PAGE 1 OF 19 PAGES

~ 

# 1

~ ~.~



### DETERMINATION OF SKIN IRRITATION POTENTIAL USING THE EPISKIN TM RECOhISTITUTED 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 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 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 MRQAPROJECT 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. EPISKIN TM M®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 OD 540 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 19PROJECTNUMBER: 

### __._ PAGE5

DETERMINATION OF SKIN IRRITATION POTENTIAL USING THE EPISKIN TM RECONSTITUTED 

### HUMAN EPIDERMIS MODEL SUMMARY lntroduction. The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN TM 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 enzymaticreduction 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 EPISKIN TM RECONSTITUTED HUMAN EPIDERMIS MODEL
1. INTRODUCTION The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN TM 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 EPISKIN TM 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. EPISKtFI TM 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 onreduction 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% CO Z 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 EPISKIN TM 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 OD 540 of 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 OD 540 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 OD 54 0 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 EPISKIN rM RECOhISTITUTED HUMAN EPIDERMIS MODEL 

### Table 1 Mean OD 540 Values and Percentage Viabilities for the Negative

Control Material, Positive Control Material and Test Material

Material OD 54o of tissues Mean ODS ~

oftriplicate tissues ± SD of

OD SQO 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 0.890 98.5 Test Material 0.884 0.053 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 EPISKIN TM 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

t 9 

# ~

l3

~ 

### tic 2211012009 Tesr7F3 R390109 BrrtEh PrurFuef Testl4lethod Test7lepeription L1P&5 pH CPX1i H Rsts RPM1+1t5 Cslout 6Ft138 Tatd3 $WWA E1C2/2 N"n 

### casfpnrrr 19Kiame 12 & D Cu,wtnmer ftrrder rVia

1fdn. hfax. 

### Ta st ltesuit 42 2; i 50 2A.{t 2.d



### 3'i 

### nrd ~ ,t. E. Yary

~ ey ,ua t~ rtnraSl f= [Y

~ ca ~ t ,N,45c {rzctanir.

~ t

~ rp„
~ gzr! Pmre l qJ"fPROJECT 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