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### HFCPE: Eye Irritation to the Rabbit

HLS study number: VHJ0005

Version ID: Final

Issue date: 28 September 2010Huntingdon Life Sciences VHJ0005

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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 VHJ0005

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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 ...................................................................................................................

. 9 2. Test substance............................................................................................................. 10 3. Experimental Procedure............................................................................................... 11 3.1 Animal management ........................................................................................ 11 3.2 Test substance preparation.............................................................................. 11 3.3 Treatment procedure........................................................................................ 11 3.4 Serial observations........................................................................................... 12 3.4.1 Clinical signs......................................................................................... 12 3.4.2 Ocular responses ................................................................................. 13 3.4.3 Interpretation of responses................................................................... 14 3.4.4 Termination........................................................................................... 16 3.5 Archives............................................................................................................ 16 3.6 Deviations from protocol................................................................................... 16 4. Results ........................................................................................................................

. 17 4.1 Clinical signs .................................................................................................... 17 4.2 Ocular responses ............................................................................................. 17 5. Conclusion ................................................................................................................... 18


## List of Tables

Table 1 Mean values for ocular lesions for 2001/59/EC and GHS classification........ 19 Table 2 Mean values for ocular lesions for Kay and Calandra classification.............. 20 Table 3 Grades for ocular irritation responses following instillation of HFCPE .......... 21


## List of Annexes

Annex 1 Weight of Evidence ....................................................................................... 24 Annex 2 GLP Compliance Statement.......................................................................... 25Huntingdon Life Sciences VHJ0005

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## Contributing Scientist
HFCPE: Eye irritati on to the rabbit 

### Study management P B Rees BSc (Hons) CBiol MSB Study DirectorHuntingdon Life Sciences VHJ0005

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## Summary

A study was performed to assess the eye 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, Method B.5 Acute Toxicity: Eye irritation/corrosion. OECD Guideline for Testing of Chemicals No.405 “Acute Eye Irritation/Corrosion”. Adopted 24 April 2002.

EPA Health Effects Test Guidelin es OPPTS 870.2400 Acute Eye Irritation EPA 712-C-98-195. August 1998. Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Eye Irritation (2-1-5), 12 N ohsan No. 8147, Agricultural Production Bureau, November 24, 2000. Three rabbits were each administered a single oc ular dose of a volume of 0.1 mL of the test substance and observed for 72 hours after instillation. Injection of the conjunctival blood vessels wa s apparent in all anim als throughout the first 24 hours after instillation; slight discharge wa s additionally evident in one animal one hour after instillation. The treated eye of e ach animal was overtly normal 48 hours after instillation. Instillation of the test material gave rise to practically no or a slight initial pain response.
The total mean scores accordi ng to the system of Kay and Calandra (1962) are summarised below:

Time after instillation of HFCPE Area of eye 1 hour 24 hours 48 hours 72 hours

Cornea 0.0 0.0 0.0 0.0 Iritis 0.0 0.0 0.0 0.0 Conjunctiva 2.7 2.0 0.0 0.0 Total mean score 2.7 2.0 0.0 0.0

The highest total mean score was 2.7 occurri ng at the 1 hour observation; accordingly under the criteria of Kay and Calandra (1962) HFCPE was classified as “minimally irritating” to the eye.Huntingdon Life Sciences VHJ0005

8 The means of scores for the ocular reactions at approximately 24, 48 and 72 hours after administration, calculated separately for each animal, are summarised below:
Animal number Corneal Iridial Conjunctival opacity lesions Redness Chemosis 104 0.0 0.0 0.3 0.0 105 0.0 0.0 0.3 0.0 106 0.0 0.0 0.3 0.0 EC R36 trigger values* ≥ 2, < 3 ≥ 1, < 2 ≥ 2.5 ≥ 2 EC R41 trigger values* ≥ 3 = 2
* Classification triggered if any value is attained by two or more animals.

HFCPE did not require labelling wi th the risk phrase R36, “Irritating to eyes”, in accordance with Commission Directive 2001/59/EC. HFCPE (F-07095) was not classified in accord ance to the Globally Harmonised System (GHS), (United Nations, 2005).Huntingdon Life Sciences VHJ0005

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## 1. Introduction
The study was designed to assess eye irrita tion potential of HFCPE following a single instillation into the eye of the rabbit. The test substance may come into contact with the eye 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, Method B.5 Acute Toxicity: Eye irritation/corrosion. OECD Guideline for Testing of Chemicals No.405 “Acute Eye Irritation/Corrosion”. Adopted 24 April 2002.

