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Working for a better future

HFCPE: Eye Irritation to the Rabbit 

HLS study number: VHJ0005 

Version ID: Final 

Issue date: 28 September 2010 
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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 

 UK 

 
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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.......................................................................... 25


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Contributing Scientist 

HFCPE: Eye irritation to the rabbit 

Study management 

P B Rees BSc (Hons) CBiol MSB 

Study Director 
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Summary 

A study was performed to assess the eye irritation potential of HFCPE to the rabbit. The 

method followed was that described in: 

EEC Methods for the determination of toxicity 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 Guidelines 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 Nohsan No. 8147, Agricultural 

Production Bureau, November 24, 2000. 

Three rabbits were each administered a single ocular 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 was apparent in all animals throughout the first 

24 hours after instillation; slight discharge was additionally evident in one animal one hour 

after instillation. The treated eye of each 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 according 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 occurring at the 1 hour observation; accordingly under 

the criteria of Kay and Calandra (1962) HFCPE was classified as “minimally irritating” to the 

eye. 
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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 with the risk phrase R36, “Irritating to eyes”, in accordance 

with Commission Directive 2001/59/EC. 

HFCPE (F-07095) was not classified in accordance to the Globally Harmonised System 

(GHS), (United Nations, 2005). 
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1. Introduction 

The study was designed to assess eye irritation 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 toxicity 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 Guidelines 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 Nohsan No. 8147, Agricultural 

Production Bureau, November 24, 2000. 

The albino rabbit was chosen as it has been shown 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. 
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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% 
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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 rabbits were acclimatised to the experimental 

environment for a period of 31 weeks prior to the start of the study. 

Each animal was housed individually in a plastic cage with perforated floors at the 

Eye Research Centre, Eye, Suffolk, IP23 7PX and 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 contaminants 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 supplement of hay was offered during acclimatisation 

until two days prior to dose instillation, for the remainder of acclimatisation and throughout 

the study observation period wholemeal bread was offered. 

During the acclimatisation and study period the animals were given small soft white 

untreated wood blocks for environmental enrichment. 

Results of routine physical and chemical examination 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 displaying 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 substance received, its stability under the storage 

conditions and the conditions of administration 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, iridial 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 before releasing; the left eye 

remained untreated. 
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3.4 Serial observations 

3.4.1 Clinical signs 

The behaviour of each rabbit was observed immediately 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 checked at least twice during the first hour after 

dosing and at regular intervals 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. Reactions not included overleaf were described in 

detail. 
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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 (area 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 visible, 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 designated + (positive) or - 

(negative). 

Iris: 

Normal 0 

Markedly deepened rugae, congestion, swelling, moderate 

circumcorneal hyperaemia or injection, any of these or combination of 

any thereof, iris still reacting to light (sluggish reaction is positive) 1 

No reaction to light, haemorrhage, gross destruction (any or all of these) 2 

Conjunctivae Redness: (refers to the most 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 (includes nictating membranes) 1 

Obvious swelling with partial eversion of lids 2 

Swelling with lids about half-closed 3 

Swelling with lids more than half-closed 4 
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The presence or absence of ulceration or necrosis was designated + (positive) 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 available 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 ocular lesions are caused which are present 24 hours or more after 

instillation of the test substance. Ocular lesions are considered significant 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 using 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). 
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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 (theoretical maximum = 10) 

Conjunctival score using the grade for conjunctival 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 Classification and Labelling of Chemicals (GHS) of the 

United Nations (2005) contains the following criteria 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 instillation of the test substance was reported 

separately. 
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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 together 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 end of the retention period the Sponsor will be 

contacted and advice sought on the return, disposal or further 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 Protocol indicates that the animals to be 

employed on this study would be within the approximate 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. 
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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 was apparent in all animals throughout the first 

24 hours after instillation; slight discharge was additionally evident in one animal one hour 

after instillation. The treated eye of each animal was overtly normal 48 hours after 

instillation. 

Instillation of the test material gave rise to practically no or slight initial pain response. 
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5. Conclusion 

The highest total mean score was 2.7 occurring 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 with the risk phrase R36, “Irritating to eyes”, in accordance 

with Commission Directive 2001/59/EC. 

HFCPE (F-07095) was not classified in accordance to the Globally Harmonised System 

(GHS), (United Nations, 2005). 
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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 Male 
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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 0.0 
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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 Male 
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Table 3-continued Grades for ocular irritation 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 Male 
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Table 3 continued Grades for ocular irritation 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 Male 
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Annex 1 Weight of Evidence 

Prior to undertaking in-vivo eye 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 applications. 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 animal data (including dermal toxicity) 

Dermal irritation in the rabbit (VHJ0003): no dermal irritation 

Searches for the test material 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 irritation 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 irritation 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): predicted as non-irritant to the skin 

BCOP (HLS study VHJ0004): no severe eye irritancy or corrosivity prediction 
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Annex 2 GLP Compliance Statement