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### REPORT

EPISKIN TM 

### S kin I rritation T est 15 m in - 42 h ours

HLS s tudy number: CEF0004

Version ID: Audited Draft 2 report

Issue date: 27 June 2013Huntingdon Life Sciences
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## Sponsor and Test Facility Details

Sponsor

Test facility Huntingdon Life Sciences Huntingdon Research Centre Woolley Road Alconbury Huntingdon Cambridgeshire
PE28 4HS

UKHuntingdon Life Sciences
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## Table of Contents

Sponsor and Test Facility Details

................................ ................................ .......................... 2

Compliance with Good Laboratory Practice

................................ ................................ .......... 5

Quality Assurance Statement

................................ ................................ ................................ 6

Contributing Scientists

................................ ................................ ................................ .......... 7

Summary

................................ ................................ ................................ .............................. 8

1. Introduction

................................ ................................ ................................ .................. 9

1.1 Objective

................................ ................................ ................................ ........... 9

1.2 Test procedure

................................ ................................ ................................ .. 9

2. Experimental Procedure

................................ ................................ ............................. 10

2.1 Study schedule

................................ ................................ ............................... 10

2.2 Test substance

................................ ................................ ............................... 10

2.3 Reduction of MTT by test substance

................................ ............................... 10

2.4 Check for colouring potential of test substance

................................ ............... 10

2.5 Receipt of tissu es

................................ ................................ ........................... 11

2.6 Test substance pH

................................ ................................ .......................... 11

2.7 Controls

................................ ................................ ................................ .......... 11

2.8 Preparation/application of samples

................................ ................................ . 11

2.9 Test procedure

................................ ................................ ................................ 12

3. Assessment of Results

................................ ................................ ............................... 13

3.1 Assessment of viability

................................ ................................ .................... 13

3.2 Assay acceptance criteria

................................ ................................ ............... 13

3.2.1 Negative control

................................ ................................ .................. 13

3.2.2 Positiv e control

................................ ................................ .................... 13

3.2.3 Data interpretation

- Prediction model

................................ ................. 13

4. Computer Systems

................................ ................................ ................................ ..... 14

5. Maintenance of Records

................................ ................................ ............................. 14

6. Deviations from Prot ocol

................................ ................................ ............................ 14

7. Results

................................ ................................ ................................ ....................... 15

7.1 Reduction of MTT by test substance

................................ ............................... 15

7.2 Check for colouring potential of test substance

................................ ............... 15

7.3 Test substanc e pH

................................ ................................ .......................... 15

7.4 Assay validity

................................ ................................ ................................ .. 15

7.4.1 Negative control

................................ ................................ .................. 15

7.4.2 Positive control

................................ ................................ .................... 15

7.5 EPISKIN TM results

................................ ................................ ............................ 15

8. Conclusion

................................ ................................ ................................ ................. 16

9. References

................................ ................................ ................................ ................. 17Huntingdon Life Sciences
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## List of Tables

Table 1 EPISKIN TM study data

................................ ................................ ................... 18



## List of A nnexes

Annex 1 Technical Data, Safety Sheet and Certificate of Analysis for the EPISKIN TM

construct

................................ ................................ ................................ ...... 19

Annex 2 Huntingdon Research Centre GLP Compliance Statement 2012

................. 20Huntingdon Life Sciences
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## Compliance with Good Laboratory Pract ice

EPISKIN TM 

### S kin Irritation T est 15 m in - 42 h ours

The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I consider the data generated to be valid.

The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 3106, as amended by Statutory Instrument 2004 No. 994).

OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98) 17.

EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal No L 50/44).

These principles of Good Laboratory Practice are accepted by the regulatory authorities of the United States of America and Japan on the basis of intergovernmental agreements.

___________________________________________ ________________________ Jo anne Hubbard, HNC (Applied Biology) Date Study Director Huntingdon Life SciencesHuntingdon Life Sciences
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## Quality Assurance Statement

EPISKIN TM 

### S kin Irritation T est 15 m in - 42 h ours

The following inspections and audits have been carried out in relation to this study:

Study Phase Date(s) of Inspection Date of Reportin g to Study Director and Management

Protocol Audit 08 May 2013 08 May 2013

Report Audit 20 Jun 2013

- 21 Jun 2013 21 Jun 2013

Process based inspections : At or about the time this study was in progress inspections of procedures employed on thi s type of study were carried out. These were conducted and reported to appropriate Company Management as indicated below:

Process Based Inspections Date(s) of Inspection Date of Reporting to Management

Test Item Preparation and

Treatment 16 Nov 2012 19 Nov 2012

Transfer of Extracts and Plate Reading 21 Jan 2013 28 Jan 2013

Study Management and

Conduct 31 May 2013 03 Jun 2013

In addition, an inspection of the facility where this study was conducted was carried out on an annual basis. These inspections were reported to Company Management

