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### HFCPE
THE BOVINE CORNEAL OPACITY AND PERMEABILITY ASSAY (BCOP)

HLS study number: VHJ0004

Version ID: Final Report

Issue date: 8 January 2010Huntingdon Life Sciences VHJ0004

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## Sponsor and Test Facility Details

Sponsor Zeon Corporation Shin Marunouchi Center Building 1-6-2 Marunouchi Chiyoda-ku Tokyo 100-8246 JAPAN
Test facility Huntingdon Life Sciences Ltd Huntingdon Research Centre Woolley Road Alconbury Huntingdon Cambridgeshire PE28 4HS UKHuntingdon Life Sciences VHJ0004

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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........................................... ..................................................1 0 2.3.2 Preparation of cMEM.................................... .......................................10 2.3.3 Controls............................................

................................................... 11 2.3.4 Preparation and quality control of sodium fluorescein solutio n.............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 (OD 490 ) .................................................. ..........................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............................................

................................................... ..................18Huntingdon Life Sciences VHJ0004

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## List of Appendices

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

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



## List of Annexes

Annex 1 Huntingdon Research Centre GLP compliance statement 2008... ................21Huntingdon Life Sciences VHJ0004

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## Compliance with Good Laboratory PracticeHuntingdon Life Sciences VHJ0004

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## Quality Assurance StatementHuntingdon Life Sciences VHJ0004

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## Contributing Scientists

HFCPE: Bovine Corneal Opacity and Permeability assa y (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 TechnologiesHuntingdon Life Sciences VHJ0004

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## Summary

The Bovine Corneal Opacity and Permeability Assay (BCOP) was perfor med to assess the ocular irritancy potential in vitro of the test substance. Ethanol was tested in pa rallel as a positive control.
The assay uses isolated bovine corneas as a means of assessing the ocular corros ivity or severe irritancy potential of test substances in vitro. The isolated cornea s 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 Sc ore which can be used to classify and rank test substances as potential eye irritants accordin g 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.Huntingdon Life Sciences VHJ0004

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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 fluorescei n in an excised bovine cornea, in vitro

.



### 1.2 Test system
The test substance, HFCPE, was tested using the Bovine Cornea l 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 cornea s as a means of assessing the ocular irritancy potential of test substances in vitro.Huntingdon Life Sciences VHJ0004

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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 μ g/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 serumHuntingdon Life Sciences VHJ0004

11 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 flu orescein 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 μ l of Sodium Fluorescein stock + 950 μ l of cMEM b. 50 μ l of Dilution a. + 950 μ l 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 OD **490** 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 chamber s 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 rang e of 71- 79 (value=73). The other two calibrators were checked in the right hand calibration bl ock. Calibrator No. 2 fell within the range of 143-158 (value =155) and Calibrator No. 3 fell in t he range of 214-236 (value = 222). Once calibrated, the unit remained switched on for the duration of the assay.Huntingdon Life Sciences VHJ0004

12 2.3.6 Bovine eyes
Bovine eyes, excised by an abattoir employee, were collected as soon after s laughter 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 t ransported to the laboratory, at ambient temperature, in sufficient HBSS, containing 1% (v/v) Penicillin/Streptomycin solution, to cover all the eyes in the receptacle. Th e 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 (opa city, scratches, pigmentation, cuts, etc.) and those exhibiting defects were discarded. The tiss ue surrounding the eyeball was carefully pulled away and the cornea dissected leaving appr oximately 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 b een dissected. Once all the corneas had been dissected, they were rinsed in fresh HBS S 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 ha lf 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 bubble s 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 en sure no air bubbles were present within the holders. The posterior compartment was then plugged a nd the basal opacity measurements performed. 2.4.2 Opacity measurement
The opacitometer measured the light transmission through the centre of each mounte d cornea, displaying a numerical opacity value (arbitrary unit). The opacity of eac h cornea was measured by reading each holder in the right-hand chamber of the calibrated opacit ometer. Once the basal opacity of all the corneas had been measured, the mean basal opacity v alue was calculated. Corneas with an opacity value greater than 7 units were discar ded. The mean basal opacity value of the corneas was then calculated and three cornea s with opacity values close to the mean value were chosen for use as negative control corneas.Huntingdon Life Sciences VHJ0004

13 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 c ontrol (ethanol) or negative control (0.9% sodium chloride solution).
The controls and their results were shared with several studies performed in t he same assay.

