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### HFCPE: Skin Irritation to the Rabbit

HLS study number: VHJ0003
Version ID: Final
Issue date: 28 September 2010Huntingdon Life Sciences VHJ0003

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## Details of Sponsor and Test Facility
Sponsor ZEON Corporation Shin Marunouchi Center Building 1-6-2 Marunouchi Chiyoda-ku Tokyo, 100-8246 JAPAN

Test facility Huntingdon Life Sciences Eye Research Centre Eye Suffolk IP23 7PX UKHuntingdon Life Sciences VHJ0003

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## Table of Contents
Details of Sponsor and Test Facility ....................................................................................... 2 Compliance with Good Laboratory Practice............................................................................ 4 Quality Assurance Statement ................................................................................................. 5 Contributing Scientist.........................................................................................................

..... 6 Summary................................................................................................................................. 7 1. Introduction ...................................................................................................................

. 8 2. Test substance............................................................................................................... 9 3. Experimental Procedure............................................................................................... 10 3.1 Animal management ........................................................................................ 10 3.2 Test substance preparation.............................................................................. 10 3.3 Treatment procedure........................................................................................ 10 3.4 Serial observations........................................................................................... 11 3.4.1 Clinical signs......................................................................................... 11 3.4.2 Dermal responses ................................................................................ 11 3.4.3 Interpretation of responses................................................................... 12 3.4.4 Termination........................................................................................... 13 3.5 Archives............................................................................................................ 13 3.6 Deviations from protocol................................................................................... 13 4. Results ........................................................................................................................

. 14 4.1 Clinical signs .................................................................................................... 14 4.2 Dermal responses ............................................................................................ 14 5. Conclusion ................................................................................................................... 15 

## List of Tables

Table 1 Mean values for erythema and oedema........................................................ 16 Table 2 Individual values for erythema and oedema.................................................. 17 

## List of Annexes

Annex 1 Weight of Evidence ....................................................................................... 18 Annex 2 GLP Compliance Statement.......................................................................... 19Huntingdon Life Sciences VHJ0003

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## Contributing Scientist
HFCPE: Skin irritation to the rabbit 

### Study management P B Rees BSc (Hons) CBiol MSB Study DirectorHuntingdon Life Sciences VHJ0003

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## Summary
A study was performed to assess the skin irritati on potential of HFCPE to the rabbit. The method followed was that described in: EEC Methods for the determination of toxi city and other health effects. Council Regulation No. 440/2008. Part B, Me thod B.4 Acute Toxicity: Dermal irritation/corrosion. OECD Guideline for Testing of Chemicals No.404 “Acute Dermal Irritation/Corrosion”. Adopted 24 April 2002.
EPA Health Effects Test Guidelines OPPTS 870.2500 Acute Dermal Irritation EPA 712-C-98-196. August 1998. Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Skin Irritati on (2-1-4), 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000. Three rabbits received a single four hour, semi-occlusive, dermal administration of approximately 0.5 mL of the test substance as supplied and were observed for four days. Very slight erythema was apparent one hour after bandage removal in a single animal; no other dermal response was apparent at any time during the study. The means of scores for these reactions at approximately 24, 48 and 72 hours after administration, calculated separately for each animal, are summarised below: Means of scores at approximately 24, 48 and 72 hours Animal number Erythema Oedema
93 0.0 0.0 96 0.0 0.0 97 0.0 0.0 EC trigger values* ≥ 2 ≥ 2 * Classification is triggered if mean s of scores for either effect are ≥ 2 for two or three animals (or if effects pers ist to Day 14 in at least two animals). The Primary Irritation Index was cal culated to be 0.0; HFCPE was classified as ‘non-irritant’ according to the criteria of the ECETOC and did not require labe lling with the risk phrase R38, “Irritating to skin”, in accordance with Commission Directive 2001/59/EC.Huntingdon Life Sciences VHJ0003

