDERMIS MODEL 

SUMMARY 

lntroduction. The purpose of this test was to evaluate the skin irritation potential of the 

test material using the EPISKINTM reconstituted human epidermis model after a 

treatment period of 15 minutes followed by a post-exposure incubation period of 

42 hours. The principle of the assay was based on the measurement of cytotoxicity in 

reconstituted human epidermal cultures following topical exposure to the test material by 

means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic 

reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in 

the test material treated tissues relative to the negative controls. The concentration of 

the inflammatory mediator IL-1q in the culture medium retained following the 42 hour 

post-exposure incubation period is also determined for test materials which are found to 

be borderline non-irritant based upon the MTT reduction endpoint. This complimentary 

end-point will be used to either confirm a non-irritant result or will be used to override the 

non-irritant result. 

Triplicate tissues were treated with the test material for an exposure period of 

15 minutes. At the end of the exposure period each tissue was rinsed before incubating 

for approximately 42 hours. At the end of the post-exposure incubation period each 

tissue was taken for MTT-loading. The maintenance medium from beneath each tissue 

was transferred to pre-labelled micro tubes and stored in a freezer for possible 

inflammatory mediator determination. After MTT loading a total biopsy of each epidermis 

was made and placed into micro tubes containing acidified isopropanol for extraction of 

formazan crystals out of the MTT-loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and 

duplicate 200 pl samples were transferred to the appropriate wells of a pre-labelled 

96-well plate. The optical density was measured at 540 nm. 

Data are presented in the form of percentage viability (MTf reduction in the test material 

treated tissues relative to negative control tissues). 
PROJECT NUMBER: PA6E 6 

Resu/fs: The relative mean viability of the test material treated tissues was 97.8% after 

a 15-minute exposure. 

Qualify criteria: The quality criteria required for acceptance of results in the test were 

satisfied. 

Conclusion: The test material was considered to be Non-Irritant. 
PROJECT NUMBER: _ . _ PAGE 7 

DETERMINATION OF SKIN IRRITATION POTENTIAL USING 

THE EPISKINTM RECONSTITUTED HUMAN EPIDERMIS MODEL 

1. INTRODUCTION 

The purpose of this test was to evaluate the skin irritation potential of the test material 

using the EPISKINTM reconstituted human epidermis model after a treatment period of 

15 minutes followed by a post-exposure incubation period of 42 hours (1,2,3,4) The 

principle of the assay is based on the measurement of cytotoxicity in reconstituted 

human epidermal cultures following topical exposure to the test material by means of the 

colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of 

the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium 

bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test 

material treated tissues relative to the negative controls. The concentration of the 

inflammatory mediator IL-1a in the culture medium retained following the 42 hour 

post-exposure incubation period is also determined for test materials which are found to 

be borderline non-irritant based upon the MTT reduction endpoint. This complimentary 

end-point will be used to either confirm a non-irritant result or will be used to override the 

non-irritant result. 

The EPISKINTM model is a three-dimensional reconstituted human epidermis model 

consisting of adult human-derived epidermal keratinocytes seeded on a dermal 

substitute consisting of a collagen type I matrix coated with type IV collagen. A highly 

differentiated and stratified epidermis model is obtained after a 13 day culture period 

comprising of the main basal, supra basal, spinous and granular layers and a functional 

stratum corneum. 

The study was perFormed between 04 November 2009 and 09 November 2009. 
PROJECT NUMBER: PAGE 8 

2. TEST MATERIAL 

2.1 Description, Identification and Storage Conditions 

Sponsor's identification : 

Description pale amber coloured liquid 

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 1 

2.2 Preparation of Test Material 

The test material was used as supplied. 

3. NEGATIVE AND POSITIVE CONTROL MATERIALS 

Dulbecco's Phosphate Buffered Salfne (PBS) with Ca" and Mg` ~ was used as the 

negative control. 

Sodium Dodecyl Sulphate (SDS) was used as the positive control. 

3.1 Preparation of Negative and Positive Control Materials, MTT and Acidified 

Isopropanol 

The negative control materiaf was used as supplied. 

The positive control material was prepared as a 5% w/v aqueous dilution. 

A 3 mg/ml MTT stock solution was prepared in PBS. _ The stock solution was diluted to 

0.3 mg/ml with assay medium when required. 

