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HFCPE 

EPISKIN Skin Irritation Test-42 Hours

HLS study number: VHJ0002 

Version ID: Final report 

Issue date: 8 January 2010 
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Sponsor and Test Facility Details 

Sponsor Zeon Corporation 

 Shin Marunouchi Center Building 

 1-6-2 Marunouchi 

 Chiyoda-ku 

 Tokyo 

 100-8246 

 JAPAN 

Test facility Huntingdon Life Sciences Ltd 

 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 Receipt of tissues............................................................................................11

2.5 Controls ..........................................................................................................11

2.6 Preparation/application of samples .................................................................11

2.7 Test procedure................................................................................................11

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

4.1 Deviations from Protocol .................................................................................14

5. Results .......................................................................................................................15

5.1 Possible reduction of MTT by test substance ..................................................15

5.2 Assay validity ..................................................................................................15

5.2.1 Negative control...................................................................................15

5.2.2 Positive control ....................................................................................15

5.3 Episkin results.................................................................................................15

6. Conclusion..................................................................................................................16

7. References.................................................................................................................17

List of Tables 

Table 1 Episkin study data........................................................................................18
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List of Annexes 

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

Construct......................................................................................................19

Annex 2 Huntingdon Research Centre GLP Compliance Statement 2008..................20
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Compliance with Good Laboratory Practice 
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Quality Assurance Statement 
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Contributing Scientists 

HFCPE: EPISKIN Skin Irritation Test-42 Hours

Study management 

Joanne O Hubbard, Higher National Certificate (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 irritancy, in vitro, of the test substance, 

HFCPE. 

The test substance was applied to EPISKIN 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 of the multi layers was determined by 

mitochondrial dehydrogenase activity. The prediction model uses the percentage viability 

values (compared to negative control viability) to identify irritant and non-irritant substances. 

The test substance, HFCPE, elicited a mean tissue viability of 80.2% and was predicted as 

non-irritant. 
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1. Introduction 

1.1 Objective 

The aim of this study was to determine if the test substance is a skin irritant using the in vitro 

EPISKIN Skin Irritation Test-42 hours.

1.2 Test procedure 

The EPISKIN Skin Irritation Test-42 hours using human epidermis skin constructs supplied by 

EPISKIN, SNC, 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 objective of this study was to assess the skin irritation potential, in 

vitro, of the test substance. The test was conducted in accordance with the Standard 

Operating Procedure, In Vitro Skin Irritation Test: Human Epidermis Model (L’Oreal 2005), 

supplied by L’Oreal (leading laboratory in the validation of the test for ECVAM) and the 

OECD Guideline For The Testing of Chemicals (OECD 2008). 

The test involves the application of the test substance for 15 minutes to the EPISKIN 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 EPISKIN 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 is 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 (L’Oreal 2005) 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 1 October 2009 and by Huntingdon Life 

Sciences Management on 21 September 2009. 

Study initiation: 

(protocol signed by Study Director) 

21 September 2009 

Experimental start: 30 September 2009 

Experimental completion: 6 October 2009 

2.2 Test substance 

Identity: HFCPE 

Chemical name: 1,3,3,4,4,5,5 - Heptafluorocyclopentene

Lot number: 20090601 

Intended use: Electronic applications 

Expiry: End June 2010 

Appearance: Colourless liquid 

Storage conditions: Room temperature 

Purity/Assay: 99.3% 

2.3 Reduction of MTT by test substance 

It is possible that a test substance may reduce MTT, resulting in the production of a blue 

colour without any involvement of cellular mitochondrial dehydrogenase. Since the test 

substance is rinsed off the tissue before the MTT assay, this is usually avoided. However, it 

is possible that small amounts of test substance may be present after washing or be released 

through the tissue into the MTT medium. If the mixture with the test substance turned 

blue/purple after approximately 3 hour incubation at 37 ±2oC in 5% CO2 in air, MTT 

reduction may have occurred. 

The MTT reducing capability of the test substance, HFCPE, was investigated by mixing 

approximately 10 μl with 2 ml of 0.3 mg/ml MTT solution in duplicate. A control of 2 ml of 

0.3 mg/ml MTT solution was also included in duplicate. 
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2.4 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 (within 3 to 4 days of receipt). The maintenance medium was pre-warmed to 37o C. 

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. 

2.5 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. 