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## 1. Introduction
The study was designed to assess skin irr itation potential of HFCPE following a single dermal application to rabbits. The test subs tance may come into contact with skin during handling or use. The study was conducted in compliance with the following guidelines: EEC Methods for the determination of toxi city and other health effects. Council Regulation No. 440/2008. Part B, Me thod B.4 Acute Toxicity: Dermal irritation/corrosion. OECD Guideline for Testing of Chemicals No.404 “Acute Dermal Irritation/Corrosion”. Adopted 24 April 2002.
EPA Health Effects Test Guidelines OPPTS 870.2500 Acute Dermal Irritation EPA 712-C-98-196. August 1998. Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Skin Irritati on (2-1-4), 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.
The albino rabbit was chosen as it has been show n to be a suitable model for skin irritation studies and is the animal recommended in the test guidelines. The amount of test substance administered wa s chosen in compliance with the guidelines. The protocol was approved by the Study Director and Huntingdon Life Sciences Management on 11 June 2010 and by the Sponsor on 15 June 2010. The experimental start date was 18 June 2010 and the experimental completion date was 29 June 2010.Huntingdon Life Sciences VHJ0003

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## 2. Test substance

Identification: HFCPE Intended use: Electronic applications Description: Colourless liquid Storage conditions: Room temperature Batch number: Lot No 20090601 Date of receipt: 22 June 2009 Expiry date: End June 2010 Purity: 99.3%Huntingdon Life Sciences VHJ0003

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## 3. Experimental Procedure 3.1 Animal management Animals for this study were selected from a stock supply of healthy adult rabbits of the New Zealand White strain. They were in the weight range of 3.65 to 3.93 kg and approximately 41 weeks of age, prior to treatment (Day 1). All rabbits were acclimatised to the experimental environment for a period of 30 weeks prior to the start of the study. The rabbits were housed indivi dually in plastic cages with perforated floors at the Eye Research Centre, Eye, Suffolk, IP23 7PX. Each rabbit was offered 125 g of a standard labor atory rabbit diet per day; drinking water was provided ad libitum . The batch of diet used for the study was analysed for nutrients, possible contaminants and micro-organisms likely to be pr esent in the diet and which, if in excess of specified amounts, might have an undesirable e ffect on the test system. The animals were given a dietary supplement of hay. During the acclimatisation a nd study period the animals were given small soft white untreated wood blocks for environmental enrichment. Results of routine physical and chemical examin ation of drinking water, as conducted by the supplier are made available to H untingdon Life Sciences Limited. Animal room environmental controls were set to maintain temperature within the range 16 to 20°C, and relative humidity within 40 to 70%. These environmental parameters were recorded and the permanent record archived with other departmental raw data. Lighting was controlled by means of a time switch to give 12 hours of artificial light (06:00 to 18:00 GMT) in each 24 hour period. Each animal was identified by a numbered tag placed through the edge of one ear. This identification was unique within the Department throughout the duration of the study. Each cage was identified by a coloured label displa ying the study number and animal number. 

### 3.2 Test substance preparation HFCPE was administered as supplied by the Sponsor. The absorption of HFCPE was not determined. The identity, strength and purity of the test s ubstance received, its stability under the storage conditions and the conditions of administrati on were the responsibility of the Sponsor. 

### 3.3 Treatment procedure On the day before application of the test subs tance, hair was removed with clippers from the dorso-lumbar region of each rabbit exposi ng an appropriate sized area of skin. Approximately 0.5 mL of the test substa nce was applied under a 2-ply 25 mm x 25 mm porous gauze pad secured with ‘blenderm’ surgical tape to intact skin sites on three animals.Huntingdon Life Sciences VHJ0003

11 An additional site was similarly treated with the exception of test substance and acted as a control. A single animal (number 93) received three e xposures of three minutes, one or four hours duration in a step-wise manner and acted as a pre liminary screen. In the absence of a severe effect on removal of the dressing s the next exposure was initiated. In the absence of a severe response in this animal, two further animal s (numbers 96 and 97) were committed to the study each receiving a single four hour exposure. For exposures of one hour or more each treatme nt site was covered with cotton wool and "Tubigrip" elasticated bandage dressing for the duration of the exposure period. The animals were returned to their cages immediately after treatment. At the end of the exposure period the semi-o cclusive dressing and gauze pad were removed and the treatment site was washed with lukewarm water (30-40°C) to remove any residual test substance. The treated area wa s blotted dry with absorbent paper. 

