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REPORT 

EPISKINTM Skin Irritation Test 15 min - 42 hours

HLS study number: CEF0004

Version ID: Audited Draft 2 report

Issue date: 27 June 2013
Huntingdon 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

UK
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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 tissues ........................................................................................... 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 Positive control.................................................................................... 13

3.2.3 Data interpretation - Prediction model ................................................. 13

4. Computer Systems..................................................................................................... 14

5. Maintenance of Records............................................................................................. 14

6. Deviations from Protocol ............................................................................................ 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 substance pH.......................................................................................... 15

7.4 Assay validity.................................................................................................. 15

7.4.1 Negative control .................................................................................. 15

7.4.2 Positive control.................................................................................... 15

7.5 EPISKINTM results............................................................................................ 15

8. Conclusion ................................................................................................................. 16

9. References................................................................................................................. 17
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List of Tables

Table 1 EPISKINTM study data...................................................................................18

List of Annexes

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

construct ......................................................................................................19

Annex 2 Huntingdon Research Centre GLP Compliance Statement 2012 .................20
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Compliance with Good Laboratory Practice 

EPISKINTM Skin Irritation Test 15 min - 42 hours

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.

___________________________________________ ________________________ 

Joanne Hubbard, HNC (Applied Biology) Date

Study Director

Huntingdon Life Sciences 
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Quality Assurance Statement

EPISKINTM Skin Irritation Test 15 min - 42 hours

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

Study Phase Date(s) of Inspection Date of Reporting 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 this 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 Sciences 
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Contributing Scientists 

EPISKINTM Skin Irritation Test 15 min - 42 hours

Study management

Joanne Hubbard HNC (Applied Biology) 

Study Director

Department of In Vitro Technologies

Leslie C Akhurst BSc (Hons) MSc CBiol MSB

Head of In Vitro Safety Assessment

Department of In Vitro Technologies
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Summary

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

substance, 

The test substance was applied to EPISKINTM human epidermis skin constructs. The 

constructs consisted of normal, human-derived epidermal keratinocytes, which had been 

cultured to form a multilayered, highly differentiated model of the human epidermis with a 

functional multilayered stratum corneum. The cell viability was 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 substances.

The test substance, elicited a mean tissue viability of 97.2 ± 4.7% and was 

predicted as non-irritant to the skin. 
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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 EPISKINTM Skin Irritation Test 15 Min-42 Hours.

1.2 Test procedure

The EPISKINTM Skin Irritation Test 15 Min - 42 Hours using human epidermis skin constructs 

supplied by SkinEthic Laboratories, Lyon, France, has been accepted as a replacement to the 

in vivo test, Draize Skin Irritation Test, by the European Centre for the Validation of 

Alternative Methods (ECVAM, 2007). The test was conducted in accordance with the 

Standard Operating Procedure, 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 EPISKINTM threedimensional human skin model. The model consists of normal, human-derived epidermal 

keratinocytes which have been seeded on a dermal 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 surface area supplied is 0.38cm2. The EPISKINTM 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 substances are sufficiently cytotoxic to cause cell 

death in the cell layers. The cell viability was 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 439) uses the percentage viability values (compared to negative control 

viability) to identify irritant and non-irritant substances. The test includes acceptance criteria 

for both negative and positive controls.
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2. Experimental Procedure 

2.1 Study schedule

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

Experimental completion: 13 May 2013

2.2 Test substance

Identity:

Chemical name

Intended use:

Lot number: 12342 

Expiry: Considered 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 

solution 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 achieved 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. 
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2.5 Receipt of tissues

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

at room temperature until use. The kit was used within the expiry date indicated by the 

supplier (expiry date: 13 May 2013). The 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 incubated for a minimum 

of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 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 studies 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 μL over each tissue using a positive displacement pipette. 

The test substance, was a white crystalline 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. 
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2.9 Test procedure

After incubation for at least 24 hours in maintenance medium, triplicate 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 the 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 remaining 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% CO2 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% CO2 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 biopsy 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 μL aliquots of the extractant from each micro tube 

were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed 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.
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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 viability of each tissue was expressed as a percentage of the mean negative control value.

3.2 Assay acceptance criteria

3.2.1 Negative control

The OD from the negative 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. The 

Standard Deviation (SD) value of the % viability should be <18.

3.2.2 Positive control

The OD of the positive control is an indicator of the sensitivity of the tissues. The mean 

viability should be ≤40% of the 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 sample was classed as Irritant R38 (EU classification) or Category 2 (GHS classification). 

Criteria for in vitro interpretation EU Classification Globally Harmonized System

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

Mean tissue viability is > 50 % Non-irritant (NI) No category
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4. Computer Systems

Xybion Pristima (version 6.3.2) – used for test substance management.

5. Maintenance of Records

Records and documentation relating 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 substance 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 used 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 study.
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7. Results 

7.1 Reduction 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% CO2 in air. The test substance had not interacted with the MTT.

7.2 Check for colouring potential of test substance

The test substance, /water solution and water control were colourless after the 15 

minute shaking period. The test substance, , had not shown any potential 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 negative 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 40% viability and SD of 18. 

7.5 EPISKINTM results

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

Sample

Tissue viability as percentage of mean OD negative 

control Prediction

MTT endpoint Replicate Tissues Mean ±

SD

a b c 

Negative Control 100.6 97.9 101.5 100.0 ± 1.8 Not applicable

Positive Control 45.0 32.2 37.9 38.4 ± 6.5 Irritant

99.1 91.9 100.6 97.2 ± 4.7 Non-irritant
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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. 
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9. References

L’Oreal. In Vitro Skin Irritation Test: Human Epidermis Model EPISKINTM, EPISKINTM Skin

Irritation Test 15mins-42 Hours, Standard Operating Procedure: February 2009 Version 1.8.

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

Reconstructed Human Epidermis Test Method. Adopted 22 July 2010.

European Centre for the Validation of Alternative Methods (ECVAM) Statement of the 

Validity of In Vitro tests for Skin Irritation, 27 April 2007.

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.
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Table 1 EPISKINTM 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.
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Annex 1 Technical Data, Safety Sheet and Certificate of Analysis for the 

EPISKINTM construct
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Annex 2 Huntingdon Research Centre GLP Compliance Statement 2012