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Working for a better future

HFCPE 

THE BOVINE CORNEAL OPACITY AND PERMEABILITY ASSAY 

(BCOP) 

HLS study number: VHJ0004 

Version ID: Final Report 

Issue date: 8 January 2010 
 Huntingdon Life Sciences 

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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 system.......................................................................................................9

2. Experimental Procedure .............................................................................................10

2.1 Study schedule ...............................................................................................10

2.2 Test substance details ....................................................................................10

2.2.1 Test substance ....................................................................................10

2.3 Materials and reagents....................................................................................10

2.3.1 Reagents.............................................................................................10

2.3.2 Preparation of cMEM...........................................................................10

2.3.3 Controls...............................................................................................11

2.3.4 Preparation and quality control of sodium fluorescein solution.............11

2.3.5 Calibration of opacitometer..................................................................11

2.3.6 Bovine eyes.........................................................................................12

2.4 Method............................................................................................................12

2.4.1 Preparation of corneas ........................................................................12

2.4.2 Opacity measurement..........................................................................12

2.4.3 Test substance pH...............................................................................13

2.4.4 Treatment groups ................................................................................13

2.4.5 Treatment of corneas...........................................................................13

2.4.6 Permeability determination ..................................................................14

2.4.7 Criteria for determination of a valid test ...............................................14

2.5 Assessment of results.....................................................................................14

2.5.1 Opacity ................................................................................................14

2.5.2 Permeability (OD490) ............................................................................14

2.5.3 In Vitro Irritancy score calculation ........................................................15

2.5.4 Classification criteria............................................................................15

2.5.5 Archives ..............................................................................................15

2.6 Deviations from protocol..................................................................................15

3. Results .......................................................................................................................16

3.1 Assay validity ..................................................................................................16

3.2 pH determination.............................................................................................16

3.3 BCOP results ..................................................................................................16

4. Conclusion..................................................................................................................17

5. References.................................................................................................................18
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List of Appendices 

Appendix 1 BCOP data...................................................................................................19

List of Annexes 

Annex 1 Huntingdon Research Centre GLP compliance statement 2008...................21
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Compliance with Good Laboratory Practice 
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Quality Assurance Statement 
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Contributing Scientists 

HFCPE: Bovine Corneal Opacity and Permeability assay (BCOP) 

Study management 

Joanne O. Hubbard, Higher National Certificate (Applied Biology) 

Study Manager 

In Vitro Safety Assessment 

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 Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the 

ocular irritancy potential in vitro of the test substance. Ethanol was tested in parallel as a 

positive control. 

The assay uses isolated bovine corneas as a means of assessing the ocular corrosivity or 

severe irritancy potential of test substances in vitro. The isolated corneas were obtained as a 

by-product of the meat production industry. Two endpoints, corneal opacity and 

permeability, were measured and combined to give an In Vitro Irritancy Score which can be 

used to classify and rank test substances as potential eye irritants according to OECD 

guideline 437 (OECD 437). 

The test substance, HFCPE, elicited an In Vitro Irritancy Score of 3.0 ± 1.0 and was predicted 

to be a non corrosive or severe eye irritant. 
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1. Introduction 

1.1 Objective 

The objective of the test was to evaluate the ocular irritancy potential of the test substance as 

measured by its ability to induce opacity and permeability to sodium fluorescein in an excised 

bovine cornea, in vitro. 

1.2 Test system 

The test substance, HFCPE, was tested using the Bovine Corneal Opacity & Permeability 

Assay (BCOP) which was developed by Pierre Gautheron 1992 as an in vitro alternative to 

the in vivo Draize Eye Irritation test. The assay uses isolated bovine corneas as a means of 

assessing the ocular irritancy potential of test substances in vitro. 
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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 date: 6 October 2009 

Experimental completion date: 7 October 2009 

2.2 Test substance details 

2.2.1 Test substance 

Identity: HFCPE 

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

Intended use: Electronic applications 

Lot number: 20090601 

Expiry: End June 2010 

Appearance: Colourless liquid 

Storage conditions: Room temperature 

Purity/ Assay: 99.3% 

2.3 Materials and reagents 

2.3.1 Reagents 

Minimal Essential Medium Eagles without Phenol Red (MEM) 

