PAGE 1 OF 20 PAGES 

~ 

DETERMINATION OF SKIN IRRITATION POTENTIAL USING 

THE EPISKINTMRECONSTITUTED 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 additian, 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 Standard Test Method Compliance Audit 

16 April 2009 Test Material Preparation 

15 April 2009 Test System Preparation 

16 April 2009 Exposure 

21 April 2009 Assessment of Response 

§ 26 May 2009 Draft Report Audit 

§ Date of QA Signature Final Report Audit 

§ Evaluation specific to this study 

.: 

..................... ..C... ~~,.....~:~-.~--, ........ ................. DATE: . ............ 

 09 

....... 

JUN 2009 

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

Forthe QualityAssurance Unit' 

*Authorised QA S§gnatures: 

Manager, Quality Assurance: J G Riley BSc (Hons) MRQA 

Deputy Head of Department: JM Crowther MIScT MRQA 

Senior Audit Staff: G Wren ONC MRQA 
PROJECT NUMBER' PAGE 3 

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 2004/0994)). These Regufations 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 2004/101EC. 

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

................... Z. ........................ ...... .......... DATE: ........... MVJ.&.t /....9......... 

N Warren MIAT 

Study Director 

This report may be presented in final form as a digital (pdf) document. Such documents are preparetl by scanning the paper originai, 

and are considered of equivaient 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 documenL 
PROJECT NUM6ER: ' PAGE 4 

CONTENTS 

QUALITY ASSURANCE REPORT 2 

GLP COMPL(ANCE STATEMENT 3 

CONTENTS 4 

SUMMARY 5 

1. INTRODUCTION 7 

2. TEST MATERIAL a 

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. EPfSKINTM Model Kit 9 

5. PROCEDURE 9 

5.1 Pre-Test 9 

5.2 Main Test 10 

6. [NTERPRETATION 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, Positive Control Material and Negative Control Material 14 

83 Quality Criteria 14 

9. CONCLUSION 14 

10, REFERENCES 15 

Table 1 Mean OD$40 Values and % 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 

Appendfx 1 Certificate of Analysis 18 

Appendix 2 Statement of GLP Compliance in Accordance with Directive 200419/EC 20 
PROdECT NUMBER: ' PAGE 5 

DETERMINATION OF SKIN IRRITATION POTENTIAL USING 

THE EPISKINTM RECONSTITUTED HUMAN EPIDERMIS MODEL 

SUMMARY 

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. 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-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 borderfine 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 beneeth 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 % viability (MTT reduction in the test material treated 

tissues relative to negative control tissues). 
PROJECT tdUM6ER: ; PAGE 6 

Results: The relative mean viability of the test material treated tissues was 86.6% after 

a 15-minute exposure. 

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

satisfied. 

Conclusion: The test material was considered to be Non-Irritant. 
PROJECTNUMBER: 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, Z. 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 EPISKINTDA 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 

difPerentiated and stratified epidermis model is obtained after 13 day culture period 

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

stratum corneum. 

The control group served as a common control with Harlan Laboratories Ltd Project 

number 2724J0007. 

The study was performed between 22 April 2009 and 28 April 2009. 
PROJECT NUMBER: PAGE 8 

2. TEST MATERIAL 

2.1 Description, Identification and Storage Conditions 

Sponsor's identification 

Description ambercoloured 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 stability of the test 

materiaf 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 Saline (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 Controi Materials, MTT and Acidified 

Isopropanol 

The negative control material 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 lsopropanol was prepared when required. 
PROJECT NUMBER: 1 PAGE 9 

4. EPISKINTM Model Kit 

Date received : 21 April 2009 

5. PROCEDURE 

5.1 Pre-Test 

5.1.1 Assessment of Direct Test Material Reduction of MTT 

5.9.1.1 MTT dye metabo/ism, cel8 viabffity assay 

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

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

dehydrogenase in viable cells. 

5.9.1.2 Assessment of Direct Test Material Reduction of MTT 

One limitation of the assay is possible interference of the test substance with M1T. 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 is 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 

below. 

5.1.9.3 Test for ®frect 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 μl of the test material was added to 2 ml of a 0.3 mg/ml MTT solution freshly prepared 

in assay medium. The sofution was incubated in the dark at 37°C, 5% CO2 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 blue/purple, the test substance is 

presumed to have reduced the MTT. 