A 0.04 N concentration of hydrochloric acid in Isopropanol was prepared when required. 
PROJECT NUMBER: PAGE 9 

4. EPISKtFITM Model 6Cit 

Date received : 03 November 2009 

5. PR®CEDURE 

5.1 Pre-Test 

5.1.1 Assessment of Direct Test Material Reduction of MTT 

5.1,1.1 MTT dye metabolism, ceU viability assay 

The MTT assay, a colourimetric method of determining cell viability, is based on 

reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a purple formazan dye by mitochondrial succinate 

dehydrogenase in viable cells. 

5,1.1,2 Assessment of Direct Test Materia! Reduction of MTT 

One limitation of the assay is possible interFerence of the test substance with MTT. A 

test substance may directly reduce MTT, thus mimicking dehydrogenase activity of the 

cellular mitochondria. This property of the test substance is only a problem, if at the time 

of the MTT test (after rinsing) there are still sufficient amounts of the test substance 

present on or in the tissues. In this case, the true metabolic MTT reduction and the false 

direct MTT reduction can be differentiated and quantified by the procedure described as 

follows: 

5.1.1.3 Test for Direct MTT Reduction 

As specified, a test substance may interFere with the MTT endpoint, if it is able to directly 

reduce MTT and at the same time is present on or in the tissues when the MTT viability 

test is perFormed. To identify this possible interference, each test substance is checked 

for the ability to directly reduce MTT according to the following procedure: 

10 NI of the test material was added to 2 mi of a 0.3 mg/ml MTT solution freshly prepared 

in assay medium. The solution was incubated in the dark at 37°C, 5% CO Z in air for 

3 hours. Untreated MTT solution was used as a control. 
PROJECT NUMBER: PAGE 10 

If the MTT solution containing the test substance turns bluelpurple, the test substance is 

presumed to have reduced the MTT. 

5.1.2 Pre-Incubation (Day 0: tissue arrival) 

2 mI of maintenance medium, warmed to approximately 37°C, was pipetted into the first 

column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred 

into the maintenance medium filled wells (3 units per plate). A different 12-well plate was 

used for the test material and each control material. The tissues were incubated at 37°C, 

5% COZ in air for at least 24 hours. 

5.2 Main Test 

5.2.1 Application of Test Material and Rinsing (Day 1) 

2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the 

second column of 3 wells of the 12-well plate. 

Triplicate tissues were treated with the test material for an exposure period of 

15 minutes. The test material was applied topically to the corresponding tissues 

ensuring uniform covering. 10 NI of the test material was applied to the epidermis 

surface. Triplicate tissues treated with 10 NI of PBS served as the negative controls and 

triplicate tissues treated with 10 NI of SDS 5% w/v served as the positive controls. To 

ensure satisfactory contact with the positive control material the SDS solution was 

spread over the entire surface of the epidermis using a pipette tip (taking particular care 

to cover the centre). After 7 minutes contact time the SDS solution was re-spread with a 

pipette tip to maintain the distribution of the SDS for the remainder of the contact period. 

The plate(s) were kept in the biological safety cabinet at room temperature for 

15 ± 0.5 minutes. 

At the end of the exposure period, each tissue was removed from the well using forceps 

and rinsed using a wash bottle containing PBS with Ca" and Mg". Rinsing was 

achieved by filling and emptying each tissue insert for approximately 40 seconds using a 

constant soft stream of PBS to gently remove any residual test material. The rinsed 

tissues were transferred to the second column of 3 wells containing 2 ml of maintenance 

medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 

approximately 42 hours. 
PROJECT NUMBER* PAGE11 

5.2.2 MTT Loading/Formazan Extraction (Day 3) 

Following the 42-hour post-exposure incubation period each 12-well plate was placed 

onto a plate shaker for 15 ±2 minutes to homogenise the released mediators in the 

maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was 

transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30°C for possible 

inflammatory mediator determination. 

2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into 

the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the 

MTT filled wells, being careful to remove any excess maintenance medium from the 

boftom of the tissue insert by blotting on absorbent paper. The tissues were incubated 

for 3 hours at 37°C, 5% CO2 in air. At the end of the 3 hour incubation period each tissue 

was placed onto absorbent paper to dry. The tissues were examined and the degree of 

MTT staining evaluated (qualitative evaluation of cell viability) using the MTT Visual 

Scoring scheme. Following qualitative evaluation of tissue viability a total biopsy of the 

epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully 

separated from the collagen matrix using forceps and both parts (epidermis and collagen 

matrix) placed into labelled 1.5 ml micro tubes containing 500 NI of acidified isopropanol, 

ensuring that both the epidermis and collagen matrix were fully immersed. Each tube 

was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes 

were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of 

formazan crystals out of the MTT-loaded tissues. 