......................................................................... ............................................ Dilys Smith, MRQA, Date Lead Auditor, Department of Quality Assurance Huntingdon Life SciencesHuntingdon Life Sciences
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## Contributing Scientist s

EPISKIN TM 

### S kin Irritation T est 15 m in - 42 h ours

Study manage ment

Joanne Hubbard HNC (Applied Biology) Study Director Department of In Vitro Technologies

Leslie C Akhu rst BSc (Hons) MSc CBiol MSB

Head of In Vitro Safety Assessment Department of In Vitro TechnologiesHuntingdon Life Sciences
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## Summary

The objective of th is test was to assess the skin irritation potential

, in vitro , of the test substance,

The test substance was applied to EPISKIN TM human epidermis skin constructs. The constructs consisted of normal, human-derived epidermal keratinocytes, which had been

cultured to fo rm a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum. The cell viability wa s determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3 -(4,5- dimethylthiazol -2- yl) -2, 5-diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction

model (OECD guideline 439) uses the percentage viability values (compared to negative control viability) to identify irritant and non- irritant substance s.

The test substance, elicited a mean tissue viability of 97.2 ± 4.7 % and was predicted as non- irritant to the skin.Huntingdon Life Sciences
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## 1. Introduction

1.1 Objective

The objective of this study was to assess the skin irritation potential, in vitro, of the test

substance EPISKIN TM Sk in Irritation Test 15 M in

- 42 Hours

.



### 1.2 Test procedure

The EPISKIN TM S kin Irritation T est 15 M in

- 42 H ours using human e pidermis skin constructs supplied by SkinEthic Laboratories , Lyon, France, has been accepted as a replacement to the

in vivo test, Draize S k in I rritation T est, by the European Centre for the Validation of Alternative Methods ( ECVAM, 2007 )

. The test was conducted in accordance with the Standard Operating P rocedure, In Vitro Skin Irritation Test: Human Epidermis Model ( L’Oreal 2009 ) , supplied by L’Oreal (leading laboratory in the validation of the test for ECVAM) and the OECD 439 Guideline For The Testing of Chemicals: In Vitro Skin

Irritation: Reconstructed Human Epidermis Test Method ( OECD 439 ).

The test involves the application of the test substance for 15 minutes to the E PISKIN TM three

dimensional human skin model. The model consists of normal, human

- derived epidermal keratinocytes which have been seeded on a derm al substitute consisting of a collagen type 1 matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi

- layered stratum corneum has formed. The epidermis surfa ce area supplied is 0.38cm 2

. The EPISKIN TM kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar.

The principle of the assay is that irritant substance s are sufficiently cytotoxic to cause cell death in the cell layers. The cell viability wa s determined by mitochondrial dehydrogenase activity, a ssessed by the reduction of MTT (3

- (4,5

- dimethylthiazol

- 2

- yl)

- 2,

5

- diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction

model ( OECD 439 ) uses the percentage viability values (compared to negative control viability) to identify irritant and non

- irritant substance s. The test includes acceptance criteria for both negative and positive controls.Huntingdon Life Sciences
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## 2. Experimental Procedure

2.1 Study s chedule

The protocol was approved by the Sponsor on 7 May 2013 and by Huntingdon Life Sciences Management on 7 May 2013.

Study initiation (protocol signed by Study Director): 7 May 2013

Experimental start: 7 May 2013

Experiment al completion: 13 May 2013



### 2.2 Test substance

Identity:

Chemical name

Intended use:

Lot number : 12342

Expiry: Co nsidered suitable for use (expiry date will be documented in raw data and final report)

Appearance: White crystalline powder

Storage conditions: Room temperature (ca 20°C), in the dark

Purity/Assay: Purity will be documented in the raw data and final report



### 2.3 Reduction of MTT by test substance

The MTT reducing capability of the test substance, was investigated by mixing approximately 10 mg of the test substance with 2 mL of 0.3 mg/ mL MTT solution in duplicate. A control of 10 μL of distilled water, mixed with 2 mL of 0.3 mg/ mL MTT

solutio n was also included in duplicate.



### 2.4 Check for colouring potential of test substance

The test substance, was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). Evaluation was achie ved by mixing 13.7 mg of the test substance, with 123 μL of distilled water in a transparent container to give 10 mg/90 μL . 100 μL of distilled water was included as a control. The solution was mixed for 15 minutes on a shaker. At the end of the shaking period the colour of the solution was assessed by eye.Huntingdon Life Sciences
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### 2.5 Receipt of tissues

On r eceipt, the kit contents were checked and the inserts with tissues on agarose were stored

at room tempera ture until use. The kit w as used within the expiry date indicated by the supplier (e xpiry date: 13 May 2013) . Th e maintenance medium was pre -warmed to 37oC.