EPA Health Effects Test Guidelin es OPPTS 870.2400 Acute Eye Irritation EPA 712-C-98-195. August 1998. Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Eye Irritation (2-1-5), 12 N ohsan No. 8147, Agricultural Production Bureau, November 24, 2000.

The albino rabbit was chosen as it has been s hown to be a suitable model for eye irritation studies and is the animal recommended in the test guidelines.
The amount of test substance instilled was chosen in compliance with the guidelines. The protocol was approved by the Study Director and Huntingdon Life Sciences Management on 24 June 2010 and by the Sponsor on 25 June 2010. The experimental start date was 28 June 2010 and the experimental completion date was 6 July 2010.Huntingdon Life Sciences VHJ0005

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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 VHJ0005

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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 3.87 to 4.20 kg and 41 weeks of age, prior to treatment (Day 1). All rabb its were acclimatised to the experimental environment for a period of 31 week s prior to the start of the study. Each animal was housed individually in a pl astic cage with perfor ated floors at the Eye Research Centre, Eye, Suffolk, IP23 7PX an d was offered 125 g of a standard laboratory rabbit diet per day; drinking water was provided ad libitum . The batch of diet used for the study is analysed for nutrients, possible cont aminants and micro-organisms likely to be present in the diet and which, if in excess of specified amounts, might have an undesirable effect on the test system. A dietary supplem ent of hay was offered during acclimatisation until two days prior to dose in stillation, for the remainder of acclimatisation and throughout the study observation period w holemeal bread was offered. 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 Huntingdon Life Sciences.
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 The eyes of each animal were examined prior to instillation of the test substance to ensure that there was no pre-existing corneal damage, ir idial inflammation or conjunctival irritation. Each animal was gently restrained. The dose was instilled into the right eye by pulling the lower eyelid away from the eye ball to form a cup into which the test substance was dropped. The eyelids were then gently held together for one second be fore releasing; the left eye remained untreated.Huntingdon Life Sciences VHJ0005

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### 3.4 Serial observations 3.4.1 Clinical signs
The behaviour of each rabbit was observed imme diately following instillation of the test substance to allow assessment of the initial pain response by the criteria detailed below. Criteria for assessment of pain response (not a component of the regulatory guidelines)
Reaction of animal to instillation of test substance Class Descriptive Rating

No Response 0 No initial pain A few blinks only; normal within one or two minutes 1 Practically no initial pain

Rabbit blinks and tries to open eye but the reflexes close it 2 Slight initial pain

Rabbit holds eye shut and puts pressure on lids; may rub eye with paw, may vocalise 3 Moderate initial pain

Rabbit holds eye shut vigorously, may tilt head, may have reluctance to rub eye; may vocalise 4 Severe initial pain

Rabbit holds eye shut very firmly; may vocalise, claws at eye, jumps and tries to escape 5 Very severe initial pain

The animals were returned to their cages and ch ecked at least twice durin g the first hour after dosing and at regular interval s throughout the day to ensure no severe injury passed unnoticed. Ocular reactions to treatment were assessed 1, 24, 48 and 72 hours after treatment, according to the criteria below. R eactions not included overleaf were described in detail.Huntingdon Life Sciences VHJ0005

13 3.4.2 Ocular responses The untreated eye was used as a comparison with the treated eye during assessment of ocular lesions. Ocular irritation was assessed using the prescribed numerical system: Cornea Opacity: degree of density (a rea most dense taken for reading) No opacity 0 Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible 1 Easily discernible translucent areas, details of iris slightly obscured 2 Nacreous areas, no details of iris visi ble, size of pupil barely discernible 3 Opaque cornea, iris not discernible through the opacity 4