5.2.3 AbsorbancelOptical Density nfleasurements (Day 6) 

At the end of the formazan extraction period each tube was mixed thoroughly on a vortex 

mixer to produce a homogenous coloured solution. 

For each tissue, duplicate 200 NI samples were transferred to the appropriate wells of a 

pre-labelled 96-well plate. 200 pl of acidified isopropanol alone was added to the two 

wells designated as `blanks'. The optical density was measured (quantitative viability 

analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader. 
PROJECT NUMBEF PAGE 12 

6. INTERPRETATION OF RESULTS 

6.1 Quantitative MTT Assessment (percentage tissue viability) 

For the test material the relative mean tissue viabilities obtained after the 15 minute 

treatment followed by the 42 hour post-exposure incubation period were compared to the 

mean of the negative control treated tissues (n=3). The relative mean viabilities were 

calculated in the following way: 

Relative mean viability (%) = mean OD540of test material 

 x 100 

mean OD54o of negative control 

Classification of irritation potential is based upon relative tissue viability following the 

15 minute exposure period followed by the 42 hour post-exposure incubation period 

according to the following table: 

Criteria for in vitro interpretation Classification 

Mean tissue viability is <_50% Irritant (1) R38 

Mean tissue viability is >50% Non-Irritant (NI)• 

61 Quality Criteria 

The results of the assay are considered acceptable if the following assay acceptance 

criterion is achieved: 

Positive Control 

The assay establishes the acceptance criterion for an acceptable test if the relative mean 

tissue viability for the positive control treated tissues was :540% relative to the negative 

control treated tissues, and the Standard Deviation (SD) value of the percentage viability 

is <_20%. 

•= The concentration of the inflammatory mediator IL-1a in the culture medium retained following the 

42 hour post-exposure incubation will be determined for test materials which are found to be 

borderline non-irritant based upon the MTT cell vlability endpoint (mean tissue viability 51 - 60%). 

This complimentary end-point will be used to either confirm a non-irritant result or will be used to 

override the non-irritant result. 
PROJECT NUMBER: PAGE 13 

Negative Control 

The assay establishes the acceptance criterion for an acceptable test if the mean ODsao 

for the negative control treated tissues was ?0.6, and the SD value of the percentage 

viability is <_20%. 

7. 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. 
PROJECTNUMBER:" PAGE 14 

8. RESULTS 

8.1 Direct MTT Reduction 

The MTT solution containing the test material did not turn blue/purple which indicated 

that the test material did not directly reduce MTT. 

8.2 Test Material, Positive Control Material and Negative Control Material 

The individual and mean OD540 values, standard deviations and tissue viabilities for the 

test material, negative control material and positive control material are given in Table 1. 

The mean viabilities and standard deviations of the test material and positive control, 

relative to the negative control are also given in Table 1. 

The relative mean viability of the test material treated tissues was 97.8% after a 

15-minute exposure. 

The qualitative evaluation of tissue viability is given in Table 2. 

Following the 15-minute exposure the test material treated tissues appeared blue which 

was considered indicative of viable tissue. 

8.3 Quality Criteria 

The relative mean tissue viability for the positive control treated tissues was <_40% 

relative to the negative control treated tissues and the standard deviation value of the 

percentage viability was :520%. The positive control acceptance criterion was therefore 

satisfied. 

The mean OD540 for the negative control treated tissues was >_0.6 and the SD value of 

the percentage viability was _<20%. The negative control acceptance criterion was 

therefore satisfied. 

9. CONCLUSION 

The test material was considered to be Non-irritant. 
PROJECT NUMBER: PAGE 15 

10. REFERENCES 

1) Fentem, J.H., Briggs, D., Chesne, C., Ellioft, G.R., Harbell, J.W., Heylings, J.R., 

Portes, P., van de Sandt, J.J.M. and P.A. Botham (2001) A prevalidation study on 

in vitro tests for acute Skin Irritation: results and evaluation by the Management 

Team. Toxicology in vitro 15, pp.57-93. 

2) Zuang, V., Balls, M., Botham, P.A., Coquette, A., Corsini, E., Curren, R.D., Elliot, 

G.R., Fentem, J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., van de 

Sandt, J.J.M., Wiemann, C. and A.P Worth (2002). Follow-up to the ECVAM 

prevalidation study on in vitro tests for acute Skin Irritation. ECVAM Skin Irritation 

Task Force Report 2. ATLA 30, pp. 109-129. 