5.1.2 Pre-Incubation (Day 0: tissue arrival) 

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

column of 3 welfs 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°lo 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, warrned 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 ul of the test material was applied to the epidermis 

surface. Triplicate tissues, treated with 10 NI of PBS, were used to serve as negative 

controls. Tripiicate tissues, treated with 10 NI of SDS 5% w/v, were used to serve as 

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: PAGE 11 

5.2.2 iNTT Loading/Formazan Extracfion (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 store 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 maPntenance niedium from the 

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

for 3 hours at 37°C, 5% CO 2in 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 EPISKINTMbiopsy 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 pl 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 AAeasurements (Day fi) 

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 pl samples were transferred to the appropriate wells of a 

pre-labelled 96-well plate. 200 ul 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. 
PROJECTNUMBER:_ PAGE 12 

6. 1NTERPRETATI®N 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 viability = mean OD. of test material x 100 

mean OD540 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 interpretaGon Classification 

Mean tissue viabllity is <_50% irritant (I) R38 

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

6.2 Quality Criteria 

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

criterion is achieved: 

Positive ControE 

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

tissue viability for the positive control treated tissues was s40% relative to the negative 

control treated tissues, and the Standard Deviation (SD) value of the % viability is 520%. 

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

42 hour post-exposure incubation will be determined for test mateda4s which are found to be 

borderline non-irritant based upon the MTT cell viability 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 resuft. 
pROJECT NUMBER: ' PAGE 13 

Negative Control 

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

for the negative controJ treated tissues was ?0.6, and the SD value of the % viability is 

<_2D%. 

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. 
PROJECT NUMBER:: PAGE 14 

B. 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 OD54o values, standard deviations and tissue viabilities for the 

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

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

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

The relative mean viabifity of the test material treated tissues was 86.6% 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 :540% 

relative to the negative control treated tissues and the Standard Deviation (SD) value of 

the % viability was _<20%. The positive control acceptance criterion was therefore 

satisfied. 

The mean OD540for the negative control treated tissues was ?0.6 and the SD value of 

the % viability was <-20%. The negative control acceptance criterion was therefore 

satisfied. 

9. C®NCLUSION 

The test material was considered to be Non-Irritant. 
PROJECT NUM6ER: PAGE 15 

10. REFERENCES 

1) Fentem, J.H., Briggs, D., Chesne, C., Elliott, 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. 
PROJECT NUMBER: PAGE 16 

JETERMINATION OP SKIN IRRITATION POTENTIAL US1NG 

THE EPISKINTMRECONSTITUTED HUMAN EPIDERMIS MODEL 

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

Material, Positive Control Material and Test Material 

Material OD~ of 

tissues 

Mean OD ~o 

of triplicate 

tissues 

} SD of 

OD~ 

Relative 

individual 

tissue° 

viabili /° 

Relative 

mean % 

viability 

± SD of °h 

viability 

0.927 97.5 

Negative 

Control 0.932 0.951 0.04 98.0 100* na 

Material® 

0.995 104.6 

0.079 8.3 

Positive Control 

Material~ 

0.045 0.042 0.04 4.7 4.4 4.1 

0.001 0.1 

0.814 85.6 

Test Material 0.793 0.823 0.04 83.4 86.6 3.7 

0.863 90.7 

*= The mean viability of the negaUve control tissues is set at 100% 

na = Not applicable 

®= Control group shared with Hadan Laboratories Ltd Project number 2724/0007 
PROJECT NUMBER: ; PAGE 97 

: DETERMINATION OF SKIN IRRITATION POTENTIAL USING 

THE EPISKINTMRECONSTITUTED HUMAN EPIDERMIS MODEL 

Table 2 Qualitative Eva[uation of Tissue Viabifity (MTT uptake visual 

evaluation) 

Material Tissue 1 Tissue 2 Tissue 3 

Negative Control 

Material® 

Positive Control 

Materiali]) ++ ++ ++ 

~ 

Test Material 

MTT visual scoring scheme 

= blue tissue (viable) 

+ = blue/white tissue (semi-viable) 

++ = tissue is completely white (dead) 

e= Control group shared with Harlan Laboratories Ltd Project number 272410007 
PROJECT PAGE 18 

Appendix 1 Certificate of AnaBysis 

CerEFHcete of Aneh/his 

.. a aveiurmWra98rmeMSyalem ~s~aiereabmcinranae;«, zrstandattr 

 ISOBOOIwuaace7bmanuiectueeavW[estlnismaitria~ 

 Yn9namepnm.edalUvsmdofAfz4ocumenilsenektlrMlCJi4ibWre. 

Custamer deFSiEs . . .. . . .. . . .. . ......._... 