Area of cornea involved: None 0 One quarter (or less) but not zero 1 Greater than one quarter, but less than half 2 Greater than half, but less than three quarters 3 Greater than three quarters, up to whole area 4 The presence or absence of corneal ulceration or stippling was designa ted + (positive) or - (negative).
Iris: Normal 0 Markedly deepened rugae, conge stion, swelling, moderate circumcorneal hyperaemia or injection, any of thes e or combination of any thereof, iris still reacting to light (sluggish reaction is positive) 1 No reaction to light, haemorrhage, gro ss destruction (any or all of these) 2 Conjunctivae Redness: (refers to the mo st severe reading of palpebral and bulbar conjunctivae, as compared to the control eye) Blood vessels normal 0 Some blood vessels definitely hyperaemic (injected) 1 Diffuse, crimson colour, individual vessels not easily discernible 2 Diffuse beefy red 3 Chemosis: (lids and/or nictating membranes) No swelling 0 Any swelling above normal (incl udes nictating membranes) 1 Obvious swelling with pa rtial eversion of lids 2 Swelling with lids about half-closed 3 Swelling with lids more than half-closed 4Huntingdon Life Sciences VHJ0005

14 The presence or absence of ulceration or necros is was designated + (posi tive) or - (negative). Discharge: No discharge 0 Any amount different from normal 1 Discharge with moistening of lids and hairs adjacent to lids 2 Discharge with moistening of lids and hairs and considerable area around eye 3* Extensive mucoid discharge 4 * Maximum score permissible under system of Kay and Calandra. An ophthalmoscope or pencil beam torch was ava ilable for use to facilitate inspection of the eyes. 3.4.3 Interpretation of responses

The classification criteria of the European Communities A substance or preparation is considered irritating to eyes (R36) if, when applied to the eye of the animals, significant ocul ar lesions are caused which ar e present 24 hours or more after instillation of the test substance. Ocular lesi ons are considered signifi cant if two or more of the rabbits have mean values at or above the limit values following:

Limit range Corneal Opacity (CO) 2 < CO < 3 Iris lesions (IL) 1< IL < 2 Redness of conjunctiva (RoC) RoC > 2.5 Chemosis (C) C > 2
Mean values are calculated us ing all scores recorded 24, 48 and 72 hours after treatment. If the limit values for corneal opacity are equal to or greater than 3 or iris lesions are equal to 2, the substance is considered to have the risk of serious damage to eyes (R41).Huntingdon Life Sciences VHJ0005

15 The classification system of Kay and Calandra (1962)* Corneal scoring using the grade for degree of opacity density (A) and the grade for the area of cornea involved (B): Corneal score = A x B x 5 (theoretical maximum = 80) Iridial scoring using the grades for iritis (C): Iridial score = C x 5 (the oretical maximum = 10) Conjunctival score using the grade for conjunc tival redness (D), chemosis grade (E) and grade for discharge (F): Conjunctival score = (D + E + F) x2 (theoretical maximum = 20) The scores for each area are meaned for each time point and the highest total meaned score is used for the classification thus:

Total mean score Classification 0.0 – 0.5 Non-irritating 0.5 – 2.5 Practically non-irritating 2.5 – 15 Minimally irritating 15 – 25 Mildly irritating 25 – 50 Moderately irritating 50 – 80 Severely irritating 80 – 100 Extremely irritating 100 – 110 Maximally irritating * Kay, JH & Calandra, JC (1962) Interpretation of Eye Irritation Tests.

UN GHS Classification The Globally Harmonised System of Classifica tion and Labelling of Chemicals (GHS) of the United Nations (2005) contains the following cr iteria for classification of irritants and corrosives to the eye:
Category 1 Category 2 Irreversible damage to cornea, iris, conjunctiva 21 days after exposure in at least one animal Reversible adverse effects on cornea, iris and conjunctiva

Mean Draize score in 2 of 3 animals: corneal opacity > 3, iritis > 1.5 Mean Draize score in 2 of 3 animals: corneal opacity > 1, iritis > 1, redness > 2, chemosis > 2 Subcategory 2A Reversible in 21 days Subcategory 2B Reversible in 7 days The observed pain response following instil lation of the test substance was reported separately.Huntingdon Life Sciences VHJ0005

16 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 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 104 15 September 2009 28 June 2010 41 Weeks 105 18 September 2009 30 June 2010 41 Weeks 106 16 September 2009 30 June 2010 19 Weeks The animals were healthy young adult animals and the use of slightly older than intended animals had no impact upon the study. There was no other deviation from protocol.Huntingdon Life Sciences VHJ0005