The tissues were removed from the agar and placed into wells of 12 well plates containing

2 mL pre- warmed maintenance medium per well. The tissues were incuba ted for a minimum

of 24 hours at 37 ± 2 ° C in a humidified atmosphere of 5% CO 2 in air.



### 2.6 Test substance pH

An estimate of the pH of the test substance as a 10% w/v solution in purified water,was determined using pH indicator paper and recorded.



### 2.7 Controls

The negative control was sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium.

The positive control was 5% Sodium Dodecyl Sulphate (SDS) in purified water

.

The controls and their results were shared with other stud ies performed in the same assay

.



### 2.8 Preparation/application of samples

The positive and negative controls were in liquid form and were applied by dispensing a

volume of 10 μ L over each tissue using a positive displacement pipette.

The test substance, was a w hite crys talline powder. A weight of 10 ± 2mg was dispensed over each tissue using glass weighing boats. The tissues were wetted with 5 μL of purified water prior to application of the test substance.Huntingdon Life Sciences
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### 2.9 Test procedure

After incubation for at least 24 hours in ma intenance medium , tri plicate tissues were dosed

for 15 ± 0.5 minutes with the test substance , negative or positive control at room temperature. The test substance was spread over t h e surface of the tissues with a curved spatula.

A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance

. On application of 10 μL , the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat spatula after 7 minutes (actual time 8 minutes) application time.

After 15 ± 0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbecco ’ s Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove rem aining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37 ± 2 ° C in a humidified atmosphere of 5% CO 2 in air.

After 42 ± 1 hour

, each insert was transferred to a well containing 2 mL of 0.3 mg/ mL MTT

and incubated for 3 hours ± 5 minutes at 37 ± 2 ° C in a humidified atmosphere of 5% CO 2 in

air.

At the end of 3 hours ± 5 minutes

, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a bi opsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro tube.

When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration).

The tissues were extracted by storing at 2

- 8 oC, protected from light, for 48

- 70 hours.

After formazan extraction, duplicate 200 μ L aliquots of the extractant from each micro tube were pipetted into the wells of flat

- bottomed 96

- well plates. The extractant was mi xed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm

, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.Huntingdon Life Sciences
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## 3. Assessment of Results

3.1 Assessment of viability

The mean Optical Density (OD) for the six replicate blanks was subtracted from the

individual substance and control tissues OD.

The v iability of each tissue was expressed as a percentage of the mean negative control value.



### 3.2 Assay acceptance criteria

3.2.1 Negative c ontrol

The OD from the negati ve control tissue in the MTT assay is an indicator of tissue viability after the shipping and storage procedure and under the specific conditions of the assay. The

mean absorbance of the triplicate negative control values should be ≥ 0.6 and ≤1.5. T he

Sta ndard Deviation (SD) valu e of the % viability should be < 18.

3.2.2 Positive c ontrol

The OD of the positive control is an indicator of the sensitivity of the tissues. The mean viability should be ≤4 0% of t he negative control and the SD < 18 %

.

3.2.3 Data interpretation

- Prediction model

Where the mean tissue viability was equal to or less than 50% of the negative control value,

the s ample was classed as Irritant R38 (EU classification) or Category 2 (GHS classification)

.

Criteria for in vitro interpretation EU Classif ication Globally Harmonized System

Mean tissue v iability is ≤ 50 % Irritant (I) R38 Category 2

Mean tissue viability is > 50 % Non

- irritant (NI) No categoryHuntingdon Life Sciences
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## 4. Computer Systems

X y bion Pristima (version 6. 3 .2 )

– used for test substance management

.



## 5. Maintenance of Records

Records and documentation relati ng to this study will be maintained in the archives of Huntingdon Life Sciences for a period of one year from the date on which the Study Director signs the final report. This will include but may not be limited to the Study Protocol, raw data, test subst ance and other samples and specimens generated during the course of this study. If requested, Huntingdon Life Sciences will continue to retain the substances at

additional cost.

Samples and specimens that no longer afford evaluation will be discarded in accordance with Standard Operating Procedures.

Huntingdon Life Sciences will retain in its archive the Quality Assurance records relevant to this study for a period of 20 years and a copy of the final report indefinitely.



## 6. Deviations from Protocol

Water u sed to prewet tissues prior to application of test substance.

The protocol stated that tissues are wetted with 5 μl of distilled water prior to application of the test substance. In this study, the tissues were wetted with 5 μl of purified water prior to application of the test substance.