Cuatomcrflef. 

7nepeCSFen L,oi 

CoiAPrEnMtl, 2a.1T.260B 

~ . 

OuantPly- 0.400 

OACoestaet. 

Fax NU. 

Batch DetaiYs . ProductName: - 

ProductCotle: _ 

Cu#L Praduct !leme: 

Cust Product Cude: 

9dtch N[f. 282166 

DdtaUCTe9fit 05.11.2000 

SpeciRcstictr. flEViEWED23-JUN~-IXM 

DateofiRtaaufa(:turo: 09.04.8008 

Dat®ofEspirye 04.042018 

Qua64y Ccntrol ResWq . . _ ..... 

AnalyUsai Tast SpaciGcatlon Limit 

Methar3No. CharacterisRc Lower uppar Valua Unrt Status 

 ABtlendurtl 00 Pa69 Or Fai1 PasS - P 

G6000652 DPI711BAPPEARANCE YEl.LOW Pas5 - P 

(COt<Ol/R) 

G6000052 . 04`17718APPEApANCC CLEAR Pass - P 

(CLAF2ITY) . G6000052 DP1771B APPEARACE VISCOUS LK3U1D Pass - P 

(FOtiM) 

G8000054 DPt7f3 DLIQUR C1fARACTERISTIC.FREE Paxs - P 

OFOBJECTIONALODOUR 

G6000171 IW211BASdi 0.0 2.0 1.3 % P 

G$00017$ OP14l5 CC3LOUR 0.0 7.0 42 Garrfner P 

06000774 Dp6J5 PH 4,5 ' 5.5 54 P 

G60RD177 DPi738TOTALSOUDS 20_0 2a.p 21,0 4 P 

GG010176 DC2t2NI7FiOC`EN 2.2 3,4 2F, % P 

G6020001 DM112S TQ'€AL C' ,OUNT 100 CFU/6 MA7( PasS - P 

Q&O'C 

G6020002 DM211 S YfiASTB & 100 CFUIG MAX Pass - P 

MOULI7S 0 25°C 

G60200t1 DM7611SGRAM ABSENT Pass - P 

Pepe 1.1 2 
PROJECT NUMBER: r ~ ~ . PAGE 19 

Appendix 1(continued) Certificate of Analysis 

C®r[ificate of Analyui& r 

A qualky .&ft4hltii! 3}5t8m rBg*IPrO(t t? ihe 1M2nlalto0a1 affi, ,Oflitl 

150 9CQi wYi 44Dd Iti [n8TtH9c[Vfa.-uW Mt Ihim metenai. 

Ylhe nvnc winted af titRdYW W I.hlS dact•nnnl i@a.i aWe[r~de slptaN,e. 

Custaraeae d®talls 

G"eWmar 9"tef. 

Inspectlon Eat 050000063934 

C taf A RriMad. 28:112008 

No_ 

n0. 

C7neYmty. OAO11 

QIi Cont®cK. 

Pax No. 

-- .. 

NEGATIVE eACdLLl iG 

66S)20472 DM71123STAPt4.Att®EUS Ai39ENT Pass - P 

9G 

I t iet4commended that this prOtlrrEt be used wekhln 6 montfls af deiivery 

Batc3i ^atPfus: Pass 

Condinned ny 

Stephen Hughes 

PagL20f 2 
PROJECT NUMBER:.. _ PAGE 20 

Appendix 2 Statetnent of GLP Compliance in Accordance with Directive 

2004J9fEC 

(D~ Department 

~ of Health 

THW DEPA;IZTMFNT OF IIFALTR OF THE GQVERNMEIyT 

OFTFIE UNITED KINGDOM 

GOOIi LABORiiTORY PRACCICE 

- STATEMENTOFCOb4PIdANCE . 

.. IN ACCORDANCE WITH DD2ECTIVE 200491EC 

18STFACI1ITY _ .. . TESTTYYE 

IIarlan T,aboratories Ltd: AnalyticaVClinical Chemistry 

Shardlow Business Park Environmental Fate . London Road Environmental Taxiculogy 

Shardlovv - Mutagen(city 

Dcrby . - PhyslChem - DE722GD Toxicology - 

JfAT60FII.'SPECFION 

19thAugust 20DS 

A general inspecfion for compfiance with the Principles of Good Laboratory Practice 

was camed out at the above test facility as pattofthe UK GLP Compliance Programme. 

A[ the time of inspection no deviations were found of sufficient magnitude to affect 

the vaiidity of non-clinical studies perfottned at these facilitics,