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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 Ocular responses Injection of the conjunctival blood vessels wa s apparent in all anim als throughout the first 24 hours after instillation; slight discharge wa s additionally evident in one animal one hour after instillation. The treated eye of e ach animal was overtly normal 48 hours after instillation. Instillation of the test material gave rise to pr actically no or slight in itial pain response.Huntingdon Life Sciences VHJ0005

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## 5. Conclusion

The highest total mean score was 2.7 occurri ng at the 1 hour observation; accordingly under the criteria of Kay and Calandra (1962) HFCPE was classified as “minimally irritating” to the eye. HFCPE did not require labelling wi th the risk phrase R36, “Irritating to eyes”, in accordance with Commission Directive 2001/59/EC. HFCPE (F-07095) was not classified in accord ance to the Globally Harmonised System (GHS), (United Nations, 2005).Huntingdon Life Sciences VHJ0005

19 Table 1 Mean values for ocular lesions for 2001/59/EC and GHS classification 24, 48 and 72 hours after instillation of HFCPE

Animal number and
sex Corneal Opacity Iridial lesions Redness of Conjunctiva Chemosis

104 M 0.0 0.0 0.3 0.0 105 M 0.0 0.0 0.3 0.0 106 M 0.0 0.0 0.3 0.0 M MaleHuntingdon Life Sciences VHJ0005

20 Table 2 Mean values for ocular lesions for Kay and Calandra classification
Mean irritation scores after instillation of HFCPE

Area of eye 1 hour 24 hours 48 hours 72 hours

Cornea 0.0 0.0 0.0 0.0

Iritis 0.0 0.0 0.0 0.0
Conjunctiva 2.7 2.0 0.0 0.0

Total mean score 2.7 2.0 0.0 0.0Huntingdon Life Sciences VHJ0005

21 Table 3 Grades for ocular irritation responses following instillation of HFCPE
Animal number and sex: 104 M # Pain evaluation response: 2 Region of the eye Response Grade of response at time after instillation Hours

1 24 48 72 Cornea Opacity (A) 0 0 0 0 Area (B) 0 0 0 0 Ulceration - - - - Stippling - - - - Corneal score (A x B x 5) 0 0 0 0 Iris Value (C) 0 0 0 0 Iridial score (C x 5) 0 0 0 0 Conjunctiva Redness (D) 1 1 0 0 Chemosis (E) 0 0 0 0 Discharge (F) 1 0 0 0 Necrosis - - - - Ulceration - - - - Conjunctival score ((D+E+F)x2) 4 2 0 0
# Sentinel animal M MaleHuntingdon Life Sciences VHJ0005

22 Table 3-continued Grades for ocular ir ritation responses following instillation of HFCPE
Animal number and sex: 105 M Pain evaluation response: 2 Region of the eye Response Grade of response at time after instillation Hours

1 24 48 72 Cornea Opacity (A) 0 0 0 0 Area (B) 0 0 0 0 Ulceration - - - - Stippling - - - - Corneal score (A x B x 5) 0 0 0 0 Iris Value (C) 0 0 0 0 Iridial score (C x 5) 0 0 0 0 Conjunctiva Redness (D) 1 1 0 0 Chemosis (E) 0 0 0 0 Discharge (F) 0 0 0 0 Necrosis - - - - Ulceration - - - - Conjunctival score ((D+E+F)x2) 2 2 0 0
M MaleHuntingdon Life Sciences VHJ0005

23 Table 3 continued Grades for ocular ir ritation responses following instillation of HFCPE
Animal number and sex: 106 M Pain evaluation response: 1 Region of the eye Response Grade of response at time after instillation Hours

1 24 48 72 Cornea Opacity (A) 0 0 0 0 Area (B) 0 0 0 0 Ulceration - - - - Stippling - - - - Corneal score (A x B x 5) 0 0 0 0 Iris Value (C) 0 0 0 0 Iridial score (C x 5) 0 0 0 0 Conjunctiva Redness (D) 1 1 0 0 Chemosis (E) 0 0 0 0 Discharge (F) 0 0 0 0 Necrosis - - - - Ulceration - - - - Conjunctival score ((D+E+F)x2) 2 2 0 0
M MaleHuntingdon Life Sciences VHJ0005

24 Annex 1 Weight of Evidence Prior to undertaking in-vivo eye irritation tes ting 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)
Dermal irritation in the rabb it (VHJ0003): no dermal irritation
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 study 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 VHJ0005

25 Annex 2 GLP Compliance Statement