### 3.4 Serial observations 3.4.1 Clinical signs All animals were observed daily for signs of ill health or toxicity. 3.4.2 Dermal responses
Examination of the treated skin was made on removal of the dressings (for 3 minute or one hour exposures) and approximately 1, 24, 48 and 72 hours later. Only the data for the four hour exposure are reported, the data from the three minute and one hour exposures are held in the archives. Local dermal irritation was assessed using the prescribed numerical system: Erythema and eschar formation: No erythema 0 Very slight erythema (barely perceptible) 1 Well-defined erythema 2 Moderate to severe erythema 3 Severe erythema (beet redness) or eschar formation (injuries in depth) preven ting grading of erythema 4
Oedema formation: No oedema 0 Very slight oedema (barely perceptible) 1 Slight oedema (edges of area well -defined by definite raising) 2 Moderate oedema (raised approximately 1 millimetre) 3 Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4Huntingdon Life Sciences VHJ0003

12 3.4.3 Interpretation of responses Primary Irritation Index A primary irritation index (PII) was calculate d from the erythema and oedema scores according to the following formula as described in Technical Report No. 66 “Skin irritation and Corrosion: Reference chemicals data bank” (March 1995) ECETOC, Brussels.
PII= ∑ Erythema at 24/48/72 hours + ∑ Oedema at 24/48/72 hours 3 x number of animals The maximum possible score was 8.0. The PII was then used to classify the test substance as follows: Primary Irritation Index Classification 0 non-irritant >0 - 2.0 mildly irritating 2.1 - 5.0 moderate irritant 5.1 - 6.0 moderate to severe irritant

> 6.0 severe irritant This classification system is a modification base d on “Appraisal of the Safety of Chemicals in Foods Drugs and Cosmetics” published by the As sociation of Food and Drug Officials of the United States, 1959. EU Classification The Official Journal of the European Comm unities (Directive 2001/59/EC) contains the following criteria for classification of irritants and corrosives to skin. Substances and preparations shall be classified corrosive if, when applied to healthy intact animal skin, full thickness destruction of skin tis sue on at least one animal occurs as a result of up to four hours exposure. The following ri sk phases shall be assigned in accordance with the following criteria: R35: Causes severe burns - if, when applied to healthy intact animal skin, full thickness destruction of the skin occurs as a result of up to three minutes exposure. R34: Causes burns - if, when applied to healthy intact animal skin, full thickness destruction of the skin occurs as a result of up to four hours exposure. Non-corrosive substances and preparations shall be classified irritant if, when applied to healthy intact animal skin for up to four hours, significant inflammation is caused and is present 24 hours or more after the end of the exposure period. Inflammation is significant if the mean value of the scores for either erythema (including eschar formation) or oedema formation observed in two or more animals is e quivalent to the value of two or more. All scores at 24, 48 and 72 hours should be used in calculating the resp ective mean values.Huntingdon Life Sciences VHJ0003

13 Inflammation of the skin is also significant if it persists in at least tw o animals at the end of the observation period. Particular effects e.g. hyperplasia, sca ling, discolouration, fissures, scabs and alopecia should be taken into account. The following risk phrase shall be assigned: R38: Irritating to skin 3.4.4 Termination Following completion of the observation period the animals were humanely killed by an intravenous injection of sodium pentobarbital. 

### 3.5 Archives All raw data arising from the performance of this study at Huntingdon Life Sciences is the property of the Sponsor and will be lodged togeth er with a copy of the final report in the Huntingdon Life Sciences Archive. Such records will be retained for a minimum of one year from the date on which the Study Director signs the final report. At the e nd of the retention period the Sponsor will be contacted and advice sought on th e return, disposal or furthe r retention of the records. Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and a copy of the final report in its archive indefinitely. 