Minimal Essential Medium Eagles with Phenol Red (MEM with phenol red) 

L-Glutamine (200 mM Solution) 

Hanks Balanced Salt Solution (HBSS) containing Ca++ and Mg++ without Phenol Red 

Foetal Calf Serum 

Penicillin - Streptomycin (10000 IU/ml - 10000 μg/ml) Solution 

0.9% Sodium Chloride Solution 

2.3.2 Preparation of cMEM 

To a 500 ml bottle of MEM the following were added: 

5 ml of L-Glutamine (200 mM solution) 

5 ml of Foetal calf serum 
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2.3.3 Controls 

The negative control was sodium chloride solution, 0.9% (Sigma, S8776) 

Lot number: 128K2337 

Expiry: 29 September 2011 

The positive control material was ethanol (Aldrich, 27074-1) 

Lot number: 04248PC 

Expiry: 5 September 2011 

Purity: 91.43% 

2.3.4 Preparation and quality control of sodium fluorescein solution 

A stock solution of Sodium Fluorescein (4 mg/ml) was prepared using cMEM. 

Quality Control 

Two aliquots of this solution were diluted 1:400 in cMEM, in two steps, as follows: 

a. 50 μl of Sodium Fluorescein stock + 950 μl of cMEM 

b. 50 μl of Dilution a. + 950 μl of cMEM 

The Optical Density (OD) of Dilution b was measured on a spectrophotometer at 490 nm 

after blanking on a 1 ml sample of cMEM. The two values obtained were averaged and the 

resulting mean OD490 was between 1.710 and 1.910 (mean OD value was 1.840). 

The batch of stock solution was prepared and aliquoted into suitable vials and stored at -

20°C ± 5°C in the dark until required for use. 

2.3.5 Calibration of opacitometer 

The opacitometer was calibrated at the beginning of the test as follows: 

The opacitometer was switched on and allowed to warm up for a minimum of 30 minutes 

prior to calibration. Both calibration blocks were inserted into the reading chambers and the 

“balance” control was adjusted to give a reading of zero. Calibrator No. 1 was inserted into 

the right hand calibration block and the reading was taken which fell within the range of 71-

79 (value=73). The other two calibrators were checked in the right hand calibration block. 

Calibrator No. 2 fell within the range of 143-158 (value =155) and Calibrator No. 3 fell in the 

range of 214-236 (value = 222). Once calibrated, the unit remained switched on for the 

duration of the assay. 
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2.3.6 Bovine eyes 

Bovine eyes, excised by an abattoir employee, were collected as soon after slaughter as 

possible (excised at 13.00 hours, 6 October 2009). Instructions were given to avoid 

damaging the corneas during excision. Excised eyes were maintained and transported to the 

laboratory, at ambient temperature, in sufficient HBSS, containing 1% (v/v) 

Penicillin/Streptomycin solution, to cover all the eyes in the receptacle. The eyes were used 

within 4 hours of slaughter (incubation of mounted corneas commenced at 16.41 hours, 

 6 October 2009). 

2.4 Method 

2.4.1 Preparation of corneas 

All eyes were carefully examined, macroscopically, for defects (opacity, scratches, 

pigmentation, cuts, etc.) and those exhibiting defects were discarded. The tissue surrounding 

the eyeball was carefully pulled away and the cornea dissected leaving approximately 2 to 

3 mm of sclera present around the cornea. The isolated corneas were stored in a petri dish 

containing HBSS plus 1% Penicillin/Streptomycin solution until all the corneas had been 

dissected. Once all the corneas had been dissected, they were rinsed in fresh HBSS plus 1% 

Penicillin/Streptomycin solution prior to mounting. 