2.6 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, HFCPE, a colourless liquid, was applied by dispensing a volume of 10 μl 

over each tissue using a positive displacement pipette. 

2.7 Test procedure 

After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 

15 minutes with test substance, negative or positive control at room temperature. 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 application time. 

After 15 minutes, each tissue was rinsed with 25 ml sterile Dulbeccos Phosphate Buffered 

Saline (DPBS) to remove residual test substance. Inserts were blotted on absorbent paper to 

remove remaining DPBS and the epidermal surface gently swabbed. 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. 
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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 a minimum of 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 acidified 

isopropanol solution as a blank. 
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3. Assessment of Results 

3.1 Assessment of viability 

The mean Optical Density (OD) for the 6 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 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 ≤30% of the negative control and the SD ≤18. 

3.2.3 Data interpretation - Prediction model 

If the mean tissue viability was less than 50% of the negative control value, the sample was 

classed as Irritant R38 (EU classification). 

Criteria for in vitro interpretation Classification 

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

Mean tissue viability is > 50 % Non irritant (NI) 
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4. 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, a copy of the final report, 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 the Quality Assurance records relevant to this study and 

a copy of the final report in its archive indefinitely. 

4.1 Deviations from Protocol 

There were no deviations from the protocol. 
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5. Results 

5.1 Possible reduction of MTT by test substance 

There was no colour change in the test substance, HFCPE/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. 

5.2 Assay validity 

5.2.1 Negative control 

The mean absorbance of the triplicate negative control values was 0.790 which was above the 

minimum acceptance value of 0.6. The standard deviation of the % viability was 16.7 which 

was below the maximum acceptance value of 18. 

5.2.2 Positive control 

The mean % viability of the positive control was 22.4 ± 5.5 of the negative control. These 

were below the maximum acceptance values of 30% viability and SD of 18. 

5.3 Episkin results 

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

Tissue viability as percentage of mean OD negative 

control 

Replicate Tissues 

Sample 

a b c 

Mean ± 

SD 

Prediction 

MTT endpoint 

Negative Control 81.9 103.3 114.8 100.0 ± 16.7 Not applicable 

Positive control 18.6 19.8 28.7 22.4 ± 5.5 Irritant 

HFCPE 88.2 84.9 67.3 80.2 ± 11.3 Non irritant 
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6. Conclusion 

It was concluded that the test substance, HFCPE, with a mean tissue viability of 

80.2 ± 11.3%, was predicted as a non irritant to the skin. 
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7. References 

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

Irritation Test-42 hours, Standard Operating Procedure: January 2005 Version 1.6. 

OECD Guideline For The Testing of Chemicals (2008), Draft proposal for a new guideline: 

In Vitro Skin Irritation: Human Skin Model Test. Year 2008 

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

Validity of In Vitro tests for Skin Irritation, 27 April 2007. 
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Table 1 EPISKIN study data 

Sample Tissue Optical Density OD - Blank % Negative

Replicate (OD) Control 

a 0.795 0.665

0.760 0.630 81.9

Average 0.647

sd 0.024

Negative b 0.964 0.834

Control 0.929 0.799 103.3

(NC) Average 0.816

sd 0.024

c 1.070 0.940

1.004 0.874 114.8

Average 0.907

sd 0.047

Replicates Average 0.790 100.0

a,b,c sd 0.132 16.7

a 0.278 0.148

0.276 0.146 18.6

Average 0.147

sd 0.001

Positive b 0.296 0.166

Control 0.276 0.146 19.8

(PC) Average 0.156

sd 0.015

c 0.364 0.234

0.350 0.220 28.7

Average 0.227

sd 0.010

Replicates Average 0.177 22.4

a,b,c sd 0.044 5.5

a 0.826 0.696

0.828 0.698 88.2

Average 0.697

sd 0.002

HFCPE b 0.803 0.673

(S1) 0.799 0.669 84.9

Average 0.671

sd 0.003

c 0.671 0.541

0.653 0.523 67.3

Average 0.532

sd 0.013

Replicates Average 0.633 80.2

a,b,c sd 0.089 11.3

0.131

0.130

0.128

0.127

Blank 0.130

0.133

Average 0.130

sd 0.002

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 

EPISKIN Construct 
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Annex 2 Huntingdon Research Centre GLP Compliance Statement 2008