### 3.6 Deviations from protocol The following deviation from protocol occurred: Section 3.1 (Animal management) of the Study Pr otocol indicates that the animals to be employed on this study would be within the a pproximate age range of 12 to 40 weeks of age on the day of dosing. The dates of birth and dose administration for the animals used on the study are given below:

Animal number Date of Birth Date of administration Age at administration 93 9 September 2009 21 June 2010 41 Weeks 96 8 September 2009 23 June 2010 41 Weeks 97 8 September 2009 23 June 2010 41 Weeks The animals were healthy young adult animals and the use of slightly older than intended animals had no impact upon the study.Huntingdon Life Sciences VHJ0003

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## 4. Results 4.1 Clinical signs There was no sign of toxicity or ill health in any rabbit during the observation period. 

### 4.2 Dermal responses Very slight erythema was apparent one hour after bandage removal in a single animal; no other dermal response was apparent at any time during the study.Huntingdon Life Sciences VHJ0003

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## 5. Conclusion
The Primary Irritation Index was cal culated to be 0.0; HFCPE was classified as ‘non-irritant’ according to the criteria of the ECETOC and did not require labe lling with the risk phrase R38, “Irritating to skin”, in accordance with Commission Directive 2001/59/EC.Huntingdon Life Sciences VHJ0003

16 Table 1 Mean values for erythema and oedema 24, 48 and 72 hours after removal of dressings * Erythema Oedema Animal number and sex Test Control Test Control

93 M 0.0 0.0 0.0 0.0 96 M 0.0 0.0 0.0 0.0 97 M 0.0 0.0 0.0 0.0 * 4 hour exposure M Male17 Huntingdon Life Sciences VHJ0003Table 2 Individual valuesfor erythema and oedemaTest site: 0.5 mL HFCPE } semi occluded for four hoursControl site: No treatment }Score after removal of dressings1 hour 24 hours 48 hours 72 hoursAnimal numberand sexType ofResponseTest site Control site Test site Control siteTest site Control site Test site Control siteErythema 0 0 0 0 0 0 0 093 M #Oedema 0 0 0 0 0 0 0 0Erythema 0 0 0 0 0 0 0 096 MOedema 0 0 0 0 0 0 0 0Erythema 1 0 0 0 0 0 0 097 MOedema 0 0 0 0 0 0 0 0M Male# Sentinel animalHuntingdon Life Sciences VHJ0003

18 Annex 1 Weight of Evidence Prior to undertaking in-vivo skin irritation testing the Study Director conducted a weight-ofthe-evidence analysis to ensure that the in -vivo testing was sufficiently justified. The following factors have been taken into consideration:
1. Nature of test substance. A colourless liquid for use in electronic applicat ions. The Sponsor indicated that the test substance has the following: Molecular weight: 194.05 Chemical name: 1,3,3,4,4,5,5-Heptafluorocyclopentene Log Pow: 2.4 Structure:

2. Evaluation of existing human and anima l data (including dermal toxicity) Searches for the test materi al failed to yield any data. Searches for related materials yielded: OFCPE (1,2,3,3,4,4,5,5-Octofluorocyclopentane ): skin irritation study in rabbits (HLS study ZCE037): no dermal irritation. Eye irr itation study in rabbits (HLS study ZCE038): irritating to the eye HFCPA (1,1,2,2,3,3,4-Heptafluorocyclopentane ): skin irritation st udy in rabbits (HLS study ZCE034): no dermal irritation. Eye irr itation study in rabbits (HLS study ZCE035): non-irritating to the eye
3. Analysis of structure activity relationships (SAR). None available.
4. Physicochemical properties. pH (as measured at Huntingdon Life Sciences) – 5.0
5. Results from in-vitro or ex-vivo tests. EPISKIN (HLS study VHJ0002): predicte d as non-irritant to the skin BCOP (HLS study VHJ0004): no severe eye ir ritancy or corrosivity predictionHuntingdon Life Sciences VHJ0003

19 Annex 2 GLP Compliance Statement