The corneas were mounted in the cornea holders with the endothelial side against the O-ring 

of the posterior half of the holder. Each cornea was gently flattened over the O-ring and 

holder surface with a wetted, gloved finger to expel any air. The anterior half of the holder 

was then positioned on top of the cornea and secured with screws. Both compartments of the 

holder were filled with cMEM, using a syringe. The posterior compartment was always filled 

first to return the cornea to its natural shape. Care was taken to ensure no air bubbles were 

present within the holders. The holders were then plugged and incubated, in an upright 

position, for 60 minutes ± 5 minutes at 32°C ± 1°C in a waterbath. The waterbath 

temperature remained within the limits of 32°C ± 1°C throughout the experiment. 

At the end of the 60 minute incubation period, the medium was removed from both the 

anterior and posterior compartments using a pipette tip attached to a vacuum pump. The 

compartments were refilled with fresh cMEM. Again care was taken to ensure no air bubbles 

were present within the holders. The posterior compartment was then plugged and the basal 

opacity measurements performed. 

2.4.2 Opacity measurement 

The opacitometer measured the light transmission through the centre of each mounted cornea, 

displaying a numerical opacity value (arbitrary unit). The opacity of each cornea was 

measured by reading each holder in the right-hand chamber of the calibrated opacitometer. 

Once the basal opacity of all the corneas had been measured, the mean basal opacity value 

was calculated. Corneas with an opacity value greater than 7 units were discarded. The 

mean basal opacity value of the corneas was then calculated and three corneas with opacity 

values close to the mean value were chosen for use as negative control corneas. 
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2.4.3 Test substance pH 

An estimate of the pH of the test substance was determined using pH test sticks and recorded. 

2.4.4 Treatment groups 

Corneas were treated in triplicate with either the test substance, positive control (ethanol) or 

negative control (0.9% sodium chloride solution). 

The controls and their results were shared with several studies performed in the same assay. 

2.4.5 Treatment of corneas 

The test substance, HFCPE, a colourless liquid, and the positive control (ethanol) were tested 

undiluted. 

Immediately prior to treatment, the medium was removed from the anterior compartment of 

the holder using a pipette tip attached to a vacuum pump, taking extra care to ensure all 

excess liquid had been removed. Seven hundred and fifty μl (750 μl) of test substance, 

positive control or negative control was introduced into the anterior part of each holder. 

Following application, the anterior compartment was plugged and the holder turned to a

horizontal position and slightly rotated to ensure uniform distribution of the test substance 

over the surface of the cornea. The test material or controls were in contact with the cornea 

for a total of 10 minutes (± 30 seconds). Each holder was incubated in a horizontal position 

at 32°C ± 1°C in a waterbath. 

Following incubation, the test substance, positive and negative controls were removed and 

the epithelial surface of the cornea washed, at least three times or until the wash medium 

(MEM with phenol red) was clear and there was no discolouration. The wash medium was

added via the holes on the top of the holder. After each wash, the wash medium was 

removed using a pipette tip attached to a vacuum pump. 

The anterior compartment was then filled with cMEM taking care to ensure no air bubbles 

were present in the compartment. Once all the air bubbles had been removed, the anterior 

compartment was re-plugged and the holders returned to the waterbath and incubated, in an 

upright position, for two hours ± 10 minutes at 32°C ± 1°C. 

Following completion of the two hour incubation period, the medium was removed from both 

compartments and replaced with fresh cMEM. The posterior compartment was re-plugged 

and the opacity of each cornea measured and recorded. The opacity values obtained at this 

stage were used in calculating the final In Vitro Irritancy Score. 

Throughout the assay the corneas were examined for opaque spots or other irregularities. Any 

atypical characteristics were noted in the workbook and the final report. 
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2.4.6 Permeability determination 

The sodium fluorescein stock solution (4 mg/ml) was thawed prior to commencing the 

permeability incubation. 

Following the final opacity measurement, the medium was removed from the anterior 

compartment of the holder. One ml (1ml) of the sodium fluorescein solution was added to 

the anterior compartment using a micropipette. 

Following addition of the sodium fluorescein solution to the anterior side of the holder, the 

compartment was plugged and the corneas incubated in a horizontal position at 32°C ± 1°C 

for 90 ± 5 minutes in a waterbath. 