As the purpose of prewetting the tissues with water was to improve the contact between the test substance and the surface of the tissue, using purified water was not considered to affect

the scientific conduct of the st udy.Huntingdon Life Sciences
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## 7. Results

7.1 R eduction of MTT by test substance

There was no change in the test substance, /MTT solution or the water

control/MTT solution after three hours incubation in the dark at 37 ± 2 ° C in a humidified atmosphere of 5% CO 2 in air. The test su bstance had not i nteracted with the MTT.



### 7.2 Check for colouring potential of test substance

The test substance, /water solution and water control w ere colourless after the 15 minute shaking period. The test substance, , had not shown any potent ial for colouring water.



### 7.3 Test substance pH

The pH of the test substance, , as a 10% (w/v) solution in purified water, measured using pH indicator paper, was approximately 7.0

.



### 7.4 Assay validity

7.4.1 Negative control

The mean absorbance of the triplicate ne gative control values was 0.913 which was between

the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 1.8 which was below the maximum value of 18.

7.4.2 Positive control

The percentage mean viability of the positive control was 38.4 ± 6.5 of the negative control. These were below the maximum acceptance values of 4 0% viability and SD of 18.



### 7.5 EPISKIN TM 

### results

The results of the assay are summarised in the table below.

Sample Tissue viability as percentage of mean OD n egative

control Prediction MTT endpoint Replicate Tissues Mean ±

SD

a b c

Negative Control 100.6 97.9 101.5 100.0 ± 1.8 No t applicable

Positive C ontrol 45.0 32.2 37.9 38.4 ± 6.5 Irritant

99.1 91.9 100.6 97.2 ± 4.7 Non

- irritantHuntingdon Life Sciences
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## 8. Conclusion

It was concluded that the test substance, with a mean tissue viability of
97.2 ± 4.7%, was predicted as non- irritant to the skin.Huntingdon Life Sciences
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## 9. References

L’Oreal

. In Vitro Skin Irritation Test: Human Epidermis Model E PISKIN TM

, EPISKIN TM S kin

I rritation T est 15mins

- 42 H ours

, Standard Operating P rocedure : February 200 9 Version 1. 8

.

OECD 439. OECD Guideli ne for The Testing of Chemicals : In Vitro Skin Irritation:

Reconstructed Human Epidermis Test Method

. Adopted 22 July 2010

.

European Centre f or the Validation of Alternative Methods (ECVAM) Statement of the Validity of In Vitro tests for Skin Irritation, 27 April 200 7.

ECVAM Statement on the performance under UN GHS of Three In

- Vitro Assays for Skin Irritation Testing. 9 April 2009.

Spielmann, H., Hoffmann, S., Liebsch, M., Botham, P., Fentem, J.H., Eskes, C., Roguet, R., Cotovio, J., Cole, T., Worth, A., Heylings, J., Jones, P., Robles, C., Kandarova, H., Gamer, A., Remmele, M., Curren, R., Raable, H., Cocksshott, A., Gerner, I. and Zuang, V. (2007) The ECVAM International Validation on in vitro tests for acute skin irritation: report on the validity of the EPISKIN and EpiDerm assays and on the Skin integrity function test. ATLA, 35, 559

- 601.Huntingdon Life Sciences
CEF0004

18 Table 1 EPISKIN TM study data

Sample Tissue Optical Density OD - Blank % Negative
Replicate (OD) Control

1.071 0.927

1.054 0.910 Negative 1.059 0.915

Control 1.018 0.874

1.087 0.942

1.055 0.911 Replicates Average 0.913 100.0
a,b,c sd 0.023 1.8

0.569 0.425

0.542 0.398

Positive 0.431 0.286

Control 0.445 0.301

0.510 0.366

0.470 0.326 Replicates Average 0.350 38.4
a,b,c sd 0.055 6.5

1.072 0.928

1.027 0.883

1.014 0.870

0.952 0.808

1.093 0.949

1.033 0.888 Replicates Average 0.887 97.2
a,b,c sd 0.049 4.7

0.135

0.149

Blank 0.150

0.140

0.145

0.146 Average 0.144

sd 0.006 b

c 99.1 a

b

c

a

b

c

a

91.9

100.6 100.6

97.9

101.5

45.0

32.2

37.9

sd = Standard Deviation

Note. Rounded values only are displayed; unrounded numbers are used for calculations by Excel spreadsheet.Huntingdon Life Sciences
CEF0004

19 Annex 1 Technical Data, Safety Sheet and Certificate of Analysis for the

E PISKIN TM con structHuntingdon Life Sciences
CEF0004

20 Annex 2 Huntingdon Research Centre GLP Compliance S tatement 201 2