3) Cotovio, J., Grandidier, M-H., Portes, P., Roguet, R. and G. Rubinsteen (2005) 

The in vitro acute Skin Irritation of chemicals: Optimisation of the EPISKIN 

Prediction Model within the Framework of the ECVAM Validation Process. ATLA 

33, pp.329-349. 

4) Hartung. T. (2007) ESAC statement on the validity of in vitro tests for skin 

irritation. European Commission, Directorate General, Joint Research Centre, 

Institute for Health and Consumer Protection, European Centre for the Validation 

of Alternative Methods (ECVAM). Ispra. Website: hftp://ecvam.jrc.it/index.htm. 
PROJECTNUMBER:' --- PAGE16 

DETERMINATION OF SKIN IRRITATION POTENTIAL USING 

THE EPISKINrMRECOhISTITUTED HUMAN EPIDERMIS MODEL 

Table 1 Mean OD540Values and Percentage Viabilities for the Negative 

Control Material, Positive Control Material and Test Material 

Material OD54o of 

tissues 

Mean ODS~ 

oftriplicate 

tissues 

± SD of 

ODSQO 

Relative 

individual 

tissue 

viabilit (%) 

Relative 

mean° 

viability ( /°) 

± SD of 

Relative 

mean 

viabili (% 

0.879 97.2 

Negative 

Control 0.965 0.904 0.053 106.7 100* 5.9 

Material 

0.868 96.0 

0.061 6.7 

Positive 

Control 0.044 0.054 0.009 4.9_ 6.0 1.0 

Material 

0.058 6.4 

0.828 91.6 

Test Material 0.890 0.884 0.053 98.5 97.8 5.8 

0.933 103.2 

SD = Standard deviation 

*= The mean viability of the negative control tissues is set at 100% 
PROJECT NUMBER:: PAGE 17 

DETERMINATION OF SKIN IRRITATION POTENTIAL USING 

THE EPISKINTM RECONSTITUTED HUMAN EPIDERMIS MODEL 

Table 2 Qualitative Evaluation of Tissue Viability (MTT uptake visual 

evaluation) 

Material Tissue 1 Tissue 2 Tissue 3 

Negative Control Material 

Positive Control Material ++ ++ ++ 

Test Material - - - 

MTT visual scoring scheme 

- = blue tissue (viable) 

+ = blue/white tissue (semi-viable) 

++ = tissue is completely white (dead) 
PROJECT NUMBER. PAGE 18 

Appendix 1 Certificate of Analysis 

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BrrtEh 

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Testl4lethod Test7lepeription 

L1P&5 pH 

CPX1i H Rsts 

RPM1+1t5 Cslout 

6Ft138 Tatd3 $WWA 

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casfpnrrr 19Kiame 12 & D 

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1fdn. hfax. Ta st ltesuit 

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,t. E. Yary ~ey ,ua t~rtnraSl f= [Y~ ca~t ,N,45c 

{rzctanir.~t ~rp„~gzr! Pmre l qJ"f 
PROJECT NUMBER: PAGE 19 

Appendix 2 Statement ofi GLP Compliance in Accordance with Directive 

2004f9/EC 

Department 

of Health 

THE DEPART14IE1VT OF HEALTH OF THE GOVERNIVIEN'C 

OF THE IINITED KIlVGDOM 

GOOD LABORATORY PRACTICE . " 

STATEMENT OF COMPLIANCE 

. .. IN ACCORllANCE W [TH DIRECT[VE 200dJwEC 

iFSTFACILITY .. ~. . - ~ TEStTYPE 

Harlan Laboratories Ltd, AnalyticaUClinical Chemistry , ShardlowBusinessPark EnvironmentalFate - - 

London Road ~ , ~ Environmental Tuxicology 

 Shardlow Mutsgenicity 

_ Dcrby ~ ~ . - PhysTChem 

DE722GD ~ - . Toxicology 

 . DATk OFA'SPECTION 

. . 19tbAugust 2005 

, A,general inspection for compliance with the Prrinciples of Good Laboratory Pcactice 

. wa9 can7ed out at the above mst facility as part ofihe UK GLP Compliance Programme. 

. - Atthe time of inspection no deviations were found of sufdciettt inagnitude to affec[ - 

the validityuf non-clinical studies performed atthese facilities. - 

A 

 • - - Dr.AndrewJ- Gray _ 

 - -Head,UKGLPMunitoringAutNority