Following incubation, the medium in the posterior compartment was mixed by drawing 

approximately 2.5 ml gently up and down a 5 ml syringe, with a needle attached, three times. 

An aliquot of the mixed medium from the posterior compartment was removed and 

transferred to a 1 cm path length cuvette. A spectrophotometer was adjusted to read at 

490nm (OD490) and a sample of cMEM read (OD = 0.065). The spectrophotometer was 

blanked using this solution prior to reading the permeability samples. Any solution giving an 

OD490 value above 1.8 was diluted 1 in 5 with cMEM. 

2.4.7 Criteria for determination of a valid test 

The positive control should elicit an In Vitro Irritancy Score that falls within two standard 

deviations of the historical mean for the laboratory. 

The negative control mean opacity change value should be ≤2.0 and the permeability mean 

value ≤0.1. 

2.5 Assessment of results 

2.5.1 Opacity 

The change in the opacity of each cornea was calculated by subtracting the initial basal 

opacity from the post-treatment opacity measurement. 

The mean change in opacity for the negative control corneas was calculated and was 

subtracted from the change in opacity of each treated cornea to obtain the corrected opacity 

value. 

The mean corrected opacity change value of each treatment group (of three corneas) was 

calculated from the individual corrected opacity values of the treated corneas. 

2.5.2 Permeability (OD490) 

The corrected permeability value (OD490) of each treated cornea was calculated by 

subtracting the mean negative control cornea value from the permeability value of each 

cornea. 

The mean corrected permeability value of each treatment group was calculated from the 

individual corrected permeability values of the treated corneas. 
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2.5.3 In Vitro Irritancy score calculation 

The In Vitro Irritancy Score (IVIS) was calculated using the following formula: 

 In Vitro Irritancy Score = Corrected Opacity Value + (15 x Corrected OD490 Value) 

The IVIS was calculated for each individual treatment and positive control cornea. The mean 

IVIS value for each treatment group was calculated from the IVIS of each individual cornea 

in the treatment group. 

2.5.4 Classification criteria 

A substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant. 

If the test substance is not identified as an ocular corrosive or severe irritant, additional 

testing (not part of this study) should be conducted for classification or labelling purposes. 

. 

IVIS In Vitro Classification 

≥ 55.1 Corrosive/Severe irritant 

< 55.1 Non-Corrosive/Severe irritant 

2.5.5 Archives 

Records and documentation relating to this study (including electronic records) 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. All raw data, samples and specimens 

arising from the performance of this study will remain the property of the Sponsor.

Types of sample and specimen that are unsuitable, by reason of instability, for long term 

retention and archiving may be disposed of after the periods stated in Huntingdon Life 

Sciences’ Standard Operating Procedures. 

All other samples and specimens and all raw data will be retained by Huntingdon Life 

Sciences in its archive for a period of one year from the date on which the Study Director 

signs the final report. After such time, the Sponsor will be contacted and his advice sought 

on the return, disposal or further retention of the materials. If requested, Huntingdon Life 

Sciences will continue to retain the materials subject to a reasonable fee being agreed with 

the Sponsor. 

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and 

a copy of the final report in its archive indefinitely. 

2.6 Deviations from protocol 

There were no deviations from the protocol. 
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3. Results 

3.1 Assay validity 

The positive control, ethanol, elicited an In Vitro Irritancy Score of 46.3 ± 6.6. This value 

was within the historical range (mean ± 2 x SD = 32.5 - 63.6) for the assays performed to 

date. 

The negative control, 0.9% saline, opacity mean change value was -1.333 which was below

the maximum acceptance value of 2.0. The permeability mean of the negative control was 

0.020 which was below the maximum acceptance value of 0.1. 

3.2 pH determination 

The test sample was diluted to 10% w/v with 0.9% saline to obtain an aqueous solution for 

pH measurement. The pH of the test sample, measured using universal pH sticks, was 

approximately 7.0.

3.3 BCOP results 

The study data is shown in Appendix 1. 