2.4.5 Treatment of corneas
The test substance, HFCPE, a colourless liquid, and the positive control (ethanol) wer e tested undiluted.
Immediately prior to treatment, the medium was removed from the anterior compa rtment 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 μ l (750 μ l) 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 subst ance over the surface of the cornea. The test material or controls were in contact w ith 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 remov ed and the epithelial surface of the cornea washed, at least three times or until the w ash 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 r emoved, the anterior compartment was re-plugged and the holders returned to the waterbath a nd 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 t his stage were used in calculating the final In Vitro Irritancy Score.
Throughout the assay the corneas were examined for opaque spots or other irregularit ies. Any atypical characteristics were noted in the workbook and the final report.Huntingdon Life Sciences VHJ0004

14 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 anter ior 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 t imes. 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 rea d at 490nm (OD **490** ) and a sample of cMEM read (OD = 0.065). The spectrophotometer was blanked using this solution prior to reading the permeability samples. Any solution gi ving an OD **490** 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 w ithin 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 a nd was subtracted from the change in opacity of each treated cornea to obtain the correcte d opacity value.
The mean corrected opacity change value of each treatment group (of three c orneas) was calculated from the individual corrected opacity values of the treated corneas

. 2.5.2 Permeability (OD 490 )
The corrected permeability value (OD **490** ) of each treated cornea was calculated by subtracting the mean negative control cornea value from the permeability va lue of each

cornea. The mean corrected permeability value of each treatment group was calcul ated from the individual corrected permeability values of the treated corneas.Huntingdon Life Sciences VHJ0004

15 2.5.3 In Vitro Irritancy score calculation
The In Vitro Irritancy Score (IVIS) was calculated using the followin g formula:
In Vitro Irritancy Score = Corrected Opacity Value + (15 x Correc ted OD **490** 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 eac h 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 irrita nt, additional testing (not part of this study) should be conducted for classification or labelli ng 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 spec imens 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 l ong term retention and archiving may be disposed of after the periods stated in Huntingdon Li fe Sciences’ Standard Operating Procedures.
All other samples and specimens and all raw data will be retained by Huntingdon L ife 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 sou ght on the return, disposal or further retention of the materials. If requested, Hunting don 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 releva nt 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.Huntingdon Life Sciences VHJ0004

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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 t o 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 w as 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, w as 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 irregularit ies. Following treatment with test substance, HFCPE, the corneas were not ed 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 as say 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/Sev ere irritant
Ethanol 29.667 ± 4.041 1.109 ±0.277 46.3 ± 6.6 Non C orrosive/Severe irritant 0.9% Saline -1.333 ± 0.577 0.020 ± 0.008 Not applicable No t applicableHuntingdon Life Sciences VHJ0004

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## 4. Conclusion

The test substance, HFCPE, elicited an In Vitro Irritancy Score of 3.0 ± 1.0 and w as predicted to be a non corrosive or severe eye irritant.Huntingdon Life Sciences VHJ0004

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## 5. References

Pierre Gautheron, Martine Dukic, Danielle Alix and Joseph F. Sina. Bovine Corneal Opa city and Permeability Test: An In Vitro Assay of Ocular Irritancy. Fundame ntal 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 Cor rosives and Severe Irritants.Huntingdon Life Sciences VHJ0004

19 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 0.02 1

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.Huntingdon Life Sciences VHJ0004

20 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 1.1 09

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.Huntingdon Life Sciences VHJ0004

21 Annex 1 Huntingdon Research Centre GLP compliance st atement 2008