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### HFCPE

EPISKIN Skin Irritation Test -42 Hours

HLS study number: VHJ0002

Version ID: Final report

Issue date: 8 January 2010Huntingdon Life Sciences VHJ0002

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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 UKHuntingdon Life Sciences VHJ0002

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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.................................. ..................................................1 3 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...................................

................................................... ..18Huntingdon Life Sciences VHJ0002

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## List of Annexes

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

Construct............................................

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

Annex 2 Huntingdon Research Centre GLP Compliance Statement 2008.. ................20Huntingdon Life Sciences VHJ0002

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## Compliance with Good Laboratory PracticeHuntingdon Life Sciences VHJ0002

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## Quality Assurance StatementHuntingdon Life Sciences VHJ0002

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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 TechnologiesHuntingdon Life Sciences VHJ0002

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## Summary

The objective of this test was to assess the skin irritancy, in vitro, of the tes t substance, HFCPE.
The test substance was applied to EPISKIN human epidermis skin constructs. The constr ucts consisted of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis with a f unctional multilayered stratum corneum. The cell viability of the multi layers was d etermined by mitochondrial dehydrogenase activity. The prediction model uses the percentage vi ability values (compared to negative control viability) to identify irritant and non-irr itant substances.
The test substance, HFCPE, elicited a mean tissue viability of 80.2% and was predi cted as non-irritant.Huntingdon Life Sciences VHJ0002

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## 1. Introduction

1.1 Objective

The aim of this study was to determine if the test s ubstance 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 te st, Draize Skin Irritation Test, by the European Centre for the Validation of Alternat ive 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 Standar d 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 EPISKI N threedimensional human skin model. The model consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collage n type 1 matrix coated with type IV collagen. After 13 days in culture a multilayered, hi ghly differentiated model of the human epidermis with a functional multi-layered str atum corneum has formed. The epidermis surface area supplied is 0.38cm 2 . 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.Huntingdon Life Sciences VHJ0002

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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 bl ue colour without any involvement of cellular mitochondrial dehydrogenase. Since the t est substance is rinsed off the tissue before the MTT assay, this is usually avoided. How ever, 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 tur ned blue/purple after approximately 3 hour incubation at 37 ±2oC in 5% CO 2 in air, MTT reduction may have occurred.
The MTT reducing capability of the test substance, HFCPE, was investigat ed 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.Huntingdon Life Sciences VHJ0002

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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 t o 37o C.
The tissues were removed from the agar and placed into wells of 12 well plates contai ning 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% CO 2 in air.



### 2.5 Controls

The negative control was sterile Dulbecco’s Phosphate Buffered Saline (DPB S) 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 μ l over each tissue using a positive displacement pipette

The test substance, HFCPE, a colourless liquid, was applied by dispensing a volume of 10 μ l over each tissue using a positive displacement pipette. 

### 2.7 Test procedure

After incubation of at least 24 hours in maintenance medium, triplicate tissues we re 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 μ l, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved fl at spatula after 7 minutes application time.

After 15 minutes, each tissue was rinsed with 25 ml sterile Dulbeccos Phosphate B uffered Saline (DPBS) to remove residual test substance. Inserts were blotted on abs orbent paper to remove remaining DPBS and the epidermal surface gently swabbed. Each insert wa s 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.Huntingdon Life Sciences VHJ0002

12 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 f rom the collagen matrix using forceps and both parts placed in a micro tube. When all tissue s 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 μ l aliquots of the extractant from each micro tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant wa s mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm with acidifie d isopropanol solution as a blank.Huntingdon Life Sciences VHJ0002

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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 negat ive 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 tiss ue viability after the shipping and storage procedure and under the specific conditions of the ass ay. 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 mea n 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)Huntingdon Life Sciences VHJ0002

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## 4. Maintenance of Records

Records and documentation relating to this study will be maintaine d in the archives of Huntingdon Life Sciences for a period of one year from the date on w hich the Study Director signs the final report. This will include but may not be limited t o the Study Protocol, raw data, a copy of the final report, test substance and other samples a nd specimens generated during the course of this study. If requested, Huntingdon Life Scienc es will continue to retain the substances at additional cost.
Samples and specimens that no longer afford evaluation will be disc arded in accordance with Standard Operating Procedures.
Huntingdon Life Sciences will retain the Quality Assurance recor ds 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.Huntingdon Life Sciences VHJ0002

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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% CO 2 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 w as 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. Thes e 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 appli cable

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 irritantHuntingdon Life Sciences VHJ0002

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## 6. Conclusion

It was concluded that the test substance, HFCPE, with a mean ti ssue viability of 80.2 ± 11.3%, was predicted as a non irritant to the skin.Huntingdon Life Sciences VHJ0002

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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 g uideline: 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.Huntingdon Life Sciences VHJ0002

18 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.Huntingdon Life Sciences VHJ0002

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

EPISKIN ConstructHuntingdon Life Sciences VHJ0002

20 Annex 2 Huntingdon Research Centre GLP Compliance St atement 2008