Throughout the assay the corneas were examined for opaque spots or other irregularities. 

Following treatment with test substance, HFCPE, the corneas were noted as clear. The 

corneas treated with the positive control, ethanol, were very opaque and the corneas treated 

with the negative control, 0.9% saline, were clear. The results of the BCOP assay are 

summarised in the table below. 

Sample Opacity 

± SD 

Permeability 

± SD 

In vitro irritancy 

Score 

± SD 

In vitro 

classification 

HFCPE 2.667 ± 0.577 0.021 ± 0.030 3.0 ± 1.0 Non Corrosive/Severe 

irritant 

Ethanol 29.667 ± 4.041 1.109 ±0.277 46.3 ± 6.6 Non Corrosive/Severe 

irritant 

0.9% Saline -1.333 ± 0.577 0.020 ± 0.008 Not applicable Not applicable 
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4. Conclusion 

The test substance, HFCPE, elicited an In Vitro Irritancy Score of 3.0 ± 1.0 and was predicted 

to be a non corrosive or severe eye irritant. 
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5. References 

Pierre Gautheron, Martine Dukic, Danielle Alix and Joseph F. Sina. Bovine Corneal Opacity 

and Permeability Test: An In Vitro Assay of Ocular Irritancy. Fundamental and Applied 

Toxicology 18: 442-449, 1992. 

OECD 437. 7 September 2009, OECD Guideline for the Testing of Chemicals: Bovine 

Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe 

Irritants. 
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Appendix 1 BCOP data 

SAMPLE: HFCPE

0.9% Sodium chloride solution (Negative control)

Opacity Permeability

Cornea Pre Post Change OD 490

7 5 3 -2 0.014

8 5 4 -1 0.017

9 5 4 -1 0.029

Mean change -1.333 Mean 0.020

SD 0.577 SD 0.008

Sample: HFCPE

Opacity Corrected Permeability

Cornea Pre Post Change change OD 490 Corrected

1 4 5 1 2.333 0.009 -0.011

2 3 5 2 3.333 0.068 0.048

3 6 7 1 2.333 0.047 0.027

Mean corrected change 2.667 Mean corrected OD 490 0.021

SD 0.577 SD 0.030

HFCPE

(Opacity + (15 x Corrected OD 490))

Cornea Opacity 15xOD490 In Vitro irritancy score

1 2.333 -0.165 2.168

2 3.333 0.720 4.053

3 2.333 0.405 2.738

Mean in vitro irritancy score 3.0

SD 1.0

SD = Standard Deviation

Data entered onto spreadsheet by:

In Vitro Irritancy Score of 

Note. Rounded values only are displayed, unrounded numbers are used for calculations by 

Excel spreadsheet. 
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Appendix 1 continued 

SAMPLE: Positive control ( ethanol)

0.9% Sodium chloride solution (Negative control)

Opacity Permeability

Cornea Pre Post Change OD 490

7 5 3 -2 0.014

8 5 4 -1 0.017

9 5 4 -1 0.029

Mean change -1.333 Mean 0.020

SD 0.577 SD 0.008

Sample: Positive control ( ethanol)

Opacity Corrected Permeability

Cornea Pre Post Change change OD 490 Corrected

4 6 30 24 25.333 0.926 0.906

5 4 33 29 30.333 1.444 1.424

6 3 35 32 33.333 1.016 0.996

Mean corrected change 29.667 Mean corrected OD 490 1.109

SD 4.041 SD 0.277

Positive control ( ethanol)

(Opacity + (15 x Corrected OD 490))

Cornea Opacity 15xOD490 In Vitro irritancy score

4 25.333 13.590 38.923

5 30.333 21.360 51.693

6 33.333 14.940 48.273

Mean in vitro irritancy score 46.3

SD 6.6

SD = Standard Deviation

In Vitro Irritancy Score of 

Note. Rounded values only are displayed, unrounded numbers are used for calculations by 

Excel spreadsheet. 
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Annex 1 Huntingdon Research Centre GLP compliance statement 2008